The Experts below are selected from a list of 3651 Experts worldwide ranked by ideXlab platform
Shaul Reuveny - One of the best experts on this subject based on the ideXlab platform.
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critical attributes of human early mesenchymal stromal cell laden microcarrier constructs for improved chondrogenic differentiation
Stem Cell Research & Therapy, 2017Co-Authors: Jialing Lee, Mahesh Choolani, Jessica Fang Ya Lim, Jerry Kok Ye Cha, Shaul ReuvenyAbstract:Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to Microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types. A screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs. Firstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 Microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty Microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to Microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden Microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation. This is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to Microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based Microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy.
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Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation
'Springer Science and Business Media LLC', 2017Co-Authors: Youshan Melissa Lin, Jerry Kok Yen Chan, Mahesh Choolani, Jialing Lee, Jessica Fang Yan Lim, Shaul ReuvenyAbstract:Abstract Background Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to Microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types. Methods A screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs. Results Firstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 Microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty Microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to Microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden Microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation. Conclusions This is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to Microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based Microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy
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expansion in microcarrier spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells
Cytotherapy, 2016Co-Authors: Mahesh Choolani, Shaul Reuveny, Jerry Kok Yen Chan, Steve OhAbstract:Abstract Background aims Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. Methods heMSC expanded on either collagen-coated Cytodex 3 Microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL , were investigated and compared in both conditions. Results BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β ) but not of hypertrophic genes (e.g., MMP13 and ALPL ). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. Conclusions This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.
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increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed
Cytotherapy, 2015Co-Authors: Allen Chen, Yi Kong Chew, Shaul Reuveny, Steve OhAbstract:Abstract Background aims Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. Methods MSCs were expanded in stirred cultures on Cytodex 3 Microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. Results Minimum essential medium-α–based medium supported faster MSC growth on Microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 10 6 cells/mL with confluent cell concentration of 4.7 × 10 4 cells/cm 2 was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10 6 cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10 6 cells. Conclusions We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.
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inhibition of rock myosin ii signaling pathway enables culturing of human pluripotent stem cells on Microcarriers without extracellular matrix coating
Tissue Engineering Part C-methods, 2014Co-Authors: Allen Chen, Xiaoli Chen, Yu Ming Lim, Shaul ReuvenyAbstract:Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These Microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose Microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free Microcarriers. Cells propagated on the noncoated Microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×10⁶ cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×10⁶ cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.
Steve Oh - One of the best experts on this subject based on the ideXlab platform.
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expansion in microcarrier spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells
Cytotherapy, 2016Co-Authors: Mahesh Choolani, Shaul Reuveny, Jerry Kok Yen Chan, Steve OhAbstract:Abstract Background aims Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. Methods heMSC expanded on either collagen-coated Cytodex 3 Microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL , were investigated and compared in both conditions. Results BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β ) but not of hypertrophic genes (e.g., MMP13 and ALPL ). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. Conclusions This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.
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increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed
Cytotherapy, 2015Co-Authors: Allen Chen, Yi Kong Chew, Shaul Reuveny, Steve OhAbstract:Abstract Background aims Large amounts of human mesenchymal stromal cells (MSCs) are needed for clinical cellular therapy. In a previous publication, we described a microcarrier-based process for expansion of MSCs. The present study optimized this process by selecting suitable basal media, microcarrier concentration and feeding regime to achieve higher cell yields and more efficient medium utilization. Methods MSCs were expanded in stirred cultures on Cytodex 3 Microcarriers with media containing 10% fetal bovine serum. Process optimization was carried out in spinner flasks. A 2-L bioreactor with an automated feeding system was used to validate the optimized parameters explored in spinner flask cultures. Results Minimum essential medium-α–based medium supported faster MSC growth on Microcarriers than did Dulbecco's modified Eagle's medium (doubling time, 31.6 ± 1.4 vs 42 ± 1.7 h) and shortened the process time. At microcarrier concentration of 8 mg/mL, a high cell concentration of 1.08 × 10 6 cells/mL with confluent cell concentration of 4.7 × 10 4 cells/cm 2 was achieved. Instead of 50% medium exchange every 2 days, we have designed a full medium feed that is based on glucose consumption rate. The optimal medium feed that consisted of 1.5 g/L glucose supported MSC growth to full confluency while achieving the low medium usage efficiency of 3.29 mL/10 6 cells. Finally, a controlled bioreactor with the optimized parameters achieved maximal confluent cell concentration with 16-fold expansion and a further improved medium usage efficiency of 1.68 mL/10 6 cells. Conclusions We have optimized the microcarrier-based platform for expansion of MSCs that generated high cell yields in a more efficient and cost-effective manner. This study highlighted the critical parameters in the optimization of MSC production process.
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application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy achievements and future direction
Biotechnology Advances, 2013Co-Authors: Allen Chen, Shaul Reuveny, Steve OhAbstract:Abstract Mesenchymal stem cells (MSCs) have recently made significant progress with multiple clinical trials targeting modulation of immune responses, regeneration of bone, cartilage, myocardia, and diseases like Metachromatic leukodystrophy and Hurler syndrome. On the other hand, the use of human embryonic and induced pluripotent stem cells (hPSCs) in clinical trials is rather limited mainly due to safety issues. Only two clinical trials, retinal pigment epithelial transplantation and treatment of spinal cord injury were reported. Cell doses per treatment can range between 50,000 and 6 billion cells. The current 2-dimensional tissue culture platform can be used when low cell doses are needed and it becomes impractical when doses above 50 million are needed. This demand for future cell therapy has reinvigorated interests in the use of the microcarrier platform for generating stem cells in a scalable 3-dimensional manner. Microcarriers developed for culturing adherent cell lines in suspension have been used mainly in vaccine production and research purposes. Since MSCs grow as monolayers similar to conventional adherent cell lines, adapting MSCs to a microcarrier based expansion platform has been progressing rapidly. On the other hand, establishing a robust microcarrier platform for hPSCs is more challenging as these cells grow in multilayer colonies on extracellular matrices and are more susceptible to shear stress. This review describes properties of commercially available Microcarriers developed for cultivation of anchorage dependent cells and present current achievements for expansion and differentiation of stem cells. Key issues such as microcarrier properties and coatings, cell seeding conditions, medium development and improved bioprocess parameters needed for optimal stem cell systems are discussed.
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long term microcarrier suspension cultures of human embryonic stem cells
Stem Cell Research, 2009Co-Authors: Steve Oh, Allen Chen, Angela Chin, Andre Choo, Xiaoli Chen, Shaul ReuvenyAbstract:The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces, which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated Microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable, continuous propagation of two hESC lines on Microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks, thus opening the prospect of propagation in controlled bioreactors.
Jos Malda - One of the best experts on this subject based on the ideXlab platform.
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biofabrication of tissue constructs by 3d bioprinting of cell laden Microcarriers
Biofabrication, 2014Co-Authors: Riccardo Levato, Jos Malda, Jetze Visser, Josep A Planell, Elisabeth Engel, Miguel A MateostimonedaAbstract:Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden Microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid Microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.
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adult human articular chondrocytes in a microcarrier based culture system expansion and redifferentiation
Journal of Orthopaedic Research, 2011Co-Authors: Karsten Schrobback, Travis J Klein, Zee Upton, Michael Schuetz, David I. Leavesley, Jos MaldaAbstract:xpanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use Microcarriers as substrates for chondrocyte expansion. While Microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin Microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to Microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on Microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from Microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to Microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications.
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adult human articular chondrocytes in a microcarrier based culture system expansion and redifferentiation
Journal of Orthopaedic Research, 2011Co-Authors: Karsten Schrobback, Travis J Klein, Zee Upton, Michael Schuetz, David I. Leavesley, Jos MaldaAbstract:Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use Microcarriers as substrates for chondrocyte expansion. While Microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin Microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to Microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on Microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from Microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to Microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539–546, 2011
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functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture
Journal of Biomedical Materials Research Part A, 2009Co-Authors: Danielle J Borg, Zee Upton, Rebecca A Dawson, David I. Leavesley, Dietmar Werner Hutmacher, Jos MaldaAbstract:Skin cells for transplantation are routinely prepared by growing patient keratinocytes in a semi-defined cocktail of growth factors, including serum and feeder cells. However, these reagents require substantial risk remediation and can contribute to transplant rejection. Microcarrier culture is an emerging technology that may allow the elimination of feeder cells whilst facilitating expansion of cultured keratinocytes. However, the behavior of keratinocytes in microcarrier culture and the potential of these cells to form an epidermis have been poorly defined. We characterized freshly isolated human keratinocytes cultured on CultiSpher-G® Microcarriers in the absence of murine feeder cells and assessed the potential of the keratinocytes to form an epidermis in an in vitro model. In a single passage, keratinocytes multiplied 44.9-fold in microcarrier-bioreactor culture in 17 days, whereas two-dimensional cultures reached confluence in 9 days and only expanded 7.4-fold. Histological characterization of keratinocytes on the Microcarriers revealed that the cells were randomly distributed within these porous structures, however, not all pores contained cells. High-resolution microcomputed tomography imaging of the Microcarriers confirmed limited interconnectivity of the pores. Immunoreactivity of specific epidermal markers was confirmed during cell expansion via immunohistochemistry. Despite the expression of differentiation markers, microcarrier-expanded keratinocytes retained the capacity to form an epidermis, as was evaluated using an in vitro human skin equivalent model. The epidermis formed by microcarrier-expanded keratinocytes in this model exhibited morphology similar to native skin. Significantly, the microcarrier technique successfully eliminates the need for a feeder cell layer and hence facilitates development of an improved culture system.
Christopher J Hewitt - One of the best experts on this subject based on the ideXlab platform.
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expansion of human mesenchymal stem stromal cells on temporary liquid Microcarriers
Journal of Chemical Technology & Biotechnology, 2021Co-Authors: Christopher J Hewitt, Mariana Hanga, Halina Murasiewicz, Andrzej Pacek, A W NienowAbstract:Background Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid Microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid Microcarriers for hMSC culture. Results hMSCs successfully attached to these liquid Microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid Microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. Conclusion The prospect of a temporary microcarrier that can be used to expand cells and then 'disappear' for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid Microcarriers while, very importantly, retaining their quality.
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systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors
Biotechnology Journal, 2016Co-Authors: Qasim A Rafiq, Karen Coopman, Alvin W Nienow, Christopher J HewittAbstract:Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 Microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same Microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the Microcarriers were the same as the monolayer study. Plastic Microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture. Large-scale production of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) requires expansion on Microcarriers in agitated systems. This study demonstrates the importance of microcarrier selection and presents a systematic methodology for selection of an optimal microcarrier. The study also highlights the impact of an agitated culture environment in comparison to a static system, resulting in a significantly higher hBM-MSC yield under agitated conditions.
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A potentially scalable method for the harvesting of hMSCs from Microcarriers
Biochemical Engineering Journal, 2014Co-Authors: Alvin W Nienow, Qasim A Rafiq, Karen Coopman, Christopher J HewittAbstract:The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on Microcarriers in bioreactors. Whilst much research investigating hMSC culture on Microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill Microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the Microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large Microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from Microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too.
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Expansion of human mesenchymal stem cells on Microcarriers
Biotechnology Letters, 2011Co-Authors: Christopher J Hewitt, Mark Smith, Alvin W Nienow, Robert J Thomas, Colin R. ThomasAbstract:The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 Microcarriers were not damaged when held at the minimum speed, N_JS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the Microcarriers in the flasks were 3,000 Microcarriers ml^−1 (ca. 1 g dry weight l^−1), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at N_JS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
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growth behavior of number distributed adherent mdck cells for optimization in microcarrier cultures
Biotechnology Progress, 2009Co-Authors: A. Böck, Heiner Sann, J Schulzehorsel, Yvonne Genzel, Udo Reichl, L MohlerAbstract:An assay for measuring the number of adherent cells on Microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell-to-bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of Microcarriers covered by 1-220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density-dependent growth inhibition, and basic metabolism of Madin-Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on Microcarriers after seeding was developed. With both model approaches, the fraction of growth-inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on Microcarriers after attachment.
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growth behavior of number distributed adherent mdck cells for optimization in microcarrier cultures
Biotechnology Progress, 2009Co-Authors: A. Böck, Heiner Sann, J Schulzehorsel, Yvonne Genzel, Udo Reichl, L MohlerAbstract:An assay for measuring the number of adherent cells on Microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell-to-bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of Microcarriers covered by 1–220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density-dependent growth inhibition, and basic metabolism of Madin-Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on Microcarriers after seeding was developed. With both model approaches, the fraction of growth-inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on Microcarriers after attachment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009
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establishment of a mink enteritis vaccine production process in stirred tank reactor and wave bioreactor microcarrier culture in 1 10 l scale
Vaccine, 2007Co-Authors: Boris Hundt, N Schlawin, Holger Kassner, Yvonne Genzel, C. Best, Udo ReichlAbstract:Abstract A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on Microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave® Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2 g/L Cytodex™ 1 Microcarriers and 2.0 × 105 cells/mL was successful. A maximum cell yield of 1.2 × 106 cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave® Bioreactor could not be determined accurately due to the fast sedimentation of Microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus–host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave® Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave® Bioreactor using a multiple harvest strategy. Maximum virus titres of 106.6 to 106.8 TCID50/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave® Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave® Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.
