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1
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Yang T, Wang W, Xie L, Chen S, Ye X, Shen S, Chen H, Qi L, Cui Z, Xiong W, Guo Y, Chen J. Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids. J Neurosci Methods 2024; 408:110181. [PMID: 38823594 DOI: 10.1016/j.jneumeth.2024.110181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Revised: 05/05/2024] [Accepted: 05/22/2024] [Indexed: 06/03/2024]
Abstract
BACKGROUND Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.
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Affiliation(s)
- Tingting Yang
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China; Department of Ophthalmology, Affiliated Qingyuan Hospital, Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, China
| | - Wenxuan Wang
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Linyao Xie
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Sihui Chen
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Xiuhong Ye
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Shuhao Shen
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Hang Chen
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China
| | - Ling Qi
- Central Laboratory, Affiliated Qingyuan Hospital, Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, China
| | - Zekai Cui
- Aier Eye Institute, Changsha, Hunan, China
| | - Wei Xiong
- Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China
| | - Yonglong Guo
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
| | - Jiansu Chen
- Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China; Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, China; Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China; Aier Eye Institute, Changsha, Hunan, China.
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2
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Layer PG. In a century from agitated cells to human organoids. J Neurosci Methods 2024; 405:110083. [PMID: 38387805 DOI: 10.1016/j.jneumeth.2024.110083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 02/09/2024] [Accepted: 02/18/2024] [Indexed: 02/24/2024]
Abstract
Reaching back more than a century, suspension cultures have provided major insights into processes of histogenesis; e.g., cell communication, distinction of self/nonself, cell sorting and cell adhesion. Besides studies on lower animals, the vertebrate retina served as excellent reaggregate model to analyze 3D reconstruction of a complex neural laminar tissue. Methodologically, keeping cells under suspension is essential to achieve tissue organisation in vitro; thereby, the environmental conditions direct the emergent histotypic particulars. Recent progress in regenerative medicine is based to a large extent on human induced pluripotent stem cells (hiPSCs), which are cultured under suspension. Following their genetically directed differentiation into various histologic 3D structures, organoids provide excellent multipurpose in vitro assay models, as well as tissues for repair transplantations. Historically, a nearly fully laminated retinal spheroid from avian embryos was achieved already in 1984, foreshadowing the potential of culturing stem cells under suspension for tissue reconstruction purposes.
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Affiliation(s)
- Paul Gottlob Layer
- Technical University of Darmstadt, Developmental Biology & Neurogenetics, Schnittspahnstrasse 13, Darmstadt 64297, Germany.
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3
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Eldred KC, Reh TA. Human retinal model systems: Strengths, weaknesses, and future directions. Dev Biol 2021; 480:114-122. [PMID: 34529997 DOI: 10.1016/j.ydbio.2021.09.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 09/01/2021] [Accepted: 09/02/2021] [Indexed: 10/20/2022]
Abstract
The retina is a complex neuronal structure that converts light energy into visual perception. Many specialized aspects of the primate retina, including a cone rich macula for high acuity vision, ocular size, and cell type diversity are not found in other animal models. In addition, the unique morphologies and distinct laminar positions of cell types found in the retina make this model system ideal for the study of neuronal cell fate specification. Many key early events of human retinal development are inaccessible to investigation as they occur during gestation. For these reasons, it has been necessary to develop retinal model systems to gain insight into human-specific retinal development and disease. Recent advances in culturing retinal tissue have generated new systems for retinal research and have moved us closer to generating effective regenerative therapies for vision loss. Here, we describe the strengths, weaknesses, and future directions for different human retinal model systems including dissociated primary tissue, explanted primary tissue, retinospheres, and stem cell-derived retinal organoids.
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Affiliation(s)
- Kiara C Eldred
- Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, 98195, USA
| | - Thomas A Reh
- Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, 98195, USA.
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4
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Advanced 3D Cell Culture Techniques in Micro-Bioreactors, Part II: Systems and Applications. Processes (Basel) 2020. [DOI: 10.3390/pr9010021] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems.
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5
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Advanced 3D Cell Culture Techniques in Micro-Bioreactors, Part I: A Systematic Analysis of the Literature Published between 2000 and 2020. Processes (Basel) 2020. [DOI: 10.3390/pr8121656] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Bioreactors have proven useful for a vast amount of applications. Besides classical large-scale bioreactors and fermenters for prokaryotic and eukaryotic organisms, micro-bioreactors, as specialized bioreactor systems, have become an invaluable tool for mammalian 3D cell cultures. In this systematic review we analyze the literature in the field of eukaryotic 3D cell culture in micro-bioreactors within the last 20 years. For this, we define complexity levels with regard to the cellular 3D microenvironment concerning cell–matrix-contact, cell–cell-contact and the number of different cell types present at the same time. Moreover, we examine the data with regard to the micro-bioreactor design including mode of cell stimulation/nutrient supply and materials used for the micro-bioreactors, the corresponding 3D cell culture techniques and the related cellular microenvironment, the cell types and in vitro models used. As a data source we used the National Library of Medicine and analyzed the studies published from 2000 to 2020.
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Microfluidic and Microscale Assays to Examine Regenerative Strategies in the Neuro Retina. MICROMACHINES 2020; 11:mi11121089. [PMID: 33316971 PMCID: PMC7763644 DOI: 10.3390/mi11121089] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 12/03/2020] [Accepted: 12/05/2020] [Indexed: 12/15/2022]
Abstract
Bioengineering systems have transformed scientific knowledge of cellular behaviors in the nervous system (NS) and pioneered innovative, regenerative therapies to treat adult neural disorders. Microscale systems with characteristic lengths of single to hundreds of microns have examined the development and specialized behaviors of numerous neuromuscular and neurosensory components of the NS. The visual system is comprised of the eye sensory organ and its connecting pathways to the visual cortex. Significant vision loss arises from dysfunction in the retina, the photosensitive tissue at the eye posterior that achieves phototransduction of light to form images in the brain. Retinal regenerative medicine has embraced microfluidic technologies to manipulate stem-like cells for transplantation therapies, where de/differentiated cells are introduced within adult tissue to replace dysfunctional or damaged neurons. Microfluidic systems coupled with stem cell biology and biomaterials have produced exciting advances to restore vision. The current article reviews contemporary microfluidic technologies and microfluidics-enhanced bioassays, developed to interrogate cellular responses to adult retinal cues. The focus is on applications of microfluidics and microscale assays within mammalian sensory retina, or neuro retina, comprised of five types of retinal neurons (photoreceptors, horizontal, bipolar, amacrine, retinal ganglion) and one neuroglia (Müller), but excludes the non-sensory, retinal pigmented epithelium.
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Bachmann G, Frohns F, Thangaraj G, Bausch A, Layer PG. IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling. Invest Ophthalmol Vis Sci 2020; 60:4759-4773. [PMID: 31738824 DOI: 10.1167/iovs.18-24952] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Purpose Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.
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Affiliation(s)
- Gesine Bachmann
- Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany
| | - Florian Frohns
- Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany.,Radiation Biology, Technische Universität Darmstadt, Darmstadt, Germany
| | - Gopenath Thangaraj
- Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany.,Division of Biotechnology, Faculty of Life Sciences, JSS Academy of Higher Education & Research, Mysuru, India
| | - Alexander Bausch
- Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany
| | - Paul G Layer
- Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany
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8
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Murata D, Akieda S, Misumi K, Nakayama K. Osteochondral Regeneration with a Scaffold-Free Three-Dimensional Construct of Adipose Tissue-Derived Mesenchymal Stromal Cells in Pigs. Tissue Eng Regen Med 2017; 15:101-113. [PMID: 30603538 PMCID: PMC6171634 DOI: 10.1007/s13770-017-0091-9] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Revised: 09/12/2017] [Accepted: 10/13/2017] [Indexed: 12/14/2022] Open
Abstract
Osteochondral lesion is a major joint disease in humans. Therefore, this study was designed to investigate the regeneration of articular cartilage and subchondral bone, using three-dimensional constructs of autologous adipose tissue-derived mesenchymal stromal cells without any biocompatible scaffolds. Mesenchymal stromal cells were harvested by liposuction from seven pigs, isolated enzymatically, and expanded until construct creation. The pig models had two osteochondral defects (cylindrical defects with a diameter of 5.2 mm and a depth of 5 mm) in one of their patello-femoral grooves. A columnar structure consisting of approximately 770 spheroids of 5 × 104 autologous mesenchymal stromal cells were implanted into one of the defects (implanted defect), while the other defect was not implanted (control). The defects were evaluated pathologically at 6 months (in three pigs) and 12 months (in five pigs) after implantation. At 6 months after surgery, histopathology revealed active endochondral ossification underneath the plump fibrocartilage in the implanted defects, but a deficiency of fibrocartilaginous coverage in the controls. At 12 months after surgery, the fibrocartilage was transforming into hyaline cartilage as thick as the surrounding normal cartilage and the subchondral bone was thickening in the implanted defects. The histological averages of the implanted sites were significantly higher than those in the control sites at both 6 and 12 months after surgery. The implantation of a scaffold-free three-dimensional construct of autologous mesenchymal stromal cells into an osteochondral defect can induce regeneration of hyaline cartilage and subchondral bone structures over a period of 12 months.
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Affiliation(s)
- Daiki Murata
- 1Department of Veterinary Clinical Science, Joint Faculty of Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima, 890-0065 Japan
| | - Shizuka Akieda
- Cyfuse Biomedical K.K, 1-1 Maidashi 3-chome, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Kazuhiro Misumi
- 1Department of Veterinary Clinical Science, Joint Faculty of Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima, 890-0065 Japan
| | - Koichi Nakayama
- 3Department of Regenerative Medicine and Biomedical Engineering, Faculty of Medicine, Saga University, Honjyo 1-chome, Honjyo-cho, Saga, 840-8502 Japan
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9
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Neubauer A, Nies C, Schepkin VD, Hu R, Malzacher M, Chacón-Caldera J, Thiele D, Gottwald E, Schad LR. Tracking protein function with sodium multi quantum spectroscopy in a 3D-tissue culture based on microcavity arrays. Sci Rep 2017. [PMID: 28638107 PMCID: PMC5479864 DOI: 10.1038/s41598-017-04226-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The aim of this study was to observe the effects of strophanthin induced inhibition of the Na-/K-ATPase in liver cells using a magnetic resonance (MR) compatible bioreactor. A microcavity array with a high density three-dimensional cell culture served as a functional magnetic resonance imaging (MRI) phantom for sodium multi quantum (MQ) spectroscopy. Direct contrast enhanced (DCE) MRI revealed the homogenous distribution of biochemical substances inside the bioreactor. NMR experiments using advanced bioreactors have advantages with respect to having full control over a variety of physiological parameters such as temperature, gas composition and fluid flow. Simultaneous detection of single quantum (SQ) and triple quantum (TQ) MR signals improves accuracy and was achieved by application of a pulse sequence with a time proportional phase increment (TQTPPI). The time course of the Na-/K-ATPase inhibition in the cell culture was demonstrated by the corresponding alterations of sodium TQ/SQ MR signals.
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Affiliation(s)
- Andreas Neubauer
- Computer Assisted Clinical Medicine, Centre for Biomedicine and Medical Technology Mannheim, Heidelberg University, Mannheim, Germany.
| | - Cordula Nies
- Institute of Functional Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany
| | - Victor D Schepkin
- CIMAR, National High Magnetic Field Laboratory/FSU, Tallahassee, FL, USA
| | - Ruomin Hu
- Computer Assisted Clinical Medicine, Centre for Biomedicine and Medical Technology Mannheim, Heidelberg University, Mannheim, Germany
| | - Matthias Malzacher
- Computer Assisted Clinical Medicine, Centre for Biomedicine and Medical Technology Mannheim, Heidelberg University, Mannheim, Germany
| | - Jorge Chacón-Caldera
- Computer Assisted Clinical Medicine, Centre for Biomedicine and Medical Technology Mannheim, Heidelberg University, Mannheim, Germany
| | - David Thiele
- Institute for Biological Interfaces-5, Karlsruhe Institute of Technology, Karlsruhe, Germany
| | - Eric Gottwald
- Institute of Functional Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany
| | - Lothar R Schad
- Computer Assisted Clinical Medicine, Centre for Biomedicine and Medical Technology Mannheim, Heidelberg University, Mannheim, Germany
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10
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Zhou Y. The Application of Ultrasound in 3D Bio-Printing. Molecules 2016; 21:E590. [PMID: 27164066 PMCID: PMC6274238 DOI: 10.3390/molecules21050590] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2016] [Revised: 04/20/2016] [Accepted: 04/25/2016] [Indexed: 12/21/2022] Open
Abstract
Three-dimensional (3D) bioprinting is an emerging and promising technology in tissue engineering to construct tissues and organs for implantation. Alignment of self-assembly cell spheroids that are used as bioink could be very accurate after droplet ejection from bioprinter. Complex and heterogeneous tissue structures could be built using rapid additive manufacture technology and multiple cell lines. Effective vascularization in the engineered tissue samples is critical in any clinical application. In this review paper, the current technologies and processing steps (such as printing, preparation of bioink, cross-linking, tissue fusion and maturation) in 3D bio-printing are introduced, and their specifications are compared with each other. In addition, the application of ultrasound in this novel field is also introduced. Cells experience acoustic radiation force in ultrasound standing wave field (USWF) and then accumulate at the pressure node at low acoustic pressure. Formation of cell spheroids by this method is within minutes with uniform size and homogeneous cell distribution. Neovessel formation from USWF-induced endothelial cell spheroids is significant. Low-intensity ultrasound could enhance the proliferation and differentiation of stem cells. Its use is at low cost and compatible with current bioreactor. In summary, ultrasound application in 3D bio-printing may solve some challenges and enhance the outcomes.
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Affiliation(s)
- Yufeng Zhou
- Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 639798, Singapore.
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11
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Wuchter P, Saffrich R, Giselbrecht S, Nies C, Lorig H, Kolb S, Ho AD, Gottwald E. Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells. Cell Tissue Res 2016; 364:573-584. [PMID: 26829941 DOI: 10.1007/s00441-015-2348-8] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 12/10/2015] [Indexed: 12/28/2022]
Abstract
In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 μm size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ß-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.
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Affiliation(s)
- Patrick Wuchter
- Department of Medicine V, Heidelberg University, 69120, Heidelberg, Germany. .,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany.
| | - Rainer Saffrich
- Department of Medicine V, Heidelberg University, 69120, Heidelberg, Germany.,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany
| | - Stefan Giselbrecht
- HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany.,Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands
| | - Cordula Nies
- Institute for Biological Interfaces-5, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Karlsruhe, Germany.,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany
| | - Hanna Lorig
- Institute for Biological Interfaces-5, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Karlsruhe, Germany.,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany
| | - Stephanie Kolb
- Institute for Biological Interfaces-5, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Karlsruhe, Germany.,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany
| | - Anthony D Ho
- Department of Medicine V, Heidelberg University, 69120, Heidelberg, Germany.,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany
| | - Eric Gottwald
- Institute for Biological Interfaces-5, Karlsruhe Institute of Technology (KIT), 76344, Eggenstein-Leopoldshafen, Karlsruhe, Germany. .,HEiKA - Heidelberg Karlsruhe Research Partnership, Heidelberg University and Karlsruhe Institute of Technology, Heidelberg and Karlsruhe, Germany.
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12
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Green DI, Ou Y. Towards the development of a human glaucoma disease-in-a-dish model using stem cells. EXPERT REVIEW OF OPHTHALMOLOGY 2015. [DOI: 10.1586/17469899.2015.1026329] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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13
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Gottwald E, Giselbrecht S. Organotypic tissue models in MRI method development. Z Med Phys 2014; 24:89-90. [PMID: 24636715 DOI: 10.1016/j.zemedi.2014.02.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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14
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Layer PG, Araki M, Vogel-Höpker A. New concepts for reconstruction of retinal and pigment epithelial tissues. EXPERT REVIEW OF OPHTHALMOLOGY 2014. [DOI: 10.1586/eop.10.42] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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15
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Liu M, Liu N, Zang R, Li Y, Yang ST. Engineering stem cell niches in bioreactors. World J Stem Cells 2013; 5:124-35. [PMID: 24179601 PMCID: PMC3812517 DOI: 10.4252/wjsc.v5.i4.124] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2013] [Revised: 06/05/2013] [Accepted: 07/04/2013] [Indexed: 02/06/2023] Open
Abstract
Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as "niches", to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.
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Affiliation(s)
- Meimei Liu
- Meimei Liu, Ning Liu, Ru Zang, Shang-Tian Yang, William G Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, United States
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16
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Characterization of a chip-based bioreactor for three-dimensional cell cultivation via Magnetic Resonance Imaging. Z Med Phys 2013; 23:102-10. [PMID: 23410914 DOI: 10.1016/j.zemedi.2013.01.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2012] [Revised: 01/16/2013] [Accepted: 01/23/2013] [Indexed: 11/22/2022]
Abstract
We describe the characterization of a chip-based platform (3(D)-KITChip) for the three-dimensional cultivation of cells under perfusion conditions via magnetic resonance imaging (MRI). Besides the chip, the microfluidic system is comprised of a bioreactor housing, a medium supply, a pump for generating active flow conditions as well as a gas mixing station. The closed circulation loop is ideally suited for a characterization via MRI since the small bioreactor setup with active perfusion, driven by the pump from outside the coils, not only is completely MRI-compatible but also can be transferred into the magnetic coil of an experimental animal scanner. We have found that the two halves of the chip inside the bioreactor are homogeneously perfused with cell culture medium both with and without cells inside the 3(D)-KITChip. In addition, the homogeneity of perfusion is nearly independent from the flow rates investigated in this study, and furthermore, the setup shows excellent washout characteristics after spiking with Gadolinium-DOTA which makes it an ideal candidate for drug screening purposes. We, therefore, conclude that the 3(D)-KITChip is well suited as a platform for high-density three-dimensional cell cultures, especially those requiring a defined medium flow and/or gas supply in a precisely controllable three dimensional environment, like stem cells.
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Altmann B, Ahrens R, Welle A, Dinglreiter H, Schneider M, Schober A. Microstructuring of multiwell plates for three-dimensional cell culture applications by ultrasonic embossing. Biomed Microdevices 2011; 14:291-301. [DOI: 10.1007/s10544-011-9605-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
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Thangaraj G, Greif A, Layer PG. Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors. Exp Eye Res 2011; 93:556-64. [DOI: 10.1016/j.exer.2011.07.011] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2011] [Revised: 07/18/2011] [Accepted: 07/20/2011] [Indexed: 11/29/2022]
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Altmann B, Steinberg T, Giselbrecht S, Gottwald E, Tomakidi P, Bächle-Haas M, Kohal RJ. Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate. Biomaterials 2011; 32:8947-56. [PMID: 21868090 DOI: 10.1016/j.biomaterials.2011.08.023] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2011] [Accepted: 08/09/2011] [Indexed: 01/21/2023]
Abstract
Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration.
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Affiliation(s)
- Brigitte Altmann
- Department of Prosthodontics, Dental School, University Hospital Freiburg, Freiburg, Germany
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Abstract
Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages. Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used. By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5. In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.
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Affiliation(s)
- Sebastian Gustmann
- Institute for Anatomy, Department of Neuroanatomy, University of Duisburg-Essen, Essen, Germany
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Kojima R, Yoshimoto K, Takahashi E, Ichino M, Miyoshi H, Nagasaki Y. Spheroid array of fetal mouse liver cells constructed on a PEG-gel micropatterned surface: upregulation of hepatic functions by co-culture with nonparenchymal liver cells. LAB ON A CHIP 2009; 9:1991-3. [PMID: 19568664 DOI: 10.1039/b903388b] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
A spheroid array of fetal mouse liver cells, which comprise various immature cells, was constructed on a PEG-gel micropatterned surface and its hepatic activity and degree of differentiation induction were significantly upregulated by co-culture with nonparenchymal liver cells as feeder-cells.
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Affiliation(s)
- Ryota Kojima
- Graduate School of Pure and Applied Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan
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Bazou D. Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies. Cell Biol Toxicol 2009; 26:127-41. [DOI: 10.1007/s10565-009-9123-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2008] [Accepted: 03/11/2009] [Indexed: 12/24/2022]
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Pampaloni F, Stelzer EHK. Three-Dimensional Cell Cultures in Toxicology. Biotechnol Genet Eng Rev 2009; 26:117-38. [DOI: 10.5661/bger-26-117] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
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