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Joaquim M. S. Cabral - One of the best experts on this subject based on the ideXlab platform.
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successful use of human ab serum to support the expansion of adipose tissue derived mesenchymal stem stromal cell in a microcarrier based platform
Frontiers in Bioengineering and Biotechnology, 2020Co-Authors: Francisco Moreira, Joaquim M. S. Cabral, Cláudia Lobato Da Silva, Amanda Mizukami, Lucas Eduardo Botelho De Souza, Dimas Tadeu Covas, Kamilla SwiechAbstract:Mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and for the promotion of tissue repair, hence the increase of clinical trials in a worldwide scale. In particular, adipose tissue-derived stem/stromal cells (AT MSC) present easy accessibility and a rather straightforward process of isolation, providing a clear advantage over other sources. The high demand of cell doses (millions of cells/kg), needed for infusion in clinical settings, requires a scalable and efficient manufacturing of AT MSC under xenogeneic(xeno)-free culture conditions. Here we describe the successful use of human AB serum (10%(v/v)) as a culture supplement, as well as coating substrate for the expansion of these cells in microcarriers using (i) a Spinner Flask and (ii) a 500-mL mini-bioreactor (ApplikonTM Biotechnology). Cells were characterized by immunophenotype and multilineage differentiation potential. Upon an initial cell adhesion in the Spinner Flask of 35 ± 2.5%, culture reached a maximal cell density of 2.6 ± 0.1 x 105 cells/mL at day 7, obtaining a 15 ± 1-fold increase. The implementation of the culture in the 500-mL mini-bioreactor presented an initial cell adhesion of 22 ± 5%, but it reached maximal cell density of 2.7 ± 0.4 x 105 cells/mL at day 7, obtaining a 27 ± 8-fold increase. Importantly, in both stirred systems, cells retained their immunophenotype and multilineage differentiation potential (osteo-, chondro- and adipogenic lineages). Overall, the scalability of this microcarrier-based system presented herein is of major importance for the purpose of achieving clinically meaningful cell numbers.
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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, Carlos A V Rodrigues, Simone Haupt, Oliver Brüstle, 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.
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defined essential 8 medium and vitronectin efficiently support scalable xeno free expansion of human induced pluripotent stem cells in stirred microcarrier culture systems
PLOS ONE, 2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL Spinner Flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in Spinner Flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
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hiPS cell expansion in E8 medium, in a 50 mL Spinner Flask.
2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:EDTA clump inoculation was performed using 55,000 cells/cm2 and a continuous agitation at 44 rpm (optimal values obtained solving the quadratic model). (A) Total cell numbers during the 10 days expansion. Maximum cell yield obtained solving the quadratic model is represented by the black line. Error bars represent SEM (standard error of the mean) of duplicate samples. (B) Immunostaining of hiPS cells cultured on microcarriers in the Spinner Flask with E8 medium at day 10. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm. (C) Immunostaining of hiPS cells harvested from microcarriers after 10 days expansion in Spinner Flask and re-plated on GP. Cells were stained after 3 days for intracellular pluripotency markers NANOG, OCT4 and SOX2, and nuclei counterstained with DAPI; and for surface markers SSEA4 and TRA-1-60. Scale bars– 100 μm. (D) Flow cytometry analysis of hiPS cells harvested from microcarriers after 10 days expansion in Spinner Flask. Cells were stained for OCT4 and NANOG. (E) mRNA was isolated from hiPSC at day 0 and at the end of the Spinner Flask culture (day 12) on microcarriers, and the undifferentiated hiPSC marker transcripts (OCT4 and NANOG) were analysed by RT-PCR. (F) Quantitative RT-PCR analysis of spontaneous differentiated EB of hiPSC cultured in Spinner Flask. The relative expression of each gene was measured against the same gene prior to differentiation. (G) Immunostaining showing the formation of cells expressing SOX17 (endoderm), TUJ1 (ectoderm) and α-SMA (mesoderm) after EB formation and spontaneous differentiation assay with hiPSC cultured in Spinner Flask. Nuclei were counterstained with DAPI. Scale bar: 50 μm.
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Scaling-up hiPS cell expansion in E8 medium to a 50 mL Spinner Flask, under dynamic conditions.
2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:(A) The procedure consists in 4 steps: a. EDTA+ROCKi inoculation strategy into 20 g/L VtnM; b. Attachment is performed in half of the total volume in static conditions for 24 h (in the presence of ROCKi), following by intermittent stirring overnight to allow cell- microcarrier interaction; c. Expansion is performed with a specific continuous agitation with 80% E8 medium change everyday; and d. At the end of the culture, cell pluripotency and differentiation potential are evaluated. Quadratic model relating initial cell density and agitation speed with cell yield during Spinner Flask culture of human iPS cells: 3D representation (B) and 2D heat map (C) are shown. For initial cell density: 30 000 cells/cm2 (−1 level) ≤ Initial Cell Density ≤ 70 000 cells/cm2 (1 level); for agitation speed: 30 rpm (−1 level) ≤ Agitation Speed ≤ 70 rpm (1 level).
Alvin Nienow - One of the best experts on this subject based on the ideXlab platform.
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Supplementary figures for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"
2018Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin Nienow, David Williams, Christopher HewittAbstract:Supplementary information files for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"Figure S1. Growth kinetics of hMSCs donor 2 cells using serum-free (SFM) and fetal bovine serum (FBS)-based media in both the ambr15 and Spinner Flasks with data showing the viable cell density.Figure S2. Nutrient and metabolite flux for hMSC donor 1 cells expanded on microcarriers in the serum-based and serum-free cultures in both the ambr and Spinner Flasks.Figure S3. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.ABSTRACTMicrobioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale Spinner Flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the Spinner Flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual Spinner Flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency.
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process development of human multipotent stromal cell microcarrier culture using an automated high throughput microbioreactor
Biotechnology and Bioengineering, 2017Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin NienowAbstract:Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale Spinner Flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the Spinner Flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual Spinner Flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency.
Xuegang Yuan - One of the best experts on this subject based on the ideXlab platform.
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agitation in a microcarrier based Spinner Flask bioreactor modulates homeostasis of human mesenchymal stem cells
Biochemical Engineering Journal, 2021Co-Authors: Richard Jeske, Shaquille Lewis, Angchen Tsai, Kevin Sanders, Chang Liu, Xuegang YuanAbstract:Human mesenchymal stem cells (hMSCs) are well known in cell therapy due to their secretion of trophic factors, multipotent differentiation potential, and ability for self-renewal. As a result, the number of clinical trials has been steadily increasing over the last decade highlighting the need for in vitro systems capable of producing large quantities of cells to meet growing demands. However, hMSCs are highly sensitive to microenvironment conditions, including shear stress caused by dynamic bioreactor systems, and can lead to alteration of cellular homeostasis. In this study, hMSCs were expanded on microcarriers within a 125 mL Spinner Flask bioreactor system. Our results demonstrate a three-fold expansion over seven days. Furthermore, our results show that culturing hMSCs in the microcarrier-based suspension bioreactor (compared to static planar culture) results in smaller cell size and higher levels of reactive oxidative species (ROS) and ROS regulator Sirtuin-3, which have implications on the nicotinamide adenine dinucleotide metabolic pathway and metabolic homeostasis. In addition, hMSCs in the bioreactor showed the increased Prostaglandin E2 secretion as well as reduced the Indoleamine-pyrrole 2,3-dioxygenase secretion upon stimulus with interferon gamma. The results of this study provide understanding of potential hMSC physiology alterations impacted by bioreactor microenvironment during scalable production of hMSCs for biomanufacturing and clinical trials.
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aggregation of culture expanded human mesenchymal stem cells in microcarrier based bioreactor
Biochemical Engineering Journal, 2018Co-Authors: Xuegang Yuan, Angchen Tsai, Iain K Farrance, Jon A RowleyAbstract:Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both Spinner Flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell®. The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.
Qasim A. Rafiq - One of the best experts on this subject based on the ideXlab platform.
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Supplementary figures for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"
2018Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin Nienow, David Williams, Christopher HewittAbstract:Supplementary information files for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"Figure S1. Growth kinetics of hMSCs donor 2 cells using serum-free (SFM) and fetal bovine serum (FBS)-based media in both the ambr15 and Spinner Flasks with data showing the viable cell density.Figure S2. Nutrient and metabolite flux for hMSC donor 1 cells expanded on microcarriers in the serum-based and serum-free cultures in both the ambr and Spinner Flasks.Figure S3. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.ABSTRACTMicrobioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale Spinner Flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the Spinner Flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual Spinner Flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency.
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process development of human multipotent stromal cell microcarrier culture using an automated high throughput microbioreactor
Biotechnology and Bioengineering, 2017Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin NienowAbstract:Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale Spinner Flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the Spinner Flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual Spinner Flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency.
Sara M Badenes - One of the best experts on this subject based on the ideXlab platform.
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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, Carlos A V Rodrigues, Simone Haupt, Oliver Brüstle, 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.
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defined essential 8 medium and vitronectin efficiently support scalable xeno free expansion of human induced pluripotent stem cells in stirred microcarrier culture systems
PLOS ONE, 2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL Spinner Flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in Spinner Flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
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hiPS cell expansion in E8 medium, in a 50 mL Spinner Flask.
2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:EDTA clump inoculation was performed using 55,000 cells/cm2 and a continuous agitation at 44 rpm (optimal values obtained solving the quadratic model). (A) Total cell numbers during the 10 days expansion. Maximum cell yield obtained solving the quadratic model is represented by the black line. Error bars represent SEM (standard error of the mean) of duplicate samples. (B) Immunostaining of hiPS cells cultured on microcarriers in the Spinner Flask with E8 medium at day 10. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm. (C) Immunostaining of hiPS cells harvested from microcarriers after 10 days expansion in Spinner Flask and re-plated on GP. Cells were stained after 3 days for intracellular pluripotency markers NANOG, OCT4 and SOX2, and nuclei counterstained with DAPI; and for surface markers SSEA4 and TRA-1-60. Scale bars– 100 μm. (D) Flow cytometry analysis of hiPS cells harvested from microcarriers after 10 days expansion in Spinner Flask. Cells were stained for OCT4 and NANOG. (E) mRNA was isolated from hiPSC at day 0 and at the end of the Spinner Flask culture (day 12) on microcarriers, and the undifferentiated hiPSC marker transcripts (OCT4 and NANOG) were analysed by RT-PCR. (F) Quantitative RT-PCR analysis of spontaneous differentiated EB of hiPSC cultured in Spinner Flask. The relative expression of each gene was measured against the same gene prior to differentiation. (G) Immunostaining showing the formation of cells expressing SOX17 (endoderm), TUJ1 (ectoderm) and α-SMA (mesoderm) after EB formation and spontaneous differentiation assay with hiPSC cultured in Spinner Flask. Nuclei were counterstained with DAPI. Scale bar: 50 μm.
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Scaling-up hiPS cell expansion in E8 medium to a 50 mL Spinner Flask, under dynamic conditions.
2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:(A) The procedure consists in 4 steps: a. EDTA+ROCKi inoculation strategy into 20 g/L VtnM; b. Attachment is performed in half of the total volume in static conditions for 24 h (in the presence of ROCKi), following by intermittent stirring overnight to allow cell- microcarrier interaction; c. Expansion is performed with a specific continuous agitation with 80% E8 medium change everyday; and d. At the end of the culture, cell pluripotency and differentiation potential are evaluated. Quadratic model relating initial cell density and agitation speed with cell yield during Spinner Flask culture of human iPS cells: 3D representation (B) and 2D heat map (C) are shown. For initial cell density: 30 000 cells/cm2 (−1 level) ≤ Initial Cell Density ≤ 70 000 cells/cm2 (1 level); for agitation speed: 30 rpm (−1 level) ≤ Agitation Speed ≤ 70 rpm (1 level).
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Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems
2016Co-Authors: Sara M Badenes, Maria Margarida Diogo, Tiago G Fernandes, Cláudia S. M. Cordeiro, Shayne Boucher, David Kuninger, Mohan C. Vemuri, Joaquim M. S. CabralAbstract:Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL Spinner Flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in Spinner Flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
