Large-Scale Expansion of Pluripotent Human Embryonic Stem Cells in Stirred-Suspension Bioreactors

Krawetz, Roman; Taiani, Jaymi T.; Liu, Shiying; Meng, Guoliang; Li, Xiangyun; Kallos, Michael S.; Rancourt, Derrick E.

doi:10.1089/ten.tec.2009.0228

Cited by 158 publications

(85 citation statements)

References 31 publications

(36 reference statements)

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“…Because massive numbers of hPSCs may be needed to address biomedical demands, namely, for cell therapy (e.g., 10 9 to 10 10 cells are required for heart or hepatic failure therapies) or drug-discovery pipelines (requiring ∼10 10 cells to screen a 1 million-compound library at once), the use of microcarrier-based bioreactors offers a more cost-effective system for high-production scales than planar technologies, as reported recently [9]. Our group and others have demonstrated successful expansion of hPSCs in stirred-tank bioreactors using different types of microcarriers [7,10,11]. Moreover, to meet regulatory safety concerns, Fan et al engineered xeno-free microcarriers for hPSC propagation in stirred suspension vessels, making the hPSCs more suitable for clinical applications [12].…”

Section: Introductionmentioning

confidence: 99%

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Silva

Rodrigues

Correia

et al. 2015

Stem Cells Translational Medicine

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Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:731-742 SIGNIFICANCEThe clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in welldefined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.

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Section: Introductionmentioning

confidence: 99%

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Silva

Rodrigues

Correia

et al. 2015

Stem Cells Translational Medicine

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“…Spinner flasks are one of the oldest and most widely used reactor systems and have been used extensively for the expansion of mouse as well as human ESC. Both mouse and human ESC have been expanded as aggregates in spinner flasks (Fok and Zandstra 2005;Cormier et al 2006;Abbasalizadeh et al 2012;Krawetz et al 2010;Steiner et al 2010). Extensive study in the field of large scale expansion of ES cells has given rise to novel and better systems for these cells but the field of dynamic culture of iPS cells has just started.…”

Section: Introductionmentioning

confidence: 99%

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

et al. 2014

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In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

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“…Stirred-suspension bioreactors (SSBs) [1][2][3] are an appealing culture modality for hPSC propagation and commitment given their scalability, robustly controlled operation, and widespread use in commercial production. hPSCs in these reactors can be grown as aggregates, 1,4 after encapsulation 5 or on microcarriers. 6,7 In particular, microcarrier systems afford high surface-to-volume ratio, homogenous environment, simple operation and continuous monitoring, and control of the culture environment.…”

mentioning

confidence: 99%

Facile Engineering of Xeno-Free Microcarriers for the Scalable Cultivation of Human Pluripotent Stem Cells in Stirred Suspension

Fan¹,

Hsiung²,

Chen³

et al. 2013

Tissue Engineering Part A

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A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

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Large-Scale Expansion of Pluripotent Human Embryonic Stem Cells in Stirred-Suspension Bioreactors

Cited by 158 publications

References 31 publications

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

Facile Engineering of Xeno-Free Microcarriers for the Scalable Cultivation of Human Pluripotent Stem Cells in Stirred Suspension

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