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Greaney AJ, McCarthy CM, Vethil JP, Abubaker M, Reardon EC, Crowley FD, Cunnane EM, Mulvihill JJE. A comprehensive protocol for PDMS fabrication for use in cell culture. PLoS One 2025; 20:e0323283. [PMID: 40354467 PMCID: PMC12068733 DOI: 10.1371/journal.pone.0323283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2024] [Accepted: 04/04/2025] [Indexed: 05/14/2025] Open
Abstract
Cells exhibit remarkable sensitivity to the mechanical properties of their surrounding matrix, particularly stiffness changes, a phenomenon known as cellular mechanotransduction. In vivo, tissues exhibit a wide range of stiffness, from kilopascals (kPa) to megapascals (MPa), which can alter with aging and disease. Traditional cell culture methods employ plastic substrates with stiffness in the gigapascal range, which does not accurately mimic the physiological conditions of most biological tissues. Therefore, employing substrates that can be engineered to span a wide range of stiffnesses, closely resembling the native tissue environment, is crucial for obtaining results that more accurately reflect cellular responses in vivo. Polydimethylsiloxane (PDMS) substrates are widely used in cell culture for their ability to simulate tissue stiffness, but their optimization presents several challenges. Fabrication requires precise control over mixing, weighing, and curing to ensure reproducible mechanical properties. Inconsistent preparation can lead to improperly cured PDMS substrates, compromising experimental outcomes. Additionally, PDMS's inherent hydrophobicity poses challenges for cell attachment, necessitating surface modifications to enhance adhesion. Moreover, the risk of contamination during the sterilization process necessitates stringent protocols to maintain cell culture integrity. These challenges are further compounded by substrate autofluorescence which can cause difficulties when imaging cells. The aim of this study is to develop a standardized method for fabricating PDMS substrates with tuneable stiffness, ranging from kPa to MPa, suitable for diverse cell types using standard laboratory equipment. This method aims to minimize the complexity and equipment required for PDMS fabrication, ensuring reproducibility and ease of use. Achieving consistent and contaminant-free PDMS substrates will facilitate a broader adoption of these substrates in mechanobiology research and improve the relevance of in vitro models to in vivo conditions. Ultimately, contributing to a more comprehensive understanding of cellular responses to mechanical cues in health and disease.
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Affiliation(s)
- Aisling J. Greaney
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Clíona M. McCarthy
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Jishnu Padacherri Vethil
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Mannthalah Abubaker
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Erin C. Reardon
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Frederick D. Crowley
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - Eoghan M. Cunnane
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
| | - John J. E. Mulvihill
- School of Engineering, University of Limerick, Limerick, Ireland
- Bernal Institute, University of Limerick, Limerick, Ireland
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Malysz-Cymborska I, Golubczyk D, Walczak P, Stanaszek L, Janowski M. Injectable, Manganese-Labeled Alginate Hydrogels as a Matrix for Longitudinal and Rapidly Retrievable 3D Cell Culture. Int J Mol Sci 2025; 26:4574. [PMID: 40429719 PMCID: PMC12110870 DOI: 10.3390/ijms26104574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2025] [Revised: 04/30/2025] [Accepted: 05/01/2025] [Indexed: 05/29/2025] Open
Abstract
Hydrogels are one of the most attractive biomaterials, used in both three-dimensional (3D) and in vivo cultures. They facilitate the reconstruction of tissue microenvironments by preserving the spatial arrangement of cells, cell-cell interactions, and functional dynamics in the tissue. In this work, the long-term effect of alginate hydrogel on cell culture and the possibility of rapid cell recovery by dissolving the hydrogel were investigated. Mouse glial-restricted progenitors (GRPs) and porcine mesenchymal stem cells (MSCs) were suspended in hydrogels; their metabolic activity, viability, and expression of genes, which are involved in oxidative stress, apoptosis, proliferation, migration, and differentiation, were assessed using quantitative polymerase chain reaction (qPCR). The concentration that was able to dissolve the hydrogel and was the least harmful to the cells was 0.005 M ethylenediaminetetraacetic acid (EDTA). The metabolism of both cell types was reduced from the beginning of the experiment to day 3. From day 7 to the end of the experiment, the normalization of the GRP metabolism was observed, in contrast to the MSCs. For the apoptosis-related genes, caspase 3, 7, and B-cell leukemia (Casp3, Casp 7, Bcl2) were increased in GRPs and MSCs on days 0 and 1. After 3 and 7 days, an increase in the expression of oxidative stress genes (nuclear factor of activated T-cells 5-NFAT5 and autophagy-related 14-ATG14) was observed in cells cultured in calcium chloride (CaCl2). GRPs cultured in calcium alginate (CaM) were not affected and, remarkably, showed increased Antigen Kiel 67 (Ki67) levels after 30 days. In conclusion, alginate hydrogels provide an excellent environment for stem cell culture in 3D for a longer period of time, but this is dependent on the cell type. Therefore, an individual approach to cell culture is necessary, taking into account the requirements of the cells to be used.
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Affiliation(s)
- Izabela Malysz-Cymborska
- Department of Neurology and Neurosurgery, School of Medicine, Collegium Medicum, University of Warmia and Mazury in Olsztyn, Warszawska 30, 10-082 Olsztyn, Poland
| | | | - Piotr Walczak
- Program in Image Guided Neurointerventions, Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, 670 W. Baltimore Street, Baltimore, MD 21201, USA; (P.W.); (M.J.)
| | - Luiza Stanaszek
- NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland;
| | - Miroslaw Janowski
- Program in Image Guided Neurointerventions, Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, 670 W. Baltimore Street, Baltimore, MD 21201, USA; (P.W.); (M.J.)
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3
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Meyer LO, Jérôme V, Freitag R. Custom FDM-based bioprinter with heated nozzle: optimizing slicer settings for precision printing using a print quality index. Biomed Mater 2025; 20:035030. [PMID: 40300619 DOI: 10.1088/1748-605x/add230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 04/29/2025] [Indexed: 05/01/2025]
Abstract
Bioprinting of microtissues has become a standard technique in medical and biotechnological research, offering a more accurate replication of thein vivosetting than conventional 2D cell culture. However, widespread adoption is limited by the absence of a universally accepted printing benchmark-common in standard fused deposition modeling (FDM) printing, as well as the high cost and restricted customizability of commercial bioprinters. This study introduces a method to convert a standard FDM printer into a bioprinter. All cell-contacting components are biocompatible and autoclavable, while the printer body can be UV-sanitized. Using a heated FDM printhead, we used the thermal properties of alginate-gelatin bioinks to achieve high-resolution 3D printing. A key achievement was the developed print quality index (PQI) method, which correlates nozzle temperature with bioink flow behavior, streamlining optimization of slicer settings. Guided by PQI, we reproducibly bioprinted complex alginate-gelatin structures with high quality and dimensional/geometric accuracy. A case study using recombinant HuH7EGFPcell-laden hydrogels demonstrated long-term cell proliferation, confirming high viability. Given its efficiency, the PQI method has the potential to become the missing printing benchmark for slicer optimization in bioprinting. The presented approach significantly advances the accessibility of sophisticated bioprinting technology to interested research groups worldwide.
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Affiliation(s)
- Leif O Meyer
- Process Biotechnology, University of Bayreuth, Germany
| | | | - Ruth Freitag
- Process Biotechnology, University of Bayreuth, Germany
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4
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Sapkota T, Shrestha S, Regmi BP, Bhattarai N. Fabrication of cell-laden hydrogel microcapsules of alginate and chitin fibrils using divalent and trivalent metal ions. RSC Adv 2025; 15:12876-12895. [PMID: 40264879 PMCID: PMC12013471 DOI: 10.1039/d5ra01397f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2025] [Accepted: 04/16/2025] [Indexed: 04/24/2025] Open
Abstract
Nanofiber-embedded 3D hydrogel constructs have garnered significant attention due to their versatile applications in drug delivery, cell therapy, tissue engineering, and regenerative medicine. These constructs are especially prized for their capacity to mimic the composition of the extracellular matrix (ECM) found in living tissues and organs. The unique chemical and mechanical properties of hydrogel microcapsules have made them particularly notable among various biomaterial constructs for their effectiveness in cell encapsulation, which aims to improve cell growth and proliferation. In this study, we developed alginate hydrogel microcapsules embedded with chitin nanofibrils, using divalent calcium ions and trivalent iron ions as crosslinking agents. An electrostatic encapsulation technique was utilized to create microcapsules with diameters ranging from 200-500 μm, and their physicochemical properties, rheological properties, size, and mechanical stability were evaluated. The rheological analysis demonstrated that the Fe3+ crosslinked hydrogel (AF0) and Fe3+/Ca2+ cross-linked hydrogel (AFC) have higher storage modulus than the Ca2+ crosslinked hydrogel (AC0). Additionally, FTIR analyses of AF0 and AFC demonstrated a broader -O-H stretching peak compared to that of AC0, suggesting that more hydroxyl groups of alginate chains are involved in crosslinking with ferric ions exhibiting extended mechanical stability compared to those crosslinked with calcium ions under in vitro physiological conditions. We also investigated the cellular responses to the composite hydrogels crosslinked with these divalent and trivalent metal ions through in vitro studies involving the seeding and encapsulation of NIH/3T3 fibroblast cells. Remarkably, both types of crosslinked microcapsules maintained excellent cell viability for up to 5 days. Our in vitro scratch assay demonstrated that media extracted from AF0 microcapsules facilitated faster wound closure compared to that extracted from AC0, suggesting that hydrogels crosslinked with Fe3+ ions promote enhanced cellular proliferation. These results suggest that calcium and ferric ion crosslinked alginate-chitin composite microcapsules provide a promising platform for developing 3D hydrogel constructs suitable for various biomedical applications, including wound healing models, tissue engineering, and drug toxicity testing.
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Affiliation(s)
- Thakur Sapkota
- Department of Applied Science and Technology, North Carolina A&T State University Greensboro NC 27411 USA
- Department of Chemical, Biological, and Bioengineering, North Carolina A&T State University Greensboro NC 27411 USA
| | - Sita Shrestha
- Department of Chemical, Biological, and Bioengineering, North Carolina A&T State University Greensboro NC 27411 USA
| | - Bishnu P Regmi
- Department of Chemistry, Florida Agricultural and Mechanical University Tallahassee FL 32307 USA
| | - Narayan Bhattarai
- Department of Applied Science and Technology, North Carolina A&T State University Greensboro NC 27411 USA
- Department of Chemical, Biological, and Bioengineering, North Carolina A&T State University Greensboro NC 27411 USA
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5
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Gantert B, Karakaya E, Hofmann F, Jungst T, Meinel L, Bosserhoff AK, Detsch R, Lühmann T. Alginate-Dialdehyde-Based Reporter Ink Enabling Online Detection of Matrix Metalloproteinase Activity of Encapsulated Cells. ACS Biomater Sci Eng 2025; 11:2435-2447. [PMID: 40052617 DOI: 10.1021/acsbiomaterials.4c02399] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/15/2025]
Abstract
Biofabrication and three-dimensional (3D) bioprinting enable precise spatial arrangement of cells within biomaterial scaffolds. We developed an alginate-based and Förster resonance energy transfer (FRET)-responsive "turn-on" reporter ink platform to enable real-time monitoring of matrix metalloproteinase (MMP) activity. Three distinct MMP-cleavable turn-on peptide reporters were synthesized and characterized for their cell-specific cleavage profiles using recombinant MMPs, cell-derived media, and different cell cultures (NIH3T3, HEK293, and MelHo). All turn-on reporters were covalently and site-specifically incorporated into alginate dialdehyde (ADA) to yield an MMP reporter ink. The ADA reporter ink with an MMP 13 turn-on reporter was responsive to all tested cell types over time within the cast bulk constructs. The ADA reporter ink material blended with gelatin had comparable print resolution and structural fidelity as observed for ADA. The extrusion-based bioprinted MelHo cell grids, measuring 2 × 2 cm2 and containing 1 × 106 cells/mL, exhibited MMP activity responses comparable to those of the casted reporter ink system, with a 3-fold increase observed at 24 h. This study introduces a versatile, FRET-based alginate bioink platform for the real-time monitoring of MMP activities, expanding the toolkit to understand cellular performance in bioprinted 3D constructs.
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Affiliation(s)
- Benedikt Gantert
- Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
| | - Emine Karakaya
- Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Ulrich-Schalk-Str. 3, 91056 Erlangen, Germany
| | - Florian Hofmann
- Department for Functional Materials in Medicine and Dentistry, Institute of Functional Materials and Biofabrication, University of Würzburg and KeyLab Polymers for Medicine of the Bavarian Polymer Institute (BPI), 97070 Würzburg, Germany
| | - Tomasz Jungst
- Department for Functional Materials in Medicine and Dentistry, Institute of Functional Materials and Biofabrication, University of Würzburg and KeyLab Polymers for Medicine of the Bavarian Polymer Institute (BPI), 97070 Würzburg, Germany
| | - Lorenz Meinel
- Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
- Helmholtz Institute for RNA-Based Infection Research (HIRI), Helmholtz Center for Infection Research (HZI), 97080 Würzburg, Germany
| | - Anja K Bosserhoff
- Institute of Biochemistry, Emil-Fischer-Zentrum, University of Erlangen-Nuremberg, Fahrstrasse 17, 91054 Erlangen, Germany
| | - Rainer Detsch
- Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Ulrich-Schalk-Str. 3, 91056 Erlangen, Germany
| | - Tessa Lühmann
- Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
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Shi A, Shi Y, Li J, Ye M, Ma X, Peng Y, Gai K, Chen J. Advancements in 3D gel culture systems for enhanced angiogenesis in bone tissue engineering. J Mater Chem B 2025; 13:3516-3527. [PMID: 39998426 DOI: 10.1039/d4tb01139b] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/26/2025]
Abstract
Angiogenesis-osteogenesis coupling is a crucial process in bone tissue engineering, requiring a suitable material structure for vessel growth. Recently, the 3D culture system has gained significant attention due to its benefits in cell growth, proliferation and tissue regeneration. Its most notable advantage is its ECM-like function, which supports endothelial cell adhesion and facilitates the formation of vascular-like networks-crucial for angiogenesis-osteogenesis coupling. Hydrogels, with their highly hydrophilic polymer network resembling the extracellular matrix, make the 3D gel culture system an ideal approach for angiogenesis due to its cellular integrity and adjustable properties. This article reviews the current use of 3D gel culture systems in bone tissue engineering, covering substrates, characteristics and processing technologies, thereby offering readers profound insights into these systems.
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Affiliation(s)
- Aijing Shi
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Yixin Shi
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Jie Li
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Minghan Ye
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Xiaoqing Ma
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Yuke Peng
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
| | - Kuo Gai
- Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province, Hangzhou, 310000, China.
| | - Junyu Chen
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, China.
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7
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Martinet A, Miebach L, Weltmann K, Emmert S, Bekeschus S. Biomimetic Hydrogels - Tools for Regenerative Medicine, Oncology, and Understanding Medical Gas Plasma Therapy. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2025; 21:e2403856. [PMID: 39905967 PMCID: PMC11878268 DOI: 10.1002/smll.202403856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Revised: 01/23/2025] [Indexed: 02/06/2025]
Abstract
Biomimetic hydrogels enable biochemical, cell biology, and tissue-like studies in the third dimension. Smart hydrogels are also frequently used in tissue engineering and as drug carriers for intra- or extracutaneous regenerative medicine. They have also been studied in bio-sensor development, 3D cell culture, and organoid growth optimization. Yet, many hydrogel types, adjuvant components, and cross-linking methods have emerged over decades, diversifying and complexifying such studies. Here, an evaluative overview is provided, mapping potential applications to the corresponding hydrogel tuning. Strikingly, hydrogels are ideal for studying locoregional therapy modalities, such as cold medical gas plasma technology. These partially ionized gases produce various reactive oxygen species (ROS) types along with other physico-chemical components such as ions and electric fields, and the spatio-temporal effects of these components delivered to diseased tissues remain largely elusive to date. Hence, this work outlines the promising applications of hydrogels in biomedical research in general and cold plasma science in particular and underlines the great potential of these smart scaffolds for current and future research and therapy.
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Affiliation(s)
- Alice Martinet
- Department of Dermatology and VenerologyRostock University Medical CenterStrempelstr. 1318057RostockGermany
- ZIK plasmatisLeibniz Institute for Plasma Science and Technology (INP)Felix‐Hausdorff‐Str. 217489GreifswaldGermany
| | - Lea Miebach
- ZIK plasmatisLeibniz Institute for Plasma Science and Technology (INP)Felix‐Hausdorff‐Str. 217489GreifswaldGermany
| | - Klaus‐Dieter Weltmann
- Department of Dermatology and VenerologyRostock University Medical CenterStrempelstr. 1318057RostockGermany
| | - Steffen Emmert
- Department of Dermatology and VenerologyRostock University Medical CenterStrempelstr. 1318057RostockGermany
| | - Sander Bekeschus
- Department of Dermatology and VenerologyRostock University Medical CenterStrempelstr. 1318057RostockGermany
- ZIK plasmatisLeibniz Institute for Plasma Science and Technology (INP)Felix‐Hausdorff‐Str. 217489GreifswaldGermany
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Pooja N, Ahmed NY, Mal SS, Bharath PAS, Zhuo GY, Noothalapati H, Managuli V, Mazumder N. Assessment of biocompatibility for citric acid crosslinked starch elastomeric films in cell culture applications. Sci Rep 2025; 15:6427. [PMID: 39984659 PMCID: PMC11845471 DOI: 10.1038/s41598-025-90933-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2024] [Accepted: 02/17/2025] [Indexed: 02/23/2025] Open
Abstract
This study investigates the synthesis of potato starch elastomers reinforced with silicon dioxide (SiO2) and citric acid as a crosslinking agent to enhance their mechanical and barrier properties. Surface morphology analysis using optical microscopy revealed that pure potato starch films had uneven surfaces. However, higher SiO2 concentrations increased roughness, while citric acid crosslinked films displayed smoother surfaces overall. Water vapor transmission rates (WVTR) indicated that native starch films were highly hydrophilic, while SiO2 incorporation and citric acid crosslinking significantly reduced WVTR of 17% (30% lower than native film), enhancing the barrier properties. Tensile strength testing revealed that citric acid crosslinking increased the tensile strength by 25%, while SiO2 further reinforced the films but decreased elasticity by 15%. SiO2 had little impact on degradation rates, while citric acid crosslinking delayed microbial growth, extending film longevity by 20%. Biocompatibility assays using SiHa, HT-29, and HEK 293 cell lines revealed that the films had varying degrees of cell confluency. Films with both SiO2 and citric acid showed improved confluency (20% higher) compared to films containing only SiO2. However, citric acid alone resulted in the highest confluency (95% viability), suggesting its significant role in biocompatibility. This eco-friendly approach demonstrates substantial advancements in film properties, offering potential applications in diverse biomedical industries.
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Affiliation(s)
- N Pooja
- Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India
| | - Nafisa Yeshmin Ahmed
- Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India
| | - Sib Sankar Mal
- Department of Chemistry, National Institute of Technology, Suratkal, 575025, Karnataka, India
| | - Prasad A S Bharath
- Department of Public Health Genomics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India
| | - Guan-Yu Zhuo
- Institute of Biophotonics, National Yang Ming Chiao Tung University, Taipei, 11221, Taiwan
| | - Hemanth Noothalapati
- Department of Biomedical Engineering, Chennai Institute of Technology, Chennai, 600069, Tamil Nadu, India
- Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, 502285, Telangana, India
- Faculty of Life and Environmental Sciences, Shimane University, 1060 Nishikawatsu-Cho, Matsue, 690- 8504, Japan
| | - Vishwanath Managuli
- Department of Mechanical and Manufacturing Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India
| | - Nirmal Mazumder
- Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.
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Zhang Y, Ding X, Yang Z, Wang J, Li C, Zhou G. Emerging Microfluidic Building Blocks for Cultured Meat Construction. ACS APPLIED MATERIALS & INTERFACES 2025; 17:8771-8793. [PMID: 39884858 DOI: 10.1021/acsami.4c19276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/01/2025]
Abstract
Cultured meat aims to produce meat mass by culturing cells and tissues based on the muscle regeneration mechanism, and is considered an alternative to raising and slaughtering livestock. Hydrogel building blocks are commonly used as substrates for cell culture in tissue engineering and cultured meat because of their high water content, biocompatibility, and similar three-dimensional (3D) environment to the cellular niche in vivo. With the characteristics of precise manipulation of fluids, microfluidics exhibits advantages in the fabrication of building blocks with different structures and components, which have been widely applied in tissue regeneration. Microfluidic building blocks show promising prospects in the field of cultured meat; however, few reviews on the application of microfluidic building blocks in cultured meat have been published. This review outlines the recent status and prospects of the use of microfluidic building blocks in cultured meat. Starting with the introduction of cells and materials for cultured meat tissue construction, we then describe the diverse structures of the fabricated building blocks, including microspheres, microfibers, and microsphere-microfiber hybrid systems. Next, the stacking strategies for tissue construction are highlighted in detail. Finally, challenges and future prospects for developing microfluidic building blocks for cultured meat are discussed.
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Affiliation(s)
- Yue Zhang
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
| | - Xi Ding
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
| | - Zijiang Yang
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
| | - Jie Wang
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
| | - Chunbao Li
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
| | - Guanghong Zhou
- State Key Laboratory of Meat Quality Control and Cultured Meat Development, Key Laboratory of Meat Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
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10
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Soltanmohammadi F, Mahmoudi Gharehbaba A, Alizadeh E, Javadzadeh Y. Innovative approaches to tissue engineering: Utilizing decellularized extracellular matrix hydrogels for mesenchymal stem cell transport. Int J Biol Macromol 2025; 290:138893. [PMID: 39706433 DOI: 10.1016/j.ijbiomac.2024.138893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 12/07/2024] [Accepted: 12/16/2024] [Indexed: 12/23/2024]
Abstract
In recent years, the realm of tissue regeneration experienced significant advancements, leading to the development of innovative therapeutic agents. The systemic delivery of mesenchymal stem cells (MSCs) emerged as a promising strategy for promoting tissue regeneration. However, this approach is hindered by hurdles such as poor cell survival, limited cell propagation, and inadequate cell integration. Decellularized extracellular matrix (dECM) hydrogel serves as an innovative carrier that protects MSCs from the detrimental effects of the hostile microenvironment, facilitates their localization and retention at the injection site, and preserves their viability. Regarding its low immunogenicity, low cytotoxicity, high biocompatibility, and its ability to mimic natural extracellular matrix (ECM), this natural hydrogel offers a new avenue for systemic delivery of MSCs. This review digs into the properties of dECM hydrogels (dECMHs), the methods employed for decellularization and the utilization of dECMH as carriers for various types of MSCs for tissue regeneration purposes. This review also sheds light on the benefits of hybrid hydrogels composed of dECMH and other components such as proteins and polysaccharides. By addressing the limitations of conventional hydrogels and enhancing efficacy of cell therapy, dECMH opens new pathways for the future of tissue regeneration.
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Affiliation(s)
- Fatemeh Soltanmohammadi
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Adel Mahmoudi Gharehbaba
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Effat Alizadeh
- Endocrin Research Center and Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
| | - Yousef Javadzadeh
- Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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11
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Na YJ, Choi KJ, Jung WH, Park SB, Koh B, Hoe KL, Kim KY. Development of 3D Muscle Cell Culture-Based Screening System for Metabolic Syndrome Drug Research. Tissue Eng Part C Methods 2025; 31:53-64. [PMID: 39912898 DOI: 10.1089/ten.tec.2024.0292] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2025] Open
Abstract
Developing effective drug screening methods for type 2 diabetes requires physiologically relevant models. Traditional 2D cell cultures have limitations in replicating in vivo conditions, leading to challenges in assessing drug efficacy. To overcome these issues, we developed a 3D artificial muscle model that induces insulin resistance, a hallmark of type 2 diabetes. Using C2C12 myoblasts cultured in a scaffold of 1% alginate and 1 mg/mL collagen type 1, we optimized conditions for differentiation and structural stability. Insulin resistance was induced using palmitic acid (PA), and glucose uptake was assessed using the fluorescent glucose analog 2-NBDG. The 3D model demonstrated superior glucose uptake responses compared with 2D cultures, with a threefold increase in insulin-stimulated glucose uptake on days 4 and 8 of differentiation. Induced insulin resistance was observed with 0.1 mM PA, which maintained cell viability and differentiation capacity. The model was validated through comparative drug screening using rosiglitazone and metformin, as well as 165 candidate compounds provided by Korea Chemical Bank. Drug screening revealed that three out of five hit compounds identified in both 2D and 3D models exhibited greater efficacy in 3D cultures, with results consistent with ex vivo assays using mouse soleus muscle. This model closely mimics in vivo conditions, offering a robust platform for type 2 diabetes drug discovery while supporting ethical research practices.
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Affiliation(s)
- Yoon-Ju Na
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
| | - Kyoung Jin Choi
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
| | - Won Hoon Jung
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
| | - Sung Bum Park
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
| | - Byumseok Koh
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
| | - Kwang-Lae Hoe
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
| | - Ki Young Kim
- Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea
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12
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Palacios PA, Flores I, Cereceda L, Otero FF, Müller M, Brebi P, Contreras HR, Carreño LJ. Patient-Derived Organoid Models for NKT Cell-Based Cancer Immunotherapy. Cancers (Basel) 2025; 17:406. [PMID: 39941775 PMCID: PMC11815936 DOI: 10.3390/cancers17030406] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2024] [Revised: 01/22/2025] [Accepted: 01/24/2025] [Indexed: 02/16/2025] Open
Abstract
Invariant Natural Killer T (iNKT) cells are a unique subset of T cells that bridge innate and adaptive immunity, displaying potent anti-tumor properties through cytokine secretion, direct cytotoxicity, and recruitment of immune effector cells such as CD8+ T cells and NK cells. Despite their therapeutic potential, the immunosuppressive tumor microenvironment (TME), characterized by regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), limits iNKT cell efficacy. Patient-derived organoid (PDO) platforms provide an innovative model for dissecting these complex interactions and evaluating strategies to reinvigorate iNKT cell functionality within the TME. PDOs closely mimic the genetic, phenotypic, and structural characteristics of primary tumors, enabling the study of tumor-immune dynamics. Integrating iNKT cells into PDOs offers a robust platform for investigating CD1d-mediated interactions, Th1-biased immune responses driven by glycolipid analogs like α-GalCer, and combination therapies such as immune checkpoint inhibitors. Additionally, PDO systems can assess the effects of metabolic modulation, including reducing lactic acid accumulation or targeting glutamine pathways, on enhancing iNKT cell activity. Emerging innovations, such as organoid-on-a-chip systems, CRISPR-Cas9 gene editing, and multi-omics approaches, further expand the potential of PDO-iNKT platforms for personalized immunotherapy research. Although the application of iNKT cells in PDOs is still undeveloped, these systems hold immense promise for bridging preclinical studies and clinical translation. By addressing the challenges of the TME and optimizing therapeutic strategies, PDO-iNKT platforms offer a transformative avenue for advancing cancer immunotherapy and personalized medicine.
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Affiliation(s)
- Pablo A. Palacios
- Millennium Institute on Immunology and Immunotherapy, Programa de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
| | - Iván Flores
- Department of Basic and Clinical Oncology, Faculty of Medicine, Universidad de Chile, Santiago 8350499, Chile
| | - Lucas Cereceda
- Millennium Institute on Immunology and Immunotherapy, Programa de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
| | - Francisco F. Otero
- Millennium Institute on Immunology and Immunotherapy, Programa de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
| | - Marioly Müller
- Departamento de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
| | - Priscilla Brebi
- Millennium Institute on Immunology and Immunotherapy, Laboratory of Integrative Biology (LIBi), Centro de Excelencia en Medicina Traslacional (CEMT), Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco 4811230, Chile
- Biomedical Research Consortium (BMRC), Santiago 8331150, Chile
| | - Héctor R. Contreras
- Department of Basic and Clinical Oncology, Faculty of Medicine, Universidad de Chile, Santiago 8350499, Chile
- Center for Cancer Prevention and Control (CECAN), Santiago 8350499, Chile
| | - Leandro J. Carreño
- Millennium Institute on Immunology and Immunotherapy, Programa de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
- Biomedical Research Consortium (BMRC), Santiago 8331150, Chile
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13
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Wood N, Doria EI, Rahman TT, Li W, Pei Z, Qin H. Effects of Calcium Chloride Crosslinking Solution Concentration on the Long-Term Cell Viability of 16HBE14o- Human Bronchial Cells Embedded in Alginate-Based Hydrogels. Biomimetics (Basel) 2025; 10:40. [PMID: 39851756 PMCID: PMC11763177 DOI: 10.3390/biomimetics10010040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2024] [Revised: 12/20/2024] [Accepted: 01/07/2025] [Indexed: 01/26/2025] Open
Abstract
In this preliminary study, the long-term effects of calcium chloride crosslinking concentration on viability of 16HBE14o- human bronchial epithelial cells embedded in alginate-extracellular matrix (ECM) or alginate-methylcellulose-ECM hydrogels have been investigated. There is currently a limited understanding regarding the effects of crosslinking solution concentration on lung epithelial cells embedded in hydrogel. Furthermore, the effects of calcium chloride concentration in crosslinking solutions on other cell types have not been reported regarding whether the addition of viscosity and stiffness tuning agents such as methylcellulose will alter the responses of cells to changes in calcium chloride concentration in crosslinking solutions. While there were no significant effects of calcium chloride concentration on cell viability in alginate-ECM hydrogels, there is a decrease in cell viability in alginate-methylcellulose-ECM hydrogels crosslinked with 300 mM calcium chloride crosslinking solution. These findings have implications in the maintenance of 16HBE14o- 3D cultures with respect to the gelation of alginate with high concentrations of ionic crosslinking solution.
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Affiliation(s)
- Nathan Wood
- Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA; (E.I.D.); (W.L.)
| | - Esther I. Doria
- Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA; (E.I.D.); (W.L.)
| | - Taieba Tuba Rahman
- Department of Industrial & Systems Engineering, Texas A&M University, 3127 TAMU, College Station, TX 77843, USA; (T.T.R.); (Z.P.)
| | - Wanhe Li
- Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA; (E.I.D.); (W.L.)
| | - Zhijian Pei
- Department of Industrial & Systems Engineering, Texas A&M University, 3127 TAMU, College Station, TX 77843, USA; (T.T.R.); (Z.P.)
| | - Hongmin Qin
- Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA; (E.I.D.); (W.L.)
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14
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Jeong W, Son J, Choi J, Han J, Jeon S, Kim MK, Ha W, Kang HW. Clinically Relevant and Precisely Printable Live Adipose Tissue-Based Bio-Ink for Volumetric Soft Tissue Reconstruction. Adv Healthc Mater 2025; 14:e2402680. [PMID: 39466900 DOI: 10.1002/adhm.202402680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Revised: 09/12/2024] [Indexed: 10/30/2024]
Abstract
Autologous fat is widely used in soft tissue reconstruction; however, significant volume reduction owing to necrosis and degradation of the transplanted adipose tissue (AT) remains a major challenge. To address this issue, a novel live AT micro-fragment-based bio-ink (ATmf bio-ink) compatible with precision 3D printing, is developed. Live AT micro-fragments of ≈280 µm in size are prepared using a custom tissue micronizer and they are incorporated into a fibrinogen/gelatin mixture to create the ATmf bio-ink. AT micro-fragments exhibit high viability and preserve the heterogeneous cell population and extracellular matrix of the native AT. The developed bio-ink enables precise micropatterning and provides an excellent adipo-inductive microenvironment. AT grafts produced by co-printing the bio-ink with polycaprolactone demonstrate a 500% improvement in volume retention and a 300% increase in blood vessel infiltration in vivo compared with conventional microfat grafts. In vivo engraftment of AT grafts is further enhanced by using a stem cell-laden ATmf bio-ink. Last, it is successfully demonstrated that the bio-ink is enabled for the creation of clinically relevant and patient-specific AT grafts for patients undergoing partial mastectomy. This novel ATmf bio-ink for volumetric soft tissue reconstruction offers a pioneering solution for addressing the limitations of existing clinical techniques.
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Affiliation(s)
- Wonwoo Jeong
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - Jeonghyun Son
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA
| | - Jeonghan Choi
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
| | - Jonghyeuk Han
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
- Wallace H. Coulter Department of Biomedical Engineering, Emory University School of Medicine & Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Seunggyu Jeon
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - Min Kyeong Kim
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
| | - Won Ha
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
| | - Hyun-Wook Kang
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, 50, UNIST-gil, Ulju-gun, Ulsan, 44919, South Korea
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15
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Galocha-León C, Antich C, Voltes-Martínez A, Marchal JA, Mallandrich M, Halbaut L, Souto EB, Gálvez-Martín P, Clares-Naveros B. Human mesenchymal stromal cells-laden crosslinked hyaluronic acid-alginate bioink for 3D bioprinting applications in tissue engineering. Drug Deliv Transl Res 2025; 15:291-311. [PMID: 38662335 PMCID: PMC11614963 DOI: 10.1007/s13346-024-01596-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/01/2024] [Indexed: 04/26/2024]
Abstract
Three-dimensional (3D) bioprinting is considered one of the most advanced tools to build up materials for tissue engineering. The aim of this work was the design, development and characterization of a bioink composed of human mesenchymal stromal cells (hMSC) for extrusion through nozzles to create these 3D structures that might potentially be apply to replace the function of damaged natural tissue. In this study, we focused on the advantages and the wide potential of biocompatible biomaterials, such as hyaluronic acid and alginate for the inclusion of hMSC. The bioink was characterized for its physical (pH, osmolality, degradation, swelling, porosity, surface electrical properties, conductivity, and surface structure), mechanical (rheology and printability) and biological (viability and proliferation) properties. The developed bioink showed high porosity and high swelling capacity, while the degradation rate was dependent on the temperature. The bioink also showed negative electrical surface and appropriate rheological properties required for bioprinting. Moreover, stress-stability studies did not show any sign of physical instability. The developed bioink provided an excellent environment for the promotion of the viability and growth of hMSC cells. Our work reports the first-time study of the effect of storage temperature on the cell viability of bioinks, besides showing that our bioink promoted a high cell viability after being extruded by the bioprinter. These results support the suggestion that the developed hMSC-composed bioink fulfills all the requirements for tissue engineering and can be proposed as a biological tool with potential applications in regenerative medicine and tissue engineering.
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Grants
- Ministry of Economy and Competitiveness (FEDER funds), grant number RTC-2016-5451-1; Ministry of Economy and Competitiveness, Instituto de Salud Carlos III (FEDER funds), grant numbers DTS19/00143 and DTS17/00087); Consejería de Economía, Conocimiento, Emp Ministry of Economy and Competitiveness (FEDER funds), grant number RTC-2016-5451-1; Ministry of Economy and Competitiveness, Instituto de Salud Carlos III (FEDER funds), grant numbers DTS19/00143 and DTS17/00087); Consejería de Economía, Conocimiento, Emp
- FCT-Fundação para a Ciência e a Tecnologia, I.P., Lisbon, Portugal FCT-Fundação para a Ciência e a Tecnologia, I.P., Lisbon, Portugal
- FCT—Fundação para a Ciência e a Tecnologia, I.P., Lisbon, Portugal
- Universidade do Porto
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Affiliation(s)
- Cristina Galocha-León
- Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, University Campus of Cartuja, 18071, Granada, Spain
| | - Cristina Antich
- Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research, University of Granada, 18100, Granada, Spain
- Biosanitary Institute of Granada (ibs. GRANADA), University Hospital of Granada-University of Granada, 18100, Granada, Spain
- Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18012, Spain
- Excellence Research Unit "Modeling Nature" (MNat), University of Granada, 18016, Granada, Spain
| | - Ana Voltes-Martínez
- Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research, University of Granada, 18100, Granada, Spain
- Biosanitary Institute of Granada (ibs. GRANADA), University Hospital of Granada-University of Granada, 18100, Granada, Spain
- Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18012, Spain
- Excellence Research Unit "Modeling Nature" (MNat), University of Granada, 18016, Granada, Spain
- BioFab i3D Lab - Biofabrication and 3D (Bio)printing Singular Laboratory, University of Granada, 18100, Granada, Spain
| | - Juan A Marchal
- Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research, University of Granada, 18100, Granada, Spain
- Biosanitary Institute of Granada (ibs. GRANADA), University Hospital of Granada-University of Granada, 18100, Granada, Spain
- Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18012, Spain
- Excellence Research Unit "Modeling Nature" (MNat), University of Granada, 18016, Granada, Spain
- BioFab i3D Lab - Biofabrication and 3D (Bio)printing Singular Laboratory, University of Granada, 18100, Granada, Spain
| | - Mireia Mallandrich
- Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028, Barcelona, Spain
- Institut de Nanociència i Nanotecnologia IN2UB, Universitat de Barcelona, 08028, Barcelona, Spain
| | - Lyda Halbaut
- Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028, Barcelona, Spain
- Institut de Nanociència i Nanotecnologia IN2UB, Universitat de Barcelona, 08028, Barcelona, Spain
| | - Eliana B Souto
- Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-313, Porto, Portugal.
| | - Patricia Gálvez-Martín
- Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, University Campus of Cartuja, 18071, Granada, Spain
- R&D Human and Animal Health, Bioibérica S.A.U., 08029, Barcelona, Spain
| | - Beatriz Clares-Naveros
- Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, University Campus of Cartuja, 18071, Granada, Spain.
- Biosanitary Institute of Granada (ibs. GRANADA), University Hospital of Granada-University of Granada, 18100, Granada, Spain.
- Institut de Nanociència i Nanotecnologia IN2UB, Universitat de Barcelona, 08028, Barcelona, Spain.
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16
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Jaworska K, Senior JJ, Brüning-Richardson A, Smith AM. The effect of elevating extracellular CaCl 2: Important considerations for tissue engineering applications. Tissue Cell 2024; 91:102615. [PMID: 39579735 DOI: 10.1016/j.tice.2024.102615] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2024] [Revised: 10/30/2024] [Accepted: 11/12/2024] [Indexed: 11/25/2024]
Abstract
Polysaccharides such as sodium alginate, pectin and gellan gum are widely used biomaterials, for their ability to easily form hydrogels in the presence of divalent metal ions, such as calcium - a process often cited as a mild crosslinking mechanism. However, when using these materials as substrates for tissue engineering, there is a lack of extensive studies that investigate the impact of elevated calcium concentrations on cell health and behaviour. In this study, we performed an in-depth exploration to understand the potential effects of raising extracellular CaCl2 on cell viability, proliferation, morphology and migration. We used an established glioblastoma (GBM) cell line (U251), human dermal fibroblasts (HDF), and murine osteoblasts (MC3T3) to assess the consequences of using CaCl2 in tissue engineered models to help reevaluate biomaterial suitability and enhance standardisation practices in the field of tissue engineering. Our findings revealed that the addition of CaCl2 induced notable morphological changes in GBM cells when cultured in 3D hydrogels with excess CaCl2 added, leading to a transition from mesenchymal to amoeboid phenotypes, even at a concentration as low as 8 mM. Furthermore, cell viability was reduced in a concentration-dependent manner across all cell types, and migration was also affected. Despite the widespread use of high CaCl2 concentrations to facilitate scaffold gelation, our research unveils that there can be significant risks to cell viability, proliferation, morphology, and migration when such practices are not preceded by cell line-specific experimentation and thorough standardization procedures. This highlights the importance of careful consideration and optimisation of CaCl2 concentration when used as a crosslinking agent for hydrogels intended for use in tissue engineering applications that demand accurate recapitulation of cellular responses and physiological conditions.
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Affiliation(s)
- Kayley Jaworska
- Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom
| | - Jessica J Senior
- Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom
| | - Anke Brüning-Richardson
- Department of Physical and Life Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom
| | - Alan M Smith
- Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom.
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17
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Wang Z, Han X, Sun G, Yu M, Qin J, Zhang Y, Ding D. Advances in cancer diagnosis and therapy by alginate-based multifunctional hydrogels: A review. Int J Biol Macromol 2024; 283:137707. [PMID: 39566758 DOI: 10.1016/j.ijbiomac.2024.137707] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Revised: 10/30/2024] [Accepted: 11/13/2024] [Indexed: 11/22/2024]
Abstract
The field of oncology has been changed by the application of hydrogels. These 3D polymeric networks have demonstrated significant promise in the treatment of cancer and can boost the efficacy of conventional therapeutics including chemotherapy and immunotherapy. Noteworthy, the development of biocompatible and effective hydrogels has been of interest. In this case, alginate as a biopolymer and carbohydrate polymer has been used to modify or synthesis multifunctional nanoparticles for the treatment of human diseases, especially cancer. Therefore, highlighting the function of alginate in the development of hydrogels in cancer therapy can provide new insights for improving outcome and survival rate of patients. Alginate hydrogels improve the specific and selective delivery of cargo and therefore, they reduce the systemic toxicity of drugs, while they enhance anti-cancer activity. Alginate hydrogels protect the genes against degradation by enzymes and increase blood circulation time. The alginate hydrogels can respond to the specific stimuli in the tumor microenvironment including pH, redox and light to improve the site-specific release of cargo. The nanoparticles can be incorporated in the structure of alginate hydrogels to augment their anti-cancer activity. In addition, alginate hydrogels can accelerate immunotherapy and phototherapy through delivery of immunomodulators and photosensitizers, respectively.
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Affiliation(s)
- Ziwen Wang
- Department of Radiology, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Xu Han
- Department of Emergency, Shengjing Hospital of China Medical University, Shenyang 110004, China
| | - Guowei Sun
- Interventional Center, Fengcheng Central Hospital, Fengcheng 118199, China
| | - Miao Yu
- Department of Respiratory, General Hospital of Northern Theater Command, Shenyang 110016, China
| | - Juan Qin
- Department of Endocrinology and Metabolism, Shenyang Fourth People Hospital, Shenyang 110001, China
| | - Yuting Zhang
- Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China.
| | - Ding Ding
- Department of Clinical Nutrition, Shengjing Hospital of China Medical University, Shenyang 110004, China.
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18
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Harris CG, Semprini L, Rochefort WE, Fogg KC. Statistical optimization of cell-hydrogel interactions for green microbiology - a tutorial review. RSC SUSTAINABILITY 2024; 2:3750-3768. [PMID: 39464839 PMCID: PMC11499971 DOI: 10.1039/d4su00400k] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Accepted: 10/14/2024] [Indexed: 10/29/2024]
Abstract
In this tutorial mini-review, we explore the application of Design of Experiments (DOE) as a powerful statistical tool in biotechnology. Specifically, we review the optimization of hydrogel materials for diverse microbial applications related to green microbiology, the use of microbes to promote sustainability. Hydrogels, three-dimensional polymers networks with high water retention capabilities, are pivotal in the immobilization of microorganisms and provide a customizable environment essential for directing microbial fate. We focus on the application of DOE to precisely tailor hydrogel compositions for a range of fungi and bacteria either used for the sustainable production of chemical compounds, or the elimination of hazardous substances. We examine a variety of DOE design strategies such as central composite designs, Box-Behnken designs, and optimal designs, and discuss their strategic implementation across diverse hydrogel formulations. Our analysis explores the integral role of DOE in refining hydrogels derived from a spectrum of polymers, including natural and synthetic polymers. We illustrate how DOE facilitates nuanced control over hydrogel properties that cannot be achieved using a standard one factor at a time approach. Furthermore, this review reveals a conserved finding across different materials and applications: there are significant interactions between hydrogel parameters and cell behavior. This highlights the intricacies of cell-hydrogel interactions and the impact on hydrogel material properties and cellular functions. Lastly, this review not only highlights DOE's efficacy in streamlining the optimization of cell-hydrogel processes but also positions it as a critical tool in advancing our understanding of cell-hydrogel dynamics, potentially leading to innovative advancements in biotechnological applications and bioengineering solutions.
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Affiliation(s)
- Conor G Harris
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +1 541-737-1777
| | - Lewis Semprini
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +1 541-737-1777
| | - Willie E Rochefort
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +1 541-737-1777
| | - Kaitlin C Fogg
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +1 541-737-1777
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19
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Rosales TKO, da Silva FFA, Bernardes ES, Paulo Fabi J. Plant-derived polyphenolic compounds: nanodelivery through polysaccharide-based systems to improve the biological properties. Crit Rev Food Sci Nutr 2024; 64:11894-11918. [PMID: 37585699 DOI: 10.1080/10408398.2023.2245038] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/18/2023]
Abstract
Plant-derived polyphenols are naturally occurring compounds widely distributed in plants. They have received greater attention in the food and pharmaceutical industries due to their potential health benefits, reducing the risk of some chronic diseases due to their antioxidant, anti-inflammatory, anticancer, cardioprotective, and neuro-action properties. Polyphenolic compounds orally administered can be used as adjuvants in several treatments but with restricted uses due to chemical instability. The review discusses the different structural compositions of polyphenols and their influence on chemical stability. Despite the potential and wide applications, there is a need to improve the delivery of polyphenolics to target the human intestine without massive chemical modifications. Oral administration of polyphenols is unfeasible due to instability, low bioaccessibility, and limited bioavailability. Nano-delivery systems based on polysaccharides (starch, pectin, chitosan, and cellulose) have been identified as a viable option for oral ingestion, potentiate biological effects, and direct-controlled delivery in specific tissues. The time and dose can be individualized for specific diseases, such as intestinal cancer. This review will address the mechanisms by which polysaccharides-based nanostructured systems can protect against degradation and enhance intestinal permeation, oral bioavailability, and the potential application of polysaccharides as nanocarriers for the controlled and targeted delivery of polyphenolic compounds.
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Affiliation(s)
- Thiécla Katiane Osvaldt Rosales
- Department of Food Science and Experimental Nutrition, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil
- Instituto de Pesquisa Energéticas e Nucleares - IPEN, São Paulo, SP, Brazil
| | | | | | - João Paulo Fabi
- Department of Food Science and Experimental Nutrition, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil
- Food Research Center (FoRC), CEPID-FAPESP (Research, Innovation and Dissemination Centers, São Paulo Research Foundation), São Paulo, SP, Brazil
- Food and Nutrition Research Center (NAPAN), University of São Paulo, São Paulo, SP, Brazil
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20
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Yao Z, Feng X, Wang Z, Zhan Y, Wu X, Xie W, Wang Z, Zhang G. Techniques and applications in 3D bioprinting with chitosan bio-inks for drug delivery: A review. Int J Biol Macromol 2024; 278:134752. [PMID: 39214837 DOI: 10.1016/j.ijbiomac.2024.134752] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 07/25/2024] [Accepted: 08/12/2024] [Indexed: 09/04/2024]
Abstract
Three-dimensional bioprinting leverages computer-aided design to construct tissues and organs with specialized bioinks. A notable biomaterial for this purpose is chitosan, a natural polysaccharide sourced from crustacean exoskeletons. Chitosan's biocompatibility, biodegradability, non-toxicity, and ability to promote cell adhesion and proliferation make it an excellent component for bioinks. Initially, the rheological properties of chitosan presented challenges for its use in bioprinting. Enhancements in its printability and stability were achieved by integrating it with other natural or synthetic polymers, facilitating its successful application in bioprinting. Chitosan-based bioinks are particularly promising for controlled drug delivery. Incorporating pharmaceuticals directly into the bioink enables the printed structures to serve as localized, sustained-release systems. This approach offers multiple advantages, including precise drug delivery to targeted disease sites, increased therapeutic efficiency, and reduced systemic side effects. Moreover, bioprinting allows for the customization of drug delivery mechanisms to meet individual patient requirements. Although there have been considerable advancements, the use of chitosan-based bioinks in drug delivery is still an emerging field. This review highlights chitosan's essential role in both systemic and localized drug delivery, underscoring its significance and discussing ongoing trends in its application for pharmaceutical purposes.
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Affiliation(s)
- Zhaomin Yao
- Department of Nuclear Medicine, General Hospital of Northern Theater Command, Shenyang, Liaoning 110016, China; College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, Liaoning 110167, China
| | - Xin Feng
- School of Information and Control Engineering, Jilin Institute of Chemical Technology, Jilin, Jilin, 130011, China
| | - Zheling Wang
- Department of Nuclear Medicine, General Hospital of Northern Theater Command, Shenyang, Liaoning 110016, China
| | - Ying Zhan
- Department of Nuclear Medicine, General Hospital of Northern Theater Command, Shenyang, Liaoning 110016, China; College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, Liaoning 110167, China
| | - Xiaodan Wu
- College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, Liaoning 110167, China
| | - Weiming Xie
- School of Information and Control Engineering, Jilin Institute of Chemical Technology, Jilin, Jilin, 130011, China
| | - Zhiguo Wang
- Department of Nuclear Medicine, General Hospital of Northern Theater Command, Shenyang, Liaoning 110016, China; College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, Liaoning 110167, China.
| | - Guoxu Zhang
- Department of Nuclear Medicine, General Hospital of Northern Theater Command, Shenyang, Liaoning 110016, China; College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, Liaoning 110167, China.
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21
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Chu YY, Hikita A, Asawa Y, Hoshi K. Advancements in chondrocyte 3-dimensional embedded culture: Implications for tissue engineering and regenerative medicine. Biomed J 2024; 48:100786. [PMID: 39236979 PMCID: PMC12018037 DOI: 10.1016/j.bj.2024.100786] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2024] [Revised: 07/09/2024] [Accepted: 09/02/2024] [Indexed: 09/07/2024] Open
Abstract
Cartilage repair necessitates regenerative medicine because of the unreliable healing mechanism of cartilage. To yield a sufficient number of cells for transplantation, chondrocytes must be expanded in culture. However, in 2D culture, chondrocytes tend to lose their distinctive phenotypes and functionalities after serial passage, thereby limiting their efficacy for tissue engineering purposes. The mechanism of dedifferentiation in 2D culture can be attributed to various factors, including abnormal nuclear strength, stress-induced mitochondrial impairment, chromatin remodeling, ERK-1/2 and the p38/mitogen-activated protein kinase (MAPK) signaling pathway. These mechanisms collectively contribute to the loss of chondrocyte phenotype and reduced production of cartilage-specific extracellular matrix (ECM) components. Chondrocyte 3D culture methods have emerged as promising solutions to prevent dedifferentiation. Techniques, such as scaffold-based culture and scaffold-free approaches, provide chondrocytes with a more physiologically relevant environment, promoting their differentiation and matrix synthesis. These methods have been used in cartilage tissue engineering to create engineered cartilage constructs for transplantation and joint repair. However, chondrocyte 3D culture still has limitations, such as low viability and proliferation rate, and also difficulties in passage under 3D condition. These indicate challenges of obtaining a sufficient number of chondrocytes for large-scale tissue production. To address these issues, ongoing studies of many research groups have been focusing on refining culture conditions, optimizing scaffold materials, and exploring novel cell sources such as stem cells to enhance the quality and quantity of engineered cartilage tissues. Although obstacles remain, continuous endeavors to enhance culture techniques and overcome limitations offer a promising outlook for the advancement of more efficient strategies for cartilage regeneration.
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Affiliation(s)
- Yu-Ying Chu
- Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Plastic and Reconstructive Surgery, Craniofacial Research Centre, Chang Gung Memorial Hospital at Linko, College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Atsuhiko Hikita
- Department of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan
| | - Yukiyo Asawa
- Department of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan
| | - Kazuto Hoshi
- Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan.
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22
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Wawszczak A, Kocki J, Kołodyńska D. Alginate as a Sustainable and Biodegradable Material for Medical and Environmental Applications-The Case Studies. J Biomed Mater Res B Appl Biomater 2024; 112:1-23. [PMID: 39269132 DOI: 10.1002/jbm.b.35475] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 06/19/2024] [Accepted: 08/09/2024] [Indexed: 09/15/2024]
Abstract
Alginates are salts of alginic acid derived mainly from sea algae of the genus brown algae. They are also synthesized by some bacteria. They belong to negatively charged polysaccharides exhibiting some rheological properties. High plasticity and the ability to modify the structure are the reasons for their application in numerous industries. Moreover, when in contact with the living tissue, they do not trigger an immune response, and for this reason they are the most often tested materials for medical applications. The paper discusses the latest applications, including 3D bioprinting, drug delivery systems, and sorptive properties. Recognizing alginates as biomaterials, it emphasizes the necessity for precise processing and modification to industrialize them for specific uses. This review aims to provide a thorough understanding of the advancements in alginate research, underscoring their potential for innovative applications.
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Affiliation(s)
- Alicja Wawszczak
- Department of Inorganic Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Lublin, Poland
| | - Janusz Kocki
- Department of Clinical Genetics, Medical University of Lublin, Lublin, Poland
| | - Dorota Kołodyńska
- Department of Inorganic Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Lublin, Poland
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23
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Tran DT, Yadav AS, Nguyen NK, Singha P, Ooi CH, Nguyen NT. Biodegradable Polymers for Micro Elastofluidics. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2024; 20:e2303435. [PMID: 37292037 DOI: 10.1002/smll.202303435] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Indexed: 06/10/2023]
Abstract
Micro elastofluidics is an emerging research field that encompasses characteristics of conventional microfluidics and fluid-structure interactions. Micro elastofluidics is expected to enable practical applications, for instance, where direct contact between biological samples and fluid handling systems is required. Besides design optimization, choosing a proper material is critical to the practical use of micro elastofluidics upon interaction with biological interface and after its functional lifetime. Biodegradable polymers are one of the most studied materials for this purpose. Micro elastofluidic devices made of biodegradable polymers possess exceptional mechanical elasticity, excellent bio compatibility, and structural degradability into non-toxic products. This article provides an insightful and systematic review of the utilization of biodegradable polymers in digital and continuous-flow micro elastofluidics.
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Affiliation(s)
- Du Tuan Tran
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
| | - Ajeet Singh Yadav
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
| | - Nhat-Khuong Nguyen
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
| | - Pradip Singha
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
| | - Chin Hong Ooi
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
| | - Nam-Trung Nguyen
- Queensland Micro- and Nanotechnology Centre, Griffith University, 170 Kessels Road, Nathan, QLD, 4111, Australia
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24
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Hu T, Zhou T, Goit RK, Tam KC, Chan YK, Lam WC, Lo ACY. Bioactive Glial-Derived Neurotrophic Factor from a Safe Injectable Collagen-Alginate Composite Gel Rescues Retinal Photoreceptors from Retinal Degeneration in Rabbits. Mar Drugs 2024; 22:394. [PMID: 39330275 PMCID: PMC11433152 DOI: 10.3390/md22090394] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2024] [Revised: 08/27/2024] [Accepted: 08/28/2024] [Indexed: 09/28/2024] Open
Abstract
The management of vision-threatening retinal diseases remains challenging due to the lack of an effective drug delivery system. Encapsulated cell therapy (ECT) offers a promising approach for the continuous delivery of therapeutic agents without the need for immunosuppressants. In this context, an injectable and terminable collagen-alginate composite (CAC) ECT gel, designed with a Tet-on pro-caspase-8 system, was developed as a safe intraocular drug delivery platform for the sustained release of glial-cell-line-derived neurotrophic factor (GDNF) to treat retinal degenerative diseases. This study examined the potential clinical application of the CAC ECT gel, focusing on its safety, performance, and termination through doxycycline (Dox) administration in the eyes of healthy New Zealand White rabbits, as well as its therapeutic efficacy in rabbits with sodium-iodate (SI)-induced retinal degeneration. The findings indicated that the CAC ECT gel can be safely implanted without harming the retina or lens, displaying resistance to degradation, facilitating cell attachment, and secreting bioactive GDNF. Furthermore, the GDNF levels could be modulated by the number of implants. Moreover, Dox administration was effective in terminating gel function without causing retinal damage. Notably, rabbits with retinal degeneration treated with the gels exhibited significant functional recovery in both a-wave and b-wave amplitudes and showed remarkable efficacy in reducing photoreceptor apoptosis. Given its biocompatibility, mechanical stability, controlled drug release, terminability, and therapeutic effectiveness, our CAC ECT gel presents a promising therapeutic strategy for various retinal diseases in a clinical setting, eliminating the need for immunosuppressants.
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Affiliation(s)
- Tingyu Hu
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
| | - Ting Zhou
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
| | - Rajesh Kumar Goit
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
- Jules Stein Eye Institute, Los Angeles, CA 90095, USA
| | - Ka Cheung Tam
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
| | - Yau Kei Chan
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
| | - Wai-Ching Lam
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
- Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC V5Z 3N9, Canada
| | - Amy Cheuk Yin Lo
- Department of Ophthalmology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; (T.H.); (T.Z.); (K.C.T.); (Y.K.C.); (W.-C.L.)
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25
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Yamashita M, Tamamitsu M, Kirisako H, Goda Y, Chen X, Hattori K, Ota S. High-Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures. SMALL METHODS 2024; 8:e2301318. [PMID: 38133483 DOI: 10.1002/smtd.202301318] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Revised: 11/27/2023] [Indexed: 12/23/2023]
Abstract
3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid-like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light-sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high-throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min-1. The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.
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Affiliation(s)
- Minato Yamashita
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Miu Tamamitsu
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Hiromi Kirisako
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Yuki Goda
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Xiaoyao Chen
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Kazuki Hattori
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
| | - Sadao Ota
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
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26
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Mazari‐Arrighi E, Lépine M, Ayollo D, Faivre L, Larghero J, Chatelain F, Fuchs A. Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting. Adv Healthc Mater 2024; 13:e2302830. [PMID: 38366136 PMCID: PMC11468676 DOI: 10.1002/adhm.202302830] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Revised: 01/29/2024] [Indexed: 02/18/2024]
Abstract
Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.
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Affiliation(s)
- Elsa Mazari‐Arrighi
- Université de ParisU976 HIPI, InsermParisF‐75006France
- AP‐HPHôpital Saint‐Louis1 avenue VellefauxParisF‐75010France
| | - Matthieu Lépine
- Université de ParisU976 HIPI, InsermParisF‐75006France
- AP‐HPHôpital Saint‐Louis1 avenue VellefauxParisF‐75010France
| | - Dmitry Ayollo
- Université de ParisU976 HIPI, InsermParisF‐75006France
- AP‐HPHôpital Saint‐Louis1 avenue VellefauxParisF‐75010France
| | - Lionel Faivre
- Université de ParisU976 HIPI, InsermParisF‐75006France
- AP‐HPHôpital Saint‐Louis1 avenue VellefauxParisF‐75010France
| | - Jérôme Larghero
- Université de ParisU976 HIPI, InsermParisF‐75006France
- AP‐HPHôpital Saint‐Louis1 avenue VellefauxParisF‐75010France
| | - François Chatelain
- Université de ParisU976 HIPI, InsermParisF‐75006France
- CEAIRIGGrenobleF‐38000France
| | - Alexandra Fuchs
- Université de ParisU976 HIPI, InsermParisF‐75006France
- CEAIRIGGrenobleF‐38000France
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27
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Kawamura S, Furuya K, Sasaki N, Takeoka Y, Aizawa M, Kanzawa N. Evaluation of alginate-coated β-tricalcium phosphate fiber scaffold for cell culture. J Biomed Mater Res B Appl Biomater 2024; 112:e35433. [PMID: 38817048 DOI: 10.1002/jbm.b.35433] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2024] [Revised: 04/08/2024] [Accepted: 05/18/2024] [Indexed: 06/01/2024]
Abstract
Ex vivo tissue engineering is an effective therapeutic approach for the treatment of severe cartilage diseases that require tissue replenishment or replacement. This strategy demands scaffolds that are durable enough for long-term cell culture to form artificial tissue. Additionally, such scaffolds must be biocompatible to prevent the transplanted matrix from taking a toll on the patient's body. From the viewpoint of structure and bio-absorbability, a β-tricalcium phosphate (β-TCP) fiber scaffold (βTFS) is expected to serve as a good scaffold for tissue engineering. However, the fragility and high solubility of β-TCP fibers make this matrix unsuitable for long-term cell culture. To solve this problem, we developed an alginate-coated β-TCP fiber scaffold (βTFS-Alg). To assess cell proliferation and differentiation in the presence of βTFS-Alg, we characterized ATDC5 cells, a chondrocyte-like cell line, when grown in this matrix. We found that alginate coated the surface of βTFS fiber and suppressed the elution of Ca2+ from β-TCP fibers. Due to the decreased solubility of βTFS-Alg compared with β-TCP, the former provided an improved scaffold for long-term cell culture. Additionally, we observed superior cell proliferation and upregulation of chondrogenesis marker genes in ATDC5 cells cultured in βTFS-Alg. These results suggest that βTFS-Alg is suitable for application in tissue culture.
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Affiliation(s)
- Satoshi Kawamura
- Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
| | - Kozue Furuya
- Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
| | - Nene Sasaki
- Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
| | - Yuko Takeoka
- Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
| | - Mamoru Aizawa
- Department of Applied Chemistry, School of Science and Technology, Meiji University, Tama-ku, Kanagawa, Japan
| | - Nobuyuki Kanzawa
- Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
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28
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Kaminaga M, Otomo S, Tsunozaki S, Kadonosono T, Omata T. Fabrication of a Cancer Cell Aggregate Culture Device That Facilitates Observations of Nutrient and Oxygen Gradients. MICROMACHINES 2024; 15:689. [PMID: 38930659 PMCID: PMC11205477 DOI: 10.3390/mi15060689] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 05/18/2024] [Accepted: 05/20/2024] [Indexed: 06/28/2024]
Abstract
Three-dimensional cell culture spheroids are commonly used for drug evaluation studies because they can produce large quantities of homogeneous cell aggregates. As the spheroids grow, nutrients supplied from outer spheroid regions render the inner spheroid areas hypoxic and hyponutrient, which makes them unobservable through confocal microscopy. In this study, we fabricated a cancer cell aggregate culture device that facilitates the observation of nutrient and oxygen gradients. An alginate gel fiber was created in the cell culture chamber to ensure a flow path for supplying the culture medium. A gradient of nutrients and oxygen was generated by positioning the flow channel close to the edge of the chamber. We devised a fabrication method that uses calcium carbonate as a source of Ca2+ for the gelation of sodium alginate, which has a slow reaction rate. We then cultured a spheroid of HCT116 cells, which were derived from human colorectal carcinoma using a fluorescent ubiquitination-based cell cycle indicator. Fluorescence observation suggested the formation of a hypoxic and hyponutrient region within an area approximately 500 µm away from the alginate gel fiber. This indicates the development of a cancer cell aggregate culture device that enables the observation of different nutrition and oxygen states.
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Affiliation(s)
- Maho Kaminaga
- Department of Mechanical Engineering, National Institute of Technology, Toyota Campus, 2-1 Eisei-cho, Toyota 471-0067, Aichi, Japan
| | - Shuta Otomo
- Department of Mechanical Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori ku, Yokohama 226-0026, Kanagawa, Japan (T.O.)
| | - Seisyu Tsunozaki
- Department of Mechanical Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori ku, Yokohama 226-0026, Kanagawa, Japan (T.O.)
| | - Tetuya Kadonosono
- Department of Life Science & Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori ku, Yokohama 226-0026, Kanagawa, Japan;
| | - Toru Omata
- Department of Mechanical Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori ku, Yokohama 226-0026, Kanagawa, Japan (T.O.)
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29
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Klak M, Rachalewski M, Filip A, Dobrzański T, Berman A, Wszoła M. Bioprinting of Perfusable, Biocompatible Vessel-like Channels with dECM-Based Bioinks and Living Cells. Bioengineering (Basel) 2024; 11:439. [PMID: 38790306 PMCID: PMC11117567 DOI: 10.3390/bioengineering11050439] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Revised: 04/14/2024] [Accepted: 04/24/2024] [Indexed: 05/26/2024] Open
Abstract
There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the production of stable constructs that can survive for a long time after transplantation. While the selection of the right material is crucial for bioprinting, there is another equally important issue that is currently being extensively researched-the incorporation of the vascular system into the fabricated scaffolds. Therefore, in the following manuscript, we present the results of research on bioink with unique physico-chemical and biological properties. In this article, two methods of seeding cells were tested using bioink B and seeding after bioprinting the whole model. After 2, 5, 8, or 24 h of incubation, the flow medium was used in the tested systems. At the end of the experimental trial, for each time variant, the canals were stored in formaldehyde, and immunohistochemical staining was performed to examine the presence of cells on the canal walls and roof. Cells adhered to both ways of fiber arrangement; however, a parallel bioprint with the 5 h incubation and the intermediate plating of cells resulted in better adhesion efficiency. For this test variant, the percentage of cells that adhered was at least 20% higher than in the other analyzed variants. In addition, it was for this variant that the lowest percentage of viable cells was found that were washed out of the tested model. Importantly, hematoxylin and eosin staining showed that after 8 days of culture, the cells were evenly distributed throughout the canal roof. Our study clearly shows that neovascularization-promoting cells effectively adhere to ECM-based pancreatic bioink. Summarizing the presented results, it was demonstrated that the proposed bioink compositions can be used for bioprinting bionic organs with a vascular system formed by endothelial cells and fibroblasts.
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Affiliation(s)
- Marta Klak
- Foundation of Research and Science Development, 01-242 Warsaw, Poland or (M.W.)
- Polbionica sp. z o.o., 01-242 Warsaw, Poland
| | - Michał Rachalewski
- Foundation of Research and Science Development, 01-242 Warsaw, Poland or (M.W.)
| | - Anna Filip
- Foundation of Research and Science Development, 01-242 Warsaw, Poland or (M.W.)
| | | | | | - Michał Wszoła
- Foundation of Research and Science Development, 01-242 Warsaw, Poland or (M.W.)
- Polbionica sp. z o.o., 01-242 Warsaw, Poland
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30
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Lizana-Vasquez GD, Mendez-Vega J, Cappabianca D, Saha K, Torres-Lugo M. In vitro encapsulation and expansion of T and CAR-T cells using 3D synthetic thermo-responsive matrices. RSC Adv 2024; 14:13734-13747. [PMID: 38681842 PMCID: PMC11046447 DOI: 10.1039/d4ra01968g] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Accepted: 04/17/2024] [Indexed: 05/01/2024] Open
Abstract
Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.
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Affiliation(s)
- Gaby D Lizana-Vasquez
- Deparment of Chemical Engineering, University of Puerto Rico-Mayagüez Road 108 Km. 1.0 Bo. Miradero. P.O. Box 9046 Mayagüez 00681-9046 Puerto Rico USA +1 787 832 4040 Ext. 2585
| | - Janet Mendez-Vega
- Deparment of Chemical Engineering, University of Puerto Rico-Mayagüez Road 108 Km. 1.0 Bo. Miradero. P.O. Box 9046 Mayagüez 00681-9046 Puerto Rico USA +1 787 832 4040 Ext. 2585
| | - Dan Cappabianca
- Department of Biomedical Engineering, University of Wisconsin-Madison Madison Wisconsin USA
| | - Krishanu Saha
- Department of Biomedical Engineering, University of Wisconsin-Madison Madison Wisconsin USA
| | - Madeline Torres-Lugo
- Deparment of Chemical Engineering, University of Puerto Rico-Mayagüez Road 108 Km. 1.0 Bo. Miradero. P.O. Box 9046 Mayagüez 00681-9046 Puerto Rico USA +1 787 832 4040 Ext. 2585
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Kollampally SCR, Zhang X, Moskwa N, Nelson DA, Sharfstein ST, Larsen M, Xie Y. Evaluation of Alginate Hydrogel Microstrands for Stromal Cell Encapsulation and Maintenance. Bioengineering (Basel) 2024; 11:375. [PMID: 38671796 PMCID: PMC11048715 DOI: 10.3390/bioengineering11040375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 04/09/2024] [Accepted: 04/11/2024] [Indexed: 04/28/2024] Open
Abstract
Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.
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Affiliation(s)
- Sujith Chander Reddy Kollampally
- Department of Nanoscale Science and Engineering, College of Nanotechnology, Science, and Engineering, University at Albany, State University of New York, 257 Fuller Road, Albany, NY 12203, USA; (S.C.R.K.); (X.Z.); (S.T.S.)
| | - Xulang Zhang
- Department of Nanoscale Science and Engineering, College of Nanotechnology, Science, and Engineering, University at Albany, State University of New York, 257 Fuller Road, Albany, NY 12203, USA; (S.C.R.K.); (X.Z.); (S.T.S.)
| | - Nicholas Moskwa
- Department of Biological Sciences and The RNA Institute, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA; (N.M.); (D.A.N.); (M.L.)
- The Jackson Laboratory of Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA
| | - Deirdre A. Nelson
- Department of Biological Sciences and The RNA Institute, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA; (N.M.); (D.A.N.); (M.L.)
| | - Susan T. Sharfstein
- Department of Nanoscale Science and Engineering, College of Nanotechnology, Science, and Engineering, University at Albany, State University of New York, 257 Fuller Road, Albany, NY 12203, USA; (S.C.R.K.); (X.Z.); (S.T.S.)
| | - Melinda Larsen
- Department of Biological Sciences and The RNA Institute, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222, USA; (N.M.); (D.A.N.); (M.L.)
| | - Yubing Xie
- Department of Nanoscale Science and Engineering, College of Nanotechnology, Science, and Engineering, University at Albany, State University of New York, 257 Fuller Road, Albany, NY 12203, USA; (S.C.R.K.); (X.Z.); (S.T.S.)
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32
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Anagnostidis V, Tiwari A, Gielen F. Deep learning-assisted concentration gradient generation for the study of 3D cell cultures in hydrogel beads of varying stiffness. Front Bioeng Biotechnol 2024; 12:1364553. [PMID: 38665812 PMCID: PMC11044700 DOI: 10.3389/fbioe.2024.1364553] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Accepted: 03/22/2024] [Indexed: 04/28/2024] Open
Abstract
The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50 Pa to close to 1 kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.
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Affiliation(s)
- Vasileios Anagnostidis
- Living Systems Institute, Faculty of Health and Life Sciences, University of Exeter, Exeter, United Kingdom
- Department of Physics and Astronomy, Faculty of Environment, Science and Economy, University of Exeter, Exeter, United Kingdom
| | - Anuj Tiwari
- Living Systems Institute, Faculty of Health and Life Sciences, University of Exeter, Exeter, United Kingdom
| | - Fabrice Gielen
- Living Systems Institute, Faculty of Health and Life Sciences, University of Exeter, Exeter, United Kingdom
- Department of Physics and Astronomy, Faculty of Environment, Science and Economy, University of Exeter, Exeter, United Kingdom
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Harris CG, Gedde HK, Davis AA, Semprini L, Rochefort WE, Fogg KC. The optimization of poly(vinyl)-alcohol-alginate beads with a slow-release compound for the aerobic cometabolism of chlorinated aliphatic hydrocarbons. RSC SUSTAINABILITY 2024; 2:1101-1117. [PMID: 38585330 PMCID: PMC10993105 DOI: 10.1039/d3su00409k] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/06/2023] [Accepted: 03/08/2024] [Indexed: 04/09/2024]
Abstract
Chlorinated aliphatic hydrocarbons (CAHs), such as cis-1,2-dichloroethylene (cDCE), are prevalent in groundwater at many locations throughout the United States. When immobilized in hydrogel beads with slow-release compounds, the bacteria strain Rhodococcus rhodochrous ATCC 21198 can be used for the in situ bioremediation of cDCE. These hydrogel beads must exhibit high mechanical strength and resist degradation to extend the lifetime of slow-release compounds and bioremediation. We engineered poly(vinyl)-alcohol - alginate (PVA-AG) beads to immobilize ATCC 21198 with the slow-release compound, tetrabutoxysilane (TBOS) that produces 1-butanol as a growth substrate, for high mechanical strength. We optimized three inputs (concentration of PVA, concentration of AG, and the crosslinking time) on two responses (compressive modulus and rate of oxygen utilization) for batch incubation experiments between 1 and 30 days using a design of experiments approach. The predictive models generated from design of experiments were then tested by measuring the compressive strength, oxygen utilization, and abiotic rates of hydrolysis for a predicted optimal bead formulation. The result of this study generated a hydrogel bead with immobilized R. rhodochrous ATCC 21198 and TBOS that exhibited a high compressive modulus on day 1 and day 30, which was accurately predicted by models. These hydrogel beads exhibited low metabolic activity based on oxygen rates on day 1 and day 30 but were not accurately predicted by the models. In addition, the ratio between oxygen utilization and abiotic rates of hydrolysis were observed to be roughly half of what was expected stoichiometrically. Lastly, we demonstrated the capability to use these beads as a bioremediation technology for cDCE as we found that, for all bead formulations, cDCE was significantly reduced after 30 days. Altogether, this work demonstrates the capability to capture and enhance the material properties of the complex hydrogel beads with predictive models yet signals the need for more robust methods to understand the metabolic activity that occurs in the hydrogel beads.
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Affiliation(s)
- Conor G Harris
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
| | - Hannah K Gedde
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
| | - Audrey A Davis
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
| | - Lewis Semprini
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
| | - Willie E Rochefort
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
| | - Kaitlin C Fogg
- School of Chemical, Biological, and Environmental Engineering, Oregon State University Corvallis OR 97331 USA +541-737-1777
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Vu TT, Jo SH, Kim SH, Kim BK, Park SH, Lim KT. Injectable and Multifunctional Hydrogels Based on Poly( N-acryloyl glycinamide) and Alginate Derivatives for Antitumor Drug Delivery. ACS APPLIED MATERIALS & INTERFACES 2024. [PMID: 38470564 DOI: 10.1021/acsami.4c00298] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/14/2024]
Abstract
Chemotherapy is a conventional treatment that uses drugs to kill cancer cells; however, it may induce side effects and may be incompletely effective, leading to the risk of tumor recurrence. To address this issue, we developed novel injectable thermal/near-infrared (NIR)-responsive hydrogels to control drug release. The injectable hydrogel formulation was composed of biocompatible alginates, poly(N-acryloyl glycinamide) (PNAGA) copolymers with an upper critical solution temperature, and NIR-responsive cross-linkers containing coumarin groups, which were gelated through bioorthogonal inverse electron demand Diels-Alder reactions. The hydrogels exhibited quick gelation times (120-800 s) and high drug loading efficiencies (>90%). The hydrogels demonstrated a higher percentage of drug release at 37 °C than that at 25 °C due to the enhanced swelling behavior of temperature-responsive PNAGA moieties. Upon NIR irradiation, the hydrogels released most of the entrapped doxorubicin (DOX) (97%) owing to the cleavage of NIR-sensitive coumarin ester groups. The hydrogels displayed biocompatibility with normal cells, while induced antitumor activity toward cancer cells. DOX/hydrogels treated with NIR light inhibited tumor growth in nude mice bearing tumors. In addition, the injected hydrogels emitted red fluorescence upon excitation at a green wavelength, so that the drug delivery and hydrogel degradation in vivo could be tracked in the xenograft model.
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Affiliation(s)
- Trung Thang Vu
- Department of Smart Green Technology Engineering, Pukyong National University, Busan 48513, South Korea
- Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, South Korea
| | - Sung-Han Jo
- Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, South Korea
| | - Seon-Hwa Kim
- Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, South Korea
| | - Byeong Kook Kim
- Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, South Korea
| | - Sang-Hyug Park
- Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, South Korea
| | - Kwon Taek Lim
- Department of Smart Green Technology Engineering, Pukyong National University, Busan 48513, South Korea
- Institute of Display Semiconductor Technology, Pukyong National University, Busan 48513, South Korea
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35
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Guimarães CF, Liu S, Wang J, Purcell E, Ozedirne T, Ren T, Aslan M, Yin Q, Reis RL, Stoyanova T, Demirci U. Co-axial hydrogel spinning for facile biofabrication of prostate cancer-like 3D models. Biofabrication 2024; 16:025017. [PMID: 38306674 DOI: 10.1088/1758-5090/ad2535] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Accepted: 02/01/2024] [Indexed: 02/04/2024]
Abstract
Glandular cancers are amongst the most prevalent types of cancer, which can develop in many different organs, presenting challenges in their detection as well as high treatment variability and failure rates. For that purpose, anticancer drugs are commonly tested in cancer cell lines grown in 2D tissue culture on plastic dishesin vitro, or in animal modelsin vivo. However, 2D culture models diverge significantly from the 3D characteristics of living tissues and animal models require extensive animal use and time. Glandular cancers, such as prostate cancer-the second leading cause of male cancer death-typically exist in co-centrical architectures where a cell layer surrounds an acellular lumen. Herein, this spatial cellular position and 3D architecture, containing dual compartments with different hydrogel materials, is engineered using a simple co-axial nozzle setup, in a single step utilizing prostate as a model of glandular cancer. The resulting hydrogel soft structures support viable prostate cancer cells of different cell lines and enable over-time maturation into cancer-mimicking aggregates surrounding the acellular core. The biofabricated cancer mimicking structures are then used as a model to predict the inhibitory efficacy of the poly ADP ribose polymerase inhibitor, Talazoparib, and the antiandrogen drug, Enzalutamide, in the growth of the cancer cell layer. Our results show that the obtained hydrogel constructs can be adapted to quickly obtain 3D cancer models which combine 3D physiological architectures with high-throughput screening to detect and optimize anti-cancer drugs in prostate and potentially other glandular cancer types.
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Affiliation(s)
- Carlos F Guimarães
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, Parque de Ciência e Tecnologia 4805-017 Barco, Guimarães, Portugal
- ICVS/3B's-PT Government Associate Laboratory, Braga and Guimarães, Portugal
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Shiqin Liu
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, United States of America
| | - Jie Wang
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Emma Purcell
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Tugba Ozedirne
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Tanchen Ren
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Merve Aslan
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Qingqing Yin
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
| | - Rui L Reis
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, Parque de Ciência e Tecnologia 4805-017 Barco, Guimarães, Portugal
- ICVS/3B's-PT Government Associate Laboratory, Braga and Guimarães, Portugal
| | - Tanya Stoyanova
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, United States of America
- Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, United States of America
| | - Utkan Demirci
- Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Bio-Acoustic MEMS (BAMM) in Medicine Lab, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
- Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States of America
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Jeong D, Jang G, Jung WK, Park YH, Bae H. Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat. NPJ Sci Food 2024; 8:13. [PMID: 38374073 PMCID: PMC10876650 DOI: 10.1038/s41538-024-00257-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Accepted: 02/05/2024] [Indexed: 02/21/2024] Open
Abstract
Numerous studies have explored the cultivation of muscle cells using non-animal materials for cultivated meat production. Achieving muscle cell proliferation and alignment using 3D scaffolds made from plant-based materials remains challenging. This study introduces a technique to culture and align muscle cells using only plant-based materials, avoiding toxic chemical modifications. Zein-alginate fibers (ZA fibers) were fabricated by coating zein protein onto alginate fibers (A fibers). Zein's excellent cell compatibility and biodegradability enable high cell adhesion and proliferation rates, and the good ductility of the ZA fibers enable a high strain rate (>75%). We demonstrate mature and aligned myotube formation in ZA fibers, providing a simple way to align muscle cells using plant-based materials. Additionally, cultivated meat was constructed by assembling muscle, fat, and vessel fibers. This method holds promise for the future mass production of cultivated meat.
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Affiliation(s)
- Dayi Jeong
- Department of Stem Cell and Regenerative Biotechnology, KU Convergence Science and Technology Institute, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea
| | - Goo Jang
- Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Medicine and the Research Institute of Veterinary Science, Seoul National University, Seoul, 08826, Republic of Korea
| | - Woo Kyung Jung
- NoAH Biotech Co., Ltd., Suwon-si, Gyeonggi-do, 16614, Republic of Korea
| | - Yong Ho Park
- NoAH Biotech Co., Ltd., Suwon-si, Gyeonggi-do, 16614, Republic of Korea
- Department of Microbiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea
| | - Hojae Bae
- Department of Stem Cell and Regenerative Biotechnology, KU Convergence Science and Technology Institute, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea.
- Institute of Advanced Regenerative Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea.
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Ouedraogo LJ, Trznadel MJ, Kling M, Nasirian V, Borst AG, Shirsavar MA, Makowski A, McNamara MC, Montazami R, Hashemi NN. Hydrodynamic Assembly of Astrocyte Cells in Conductive Hollow Microfibers. Adv Biol (Weinh) 2024; 8:e2300455. [PMID: 37953458 DOI: 10.1002/adbi.202300455] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 10/19/2023] [Indexed: 11/14/2023]
Abstract
The manufacturing of 3D cell scaffoldings provides advantages for modeling diseases and injuries as it enables the creation of physiologically relevant platforms. A triple-flow microfluidic device is developed to rapidly fabricate alginate/graphene hollow microfibers based on the gelation of alginate induced with CaCl2 . This five-channel microdevice actualizes continuous mild fabrication of hollow fibers under an optimized flow rate ratio of 300:200:100 µL min-1 . The polymer solution is 2.5% alginate in 0.1% graphene and a 30% polyethylene glycol solution is used as the sheath and core solutions. The biocompatibility of these conductive microfibers by encapsulating mouse astrocyte cells (C8D1A) within the scaffolds is investigated. The cells can successfully survive both the manufacturing process and prolonged encapsulation for up to 8 days, where there is between 18-53% of live cells on both the alginate microfibers and alginate/graphene microfibers. These unique 3D hollow scaffolds can significantly enhance the available surface area for nutrient transport to the cells. In addition, these conductive hollow scaffolds illustrate unique advantages such as 0.728 cm3 gr-1 porosity and two times more electrical conductivity in comparison to alginate scaffolds. The results confirm the potential of these scaffolds as a microenvironment that supports cell growth.
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Affiliation(s)
- Lionel J Ouedraogo
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - Mychal J Trznadel
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - McKayla Kling
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
- Neuroscience Graduate Program, Iowa State University, Ames, IA, 50011, USA
| | - Vahid Nasirian
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - Alexandra G Borst
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
- Neuroscience Graduate Program, Iowa State University, Ames, IA, 50011, USA
| | | | - Andrew Makowski
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - Marilyn C McNamara
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - Reza Montazami
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
| | - Nicole N Hashemi
- Department of Mechanical Engineering, Iowa State University, Ames, IA, 50011, USA
- Neuroscience Graduate Program, Iowa State University, Ames, IA, 50011, USA
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38
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Tang Y, Yang X, Hu H, Jiang H, Xiong W, Mei H, Hu Y. Elevating the potential of CAR-T cell therapy in solid tumors: exploiting biomaterials-based delivery techniques. Front Bioeng Biotechnol 2024; 11:1320807. [PMID: 38312512 PMCID: PMC10835794 DOI: 10.3389/fbioe.2023.1320807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2023] [Accepted: 12/05/2023] [Indexed: 02/06/2024] Open
Abstract
Chimeric antigen receptor (CAR) T cells exhibit promising progress in addressing hematologic malignancies. However, CAR-T therapy for solid tumors remains limited, with no FDA-approved CAR-T products available for clinical use at present. Primary reasons include insufficient infiltration, accumulation, tumor immunosuppression of the microenvironment, and related side effects. Single utilization of CAR-T cannot effectively overcome these unfavorable obstacles. A probable effective pathway to achieve a better CAR-T therapy effect would be to combine the benefits of biomaterials-based technology. In this article, comprehensive biomaterials strategies to break through these obstacles of CAR-T cell therapy at the tumor sites are summarized, encompassing the following aspects: 1) generating orthotopic CAR-T cells; 2) facilitating CAR-T cell trafficking; 3) stimulating CAR-T cell expansion and infiltration; 4) improving CAR-T cell activity and persistence; 5) reprogramming the immunosuppressive microenvironments. Additionally, future requirements for the development of this field, with a specific emphasis on promoting innovation and facilitating clinical translation, are thoroughly discussed.
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Affiliation(s)
- Yuxiang Tang
- Tongji Medical College, Union Hospital, Institute of Hematology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China
| | - Xiaoyu Yang
- Department of Pharmacy, Tongji Medical College, Union Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Hang Hu
- School of Pharmacy, ChangZhou University, Changzhou, China
| | - Huiwen Jiang
- Tongji Medical College, Union Hospital, Institute of Hematology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China
| | - Wei Xiong
- Wuhan Sian Medical Technology Co., Ltd., Wuhan, China
| | - Heng Mei
- Tongji Medical College, Union Hospital, Institute of Hematology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China
| | - Yu Hu
- Tongji Medical College, Union Hospital, Institute of Hematology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China
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Zhang S, Liu J, Feng F, Jia Y, Xu F, Wei Z, Zhang M. Rational design of viscoelastic hydrogels for periodontal ligament remodeling and repair. Acta Biomater 2024; 174:69-90. [PMID: 38101557 DOI: 10.1016/j.actbio.2023.12.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Revised: 11/14/2023] [Accepted: 12/07/2023] [Indexed: 12/17/2023]
Abstract
The periodontal ligament (PDL) is a distinctive yet critical connective tissue vital for maintaining the integrity and functionality of tooth-supporting structures. However, PDL repair poses significant challenges due to the complexity of its mechanical microenvironment encompassing hard-soft-hard tissues, with the viscoelastic properties of the PDL being of particular interest. This review delves into the significant role of viscoelastic hydrogels in PDL regeneration, underscoring their utility in simulating biomimetic three-dimensional microenvironments. We review the intricate relationship between PDL and viscoelastic mechanical properties, emphasizing the role of tissue viscoelasticity in maintaining mechanical functionality. Moreover, we summarize the techniques for characterizing PDL's viscoelastic behavior. From a chemical bonding perspective, we explore various crosslinking methods and characteristics of viscoelastic hydrogels, along with engineering strategies to construct viscoelastic cell microenvironments. We present a detailed analysis of the influence of the viscoelastic microenvironment on cellular mechanobiological behavior and fate. Furthermore, we review the applications of diverse viscoelastic hydrogels in PDL repair and address current challenges in the field of viscoelastic tissue repair. Lastly, we propose future directions for the development of innovative hydrogels that will facilitate not only PDL but also systemic ligament tissue repair. STATEMENT OF SIGNIFICANCE.
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Affiliation(s)
- Songbai Zhang
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Xi'an 710032, PR China; The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China; Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University, Xi'an 710049, PR China
| | - Jingyi Liu
- The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China; Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University, Xi'an 710049, PR China
| | - Fan Feng
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Xi'an 710032, PR China
| | - Yuanbo Jia
- The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China; Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University, Xi'an 710049, PR China
| | - Feng Xu
- The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China; Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University, Xi'an 710049, PR China
| | - Zhao Wei
- The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China; Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University, Xi'an 710049, PR China.
| | - Min Zhang
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Xi'an 710032, PR China.
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40
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Abuwatfa WH, Pitt WG, Husseini GA. Scaffold-based 3D cell culture models in cancer research. J Biomed Sci 2024; 31:7. [PMID: 38221607 PMCID: PMC10789053 DOI: 10.1186/s12929-024-00994-y] [Citation(s) in RCA: 55] [Impact Index Per Article: 55.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2023] [Accepted: 01/04/2024] [Indexed: 01/16/2024] Open
Abstract
Three-dimensional (3D) cell cultures have emerged as valuable tools in cancer research, offering significant advantages over traditional two-dimensional (2D) cell culture systems. In 3D cell cultures, cancer cells are grown in an environment that more closely mimics the 3D architecture and complexity of in vivo tumors. This approach has revolutionized cancer research by providing a more accurate representation of the tumor microenvironment (TME) and enabling the study of tumor behavior and response to therapies in a more physiologically relevant context. One of the key benefits of 3D cell culture in cancer research is the ability to recapitulate the complex interactions between cancer cells and their surrounding stroma. Tumors consist not only of cancer cells but also various other cell types, including stromal cells, immune cells, and blood vessels. These models bridge traditional 2D cell cultures and animal models, offering a cost-effective, scalable, and ethical alternative for preclinical research. As the field advances, 3D cell cultures are poised to play a pivotal role in understanding cancer biology and accelerating the development of effective anticancer therapies. This review article highlights the key advantages of 3D cell cultures, progress in the most common scaffold-based culturing techniques, pertinent literature on their applications in cancer research, and the ongoing challenges.
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Affiliation(s)
- Waad H Abuwatfa
- Materials Science and Engineering Ph.D. Program, College of Arts and Sciences, American University of Sharjah, P.O. Box. 26666, Sharjah, United Arab Emirates
- Department of Chemical and Biological Engineering, College of Engineering, American University of Sharjah, P.O. Box 26666, Sharjah, United Arab Emirates
| | - William G Pitt
- Department of Chemical Engineering, Brigham Young University, Provo, UT, 84602, USA
| | - Ghaleb A Husseini
- Materials Science and Engineering Ph.D. Program, College of Arts and Sciences, American University of Sharjah, P.O. Box. 26666, Sharjah, United Arab Emirates.
- Department of Chemical and Biological Engineering, College of Engineering, American University of Sharjah, P.O. Box 26666, Sharjah, United Arab Emirates.
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Quoniou R, Moreau E, Cachin F, Miot-Noirault E, Chautard E, Peyrode C. 3D Coculture between Cancer Cells and Macrophages: From Conception to Experimentation. ACS Biomater Sci Eng 2024; 10:313-325. [PMID: 38110331 DOI: 10.1021/acsbiomaterials.3c01437] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2023]
Abstract
A tumor is a complex cluster with many types of cells in the microenvironment that help it grow. Macrophages, immune cells whose main role is to maintain body homeostasis, represent in the majority of cancers the most important cell population. In this context, they are called tumor-associated macrophages (TAMs) because of their phenotype, which contributes to tumor growth. In order to limit the use of animals, there is a real demand for the creation of in vitro models able to represent more specifically the complexity of the tumor microenvironment (TME) in order to characterize it and evaluate new treatments. However, the two-dimensional (2D) culture, which has been used for a long time, has shown many limitations, especially in terms of tumor representation. The three-dimensional (3D) models, developed over the last 20 years, have made it possible to get closer to what happens in vivo in terms of phenotypic and functional characteristics. Due to their architectural similarity to in vivo tissues, they provide a more physiologically relevant in vitro system. Most recently, it is the development of 3D coculture models in which two or three cell lines are cultured together that has allowed a better representation of TME with cell-cell interactions. Unfortunately, there is no clear direction for the design of these models at this time. In this Review on the coculture between cancer cells and TAMs, an in-depth analysis is performed to answer multiple questions on the conception of these models: Which models to use, and with which material and cancer lineage? Which monocyte or macrophage lines should be added to the coculture? And how can these models be exploited?
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Affiliation(s)
- Rohan Quoniou
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
| | - Emmanuel Moreau
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
| | - Florent Cachin
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
- Service de Médecine Nucléaire, Centre Jean Perrin, 63000 Clermont-Ferrand, France
| | - Elisabeth Miot-Noirault
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
| | - Emmanuel Chautard
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
- Service de Pathologie, Centre Jean Perrin, 63000 Clermont-Ferrand, France
| | - Caroline Peyrode
- Imagerie Moléculaire et Stratégies Théranostiques, UMR1240, Université Clermont Auvergne, INSERM, 63000 Clermont-Ferrand, France
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42
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Badekila AK, Pai V, Vijayan V, Kini S. Engineering alginate/carboxymethylcellulose scaffolds to establish liver cancer spheroids: Evaluation of molecular variances between 2D and 3D models. Int J Biol Macromol 2024; 254:128058. [DOI: https:/doi.org/10.1016/j.ijbiomac.2023.128058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2023]
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Qi J, Wu H, Liu G. Novel Strategies for Spatiotemporal and Controlled BMP-2 Delivery in Bone Tissue Engineering. Cell Transplant 2024; 33:9636897241276733. [PMID: 39305020 PMCID: PMC11418245 DOI: 10.1177/09636897241276733] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 07/22/2024] [Accepted: 07/26/2024] [Indexed: 09/25/2024] Open
Abstract
Bone morphogenetic protein-2 (BMP-2) has been commercially approved by the Food and Drug Administration for use in bone defects and diseases. BMP-2 promotes osteogenic differentiation of mesenchymal stem cells. In bone tissue engineering, BMP-2 incorporated into scaffolds can be used for stimulating bone regeneration in organoid construction, drug testing platforms, and bone transplants. However, the high dosage and uncontrollable release rate of BMP-2 challenge its clinical application, mainly due to the short circulation half-life of BMP-2, microbial contamination in bone extracellular matrix hydrogel, and the delivery method. Moreover, in clinical translation, the requirement of high doses of BMP-2 for efficacy poses challenges in cost and safety. Based on these, novel strategies should ensure that BMP-2 is delivered precisely to the desired location within the body, regulating the timing of BMP-2 release to coincide with the bone healing process, as well as release BMP-2 in a controlled manner to optimize its therapeutic effect and minimize side effects. This review highlights improvements in bone tissue engineering applying spatiotemporal and controlled BMP-2 delivery, including molecular engineering, biomaterial modification, and synergistic therapy, aiming to provide references for future research and clinical trials.
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Affiliation(s)
- Jingqi Qi
- Department of Orthopedics, Third Xiangya Hospital of Central South University, Changsha, China
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Hongwei Wu
- Department of Orthopedics, The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, China
| | - Gengyan Liu
- Department of Orthopedics, Third Xiangya Hospital of Central South University, Changsha, China
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Xiu Z, Yang Q, Xie F, Han F, He W, Liao W. Revolutionizing digestive system tumor organoids research: Exploring the potential of tumor organoids. J Tissue Eng 2024; 15:20417314241255470. [PMID: 38808253 PMCID: PMC11131411 DOI: 10.1177/20417314241255470] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 05/02/2024] [Indexed: 05/30/2024] Open
Abstract
Digestive system tumors are the leading cause of cancer-related deaths worldwide. Despite ongoing research, our understanding of their mechanisms and treatment remain inadequate. One promising tool for clinical applications is the use of gastrointestinal tract tumor organoids, which serve as an important in vitro model. Tumor organoids exhibit a genotype similar to the patient's tumor and effectively mimic various biological processes, including tissue renewal, stem cell, and ecological niche functions, and tissue response to drugs, mutations, or injury. As such, they are valuable for drug screening, developing novel drugs, assessing patient outcomes, and supporting immunotherapy. In addition, innovative materials and techniques can be used to optimize tumor organoid culture systems. Several applications of digestive system tumor organoids have been described and have shown promising results in related aspects. In this review, we discuss the current progress, limitations, and prospects of this model for digestive system tumors.
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Affiliation(s)
- Zhian Xiu
- Department of Medical Laboratory, Clinical Medical College, Affiliated Hospital of Jiujiang University, Jiujiang, Jiangxi, China
- Jiujiang Clinical Precision Medicine Research Center, Jiujiang, Jiangxi, China
| | - Qian Yang
- Department of Otorhinolaryngology Head and Neck Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Fusheng Xie
- Department of Medical Laboratory, Clinical Medical College, Affiliated Hospital of Jiujiang University, Jiujiang, Jiangxi, China
- Jiujiang Clinical Precision Medicine Research Center, Jiujiang, Jiangxi, China
| | - Feng Han
- Department of Medical Laboratory, Clinical Medical College, Affiliated Hospital of Jiujiang University, Jiujiang, Jiangxi, China
- Jiujiang Clinical Precision Medicine Research Center, Jiujiang, Jiangxi, China
| | - Weiwei He
- Department of Medical Laboratory, Clinical Medical College, Affiliated Hospital of Jiujiang University, Jiujiang, Jiangxi, China
- Jiujiang Clinical Precision Medicine Research Center, Jiujiang, Jiangxi, China
| | - Weifang Liao
- Department of Medical Laboratory, Clinical Medical College, Affiliated Hospital of Jiujiang University, Jiujiang, Jiangxi, China
- Jiujiang Clinical Precision Medicine Research Center, Jiujiang, Jiangxi, China
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Badekila AK, Pai V, Vijayan V, Kini S. Engineering alginate/carboxymethylcellulose scaffolds to establish liver cancer spheroids: Evaluation of molecular variances between 2D and 3D models. Int J Biol Macromol 2024; 254:128058. [PMID: 37956801 DOI: 10.1016/j.ijbiomac.2023.128058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Revised: 09/15/2023] [Accepted: 11/10/2023] [Indexed: 11/15/2023]
Abstract
Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250-300 μm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/β6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.
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Affiliation(s)
- Anjana Kaveri Badekila
- Nitte (Deemed to be University), Department of Bio & Nano Technology, Nitte University Centre for Science Education and Research, Mangalore 575018, Karnataka, India
| | - Vishruta Pai
- Nitte (Deemed to be University), Department of Bio & Nano Technology, Nitte University Centre for Science Education and Research, Mangalore 575018, Karnataka, India
| | - Vijeesh Vijayan
- Nitte (Deemed to be University), Department of Mechanical Engineering, NMAM Institute of Technology (NMAMIT), Nitte 574110, India
| | - Sudarshan Kini
- Nitte (Deemed to be University), Department of Bio & Nano Technology, Nitte University Centre for Science Education and Research, Mangalore 575018, Karnataka, India.
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Abstract
Hydrogel-based three-dimensional (3D) cell culture systems mimic the salient elements of extracellular matrices and promote native cell function. However, high-resolution 3D cell imaging that can provide biological information about multiple features of individual cells is yet to be realized. In this context, we incorporated plasmonic gold nanoparticles (AuNPs) into an alginate/gelatin hydrogel to produce surface-enhanced Raman spectroscopy (SERS)-active hydrogel inks for the 3D printing and culturing of Vero cells. Dense incorporation of AuNPs enables production of a printed 3D grid structure with 3D SERS performance, but with no measurable adverse effects on cell growth. Label-free SERS spectra were collected within a hydrogel, and a random forest binary classifier was developed to discriminate Vero cell signals from the hydrogel background with an accuracy of 87.5%. The results suggest that SERS signals from cellular components, such as proteins, lipids, and carbohydrates, account for this discrimination. We demonstrate visualization of cell shape, location, and density by combining predicted binary maps with peak feature intensity maps in 2D and 3D. SERS images with a resolution of ≈3 μm match well with the microscopy images and show clear increases in intensity with incubation time. We suggest that 3D SERS cell imaging is a promising means to examine the effect of external cell stimuli on cellular behavior for diagnostic purposes.
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Affiliation(s)
- Wei Wang
- Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
- Virginia Tech Institute of Critical Technology and Applied Science (ICTAS) Sustainable Nanotechnology Center (VTSuN), Blacksburg, Virginia 24061, United States
| | - Peter J Vikesland
- Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
- Virginia Tech Institute of Critical Technology and Applied Science (ICTAS) Sustainable Nanotechnology Center (VTSuN), Blacksburg, Virginia 24061, United States
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Dzurov M, Pospíšilová Š, Krafčíková M, Trantírek L, Vojtová L, Ryneš J. A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins. JOURNAL OF BIOMOLECULAR NMR 2023; 77:203-215. [PMID: 37688760 PMCID: PMC10687187 DOI: 10.1007/s10858-023-00422-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Accepted: 07/15/2023] [Indexed: 09/11/2023]
Abstract
Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15-40 °C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.
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Affiliation(s)
- Matej Dzurov
- CEITEC - Central European Institute of Technology, Brno University of Technology, Purkyňova 656/123, Brno, 612 00, Czech Republic
| | - Šárka Pospíšilová
- CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 753/5, Brno, 625 00, Czech Republic
| | - Michaela Krafčíková
- National Centre for Biomolecular Research, Masaryk University, Kamenice 753/5, Brno, 625 00, Czech Republic
- Institute of Biophysics, Czech Academy of Sciences, Královopolská 135, Brno, 612 65, Czech Republic
- Bijvoet Centre for Biomolecular Research, Utrecht University, Padualaan 12, Utrecht, 3584 CH, The Netherlands
| | - Lukáš Trantírek
- CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 753/5, Brno, 625 00, Czech Republic
| | - Lucy Vojtová
- CEITEC - Central European Institute of Technology, Brno University of Technology, Purkyňova 656/123, Brno, 612 00, Czech Republic.
| | - Jan Ryneš
- CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 753/5, Brno, 625 00, Czech Republic.
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48
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Izanlou S, Afshar A, Zare A, Zhilisbayeva KR, Bakhshalizadeh S, Safaei Z, Sehat-Bakhsh S, Khaledi S, Asgari HR, Kazemnejad S, Ajami M, Ajami M, Dehghan Tarzjani M, Najafzadeh V, Kouchakian MR, Mussin NM, Kaliyev AA, Aringazina RA, Mahdipour M, Shirazi R, Tamadon A. Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold. Tissue Cell 2023; 85:102215. [PMID: 37716177 DOI: 10.1016/j.tice.2023.102215] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 08/17/2023] [Accepted: 09/09/2023] [Indexed: 09/18/2023]
Abstract
Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.
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Affiliation(s)
- Safoura Izanlou
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran
| | - Alireza Afshar
- Student Research Committee, Bushehr University of Medical Sciences, Bushehr, Islamic Republic of Iran
| | - Afshin Zare
- PerciaVista R&D Co., Shiraz, Islamic Republic of Iran
| | - Kulyash R Zhilisbayeva
- Department of Scientific Work, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
| | - Shabnam Bakhshalizadeh
- Reproductive Development, Murdoch Children's Research Institute, Melbourne, Victoria, Australia; Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia.
| | - Zahra Safaei
- Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland, OR, 97239, USA
| | - Soheila Sehat-Bakhsh
- Department of Anatomical Sciences, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Islamic Republic of Iran
| | - Sajed Khaledi
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran
| | - Hamid-Reza Asgari
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran
| | - Somaieh Kazemnejad
- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Islamic Republic of Iran
| | - Mansoureh Ajami
- Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Islamic Republic of Iran
| | - Monireh Ajami
- Department of Hematology, Faculty of Paramedical Sciences, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Islamic Republic of Iran
| | - Masoumeh Dehghan Tarzjani
- Department of Gynecology and Obstetrics, Imam Khomeinin Hospital, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
| | | | - Mohammad Reza Kouchakian
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran
| | - Nadiar M Mussin
- General Surgery, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
| | - Asset A Kaliyev
- General Surgery, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
| | - Raisa A Aringazina
- Department of Internal Medicine No. 1, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
| | - Mahdi Mahdipour
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran; Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Reza Shirazi
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran; Department of Anatomy, School of Biomedical Sciences, Medicine & Health, UNSW Sydney, Sydney, Australia.
| | - Amin Tamadon
- PerciaVista R&D Co., Shiraz, Islamic Republic of Iran; Department of Scientific Work, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan.
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Utagawa Y, Ino K, Takinoue M, Shiku H. Fabrication and Cell Culture Applications of Core-Shell Hydrogel Fibers Composed of Chitosan/DNA Interfacial Polyelectrolyte Complexation and Calcium Alginate: Straight and Beaded Core Variations. Adv Healthc Mater 2023; 12:e2302011. [PMID: 37478383 PMCID: PMC11468996 DOI: 10.1002/adhm.202302011] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Indexed: 07/23/2023]
Abstract
Core-shell hydrogel fibers are widely used in cell culture applications. A simple and rapid method is presented for fabricating core-shell hydrogel fibers, consisting of straight or beaded core fibers, for cell culture applications. The core fibers are prepared using interfacial polyelectrolyte complexation (IPC) with chitosan and DNA. Briefly, two droplets of chitosan and DNA are brought in contact to form an IPC film, which is dragged to prepare an IPC fiber. The incubation time and DNA concentration are adjusted to prepare straight and beaded IPC fibers. The fibers with Ca2+ are immersed in an alginate solution to form calcium alginate shell hydrogels around the core IPC fibers. To the best of the knowledge, this is the first report of core-shell hydrogel fibers with IPC fiber cores. To demonstrate cell culture, straight hydrogel fibers are applied to fabricate hepatic models consisting of HepG2 and 3T3 fibroblasts, and vascular models consisting of human umbilical vein endothelial cells and 3T3 fibroblasts. To evaluate the effect of co-culture, albumin secretion, and angiogenesis are evaluated. Beaded hydrogel fibers are used to fabricate many size-controlled spheroids for fiber and cloning applications. This method can be widely applied in tissue engineering and cell analysis.
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Affiliation(s)
| | - Kosuke Ino
- Graduate School of EngineeringTohoku UniversitySendai980–8579Japan
| | - Masahiro Takinoue
- Department of Computer ScienceTokyo Institute of TechnologyYokohama226–8502Japan
| | - Hitoshi Shiku
- Graduate School of EngineeringTohoku UniversitySendai980–8579Japan
- Graduate School of Environmental StudiesTohoku UniversitySendai980–8579Japan
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50
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Le HT, Phan HL, Lenshof A, Duong VT, Choi C, Cha C, Laurell T, Koo KI. Ultrasound standing wave spatial patterning of human umbilical vein endothelial cells for 3D micro-vascular networks formation. Biofabrication 2023; 16:015009. [PMID: 37844581 DOI: 10.1088/1758-5090/ad03be] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Accepted: 10/16/2023] [Indexed: 10/18/2023]
Abstract
Generating functional and perfusable micro-vascular networks is an important goal for the fabrication of large and three-dimensional tissues. Up to now, the fabrication of micro-vascular networks is a complicated multitask involving several different factors such as time consuming, cells survival, micro-diameter vasculature and strict alignment. Here, we propose a technique combining multi-material extrusion and ultrasound standing wave forces to create a network structure of human umbilical vein endothelial cells within a mixture of calcium alginate and decellularized extracellular matrix. The functionality of the matured microvasculature networks was demonstrated through the enhancement of cell-cell adhesion, angiogenesis process, and perfusion tests with microparticles, FITC-dextran, and whole mouse blood. Moreover, animal experiments exhibited the implantability including that the pre-existing blood vessels of the host sprout towards the preformed vessels of the scaffold over time and the microvessels inside the implanted scaffold matured from empty tubular structures to functional blood-carrying microvessels in two weeks.
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Affiliation(s)
- Huong Thi Le
- Department of Electrical, Electronic and Computer Engineering, University of Ulsan, Ulsan 44610, Republic of Korea
| | - Huu Lam Phan
- Department of Electrical, Electronic and Computer Engineering, University of Ulsan, Ulsan 44610, Republic of Korea
| | - Andreas Lenshof
- Department of Biomedical Engineering, Lund University, S-221 00 Lund, Sweden
| | - Van Thuy Duong
- Department of Electrical, Electronic and Computer Engineering, University of Ulsan, Ulsan 44610, Republic of Korea
| | - Cholong Choi
- Department of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea
| | - Chaenyung Cha
- Department of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea
| | - Thomas Laurell
- Department of Biomedical Engineering, Lund University, S-221 00 Lund, Sweden
| | - Kyo-In Koo
- Department of Electrical, Electronic and Computer Engineering, University of Ulsan, Ulsan 44610, Republic of Korea
- Basic-Clinical Convergence Research Institute, University of Ulsan, Ulsan, Republic of Korea
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