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1
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Nepal S, Casalini J, Jafek A, Gale B. Application of Inertial Microfluidics for Isolation and Removal of Round Spermatids from a Spermatogenic Cell Sample to Assist In-Vitro Human Spermatogenesis. MICROMACHINES 2025; 16:500. [PMID: 40428627 DOI: 10.3390/mi16050500] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2025] [Revised: 04/22/2025] [Accepted: 04/24/2025] [Indexed: 05/29/2025]
Abstract
In-vitro spermatogenesis holds great potential in addressing male infertility, yet one of the main challenges is separating round spermatids from other germ cells in spermatogonial stem cell cultures. STA-PUT, a method based on velocity sedimentation, has been extensively tested for this application. Though somewhat effective, it requires bulky, expensive equipment and significant time. In contrast, the method of inertial microfluidics offers a compact, cost-effective, and faster alternative. In this study, we designed, fabricated, and tested a microfluidic spiral channel for isolating round spermatids and purifying spermatogenic cells. A commercially available spiral device close to the calculated specifications was tested for rapid prototyping, achieving 79% purity for non-spermatid cells in a single pass, with ability to achieve higher purity through repeated passes. However, the commercial device's narrow outlets caused clogging, prompting the fabrication of a custom polydimethylsiloxane device matching the calculated specifications. This custom device demonstrated significant improvements, achieving 86% purity in a single pass compared to STA-PUT's 38%, and that without any clogging issues. Further purification could be attained by repeated passes, as shown in earlier studies. This work underscores the efficacy of inertial microfluidics for efficient, high-purity cell separation, with the potential to revolutionize workflows in in-vitro spermatogenesis research.
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Affiliation(s)
- Sabin Nepal
- Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA
| | - Joey Casalini
- Paterna Biosciences Inc., Salt Lake City, UT 84119, USA
| | - Alex Jafek
- Paterna Biosciences Inc., Salt Lake City, UT 84119, USA
| | - Bruce Gale
- Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA
- Paterna Biosciences Inc., Salt Lake City, UT 84119, USA
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2
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Liu B, Ren X, Xue T, Zou Q. A Slanted-Finger Interdigitated Transducer Microfluidic Device for Particles Sorting. MICROMACHINES 2025; 16:483. [PMID: 40283358 PMCID: PMC12029442 DOI: 10.3390/mi16040483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/31/2025] [Revised: 04/13/2025] [Accepted: 04/19/2025] [Indexed: 04/29/2025]
Abstract
Sorting particles or cells of specific sizes in complex systems has long been a focus of many researchers. Acoustic surface waves, which generate acoustic radiation forces on particles or cells and, thus, influence their motion, are commonly used for the non-destructive separation of particles or cells of specific sizes. In previous studies, the frequency of acoustic surface wave generation has been limited by the interdigitated transducer (IDT). To extend the effective operating frequency range of the IDT, a slanted-finger interdigitated transducer (SFIT) with a wide acoustic path and multiple operating frequencies was designed. Compared with traditional acoustic sorting devices, which suffer from a limited frequency range and narrow acoustic paths, this new design greatly expands both the operating frequency range and acoustic path width, and enables adjustable operating frequencies, providing a solution for sorting particles or cells with uneven sizes in complex environments. The optimal resonance frequency is distributed within the 32-42 MHz range, and the operating frequencies within this range can generate a standing wave acoustic path of approximately 200 μm, thus enhancing the effectiveness of the operating frequencies. The microfluidic sorting device based on SFIT can efficiently and accurately sort polystyrene (PS) with particle sizes of 20 μm, 30 μm, and 50 μm from mixed PS microspheres (5, 10, 20 μm), (5, 10, 30 μm), and (5, 10, 50 μm), with a sorting efficiency and purity exceeding 96%. Additionally, the device is capable of sorting other types of mixed microspheres (5, 10, 20, 30, 50 μm). This new wide-acoustic-path, multi-frequency sorting device demonstrates the ability to sort particlesin a high-purity, label-free manner, offering a more alternative to traditional sorting methods.
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Affiliation(s)
- Baoguo Liu
- School of Microelectronics, Tianjin University, Tianjin 300072, China; (B.L.); (X.R.)
| | - Xiang Ren
- School of Microelectronics, Tianjin University, Tianjin 300072, China; (B.L.); (X.R.)
- State Key Laboratory of Advanced Materials for Intelligent Sensing, Tianjin University, Tianjin 300072, China
| | - Tao Xue
- Center of Analysis and Testing Facilities, Tianjin University, Tianjin 300072, China;
| | - Qiang Zou
- School of Microelectronics, Tianjin University, Tianjin 300072, China; (B.L.); (X.R.)
- State Key Laboratory of Advanced Materials for Intelligent Sensing, Tianjin University, Tianjin 300072, China
- Tianjin International Joint Research Center for Internet of Things, Tianjin 300072, China
- Tianjin Key Laboratory of Imaging and Sensing Microelectronic Technology, Tianjin University, Tianjin 300072, China
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3
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Mehanna LE, Boyd JD, Walker CG, Osborne AR, Grady ME, Berron BJ. Functional assessment of migration and adhesion to quantify cancer cell aggression. SOFT MATTER 2025; 21:2946-2957. [PMID: 40151848 DOI: 10.1039/d4sm01351d] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/29/2025]
Abstract
During epithelial-to-mesenchymal transition (EMT), cancer cells lose their cell-cell adhesion junctions as they become more metastatic, altering cell motility and focal adhesion disassembly associated with increased detachment from the primary tumor and a migratory response into nearby tissue and vasculature. Current in vitro strategies characterizing a cell's metastatic potential heavily favor quantifying the presence of cell adhesion biomarkers through biochemical analysis; however, mechanical cues such as adhesion and motility directly relate to cell metastatic potential without needing to first identify a cell specific biomarker for a particular type of cancer. This paper presents a comprehensive comparison of two functional metrics of cancer aggression, wound closure migration velocity and cell detachment from a culture surface, for three pairs of epithelial cancer cell lines (breast, endometrium, tongue tissue origins). It was found that one functional metric alone was not sufficient to categorize the cancer cell lines; instead, both metrics were necessary to identify functional trends and accurately place cells on the spectrum of metastasis. On average, cell lines with low metastatic potential (MCF-7, Ishikawa, and Cal-27) were more aggressive through wound closure migration compared to loss of cell adhesion. On the other hand, cell lines with high metastatic potential (MDA-MB-231, KLE, and SCC-25) were on average more aggressive through loss of cell adhesion compared to wound closure migration. This trend was true independent of the tissue type where the cells originated, indicating that there is a relationship between metastatic potential and the predominate type of cancer aggression. Our work presents one of the first combined studies relating cell metastatic potential to functional migration and adhesion metrics across cancer cell lines from selected tissue origins, without needing to identify tissue-specific biomarkers to achieve success. Using functional metrics provides powerful clinical relevancy for future predictive tools of cancer metastasis.
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Affiliation(s)
- Lauren E Mehanna
- Department of Chemical and Materials Engineering, University of Kentucky, 177 F. Paul Anderson Tower, Lexington, KY 40506, USA.
| | - James D Boyd
- Department of Mechanical and Aerospace Engineering, University of Kentucky, 151 Ralph G. Anderson Building, Lexington, KY 40506, USA.
| | - Chloe G Walker
- Department of Chemical and Materials Engineering, University of Kentucky, 177 F. Paul Anderson Tower, Lexington, KY 40506, USA.
| | - Adrianna R Osborne
- Department of Chemical and Materials Engineering, University of Kentucky, 177 F. Paul Anderson Tower, Lexington, KY 40506, USA.
| | - Martha E Grady
- Department of Mechanical and Aerospace Engineering, University of Kentucky, 151 Ralph G. Anderson Building, Lexington, KY 40506, USA.
| | - Brad J Berron
- Department of Chemical and Materials Engineering, University of Kentucky, 177 F. Paul Anderson Tower, Lexington, KY 40506, USA.
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4
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Penjweini R, Link KA, Qazi S, Mattu N, Zuchowski A, Vasta A, Sackett DL, Knutson JR. Low dose Taxol causes mitochondrial dysfunction in actively respiring cancer cells. J Biol Chem 2025; 301:108450. [PMID: 40147771 PMCID: PMC12052845 DOI: 10.1016/j.jbc.2025.108450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2024] [Revised: 03/03/2025] [Accepted: 03/23/2025] [Indexed: 03/29/2025] Open
Abstract
Mitochondrial oxygen consumption, dynamics, and morphology play roles in the occurrence, development, and drug resistance of cancer; thus, they are main targets for many anticancer drugs. Increased mitochondrial oxygen consumption and impaired oxygen delivery creates hypoxia, which influences the balance of metabolic cofactors for biogenesis, disease progression, and response to therapeutics. We therefore investigated the effects of Taxol, a well-known anticancer drug, on mitochondrial respiration (principally via a measure of oxidative phosphorylation versus glycolysis), morphology, and dynamics. The concomitant effects of Taxol on mitochondrial ATP and reactive oxygen species production, mitochondrial membrane potential, radical-induced formation of carbonyl groups, mitochondrial release of cytochrome c, as well as cell cycle were investigated. Cells used in this study include the following: A549 (non-small-cell lung epithelial cancer cell line), A549-ρ0 (mitochondrial DNA-depleted derivative of A549), and BEAS-2B (a noncancer cell line derived from normal bronchial epithelium), as well as PC3 (prostate cancer) and HepG2 (hepatocellular carcinoma); these cell lines are known to have disparate metabolic profiles. Using a multitude of fluorescence-based measurements, we show that Taxol, even at a low dose, still adversely affects mitochondria of actively respiring (aerobic) cancer cells. We find an increase in mitochondrial ROS and cytochrome c release, suppression of ATP production and oxidative phosphorylation, fragmentation of the mitochondrial network, and disruption of mitochondria-microtubule linkage. We find these changes in oxidative, but not glycolytic, cancer cells. Noncancer cells, which are oxidative, do not show these changes.
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Affiliation(s)
- Rozhin Penjweini
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Katie A Link
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Shureed Qazi
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Nikhil Mattu
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Adam Zuchowski
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Alexandra Vasta
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA
| | - Dan L Sackett
- Cytoskeletal Dynamics Group, Division of Basic and Translational Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA.
| | - Jay R Knutson
- Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA.
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Moreno TM, Nieto-Torres JL, Kumsta C. Monitoring Autophagy in Human Aging: Key Cell Models and Insights. FRONT BIOSCI-LANDMRK 2025; 30:27091. [PMID: 40152379 PMCID: PMC12042822 DOI: 10.31083/fbl27091] [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: 10/23/2024] [Revised: 11/08/2024] [Accepted: 11/22/2024] [Indexed: 03/29/2025]
Abstract
Autophagy, a key cellular degradation and recycling pathway, is critical for maintaining cellular homeostasis and responding to metabolic and environmental stress. Evidence for age-related autophagic dysfunction and its implications in chronic age-related diseases including neurodegeneration is accumulating. However, as a complex, multi-step process, autophagy can be challenging to measure, particularly in humans and human aging- and disease-relevant models. This review describes the links between macroautophagy, aging, and chronic age-related diseases. We present three novel human cell models, peripheral blood mononuclear cells (PBMCs), primary dermal fibroblasts (PDFs), and induced neurons (iNs), which serve as essential tools for studying autophagy flux and assessing its potential as a biomarker for aging. Unlike traditional models, these cell models retain age- and disease-associated molecular signatures, enhancing their relevance for human studies. The development of robust tools and methodologies for measuring autophagy flux in human cell models holds promise for advancing our understanding of autophagy's role in aging and age-related diseases, ultimately facilitating the discovery of therapies to enhance health outcomes.
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Affiliation(s)
- Tatiana M. Moreno
- Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
| | - Jose L. Nieto-Torres
- Department of Biomedical Sciences, School of Health Sciences, Universidad Cardenal Herrera-CEU, CEU Universities, 46115 Valencia, Spain
| | - Caroline Kumsta
- Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
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6
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Rathnaweera TN, Anand RK. iDEP-based single-cell isolation in a two-dimensional array of chambers addressed by easy-to-align wireless electrodes. LAB ON A CHIP 2025; 25:1600-1610. [PMID: 39950224 PMCID: PMC11826382 DOI: 10.1039/d4lc00976b] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Accepted: 01/21/2025] [Indexed: 02/16/2025]
Abstract
Platforms capable of selective single-cell capture and enclosure in a fluidically isolated volume for subsequent analysis are crucial for unmasking cellular heterogeneity. Our research group has previously reported an approach that employs wireless bipolar electrodes (BPEs) to facilitate individual isolation of cells in large arrays of pico- to nanoliter scale chambers by dielectrophoresis (DEP). This device was leveraged for a single-cell enzymatic assay and the isolation of circulating tumor cells (CTCs) from patient-derived blood samples, which takes advantage of the selectivity of DEP. However, alignment of BPEs to the microchamber openings is nontrivial, and augmentation of the array dimensions accumulates alignment error, thereby disrupting the uniformity of cell capture across the device. Thus, tolerance-forgiving designs that are simultaneously expandable are in demand. To address this demand, we present an approach that combines BPEs with insulator DEP (iDEP) to drastically expand alignment tolerance. This iDEP-BPE device offers a vertical tolerance (the distance the BPE is recessed within each microchamber) of 80 μm while the horizontal tolerance is nearly infinite. Further, the iDEP-BPE device decreases the exposure of cells to electrode surfaces and reactive oxygen species, thereby preserving their viability. Finally, this iDEP approach can be carried out with BPEs that are easy to fabricate, lacking features that require high-resolution lithography. These advancements potentiate the broad adoption of the iDEP-BPE approach for selective single-cell capture and on-chip analysis and potentiate its commercialization upon deployment of appropriate thermoplastic materials.
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Affiliation(s)
| | - Robbyn K Anand
- Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
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7
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Kang X, Ma J, Cha H, Hansen HHWB, Chen X, Ta HT, Tian F, Nguyen NT, Klimenko A, Zhang J, Yuan D. Ultra-Stretchable Microfluidic Devices for Optimizing Particle Manipulation in Viscoelastic Fluids. ACS APPLIED MATERIALS & INTERFACES 2024; 16:61765-61773. [PMID: 39496575 DOI: 10.1021/acsami.4c15893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2024]
Abstract
Viscoelastic microfluidics leverages the unique properties of non-Newtonian fluids to manipulate and separate micro- or submicron particles. Channel geometry and dimension are crucial for device performance. Traditional rigid microfluidic devices require numerous iterations of fabrication and testing to optimize these parameters, which is time-consuming and costly. In this work, we developed a flexible microfluidic device using ultra-stretchable and biocompatible Flexdym material to overcome this issue. Our device allows for simultaneous modification of channel dimensions by external stretching. We fabricated a stretchable device with an initial square microchannel (30 μm × 30 μm), and the channel aspect ratio can be adjusted from 1 to 5 by external stretching. Next, the effects of aspect ratio, particle size, flow rate, and poly(ethylene oxide) (PEO) concentration that make the fluid viscoelastic on particle migration were investigated. Finally, we demonstrated the feasibility of our approach by testing channels with an aspect ratio of 3 for the separation of both particles and cells.
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Affiliation(s)
- Xiaoyue Kang
- School of Mechanical and Mining Engineering, University of Queensland, St. Lucia, Brisbane, Queensland 4067, Australia
| | - Jingtao Ma
- School of Engineering and Information Technology, University of New South Wales, Canberra, ACT 2600, Australia
| | - Haotian Cha
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
| | - Helena H W B Hansen
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
| | - Xiangxun Chen
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
| | - Hang T Ta
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
- Bioscience Discipline, School of Environment and Science, Griffith University, Nathan, Queensland 4111, Australia
| | - Fangbao Tian
- School of Engineering and Information Technology, University of New South Wales, Canberra, ACT 2600, Australia
| | - Nam-Trung Nguyen
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
| | - Alexander Klimenko
- School of Mechanical and Mining Engineering, University of Queensland, St. Lucia, Brisbane, Queensland 4067, Australia
| | - Jun Zhang
- Queensland Micro and Nanotechnology Centre, Griffith University, Nathan, Brisbane, Queensland 4111, Australia
- School of Engineering and Built Environment, Griffith University, Nathan, Queensland 4111, Australia
| | - Dan Yuan
- School of Mechanical and Mining Engineering, University of Queensland, St. Lucia, Brisbane, Queensland 4067, Australia
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8
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de Hemptinne A, Gelin P, Bihi I, Kinet R, Thienpont B, De Malsche W. Exploring operational boundaries for acoustic concentration of cell suspensions. Appl Microbiol Biotechnol 2024; 108:387. [PMID: 38896136 PMCID: PMC11186915 DOI: 10.1007/s00253-024-13215-1] [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: 01/08/2024] [Revised: 05/15/2024] [Accepted: 05/27/2024] [Indexed: 06/21/2024]
Abstract
The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.
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Affiliation(s)
- Amaury de Hemptinne
- Department of Chemical Engineering, µFlow Group, Vrije Universiteit Brussel, 1050, Brussels, Belgium.
| | - Pierre Gelin
- Department of Chemical Engineering, µFlow Group, Vrije Universiteit Brussel, 1050, Brussels, Belgium
| | - Ilyesse Bihi
- Department of Chemical Engineering, µFlow Group, Vrije Universiteit Brussel, 1050, Brussels, Belgium
| | | | | | - Wim De Malsche
- Department of Chemical Engineering, µFlow Group, Vrije Universiteit Brussel, 1050, Brussels, Belgium.
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9
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Hebel D, Schönherr H. Mild Quantitative One Step Removal of Macrophages from Cocultures with Human Umbilical Vein Endothelial Cells Using Thermoresponsive Poly(Di(Ethylene Glycol)Methyl Ether Methacrylate) Brushes. Macromol Biosci 2024; 24:e2300408. [PMID: 37916483 DOI: 10.1002/mabi.202300408] [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: 09/08/2023] [Revised: 10/24/2023] [Indexed: 11/03/2023]
Abstract
The authors report on a mild, label-free, and fast method for the separation of human umbilical vein endothelial cells (HUVEC), which are relevant cells, whose use is not limited to studies of endothelial dysfunction, from cocultures with macrophages to afford HUVEC in ≈100% purity. Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes with a dry thickness of (5 ± 1) nm afford the highly effective one-step separation by selective HUVEC detachment, which is based on the brushes' thermoresponsive behavior. Below the thermal transition at 32 °C the brushes swells and desorbs attached proteins, resulting in markedly decreased cell adhesion. Specifically, HUVEC and macrophages, which are differentiated from THP-1 monocytes, are seeded and attached to PDEGMA brushes at 37°C. After decreasing the temperature to 22°C, HUVEC shows a decrease in their cell area, while the macrophages are not markedly affected by the temperature change. After mild flushing with a cell culture medium, the HUVEC can be released from the surface and reseeded again with ≈100% purity on a new surface. With this selective cell separation and removal method, it is possible to separate and thereby purify HUVEC from macrophages without the use of any releasing reagent or expensive labels, such as antibodies.
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Affiliation(s)
- Diana Hebel
- Department of Chemistry and Biology, University of Siegen, Physical Chemistry I & Research Center of Micro and Nanochemistry and (Bio)Technology (Cµ), Adolf-Reichwein-Str. 2, 57076, Siegen, Germany
| | - Holger Schönherr
- Department of Chemistry and Biology, University of Siegen, Physical Chemistry I & Research Center of Micro and Nanochemistry and (Bio)Technology (Cµ), Adolf-Reichwein-Str. 2, 57076, Siegen, Germany
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10
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Shirani E, Razmjou A, Asadnia M, Nordon RE, Inglis DW. Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2024; 40:4361-4372. [PMID: 38357828 DOI: 10.1021/acs.langmuir.3c03644] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/16/2024]
Abstract
Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.
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Affiliation(s)
- Elham Shirani
- School of Engineering, Macquarie University, Sydney, New South Wales 2109, Australia
| | - Amir Razmjou
- Mineral Recovery Research Center (MRRC), School of Engineering, Edith Cowan University, Joondalup, Perth, Western Australia 6027, Australia
| | - Mohsen Asadnia
- School of Engineering, Macquarie University, Sydney, New South Wales 2109, Australia
| | - Robert E Nordon
- Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales 2052, Australia
| | - David W Inglis
- School of Engineering, Macquarie University, Sydney, New South Wales 2109, Australia
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11
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Kuru Cİ, Ulucan-Karnak F, Dayıoğlu B, Şahinler M, Şendemir A, Akgöl S. Affinity-Based Magnetic Nanoparticle Development for Cancer Stem Cell Isolation. Polymers (Basel) 2024; 16:196. [PMID: 38256995 PMCID: PMC10818538 DOI: 10.3390/polym16020196] [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: 08/29/2023] [Revised: 10/06/2023] [Accepted: 12/27/2023] [Indexed: 01/24/2024] Open
Abstract
Cancer is still the leading cause of death in the world despite the developing research and treatment opportunities. Failure of these treatments is generally associated with cancer stem cells (CSCs), which cause metastasis and are defined by their resistance to radio- and chemotherapy. Although known stem cell isolation methods are not sufficient for CSC isolation, they also bring a burden in terms of cost. The aim of this study is to develop a high-efficiency, low-cost, specific method for cancer stem cell isolation with magnetic functional nanoparticles. This study, unlike the stem cell isolation techniques (MACS, FACS) used today, was aimed to isolate cancer stem cells (separation of CD133+ cells) with nanoparticles with specific affinity and modification properties. For this purpose, affinity-based magnetic nanoparticles were synthesized and characterized by providing surface activity and chemical reactivity, as well as making surface modifications necessary for both lectin affinity and metal affinity interactions. In the other part of the study, synthesized and characterized functional polymeric magnetic nanoparticles were used for the isolation of CSC from the human osteosarcoma cancer cell line (SAOS-2) with a cancer stem cell subpopulation bearing the CD133 surface marker. The success and efficiency of separation after stem cell isolation were evaluated via the MACS and FACS methods. As a result, when the His-graft-mg-p(HEMA) nanoparticle was used at a concentration of 0.1 µg/mL for 106 and 108 cells, superior separation efficiency to commercial microbeads was obtained.
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Affiliation(s)
- Cansu İlke Kuru
- Department of Biochemistry, Faculty of Science, Ege University, 35100 İzmir, Turkey; (C.İ.K.); (S.A.)
| | - Fulden Ulucan-Karnak
- Department of Biochemistry, Faculty of Science, Ege University, 35100 İzmir, Turkey; (C.İ.K.); (S.A.)
| | - Büşra Dayıoğlu
- Department of Bioengineering, Faculty of Engineering, Ege University, 35100 İzmir, Turkey; (B.D.); (M.Ş.); (A.Ş.)
| | - Mert Şahinler
- Department of Bioengineering, Faculty of Engineering, Ege University, 35100 İzmir, Turkey; (B.D.); (M.Ş.); (A.Ş.)
| | - Aylin Şendemir
- Department of Bioengineering, Faculty of Engineering, Ege University, 35100 İzmir, Turkey; (B.D.); (M.Ş.); (A.Ş.)
| | - Sinan Akgöl
- Department of Biochemistry, Faculty of Science, Ege University, 35100 İzmir, Turkey; (C.İ.K.); (S.A.)
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12
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Yang GD, Ma DS, Ma CY, Bai Y. Research Progress on Cardiac Tissue Construction of Mesenchymal Stem Cells for Myocardial Infarction. Curr Stem Cell Res Ther 2024; 19:942-958. [PMID: 37612870 DOI: 10.2174/1574888x18666230823091017] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 07/13/2023] [Accepted: 07/26/2023] [Indexed: 08/25/2023]
Abstract
Heart failure is still the main complication affecting the prognosis of acute myocardial infarction (AMI), and mesenchymal stem cells (MSCs) are an effective treatment to replace necrotic myocardium and improve cardiac functioning. However, the transplant survival rate of MSCs still presents challenges. In this review, the biological characteristics of MSCs, the progress of mechanism research in the treatment of myocardial infarction, and the advances in improving the transplant survival rate of MSCs in the replacement of necrotic myocardial infarction are systematically described. From a basic to advanced clinical research, MSC transplants have evolved from a pure injection, an exosome injection, the genetic modification of MSCs prior to injection to the cardiac tissue engineering of MSC patch grafting. This study shows that MSCs have wide clinical applications in the treatment of AMI, suggesting improved myocardial tissue creation. A broader clinical application prospect will be explored and developed to improve the survival rate of MSC transplants and myocardial vascularization.
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Affiliation(s)
- Guo-Dong Yang
- Department of Cardiac Surgery, The First Hospital of Jilin University, Changchun, 130021, China
| | - Da-Shi Ma
- Department of Cardiac Surgery, The First Hospital of Jilin University, Changchun, 130021, China
| | - Chun-Ye Ma
- Department of Cardiac Surgery, The First Hospital of Jilin University, Changchun, 130021, China
| | - Yang Bai
- Department of Cardiac Surgery, The First Hospital of Jilin University, Changchun, 130021, China
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13
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Sammeth M, Mudra S, Bialdiga S, Hartmannsberger B, Kramer S, Rittner H. Comparative Methods for Demystifying Spatial Transcriptomics. Methods Mol Biol 2024; 2802:515-546. [PMID: 38819570 DOI: 10.1007/978-1-0716-3838-5_17] [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: 06/01/2024]
Abstract
Spatial Transcriptomics (ST), coined as the term for parallel RNA-Seq on cell populations ordered spatially on a histological tissue section, has recently become increasingly popular, especially in experiments where microfluidics-based single-cell sequencing fails, such as assays on neurons. ST platforms, like the 10x Visium technology investigated herein, therefore produce in a single experiment simultaneously thousands of RNA readouts, captured by an array of micrometer scale spots under the histological section. Therefore, a central challenge of analyzing ST experiments consists of analyzing the gene expression morphology of all spots to delineate clusters of similar cell mixtures, which are then compared to each other to identify up- or down-regulated marker genes. Moreover, another level of complexity in ST experiments, compared to traditional RNA-Seq, is imposed by staining the tissue section with protein markers of cells or cell components to identify spots providing relevant information afterward. The corresponding microscopy images need to be analyzed in addition to the RNA-Seq read mappings on the reference genome and transcriptome sequences. Focusing on the software suite provided by the Visium platform manufacturer, we break down the ST analysis pipeline into its four essential steps-the image analysis, the read alignment, the gene quantification, and the spot clustering-and compare results obtained when using reads from different subsets of spots and/or when employing alternative genome or transcriptome references. Our comparative analyses demonstrate the impact of spot selection and the choice of genome/transcriptome references on the analysis results when employing the manufacturer's pipeline.
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Affiliation(s)
- Michael Sammeth
- Coburg University of Applied Sciences and Art, Department of Applied Sciences and Health, Coburg, Germany.
- University Hospital Würzburg, Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, Würzburg, Germany.
| | - Susann Mudra
- Coburg University of Applied Sciences and Art, Department of Applied Sciences and Health, Coburg, Germany
| | - Sina Bialdiga
- Coburg University of Applied Sciences and Art, Department of Applied Sciences and Health, Coburg, Germany
| | - Beate Hartmannsberger
- University Hospital Würzburg, Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, Centre for Interdisciplinary Pain Medicine, Würzburg, Germany
| | - Sofia Kramer
- University Hospital Würzburg, Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, Centre for Interdisciplinary Pain Medicine, Würzburg, Germany
| | - Heike Rittner
- University Hospital Würzburg, Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, Centre for Interdisciplinary Pain Medicine, Würzburg, Germany
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14
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Timonen VA, Kerkelä E, Impola U, Penna L, Partanen J, Kilpivaara O, Arvas M, Pitkänen E. DeepIFC: Virtual fluorescent labeling of blood cells in imaging flow cytometry data with deep learning. Cytometry A 2023; 103:807-817. [PMID: 37276178 DOI: 10.1002/cyto.a.24770] [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: 09/30/2022] [Revised: 05/16/2023] [Accepted: 06/02/2023] [Indexed: 06/07/2023]
Abstract
Imaging flow cytometry (IFC) combines flow cytometry with microscopy, allowing rapid characterization of cellular and molecular properties via high-throughput single-cell fluorescent imaging. However, fluorescent labeling is costly and time-consuming. We present a computational method called DeepIFC based on the Inception U-Net neural network architecture, able to generate fluorescent marker images and learn morphological features from IFC brightfield and darkfield images. Furthermore, the DeepIFC workflow identifies cell types from the generated fluorescent images and visualizes the single-cell features generated in a 2D space. We demonstrate that rarer cell types are predicted well when a balanced data set is used to train the model, and the model is able to recognize red blood cells not seen during model training as a distinct entity. In summary, DeepIFC allows accurate cell reconstruction, typing and recognition of unseen cell types from brightfield and darkfield images via virtual fluorescent labeling.
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Affiliation(s)
- Veera A Timonen
- Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland
- Applied Tumor Genomics Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Erja Kerkelä
- Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Vantaa, Finland
| | - Ulla Impola
- Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland
| | - Leena Penna
- Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland
| | - Jukka Partanen
- Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland
| | - Outi Kilpivaara
- Applied Tumor Genomics Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Medical and Clinical Genetics, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- HUSLAB Laboratory of Genetics, HUS Diagnostic Center, Helsinki University Hospital, Helsinki, Finland
- iCAN Digital Precision Cancer Medicine Flagship, Helsinki, Finland
| | - Mikko Arvas
- Research and Development, Finnish Red Cross Blood Service, Helsinki, Finland
| | - Esa Pitkänen
- Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland
- Applied Tumor Genomics Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- iCAN Digital Precision Cancer Medicine Flagship, Helsinki, Finland
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15
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Wang MT, Pang SW. Enhancing Nasopharyngeal Carcinoma Cell Separation with Selective Fibronectin Coating and Topographical Modification on Polydimethylsiloxane Scaffold Platforms. Int J Mol Sci 2023; 24:12409. [PMID: 37569784 PMCID: PMC10418797 DOI: 10.3390/ijms241512409] [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: 07/06/2023] [Revised: 07/29/2023] [Accepted: 08/02/2023] [Indexed: 08/13/2023] Open
Abstract
The extracellular matrix (ECM) serves as a complex scaffold with diverse physical dimensions and surface properties influencing NPC cell migration. Polydimethylsiloxane (PDMS), a widely used biocompatible material, is hydrophobic and undesirable for cell seeding. Thus, the establishment of a biomimetic model with varied topographies and surface properties is essential for effective NPC43 cell separation from NP460 cells. This study explored how ECM surface properties influence NP460 and NPC43 cell behaviors via plasma treatments and chemical modifications to alter the platform surface. In addition to the conventional oxygen/nitrogen (O2/N2) plasma treatment, O2 and argon plasma treatments were utilized to modify the platform surface, which increased the hydrophilicity of the PDMS platforms, resulting in enhanced cell adhesion. (3-aminopropyl)triethoxysilane and fibronectin (FN) were used to coat the PDMS platforms uniformly and selectively. The chemical coatings significantly affected cell motility and spreading, as cells exhibited faster migration, elongated cell shapes, and larger spreading areas on FN-coated surfaces. Furthermore, narrower top layer trenches with 5 µm width and a lower concentration of 10 µg/mL FN were coated selectively on the platforms to limit NP460 cell movements and enhance NPC43 cell separation efficiency. A significantly high separation efficiency of 99.4% was achieved on the two-layer scaffold platform with 20/5 µm wide ridge/trench (R/T) as the top layer and 40/10 µm wide R/T as the bottom layer, coupling with 10 µg/mL FN selectively coated on the sidewalls of the top and bottom layers. This work demonstrated an innovative application of selective FN coating to direct cell behavior, offering a new perspective to probe into the subtleties of NPC cell separation efficiency. Moreover, this cost-effective and compact microsystem sets a new benchmark for separating cancer cells.
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Affiliation(s)
| | - S. W. Pang
- Department of Electrical Engineering, Centre for Biosystems, Neuroscience, and Nanotechnology, City University of Hong Kong, Hong Kong 999077, China;
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16
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Butto T, Mungikar K, Baumann P, Winter J, Lutz B, Gerber S. Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing. Cells 2023; 12:cells12071051. [PMID: 37048124 PMCID: PMC10093037 DOI: 10.3390/cells12071051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 03/22/2023] [Accepted: 03/27/2023] [Indexed: 04/03/2023] Open
Abstract
In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages, with a specific focus on their utility for transcriptomic sequencing analyses. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.
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Affiliation(s)
- Tamer Butto
- Institute for Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University, 55128 Mainz, Germany
- Correspondence: (T.B.); (S.G.); Tel.: +49-(0)6131-39-27331 (S.G.)
| | - Kanak Mungikar
- Institute of Human Genetics, University Medical Center Mainz, 55131 Mainz, Germany
| | - Peter Baumann
- Faculty of Biology, Johannes Gutenberg-University, 55128 Mainz, Germany
- Institute of Molecular Biology (IMB), 55128 Mainz, Germany
| | - Jennifer Winter
- Institute of Human Genetics, University Medical Center Mainz, 55131 Mainz, Germany
- Leibniz Institute for Resilience Research (LIR), 55122 Mainz, Germany
| | - Beat Lutz
- Leibniz Institute for Resilience Research (LIR), 55122 Mainz, Germany
- Institute of Physiological Chemistry, University Medical Center Mainz, 55128 Mainz, Germany
| | - Susanne Gerber
- Institute of Human Genetics, University Medical Center Mainz, 55131 Mainz, Germany
- Correspondence: (T.B.); (S.G.); Tel.: +49-(0)6131-39-27331 (S.G.)
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17
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Fernández-Fernández R, López-Igual R, Casadesús J, Sánchez-Romero MA. Analysis of Salmonella lineage-specific traits upon cell sorting. Front Cell Infect Microbiol 2023; 13:1146070. [PMID: 37065195 PMCID: PMC10090396 DOI: 10.3389/fcimb.2023.1146070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Accepted: 03/14/2023] [Indexed: 03/31/2023] Open
Abstract
Microbial cell individuality is receiving increasing interest in the scientific community. Individual cells within clonal populations exhibit noticeable phenotypic heterogeneity. The advent of fluorescent protein technology and advances in single-cell analysis has revealed phenotypic cell variant in bacterial populations. This heterogeneity is evident in a wide range of phenotypes, for example, individual cells display variable degrees of gene expression and survival under selective conditions and stresses, and can exhibit differing propensities to host interactions. Last few years, numerous cell sorting approaches have been employed for resolving the properties of bacterial subpopulations. This review provides an overview of applications of cell sorting to analyze Salmonella lineage-specific traits, including bacterial evolution studies, gene expression analysis, response to diverse cellular stresses and characterization of diverse bacterial phenotypic variants.
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Affiliation(s)
- Rocío Fernández-Fernández
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Seville, Spain
- Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain
| | - Rocío López-Igual
- Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and C.S.I.C., Seville, Spain
| | - Josep Casadesús
- Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Seville, Spain
| | - María Antonia Sánchez-Romero
- Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain
- *Correspondence: María Antonia Sánchez-Romero,
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18
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Starzonek C, Mhamdi-Ghodbani M, Henning S, Bender M, Degenhardt S, Chen IP, Said M, Greinert R, Volkmer B. Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts. Cells 2023; 12:cells12060949. [PMID: 36980290 PMCID: PMC10047019 DOI: 10.3390/cells12060949] [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: 01/20/2023] [Revised: 03/07/2023] [Accepted: 03/17/2023] [Indexed: 03/30/2023] Open
Abstract
Dermal stem cells (DSCs), which are progenitor cells of melanocytes, are isolated from human foreskin and cultivated as mixed cultures containing both DSCs and fibroblasts in varying proportions. These contaminating fibroblasts may have an impact on the results of experimental studies and are a serious limitation for certain applications. The aim of the present study was to purify or enrich DSCs-an indispensable step towards future investigations. Applying different methods, we demonstrated that highly enriched DSCs with a good recovery rate can be obtained through positive selection with MACS® immunomagnetic cell sorting. These DSCs remain vital and proliferate constantly in culture, maintaining a high level of purity after enrichment. Other approaches such as treatment with Geneticin or selective detachment were not suitable to purify DSC-fibroblast co-cultures. Overall, enriched DSCs represent a novel and unique model to study the effects of UV radiation on the differentiation of DSCs into melanocytes and their potential relevance in the genesis of malignant melanoma.
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Affiliation(s)
- Christin Starzonek
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - Mouna Mhamdi-Ghodbani
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - Stefan Henning
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - Marc Bender
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - Sarah Degenhardt
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - I-Peng Chen
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | | | - Rüdiger Greinert
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
| | - Beate Volkmer
- Skin Cancer Center, Division of Molecular Cell Biology, Elbe Kliniken Stade-Buxtehude, 21614 Buxtehude, Germany
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19
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Naderi MM, Barilla L, Zhou J, Papautsky I, Peng Z. Elasto-Inertial Focusing Mechanisms of Particles in Shear-Thinning Viscoelastic Fluid in Rectangular Microchannels. MICROMACHINES 2022; 13:mi13122131. [PMID: 36557430 PMCID: PMC9781382 DOI: 10.3390/mi13122131] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Revised: 11/24/2022] [Accepted: 11/26/2022] [Indexed: 06/01/2023]
Abstract
Growth of the microfluidics field has triggered numerous advances in focusing and separating microparticles, with such systems rapidly finding applications in biomedical, chemical, and environmental fields. The use of shear-thinning viscoelastic fluids in microfluidic channels is leading to evolution of elasto-inertial focusing. Herein, we showed that the interplay between the elastic and shear-gradient lift forces, as well as the secondary flow transversal drag force that is caused by the non-zero second normal stress difference, lead to different particle focusing patterns in the elasto-inertial regime. Experiments and 3D simulations were performed to study the effects of flowrate, particle size, and the shear-thinning extent of the fluid on the focusing patterns. The Giesekus constitutive equation was used in the simulations to capture the shear-thinning and viscoelastic behaviors of the solution used in the experiments. At low flowrate, with Weissenberg number Wi ~ O(1), both the elastic force and secondary flow effects push particles towards the channel center. However, at a high flowrate, Wi ~ O(10), the elastic force direction is reversed in the central regions. This remarkable behavior of the elastic force, combined with the enhanced shear-gradient lift at the high flowrate, pushes particles away from the channel center. Additionally, a precise prediction of the focusing position can only be made when the shear-thinning extent of the fluid is correctly estimated in the modeling. The shear-thinning also gives rise to the unique behavior of the inertial forces near the channel walls which is linked with the ‘warped’ velocity profile in such fluids.
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Affiliation(s)
| | | | | | - Ian Papautsky
- Correspondence: (I.P.); (Z.P.); Tel.: +1-312-413-3800 (I.P.); +1-312-996-7467 (Z.P.)
| | - Zhangli Peng
- Correspondence: (I.P.); (Z.P.); Tel.: +1-312-413-3800 (I.P.); +1-312-996-7467 (Z.P.)
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20
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Scheuermann S, Lehmann JM, Ramani Mohan R, Reißfelder C, Rückert F, Langejürgen J, Pallavi P. TissueGrinder, a novel technology for rapid generation of patient-derived single cell suspensions from solid tumors by mechanical tissue dissociation. Front Med (Lausanne) 2022; 9:721639. [PMID: 36582292 PMCID: PMC9793748 DOI: 10.3389/fmed.2022.721639] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2021] [Accepted: 11/14/2022] [Indexed: 12/04/2022] Open
Abstract
Introduction Recent advances hold promise of making personalized medicine a step closer to implementation in clinical settings. However, traditional sample preparation methods are not robust and reproducible. In this study, the TissueGrinder, a novel mechanical semi-automated benchtop device, which can isolate cells from tissue in a very fast and enzyme-free way is tested for cell isolation from surgically resected tumor tissues. Methods Thirty-three surgically resected tumor tissues from various but mainly pancreatic, liver or colorectal origins were processed by both novel TissueGrinder and explant method. An optimized processing program for tumors from pancreatic, liver or colorectal cancer was developed. The viability and morphological characteristics of the isolated cells were evaluated microscopically. Expression of pancreatic cancer markers was evaluated in cells isolated from pancreatic tumors. Finally, the effect of mechanical stress on the cells was evaluated by assessing apoptosis markers via western blotting. Results TissueGinder was more efficient in isolating cells from tumor tissue with a success rate of 75% when compared to explant method 45% in terms of cell outgrowth six weeks after processing. Cells isolated with TissueGinder had a higher abundance and were more heterogeneous in composition as compared to explant method. Mechanical processing of the cells with TissueGrinder does not lead to apoptosis but causes slight stress to the cells. Discussion Our results show that TissueGrinder can process solid tumor tissues more rapidly and efficiently and with higher success rate compared to the conventionally used explant method. The results of the study suggest that the TissueGrinder might be a suitable method for obtaining cells, which is important for its application in individualized therapy. Due to the great variance in different tumor entities and the associated individual tissue characteristics, a further development of the dissociation protocol for other types of tumors and normal tissue will be targeted.
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Affiliation(s)
| | | | - Ramkumar Ramani Mohan
- Department of Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Christoph Reißfelder
- Department of Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Felix Rückert
- Department of Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | | | - Prama Pallavi
- Department of Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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21
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Bakhshi MS, Rizwan M, Khan GJ, Duan H, Zhai K. Design of a novel integrated microfluidic chip for continuous separation of circulating tumor cells from peripheral blood cells. Sci Rep 2022; 12:17016. [PMID: 36220844 PMCID: PMC9554048 DOI: 10.1038/s41598-022-20886-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Accepted: 09/20/2022] [Indexed: 12/29/2022] Open
Abstract
Cancer is one of the foremost causes of death globally. Late-stage presentation, inaccessible diagnosis, and treatment are common challenges in developed countries. Detection, enumeration of Circulating Tumor Cells (CTC) as early as possible can reportedly lead to more effective treatment. The isolation of CTC at an early stage is challenging due to the low probability of its presence in peripheral blood. In this study, we propose a novel two-stage, label-free, rapid, and continuous CTC separation device based on hydrodynamic inertial focusing and dielectrophoretic separation. The dominance and differential of wall-induced inertial lift force and Dean drag force inside a curved microfluidic channel results in size-based separation of Red Blood Cells (RBC) and platelets (size between 2-4 µm) from CTC and leukocytes (9-12.2 µm). A numerical model was used to investigate the mechanism of hydrodynamic inertial focusing in a curvilinear microchannel. Simulations were done with the RBCs, platelets, CTCs, and leukocytes (four major subtypes) to select the optimized value of the parameters in the proposed design. In first stage, the focusing behavior of microscale cells was studied to sort leukocytes and CTCs from RBCs, and platelets while viable CTCs were separated from leukocytes based on their inherent electrical properties using dielectrophoresis in the second stage. The proposed design of the device was evaluated for CTC separation efficiency using numerical simulations. This study considered the influence of critical factors like aspect ratio, dielectrophoretic force, channel size, flow rate, separation efficiency, and shape on cell separation. Results show that the proposed device yields viable CTC with 99.5% isolation efficiency with a throughput of 12.2 ml/h.
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Affiliation(s)
- Maliha Saleem Bakhshi
- grid.444938.60000 0004 0609 0078Mechatronics and Control Engineering Department, University of Engineering and Technology, Lahore, Pakistan
| | - Mohsin Rizwan
- grid.444938.60000 0004 0609 0078Mechatronics and Control Engineering Department, University of Engineering and Technology, Lahore, Pakistan
| | - Ghulam Jilany Khan
- grid.444936.80000 0004 0608 9608Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, University of Central Punjab, Lahore, Pakistan
| | - Hong Duan
- grid.263761.70000 0001 0198 0694School of Biological and Food Engineering, Engineering Research Center for Development and High Value Utilization of Genuine Medicinal Materials in North Anhui Province, Suzhou University, Suzhou, Anhui 234000 China
| | - Kefeng Zhai
- grid.263761.70000 0001 0198 0694School of Biological and Food Engineering, Engineering Research Center for Development and High Value Utilization of Genuine Medicinal Materials in North Anhui Province, Suzhou University, Suzhou, Anhui 234000 China ,grid.459584.10000 0001 2196 0260Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Guilin, 541004 People’s Republic of China
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22
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Buenaventura RG, Harvey AC, Burns MP, Main BS. Sequential Isolation of Microglia and Astrocytes from Young and Aged Adult Mouse Brains for Downstream Transcriptomic Analysis. Methods Protoc 2022; 5:77. [PMID: 36287049 PMCID: PMC9610580 DOI: 10.3390/mps5050077] [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: 08/12/2022] [Revised: 09/21/2022] [Accepted: 09/23/2022] [Indexed: 11/17/2022] Open
Abstract
In aging, the brain is more vulnerable to injury and neurodegenerative disease, but the mechanisms responsible are largely unknown. Evidence now suggests that neuroinflammation, mediated by resident brain astrocyte and microglia populations, are key players in the generation of inflammatory responses and may influence both age related processes and the initiation/progression of neurodegeneration. Consequently, targeting these cell types individually and collectively may aid in the development of novel disease-modifying therapies. We have optimized and characterized a protocol for the effective sequential isolation of both microglia and astrocytes from the adult mouse brain in young and aged mice. We demonstrate a technique for the sequential isolation of these immune cells by using magnetic beads technology, optimized to increase yield and limit potential artifacts in downstream transcriptomic applications, including RNA-sequencing pipelines. This technique is versatile, cost-effective, and reliable for the study of responses within the same biological context, simultaneously being advantageous in reducing mice numbers required to assess cellular responses in normal and age-related pathological conditions.
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Affiliation(s)
| | | | | | - Bevan S. Main
- Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA
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23
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Wüst R, Terrie L, Müntefering T, Ruck T, Thorrez L. Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle. Front Bioeng Biotechnol 2022; 10:964705. [PMID: 36213083 PMCID: PMC9534561 DOI: 10.3389/fbioe.2022.964705] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 07/18/2022] [Indexed: 12/03/2022] Open
Abstract
Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.
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Affiliation(s)
- Rebecca Wüst
- Tissue Engineering Lab, Dep. Development and Regeneration, KU Leuven Kulak, Kortrijk, Belgium
| | - Lisanne Terrie
- Tissue Engineering Lab, Dep. Development and Regeneration, KU Leuven Kulak, Kortrijk, Belgium
| | - Thomas Müntefering
- Department of Neurology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany
| | - Tobias Ruck
- Department of Neurology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany
| | - Lieven Thorrez
- Tissue Engineering Lab, Dep. Development and Regeneration, KU Leuven Kulak, Kortrijk, Belgium
- *Correspondence: Lieven Thorrez,
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Kranz E, Kuhlmann CJ, Chan J, Kim PY, Chen ISY, Kamata M. Efficient derivation of chimeric-antigen receptor-modified TSCM cells. Front Immunol 2022; 13:877682. [PMID: 35967430 PMCID: PMC9366550 DOI: 10.3389/fimmu.2022.877682] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Accepted: 07/07/2022] [Indexed: 11/13/2022] Open
Abstract
Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.
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Affiliation(s)
- Emiko Kranz
- Division of Hematology-Oncology, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, United States
| | - Charles J. Kuhlmann
- Department of Microbiology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Joshua Chan
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, United States
| | - Patrick Y. Kim
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, United States
| | - Irvin S. Y. Chen
- Division of Hematology-Oncology, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, United States
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, CA, United States
| | - Masakazu Kamata
- Department of Microbiology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States
- *Correspondence: Masakazu Kamata,
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Yang L, Cui L, Ma S, Zuo Q, Huang Q. A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag. Front Bioeng Biotechnol 2022; 10:930966. [PMID: 35782508 PMCID: PMC9244562 DOI: 10.3389/fbioe.2022.930966] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 05/30/2022] [Indexed: 11/13/2022] Open
Abstract
Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.
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26
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Abedini-Nassab R, Ding X, Xie H. A novel magnetophoretic-based device for magnetometry and separation of single magnetic particles and magnetized cells. LAB ON A CHIP 2022; 22:738-746. [PMID: 35040849 DOI: 10.1039/d1lc01104a] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
The use of magnetic micro- and nanoparticles in medicine and biology is expanding. One important example is the transport of magnetic microparticles and magnetized cells in lab-on-a-chip systems. The magnetic susceptibility of the particles is a key factor in determining their response to the externally applied magnetic field. Typically, to measure this parameter, their magnetophoretic mobility is studied. However, the particle tracking system for accurately determining the traveled distance in a certain time may be too complicated. Here, we introduce a lithographically fabricated chip composed of an array of thin magnetic micro-disks for evaluating the magnetic susceptibility of numerous individual magnetic particles simultaneously. The proposed novel magnetometer works based on the phase change in the trajectory of microparticles circulating around the disks in a rotating in-plane magnetic field. We explain that the easily detectable transition between the "phase-locked" and the "phase-slipping" regimes and the frequency at which it happens are appropriate parameters for measuring the magnetic susceptibility of the magnetic particles at the single-particle level. We show that this high-throughput (i.e., ∼ten thousand particles on a 1 cm2 area) single-particle magnetometry method has various crucial applications, including i) magnetic characterization of magnetic beads as well as magnetically labeled living cells, ii) determining the magnetization rate of the cells taking up magnetic nanoparticles with respect to time, iii) evaluating the rate of degradation of magnetic nanoparticles in cells over time, iv) detecting the number of target cells in a sample, and v) separating particles based on their size and magnetic susceptibility.
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Affiliation(s)
- Roozbeh Abedini-Nassab
- Faculty of Mechanical Engineering, Tarbiat Modares University, P.O. Box: 14115-111, Tehran, Iran.
| | - Xianting Ding
- School of Biomedical Engineering, Institute for Personalized Medicine, Shanghai Jiao Tong University, 200030, China
| | - Haiyang Xie
- School of Biomedical Engineering, Institute for Personalized Medicine, Shanghai Jiao Tong University, 200030, China
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27
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Sabirova A, Florica CF, Pisig F, Syed A, Buttner U, Li X, Nunes SP. Nanoporous membrane fabrication by nanoimprint lithography for nanoparticle sieving. NANOSCALE ADVANCES 2022; 4:1119-1124. [PMID: 36131770 PMCID: PMC9417922 DOI: 10.1039/d1na00812a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 12/22/2021] [Indexed: 06/15/2023]
Abstract
An isoporous membrane with strictly controlled pore size, shape and distribution could provide an efficient, precise and mild sieving of particles in nanotechnology and biomedical applications. However there is a lack of highly porous polymeric membranes combining isoporosity and high permeance in the range below 500 nm. Track-etched membranes are practically the only commercial option. Membranes prepared by phase inversion typically have a broad pore size distribution. Most nanofabrication methods have limited the preparation of membranes with pores in the micrometer range. In this work, we present a nanotechnology-based fabrication methodology to manufacture a stable and flexible nanoporous polymeric membrane with 300 nm isopores using UV nanoimprint lithography. The highly porous membrane has a pore density of 4 × 109 pores per cm2 and stable permeance of 108 000 L m-2 h-1 bar-1. Uniform ZIF-8 nanoparticles were synthesized and the isoporous membrane successfully demonstrated as high as 100% rejection and size-based sieving performance of nanoparticles.
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Affiliation(s)
- Ainur Sabirova
- King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering (BESE) Division, Advanced Membranes and Porous Materials Center 23955-6900 Thuwal Saudi Arabia
| | - Camelia F Florica
- King Abdullah University of Science and Technology (KAUST), Nanofabrication Core Laboratory 23955-6900 Thuwal Saudi Arabia
| | - Florencio Pisig
- King Abdullah University of Science and Technology (KAUST), Nanofabrication Core Laboratory 23955-6900 Thuwal Saudi Arabia
| | - Ahad Syed
- King Abdullah University of Science and Technology (KAUST), Nanofabrication Core Laboratory 23955-6900 Thuwal Saudi Arabia
| | - Ulrich Buttner
- King Abdullah University of Science and Technology (KAUST), Nanofabrication Core Laboratory 23955-6900 Thuwal Saudi Arabia
| | - Xiang Li
- King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering (BESE) Division, Advanced Membranes and Porous Materials Center 23955-6900 Thuwal Saudi Arabia
| | - Suzana P Nunes
- King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering (BESE) Division, Advanced Membranes and Porous Materials Center 23955-6900 Thuwal Saudi Arabia
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Menze L, Duarte PA, Haddon L, Chu M, Chen J. Selective Single-Cell Sorting Using a Multisectorial Electroactive Nanowell Platform. ACS NANO 2022; 16:211-220. [PMID: 34559518 DOI: 10.1021/acsnano.1c05668] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Current approaches in targeted patient treatments often require the rapid isolation of specific rare target cells. Stream-based dielectrophoresis (DEP) based cell sorters have the limitation that the maximum number of sortable cell types is equivalent to the number of output channels, which makes upscaling to a higher number of different cell types technically challenging. Here, we present a microfluidic platform for selective single-cell sorting that bypasses this limitation. The platform consists of 10 000 nanoliter wells which are placed on top of interdigitated electrodes (IDEs) that facilitate dielectrophoresis-driven capture of cells. By use of a multisectorial design formed by 10 individually addressable IDE structures, our platform can capture a large number of different cell types. The sectorial approach allows for fast and straightforward modification to sort complex samples as different cell types are captured in different sectors and therefore removes the need for individual output channels per cell type. Experimental results obtained with a mixed sample of benign (MCF-10A) and malignant (MDA-MB-231) breast cells showed a target to nontarget sorting accuracy of over 95%. We envision that the high accuracy of our platform, in addition to its versatility and simplicity, will aid clinical environments where reliable sorting of varying complex samples is essential.
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Affiliation(s)
- Lukas Menze
- Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
| | - Pedro A Duarte
- Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
| | - Lacey Haddon
- Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
| | - Michael Chu
- Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
| | - Jie Chen
- Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta T6G 1H9, Canada
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Cheng J, Zhang L, Zhang Y, Ye Y, Zhao W, Zhang L, Li Y, Liu Y, Zhang W, Guo H, Li M, Zhao Y, Huang C. 3D spiral channels combined with flexible micro-sieve for high-throughput rare tumor cell enrichment and assay from clinical pleural effusion samples. Biodes Manuf 2022. [DOI: 10.1007/s42242-021-00167-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
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Abstract
Magnetic cell separation has become a key methodology for the isolation of target cell populations from biological suspensions, covering a wide spectrum of applications from diagnosis and therapy in biomedicine to environmental applications or fundamental research in biology. There now exists a great variety of commercially available separation instruments and reagents, which has permitted rapid dissemination of the technology. However, there is still an increasing demand for new tools and protocols which provide improved selectivity, yield and sensitivity of the separation process while reducing cost and providing a faster response. This review aims to introduce basic principles of magnetic cell separation for the neophyte, while giving an overview of recent research in the field, from the development of new cell labeling strategies to the design of integrated microfluidic cell sorters and of point-of-care platforms combining cell selection, capture, and downstream detection. Finally, we focus on clinical, industrial and environmental applications where magnetic cell separation strategies are amongst the most promising techniques to address the challenges of isolating rare cells.
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Liu D, Xu W, Tang Y, Cao J, Chen R, Wu D, Chen H, Su B, Xu J. Nebulization of risedronate alleviates airway obstruction and inflammation of chronic obstructive pulmonary diseases via suppressing prenylation-dependent RAS/ERK/NF-κB and RhoA/ROCK1/MLCP signaling. Respir Res 2022; 23:380. [PMID: 36575527 PMCID: PMC9795678 DOI: 10.1186/s12931-022-02274-5] [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: 07/19/2022] [Accepted: 12/02/2022] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND Chronic obstructive pulmonary disease (COPD) is a progressive disorder that causes airway obstruction and lung inflammation. The first-line treatment of COPD is the bronchodilators of β2-agonists and antimuscarinic drugs, which can help control the airway obstruction, but the long-term use might render the drug tolerance. Bisphosphonates are widely used in osteoclast-mediated bone diseases treatment for decades. For drug repurposing, can delivery of a third generation of nitrogen-containing bisphosphonate, risedronate (RIS) ameliorate the progression of COPD? METHODS COPD rats or mice models have been established through cigarette-smoking and elastase injection, and then the animals are received RIS treatment via nebulization. Lung deposition of RIS was primarily assessed by high-performance liquid chromatography (HPLC). The respiratory parameters of airway obstruction in COPD rats and mice were documented using plethysmography method and resistance-compliance system. RESULTS High lung deposition and bioavailability of RIS was monitored with 88.8% of RIS input dose. We found that RIS could rescue the lung function decline of airspace enlargement and mean linear intercept in the COPD lung. RIS could curb the airway obstruction by suppressing 60% of the respiratory resistance and elevating the airway's dynamic compliance, tidal volume and mid-expiratory flow. As an inhibitor of farnesyl diphosphate synthase (FDPS), RIS suppresses FDPS-mediated RAS and RhoA prenylation to obstruct its membrane localization in airway smooth muscle cells (ASMCs), leading to the inhibition of downstream ERK-MLCK and ROCK1-MLCP pathway to cause ASMCs relaxation. Additionally, RIS nebulization impeded pro-inflammatory cell accumulation, particularly macrophages infiltration in alveolar parenchyma. The NF-κB, tumor necrosis factor-alpha, IL-1β, IL-8, and IL-6 declined in microphages following RIS nebulization. Surprisingly, nebulization of RIS could overcome the tolerance of β2-agonists in COPD-rats by increasing the expression of β2 receptors. CONCLUSIONS Nebulization of RIS could alleviate airway obstruction and lung inflammation in COPD, providing a novel strategy for treating COPD patients, even those with β2-agonists tolerance.
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Affiliation(s)
- Di Liu
- grid.24516.340000000123704535Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China ,grid.24516.340000000123704535Department of Radiation Oncology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China
| | - Wen Xu
- grid.24516.340000000123704535Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China
| | - Yuan Tang
- grid.24516.340000000123704535Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China ,grid.252957.e0000 0001 1484 5512Basic Medical College, Bengbu Medical College, Bengbu, People’s Republic of China
| | - Jingxue Cao
- grid.24516.340000000123704535Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China ,grid.24516.340000000123704535Department of Radiology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China
| | - Ran Chen
- grid.24516.340000000123704535Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China
| | - Dingwei Wu
- Zhejiang Xianju Pharmaceutical Co., Ltd, Xianju, People’s Republic of China
| | - Hongpeng Chen
- Zhejiang Xianju Pharmaceutical Co., Ltd, Xianju, People’s Republic of China
| | - Bo Su
- grid.24516.340000000123704535Central Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China ,grid.252957.e0000 0001 1484 5512School of Life Sciences, Bengbu Medical College, Bengbu, People’s Republic of China
| | - Jinfu Xu
- grid.24516.340000000123704535Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China
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Kang B, Han S, Son HY, Mun B, Shin MK, Choi Y, Park J, Min JK, Park D, Lim EK, Huh YM, Haam S. Immunomagnetic microfluidic integrated system for potency-based multiple separation of heterogeneous stem cells with high throughput capabilities. Biosens Bioelectron 2021; 194:113576. [PMID: 34454345 DOI: 10.1016/j.bios.2021.113576] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 07/27/2021] [Accepted: 08/19/2021] [Indexed: 12/26/2022]
Abstract
Multipotent adult stem cells (MASCs) derived from Pluripotent stem cells (PSCs) have found widespread use in various applications, including regenerative therapy and drug screening. For these applications, highly pluripotent PSCs need to be selectively separated from those that show low pluripotency for reusage of PSCs, and MASCs need to be collected for further application. Herein, we developed immunomagnetic microfluidic integrated system (IM-MIS) for separation of stem cells depending on potency level. In this system, each stem cell was multiple-separated in microfluidics chip by magnetophoretic mobility of magnetic-activated cells based on the combination of two sizes of magnetic nanoparticles and two different antibodies. Magnetic particles had a difference in the degree of magnetization, and antibodies recognized potency-related surface markers. IM-MIS showed superior cell separation performance than FACS with high throughput (49.5%) in a short time (<15 min) isolate 1 × 107 cells, and higher purity (92.1%) than MACS. IM-MIS had a cell viability of 89.1%, suggesting that IM-MIS had no effect on cell viability during isolation. Furthermore, IM-MIS did not affect the key characteristics of stem cells including its differentiation potency, phenotype, genotype, and karyotype. IM-MIS may offer a new platform for the development of multi-separation systems for diverse stem cell applications.
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Affiliation(s)
- Byunghoon Kang
- Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Seungmin Han
- Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; Division of Cardio-Thoracic Surgery, Department of Surgery, College of Medicine, University of Arizona, Tucson, AZ, USA
| | - Hye Young Son
- Department of Radiology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; Severance Biomedical Science Institute, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
| | - Byeonggeol Mun
- Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
| | - Moo-Kwang Shin
- Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
| | - Yuna Choi
- Department of Radiology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea
| | - Jongjin Park
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jeong-Ki Min
- Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Department of Biomolecular Science, KRIBB School of Bioscience, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Daewon Park
- Bioengineering Department, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, USA
| | - Eun-Kyung Lim
- BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; YUHS-KRIBB Medical Convergence Research Institute, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; Department of Nanobiotechnology, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea.
| | - Yong-Min Huh
- Department of Radiology, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; Severance Biomedical Science Institute, College of Medicine, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea; YUHS-KRIBB Medical Convergence Research Institute, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
| | - Seungjoo Haam
- Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
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Cai K, Mankar S, Ajiri T, Shirai K, Yotoriyama T. An integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting system (μ-CFACS) for the enrichment of rare cells. LAB ON A CHIP 2021; 21:3112-3127. [PMID: 34286793 DOI: 10.1039/d1lc00298h] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation cell sorters, microfluidic fluorescence-activated cell sorting (μ-FACS) devices have been developed to avoid cross-contamination, minimize device footprint, and eliminate bio-aerosols. However, due to highly precise flow control, the achievable throughput of the μ-FACS system is generally lower than the throughput of conventional FACS devices. Here, we report a fully integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting (μ-CFACS) system for the enrichment of clinical rare cells. A microfluidic sorting cartridge has been developed for enriching samples through a sequential sorting process, which was further realized by the integration of both fast amplified piezoelectrically actuated on-chip valves and compact pneumatic cylinders actuated on-chip valves. At an equivalent throughput of ∼8000 events per second (eps), the purity of rare fluorescent microparticles has been significantly increased from ∼0.01% to ∼27.97%. An enrichment of ∼9400-fold from 0.009% to 81.86% has also been demonstrated for isolating fluorescently labelled MCF-7 breast cancer cells from Jurkat cells at an equivalent sorting throughput of ∼6400 eps. With the advantages of high throughput and contamination-free design, the proposed integrated μ-CFACS system provides a new option for the enrichment of clinical rare cells.
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Affiliation(s)
- Kunpeng Cai
- Central Research Laboratories, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
| | - Shruti Mankar
- Central Research Laboratories, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
| | - Taiga Ajiri
- Central Research Laboratories, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
| | - Kentaro Shirai
- Central Research Laboratories, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
| | - Tasuku Yotoriyama
- Central Research Laboratories, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
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Jiang D, Liu J, Pan Y, Zhuang L, Wang P. Surface acoustic wave (SAW) techniques in tissue engineering. Cell Tissue Res 2021; 386:215-226. [PMID: 34390407 DOI: 10.1007/s00441-020-03397-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Accepted: 12/11/2020] [Indexed: 01/09/2023]
Abstract
Recently, the introduction of surface acoustic wave (SAW) technique for microfluidics has drawn a lot of attention. The pattern and mutual communication in cell layers, tissues, and organs play a critical role in tissue homeostasis and regeneration and may contribute to disease occurrence and progression. Tissue engineering aims to repair and regenerate damaged organs, depending on biomimetic scaffolds and advanced fabrication technology. However, traditional bioengineering synthesis approaches are time-consuming, heterogeneous, and unmanageable. It is hard to pattern cells in scaffolds effectively with no impact on cell viability and function. Here, we summarize a biocompatible, easily available, label-free, and non-invasive tool, surface acoustic wave (SAW) technique, which is getting a lot of attention in tissue engineering. SAW technique can realize accurate sorting, manipulation, and cells' pattern and rapid formation of spheroids. By integrating several SAW devices onto lab-on-a-chip platforms, tissue engineering lab-on-a-chip system was proposed. To the best of our knowledge, this is the first report to summarize the application of this novel technique in the field of tissue engineering.
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Affiliation(s)
- Deming Jiang
- Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Jingwen Liu
- Department of Gastroenterology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, China
| | - Yuxiang Pan
- Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Liujing Zhuang
- Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Ping Wang
- Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, 310027, China. .,State Key Laboratory for Sensor Technology, Chinese Academy of Sciences, Shanghai, 200050, China.
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Ryan L, Wong Y, Dwyer KM, Clarke D, Kyprian L, Craig JM. Coprocytobiology: A Technical Review of Cytological Colorectal Cancer Screening in Fecal Samples. SLAS Technol 2021; 26:591-604. [PMID: 34219541 DOI: 10.1177/24726303211024562] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
GRAPHICAL ABSTRACT
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Affiliation(s)
- Liam Ryan
- Deakin University, Waurn Ponds, Victoria, Australia
| | - YenTing Wong
- Deakin University, Waurn Ponds, Victoria, Australia
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Adil A, Kumar V, Jan AT, Asger M. Single-Cell Transcriptomics: Current Methods and Challenges in Data Acquisition and Analysis. Front Neurosci 2021; 15:591122. [PMID: 33967674 PMCID: PMC8100238 DOI: 10.3389/fnins.2021.591122] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 03/19/2021] [Indexed: 11/17/2022] Open
Abstract
Rapid cost drops and advancements in next-generation sequencing have made profiling of cells at individual level a conventional practice in scientific laboratories worldwide. Single-cell transcriptomics [single-cell RNA sequencing (SC-RNA-seq)] has an immense potential of uncovering the novel basis of human life. The well-known heterogeneity of cells at the individual level can be better studied by single-cell transcriptomics. Proper downstream analysis of this data will provide new insights into the scientific communities. However, due to low starting materials, the SC-RNA-seq data face various computational challenges: normalization, differential gene expression analysis, dimensionality reduction, etc. Additionally, new methods like 10× Chromium can profile millions of cells in parallel, which creates a considerable amount of data. Thus, single-cell data handling is another big challenge. This paper reviews the single-cell sequencing methods, library preparation, and data generation. We highlight some of the main computational challenges that require to be addressed by introducing new bioinformatics algorithms and tools for analysis. We also show single-cell transcriptomics data as a big data problem.
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Affiliation(s)
- Asif Adil
- Department of Computer Sciences, Baba Ghulam Shah Badshah University, Rajouri, India
| | - Vijay Kumar
- Department of Biotechnology, Yeungnam University, Gyeongsan, South Korea
| | - Arif Tasleem Jan
- School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, India
| | - Mohammed Asger
- Department of Computer Sciences, Baba Ghulam Shah Badshah University, Rajouri, India
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Latest Updates on the Advancement of Polymer-Based Biomicroelectromechanical Systems for Animal Cell Studies. ADVANCES IN POLYMER TECHNOLOGY 2021. [DOI: 10.1155/2021/8816564] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Biological sciences have reached the fundamental unit of life: the cell. Ever-growing field of Biological Microelectromechanical Systems (BioMEMSs) is providing new frontiers in both fundamental cell research and various practical applications in cell-related studies. Among various functions of BioMEMS devices, some of the most fundamental processes that can be carried out in such platforms include cell sorting, cell separation, cell isolation or trapping, cell pairing, cell-cell communication, cell differentiation, cell identification, and cell culture. In this article, we review each mentioned application in great details highlighting the latest advancements in fabrication strategy, mechanism of operation, and application of these tools. Moreover, the review article covers the shortcomings of each specific application which can open windows of opportunity for improvement of these devices.
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Liu Y, Xu H, Li T, Wang W. Microtechnology-enabled filtration-based liquid biopsy: challenges and practical considerations. LAB ON A CHIP 2021; 21:994-1015. [PMID: 33710188 DOI: 10.1039/d0lc01101k] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Liquid biopsy, an important enabling technology for early diagnosis and dynamic monitoring of cancer, has drawn extensive attention in the past decade. With the rapid developments of microtechnology, it has been possible to manipulate cells at the single-cell level, which dramatically improves the liquid biopsy capability. As the microtechnology-enabled liquid biopsy matures from proof-of-concept demonstrations towards practical applications, a main challenge it is facing now is to process clinical samples which are usually of a large volume while containing very rare targeted cells in complex backgrounds. Therefore, a high-throughput liquid biopsy which is capable of processing liquid samples with a large volume in a reasonable time along with a high recovery rate of rare targeted cells from complex clinical liquids is in high demand. Moreover, the purity, viability and release feasibility of recovered targeted cells are the other three key impact factors requiring careful considerations. To date, among the developed techniques, micropore-type filtration has been acknowledged as the most promising solution to address the aforementioned challenges in practical applications. However, the presently reported studies about micropore-type filtration are mostly based on trial and error for device designs aiming at different cancer types, which requires lots of efforts. Therefore, there is an urgent need to investigate and elaborate the fundamental theories of micropore-type filtration and key features that influence the working performances in the liquid biopsy of real clinical samples to promote the application efficacy in practical applications. In this review, the state of the art of microtechnology-enabled filtration is systematically and comprehensively summarized. Four key features of the filtration, including throughput, purity, viability and release feasibility of the captured targeted cells, are elaborated to provide the guidelines for filter designs. The recent progress in the filtration mode modulation and sample standardization to improve the filtration performance of real clinical samples is also discussed. Finally, this review concludes with prospective views for future developments of filtration-based liquid biopsy to promote its application efficacy in clinical practice.
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Affiliation(s)
- Yaoping Liu
- Institute of Microelectronics, Peking University, Beijing, 100871, China.
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Campbell KA, Colacino JA, Park SK, Bakulski KM. Cell Types in Environmental Epigenetic Studies: Biological and Epidemiological Frameworks. Curr Environ Health Rep 2021; 7:185-197. [PMID: 32794033 DOI: 10.1007/s40572-020-00287-0] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE OF REVIEW This article introduces the roles of perinatal DNA methylation in human health and disease, highlights the challenges of tissue and cellular heterogeneity to studying DNA methylation, summarizes approaches to overcome these challenges, and offers recommendations in conducting research in environmental epigenetics. RECENT FINDINGS Epigenetic modifications are essential for human development and are labile to environmental influences, especially during gestation. Epigenetic dysregulation is also a hallmark of multiple diseases. Environmental epigenetic studies routinely measure DNA methylation in readily available tissues. However, tissues and cell types exhibit specific epigenetic patterning and heterogeneity between samples complicates epigenetic studies. Failure to account for cell-type heterogeneity limits identification of biological mechanisms and biases study results. Tissue-level epigenetic measures represent a convolution of epigenetic signals from individual cell types. Tissue-specific epigenetics is an evolving field and the use of disease-affected target, surrogate, or multiple tissues has inherent trade-offs and affects inference. Likewise, experimental and bioinformatic approaches to accommodate cell-type heterogeneity have varying assumptions and inherent trade-offs that affect inference. The relationships between exposure, disease, tissue-level DNA methylation, cell type-specific DNA methylation, and cell-type heterogeneity must be carefully considered in study design and analysis. Causal diagrams can inform study design and analytic strategies. Properly addressing cell-type heterogeneity limits sources of potential bias, avoids misinterpretation of study results, and allows investigators to distinguish shifts in cell-type proportions from direct changes to cellular epigenetic programming, both of which provide insights into environmental disease etiology and aid development of novel methods for prevention and treatment.
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Affiliation(s)
- Kyle A Campbell
- Department of Epidemiology, University of Michigan School of Public Health, University of Michigan, Ann Arbor, MI, USA.
| | - Justin A Colacino
- Department of Environmental Health Sciences, University of Michigan School of Public Health, University of Michigan, Ann Arbor, MI, USA
| | - Sung Kyun Park
- Department of Epidemiology, University of Michigan School of Public Health, University of Michigan, Ann Arbor, MI, USA.,Department of Environmental Health Sciences, University of Michigan School of Public Health, University of Michigan, Ann Arbor, MI, USA
| | - Kelly M Bakulski
- Department of Epidemiology, University of Michigan School of Public Health, University of Michigan, Ann Arbor, MI, USA
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40
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Surendran AN, Zhou R, Lin Y. Microfluidic Devices for Magnetic Separation of Biological Particles: A Review. J Med Device 2020. [DOI: 10.1115/1.4048912] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Abstract
Separation of microparticles and cells serves a critical step in many applications such as in biological analyses, food production, chemical processing, and medical diagnostics. Sorting on the microscale exhibits certain advantages in comparison with that on the macroscale as it requires minuscule sample or reagents volume and thus reduced analysis cycle time, smaller size of devices, and lower fabrication costs. Progresses have been made over time to improve the efficiency of these microscale particle manipulation techniques. Many different techniques have been used to attain accurate particle sorting and separation in a continuous manner on the microscale level, which can be categorized as either passive or active methods. Passive techniques achieve accurate manipulation of particles through their interaction with surrounding flow by carefully designed channel structures, without using external fields. As an alternative, active techniques utilize external fields (e.g., acoustic, electronic, optical, and magnetic field, etc.) to realize desired pattern of motion for particles with specific properties. Among numerous active methods for microfluidic particle sorting, the magnetic field has been widely used in biomedical and chemical applications to achieve mixing, focusing, and separating of reagents and bioparticles. This paper aims to provide a thorough review on the classic and most up-to-date magnetic sorting and separation techniques to manipulate microparticles including the discussions on the basic concept, working principle, experimental details, and device performance.
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Affiliation(s)
- Athira N. Surendran
- Department of Mechanical and Civil Engineering, Purdue University Northwest, 2200 169th Street, Hammond, IN 46323
| | - Ran Zhou
- Department of Mechanical and Civil Engineering, Purdue University Northwest, 2200 169th Street, Hammond, IN 46323
| | - Yang Lin
- Department of Mechanical, Industrial and Systems Engineering, University of Rhode Island, 2 East Alumni Avenue, Kingston, RI 02881
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Cai K, Mankar S, Maslova A, Ajiri T, Yotoriyama T. Amplified piezoelectrically actuated on-chip flow switching for a rapid and stable microfluidic fluorescence activated cell sorter. RSC Adv 2020; 10:40395-40405. [PMID: 35520855 PMCID: PMC9057478 DOI: 10.1039/d0ra04919k] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Accepted: 10/25/2020] [Indexed: 01/22/2023] Open
Abstract
With the potential to avoid cross-contamination, eliminate bio-aerosols, and minimize device footprints, microfluidic fluorescence-activated cell sorting (μ-FACS) devices could become the platform for the next generation cell sorter. Here, we report an on-chip flow switching based μ-FACS mechanism with piezoelectric actuation as a fast and robust sorting solution. A microfluidic chip with bifurcate configuration and displacement amplified piezoelectric microvalves has been developed to build the μ-FACS system. Rare fluorescent microparticles of different sizes have been significantly enriched from a purity of ∼0.5% to more than 90%. An enrichment of 150-fold from ∼0.6% to ∼91% has also been confirmed for fluorescently labeled MCF-7 breast cancer cells from Jurkat cells, while viability after sorting was maintained. Taking advantage of its simple structure, low cost, fast response, and reliable flow regulation, the proposed μ-FACS system delivers a new option that can meet the requirements of sorting performance, target selectivity, device lifetime, and cost-effectiveness of implementation.
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Affiliation(s)
- Kunpeng Cai
- Central Research Laboratories, Sysmex Corporation 4-4-4 Takatsukadai, Nishi-ku Kobe 651-2271 Japan
| | - Shruti Mankar
- Central Research Laboratories, Sysmex Corporation 4-4-4 Takatsukadai, Nishi-ku Kobe 651-2271 Japan
| | - Anastasia Maslova
- Central Research Laboratories, Sysmex Corporation 4-4-4 Takatsukadai, Nishi-ku Kobe 651-2271 Japan
| | - Taiga Ajiri
- Central Research Laboratories, Sysmex Corporation 4-4-4 Takatsukadai, Nishi-ku Kobe 651-2271 Japan
| | - Tasuku Yotoriyama
- Central Research Laboratories, Sysmex Corporation 4-4-4 Takatsukadai, Nishi-ku Kobe 651-2271 Japan
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Chu W, Prodromou R, Day KN, Schneible JD, Bacon KB, Bowen JD, Kilgore RE, Catella CM, Moore BD, Mabe MD, Alashoor K, Xu Y, Xiao Y, Menegatti S. Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics. J Chromatogr A 2020; 1635:461632. [PMID: 33333349 DOI: 10.1016/j.chroma.2020.461632] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 10/14/2020] [Accepted: 10/15/2020] [Indexed: 02/08/2023]
Abstract
Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as β-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.
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Affiliation(s)
- Wenning Chu
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Raphael Prodromou
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Kevin N Day
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - John D Schneible
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Kaitlyn B Bacon
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - John D Bowen
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Ryan E Kilgore
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Carly M Catella
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Brandyn D Moore
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Matthew D Mabe
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606
| | - Kawthar Alashoor
- Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY 14642
| | - Yiman Xu
- College of Material Science and Engineering, Donghua University, 201620 Shanghai, People's Republic of China
| | - Yuanxin Xiao
- College of Textile, Donghua University, Songjiang District, Shanghai, 201620, People's Republic of China
| | - Stefano Menegatti
- Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, NC 27606.
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Gabr H, El Ghamrawy MK, Almaeen AH, Abdelhafiz AS, Hassan AOS, El Sissy MH. CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation. Stem Cell Res Ther 2020; 11:390. [PMID: 32912325 PMCID: PMC7488347 DOI: 10.1186/s13287-020-01876-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2020] [Revised: 05/19/2020] [Accepted: 08/05/2020] [Indexed: 11/24/2022] Open
Abstract
Background β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation. Methods and results We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin. Conclusion CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.
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Affiliation(s)
- Hala Gabr
- Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
| | | | | | | | - Aya Osama Saad Hassan
- Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
| | - Maha Hamdi El Sissy
- Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt.
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Mesenchymal Stem/Progenitor Cells: The Prospect of Human Clinical Translation. Stem Cells Int 2020; 2020:8837654. [PMID: 33953753 PMCID: PMC8063852 DOI: 10.1155/2020/8837654] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Revised: 06/19/2020] [Accepted: 07/20/2020] [Indexed: 12/13/2022] Open
Abstract
Mesenchymal stem/progenitor cells (MSCs) are key players in regenerative medicine, relying principally on their differentiation/regeneration potential, immunomodulatory properties, paracrine effects, and potent homing ability with minimal if any ethical concerns. Even though multiple preclinical and clinical studies have demonstrated remarkable properties for MSCs, the clinical applicability of MSC-based therapies is still questionable. Several challenges exist that critically hinder a successful clinical translation of MSC-based therapies, including but not limited to heterogeneity of their populations, variability in their quality and quantity, donor-related factors, discrepancies in protocols for isolation, in vitro expansion and premodification, and variability in methods of cell delivery, dosing, and cell homing. Alterations of MSC viability, proliferation, properties, and/or function are also affected by various drugs and chemicals. Moreover, significant safety concerns exist due to possible teratogenic/neoplastic potential and transmission of infectious diseases. Through the current review, we aim to highlight the major challenges facing MSCs' human clinical translation and shed light on the undergoing strategies to overcome them.
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Bacon K, Lavoie A, Rao BM, Daniele M, Menegatti S. Past, Present, and Future of Affinity-based Cell Separation Technologies. Acta Biomater 2020; 112:29-51. [PMID: 32442784 PMCID: PMC10364325 DOI: 10.1016/j.actbio.2020.05.004] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 04/29/2020] [Accepted: 05/05/2020] [Indexed: 02/06/2023]
Abstract
Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.
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Affiliation(s)
- Kaitlyn Bacon
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA
| | - Ashton Lavoie
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA
| | - Balaji M Rao
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695-7928, USA
| | - Michael Daniele
- Joint Department of Biomedical Engineering, North Carolina State University - University of North Carolina Chapel Hill, North Carolina, United States
| | - Stefano Menegatti
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695-7928, USA.
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Ion Channel Profiling in Prostate Cancer: Toward Cell Population-Specific Screening. Rev Physiol Biochem Pharmacol 2020; 181:39-56. [PMID: 32737754 DOI: 10.1007/112_2020_22] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
In the last three decades, a growing number of studies have implicated ion channels in all essential processes of prostate carcinogenesis, including cell proliferation, apoptosis, migration, and angiogenesis. The changes in the expression of individual ion channels show a specific profile, making these proteins promising clinical biomarkers that may enable better molecular subtyping of the disease and lead to more rapid and accurate clinical decision-making. Expression profiles and channel function are mainly based on the tumoral tissue itself, in this case, the epithelial cancer cell population. To date, little data on the ion channel profile of the cancerous prostate stroma are available, even though tumor interactions with the microenvironment are crucial in carcinogenesis and each distinct population plays a specific role in tumor progression. In this review, we describe ion channel expression profiles specific for the distinct cell population of the tumor microenvironment (stromal, endothelial, neuronal, and neuroendocrine cell populations) and the technical approaches used for efficient separation and screening of these cell populations.
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47
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Gray BP, Requena MD, Nichols MD, Sullenger BA. Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State. Cell Chem Biol 2020; 27:232-244.e7. [PMID: 31879266 PMCID: PMC7038790 DOI: 10.1016/j.chembiol.2019.12.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 11/01/2019] [Accepted: 12/05/2019] [Indexed: 12/13/2022]
Abstract
Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic.
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Affiliation(s)
- Bethany Powell Gray
- Department of Surgery, Duke University Medical Center, 2 Genome Ct, Durham, NC 27710, USA
| | - Martin D Requena
- Department of Surgery, Duke University Medical Center, 2 Genome Ct, Durham, NC 27710, USA
| | - Michael D Nichols
- Department of Biomedical Engineering, Duke University, 101 Science Dr, Durham, NC 27710, USA
| | - Bruce A Sullenger
- Department of Surgery, Duke University Medical Center, 2 Genome Ct, Durham, NC 27710, USA; Department of Biomedical Engineering, Duke University, 101 Science Dr, Durham, NC 27710, USA.
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48
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Kikkeri K, Kerr BA, Bertke AS, Strobl JS, Agah M. Passivated-electrode insulator-based dielectrophoretic separation of heterogeneous cell mixtures. J Sep Sci 2020; 43:1576-1585. [PMID: 31991043 DOI: 10.1002/jssc.201900553] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 01/21/2020] [Accepted: 01/22/2020] [Indexed: 01/11/2023]
Abstract
Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label-free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes' drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 μm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated-electrode insulator-based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.
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Affiliation(s)
- Kruthika Kikkeri
- The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA, USA
| | - Bethany A Kerr
- Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Andrea S Bertke
- Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA
| | - Jeannine S Strobl
- The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA, USA
| | - Masoud Agah
- The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA, USA
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El-Jawhari JJ, Ganguly P, Churchman S, Jones E, Giannoudis PV. The Biological Fitness of Bone Progenitor Cells in Reamer/Irrigator/Aspirator Waste. J Bone Joint Surg Am 2019; 101:2111-2119. [PMID: 31800424 DOI: 10.2106/jbjs.19.00133] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
BACKGROUND The biological waste collected during use of the Reamer/Irrigator/Aspirator (RIA; DePuy Synthes) has been described as an abundant source of bone progenitor cells with a comparable osteogenic gene profile to donor-matched iliac crest bone marrow (IC-BM). However, it is not clear whether these RIA-waste (RIA-W) cells are biologically fit. We aimed to evaluate the stress levels and functions of RIA-W progenitor cells. METHODS Reactive oxygen species (ROS) levels were tested in freshly collected bone progenitor cells (defined as CD45CD271 cells) using flow cytometry. ROS levels induced in these cells by hypoxia and/or oxidative stress as well as by an experimental simulation of the RIA procedure were also measured. Furthermore, the alkaline phosphatase (ALP) expression levels, proliferation, and senescence of culture-expanded RIA-W and IC-BM mesenchymal stromal cells (MSCs) were compared. RESULTS RIA-W and donor-matched IC-BM CD45CD271 cells were 97% and 98% viable, but the ROS levels were significantly higher for RIA-W cells than for IC-BM cells (p = 0.0020). Also, ROS induced by hypoxia, oxidative stress, and both were higher for RIA-W cells (p = 0.0312, 0.0156, and 0.0703, respectively). Dilution with saline solution, suction pressure, and irrigation reduced cell viability, with a positive correlation with the ROS level (p = 0.0035). The RIA-W and IC-BM colony-forming cells (average, 96,100 and 11,500, respectively) showed comparable ALP levels. Furthermore, culture-expanded RIA-W and IC-BM MSCs showed comparable ROS levels, ALP levels, susceptibility to death, and proliferation. CONCLUSIONS Although freshly collected RIA-W bone progenitor cells appeared to be transiently stressed, these cells were as viable as IC-BM cells and present in greater numbers. The proliferation and osteogenesis of both cell types were comparable. CLINICAL RELEVANCE The RIA waste bag contains bone progenitor cells with promising potential for regenerative applications, and should not be wasted.
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Affiliation(s)
- Jehan J El-Jawhari
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
- Clinical Pathology Department, Mansoura University, Mansoura, Egypt
| | - Payal Ganguly
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
| | - Sarah Churchman
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
| | - Elena Jones
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
| | - Peter V Giannoudis
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
- Academic Unit of Trauma and Orthopaedic Surgery, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom
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Crowther CV, Sanderlin V, Hayes MA, Gile GH. Effects of surface treatments on trapping with DC insulator-based dielectrophoresis. Analyst 2019; 144:7478-7488. [PMID: 31720589 PMCID: PMC6909249 DOI: 10.1039/c9an01186b] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
A central challenge in measuring the biophysical properties of cells with electrokinetic approaches is the assignment of these biophysical properties to specific biological characteristics. Changes in the electrokinetic behavior of cells may come from mutations, altered gene expression levels, post-translation modifications, or environmental effects. Here we assess the electrokinetic behavior of chemically surface-modified bacterial cells in order to gain insight into the biophysical properties that are specifically affected by changes in surface chemistry. Using E. coli as a scaffold, an amine coupling reaction was used to covalently attach glycine, spermine, bovine serum albumin (protein), or 7-amino-4-methyl-3-coumarinylacetic acid (fluorescent dye) to the free carboxylic acid groups on the surface of the cells. These populations, along with unlabeled control cells, were subject to electrokinetic and dielectrophoretic measurements to quantify any changes in the biophysical properties upon alteration. The properties associated with each electrokinetic force are discussed relative to the specific reactant used. We conclude that relatively modest and superficial changes to cell surfaces can cause measurable changes in their biophysical properties.
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Affiliation(s)
- Claire V Crowther
- School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
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