Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control

Serra, Margarida; Brito, Catarina; Sousa, Marcos F. Q.; Jensen, Janne; Tostoes, Rui; Clemente, João J.; Strehl, Raimund; Hyllner, Johan; Carrondo, Manuel J.T.; Alves, Paula M.

doi:10.1016/j.jbiotec.2010.06.015

Cited by 133 publications

(85 citation statements)

References 25 publications

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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: 84%

“…hESC-C propagation was performed as described by Serra et al [20]. Extracellular components were also evaluated for the expansion of hESC-C: Matrigel Technologies) and MEF-CM [7], both supplemented with 10 mM Rock inhibitor (Calbiochem; EMD Millipore, Billerica, MA, http://www.emdmillipore. com).…”

Section: Human Embryonic Stem Cell Culturementioning

confidence: 99%

“…Concerning suspension culture systems, different strategies for expansion and/or differentiation of hPSCs have been explored, including the cultivation of cells as aggregates [6] or immobilized on microcarriers [7] or cultivation using hydrogels [8]. With well-designed robotic automated systems or environmentally controlled bioreactors (e.g., stirred-tank bioreactors), the production of clinically relevant quantities of hPSCs will hopefully be achieved in the near future.…”

Section: Introductionmentioning

confidence: 99%

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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: 84%

Section: Human Embryonic Stem Cell Culturementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Commercially available microcarriers-such as Cultisphere S, Cytodex 3, Solohill carriers and Hillex II have been widely used for the expansion of ESC, with Cytodex 3 being the most common. The speed used for mouse ESC, varies between 40 and 70 RPM (Fok and Zandstra 2005;Abranches et al 2007;Alfred et al 2011;Fernandes et al 2007;Marinho et al 2010;Storm et al 2010;Tielens et al 2007), while the range for human ESC lies within 24-80 RPM (Storm et al 2010;Chen et al 2011;Fernandes et al 2009;Kehoe et al 2010;Leung et al 2011;Lock and Tzanakakis 2009;Marinho et al 2013;Nie et al 2009;Oh et al 2009;Phillips et al 2008;Serra et al 2010). iPSC are by nature, delicate, making their large scale culture in spinner flasks using microcarriers a much more difficult proposition.…”

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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“…The atmospheric oxygen level, however, may cause the oxidative stress of the cells, thereby decreasing their viability [43]. It has been reported that MSCs expand more efficiently at 2 % O 2 [44,45], whereas hESCs grow better at 20 to 30 % oxygen levels [46]. Moreover, Foldager et al [47] demonstrated that oxygen tension (pO 2 ) as low as 9 % in a 3D culture stimulates the expression of genes that encode chondrogenic markers (Sox9, aggrecan and Col2a1).…”

Section: Culture Conditionsmentioning

confidence: 99%

Application of cell and biomaterial-based tissue engineering methods in the treatment of cartilage, menisci and ligament injuries

Trzeciak

Richter

Suchorska

et al. 2016

International Orthopaedics (SICOT)

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Over 20 years ago it was realized that the traditional methods of the treatment of injuries to joint components: cartilage, menisci and ligaments, did not give satisfactory results and so there is a need of employing novel, more effective therapeutic techniques. Recent advances in molecular biology, biotechnology and polymer science have led to both the experimental and clinical application of various cell types, adapting their culture conditions in order to ensure a directed differentiation of the cells into a desired cell type, and employing non-toxic and non-immunogenic biomaterial in the treatment of knee joint injuries. In the present review the current state of knowledge regarding novel cell sources, in vitro conditions of cell culture and major important biomaterials, both natural and synthetic, used in cartilage, meniscus and ligament repair by tissue engineering techniques are described, and the assets and drawbacks of their clinical application are critically evaluated.

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Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control

Cited by 133 publications

References 25 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

Application of cell and biomaterial-based tissue engineering methods in the treatment of cartilage, menisci and ligament injuries

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