The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Steve Oh - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
increasing efficiency of human mesenchymal stromal cell culture by optimization of Microcarrier concentration and design of medium feed
Cytotherapy, 2015Co-Authors: Allen Chen, Shaul Reuveny, Yi Kong Chew, 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.
-
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.
-
scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended Microcarrier cultures
Tissue Engineering Part C-methods, 2010Co-Authors: Marti Lecina, Shaul Reuveny, Andre Choo, Sherwin Ting, Steve OhAbstract:A scalable platform for human embryonic stem cell (hESC)-derived cardiomyocyte (CM) production can provide a readily available source of CMs for cell therapy, drug screening, and cardiotoxicity tests. We have designed and optimized a scalable platform using Microcarrier cultures in serum-free media supplemented with SB203580 mitogen-activated protein kinase-inhibitor. Different Microcarriers (DE-53, Cytodex-1 and 3, FACT, and TOSOH-10) were used to investigate the effects of type, size, shape, and Microcarrier concentrations on the differentiation efficiency. hESCs propagated on TOSOH-10 (protamine derivatized 10-μm beads) at the concentration of 0.125 mg/mL produced 80% beating aggregates, threefold cell expansion, and 20% of CMs (determined by fluorescence-activated cell sorting for myosin heavy chain and α-actinin expression). The ratio of CM/hESC seeded in this system was 0.62 compared to 0.22 in the embryoid body control cultures. The platform robustness has been tested with HES-3 and H1 cell lines, ...
-
long term Microcarrier suspension cultures of human embryonic stem cells
Stem Cell Research, 2009Co-Authors: Steve Oh, Xiaoli Chen, Allen Chen, Angela Chin, Andre Choo, 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.
Shaul Reuveny - One of the best experts on this subject based on the ideXlab platform.
-
Expansion of human embryonic stem cells on cellulose Microcarriers.
Current protocols in stem cell biology, 2020Co-Authors: Allen K Chen, Shaul Reuveny, Xiaoli Chen, Andre B H Choo, Steve K W OhAbstract:This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose Microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrix-coated two-dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three-dimensional suspension culture using Matrigel-coated cellulose Microcarriers. hESCs from a tissue culture plate can be seeded directly onto the Microcarriers; hESC Microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100-ml spinner flask is also described. Long-term culture of hESC on the Microcarriers maintains typical pluripotent markers (OCT-4, Tra-1-60, and SSEA-4) and stable karyotype. Spontaneous differentiations of Microcarrier-maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells.
-
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.
-
increasing efficiency of human mesenchymal stromal cell culture by optimization of Microcarrier concentration and design of medium feed
Cytotherapy, 2015Co-Authors: Allen Chen, Shaul Reuveny, Yi Kong Chew, 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.
-
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.
-
scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended Microcarrier cultures
Tissue Engineering Part C-methods, 2010Co-Authors: Marti Lecina, Shaul Reuveny, Andre Choo, Sherwin Ting, Steve OhAbstract:A scalable platform for human embryonic stem cell (hESC)-derived cardiomyocyte (CM) production can provide a readily available source of CMs for cell therapy, drug screening, and cardiotoxicity tests. We have designed and optimized a scalable platform using Microcarrier cultures in serum-free media supplemented with SB203580 mitogen-activated protein kinase-inhibitor. Different Microcarriers (DE-53, Cytodex-1 and 3, FACT, and TOSOH-10) were used to investigate the effects of type, size, shape, and Microcarrier concentrations on the differentiation efficiency. hESCs propagated on TOSOH-10 (protamine derivatized 10-μm beads) at the concentration of 0.125 mg/mL produced 80% beating aggregates, threefold cell expansion, and 20% of CMs (determined by fluorescence-activated cell sorting for myosin heavy chain and α-actinin expression). The ratio of CM/hESC seeded in this system was 0.62 compared to 0.22 in the embryoid body control cultures. The platform robustness has been tested with HES-3 and H1 cell lines, ...
Joaquim M S Cabral - One of the best experts on this subject based on the ideXlab platform.
-
long term expansion of human induced pluripotent stem cells in a Microcarrier based dynamic system
Journal of Chemical Technology & Biotechnology, 2017Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Claudia C Miranda, Annette Puschklein, Simone Haupt, Carlos A V Rodrigues, Oliver Brustle, Joaquim M S CabralAbstract:BACKGROUND Human induced pluripotent stem (hiPS) cells provide a fascinating tool for exploring disease mechanisms, compound screening in pharmaceutical drug development, and might also represent a renewable source of cells for regenerative medicine applications. This requires increased cell quantities, generated under Good Manufacturing Practice-compatible conditions in a scalable system. RESULTS A Microcarrier-based suspension culture was explored for scaling-up of hiPS cell expansion in serum-free medium using synthetic peptide-acrylate surface Microcarriers, developed for long-term support of hiPS cell self-renewal. After a 7 days-culture in spinner flask, cells maintained their typical morphology, pluripotency-associated marker expression and their differentiation capability. Envisaging the improvement of the scalability of the culture, long-term expansion on the Microcarriers was attained using confluent Microcarriers as the inoculum of successive spinner flask cultures. Importantly, bead-to-bead cell transfer allowed 4 consecutive sub-culture procedures and a cumulative 241-fold expansion was achieved within 15 days, leading to a total viable cell number of 3.3x108 cells. CONCLUSION This work is expected to enable the scale-up of hiPS cell culture under defined conditions and potentially leading to the use of pluripotent stem cell derivatives in cell replacement therapies.
-
a xeno free Microcarrier based stirred culture system for the scalable expansion of human mesenchymal stem stromal cells isolated from bone marrow and adipose tissue
Biotechnology Journal, 2015Co-Authors: Joana G Carmelo, Ana Fernandesplatzgummer, Maria Margarida Diogo, Claudia Lobato Da Silva, Joaquim M S CabralAbstract:: Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of Microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free Microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use Microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic Microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings.
-
scalable ex vivo expansion of human mesenchymal stem stromal cells in Microcarrier based stirred culture systems
Methods of Molecular Biology, 2014Co-Authors: Joana G Carmelo, Ana Fernandesplatzgummer, Joaquim M S Cabral, Claudia Lobato Da SilvaAbstract:: The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a Microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different Microcarriers and xeno-free medium formulations.
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
-
growth behavior of number distributed adherent mdck cells for optimization in Microcarrier cultures
Biotechnology Progress, 2009Co-Authors: A Bock, Yvonne Genzel, Udo Reichl, Heiner Sann, J Schulzehorsel, 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
-
segregated mathematical model for growth of anchorage dependent mdck cells in Microcarrier culture
Biotechnology Progress, 2008Co-Authors: L Mohler, Udo Reichl, A BockAbstract:To describe the growth behavior of anchorage-dependent mammalian cells in Microcarrier systems, various approaches comprising deterministic and stochastic single cell models as well as automaton-based models have been presented in the past. The growth restriction of these often contact-inhibited cells by spatial effects is described at levels with different complexity but for the most part not taking into account their metabolic background. Compared to suspension cell lines these cells have a comparatively long lag phase required for attachment and start of proliferation on the Microcarrier. After an initial phase of exponential growth only a moderate specific growth rate is achieved due to restrictions in space available for cell growth, limiting medium components, and accumulation of growth inhibitors. Here, a basic deterministic unstructured segregated cell model for growth of Madin Darby Canine Kidney (MDCK) cells used in influenza vaccine production is described. Four classes of cells are considered: cells on Microcarriers, cells in suspension, dead cells, and lysed cells. Based on experimental data, cell attachment and detachment is taken explicitly into account. The model allows simulation of the overall growth behavior in Microcarrier culture, including the lag phase. In addition, it describes the time course of uptake and release of key metabolites and the identification of parameters relevant for the design and optimization of vaccine manufacturing processes.
-
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, C Best, N Schlawin, H Kassner, Yvonne Genzel, 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.
Allen Chen - One of the best experts on this subject based on the ideXlab platform.
-
increasing efficiency of human mesenchymal stromal cell culture by optimization of Microcarrier concentration and design of medium feed
Cytotherapy, 2015Co-Authors: Allen Chen, Shaul Reuveny, Yi Kong Chew, 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.
-
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.
-
long term Microcarrier suspension cultures of human embryonic stem cells
Stem Cell Research, 2009Co-Authors: Steve Oh, Xiaoli Chen, Allen Chen, Angela Chin, Andre Choo, 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.