Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Fernandes, Aline Marie; Fernandes, Tiago G.; Diogo, Maria Margarida; Silva, C. Lobato da; Henrique, Domingos; Cabral, Joaquim M. S.

doi:10.1016/j.jbiotec.2007.05.031

Cited by 145 publications

(115 citation statements)

References 33 publications

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“…b The cost given for FGF-2 was the market price in Brazil for the imported product used in this study. Stem cell therapies require large amounts of cells (35,36) and several protocols have been proposed in recent years for the expansion of adult stem cell, (8,10,12,13,19,32,(37)(38)(39)(40). The use of hES cells in cell therapy implies that a considerable number of cells are available for cell differentiation and subsequent transplantation.…”

Section: Resultsmentioning

confidence: 99%

“…Although microcarriers have been described for culturing stem cells from both animals (12,19) and humans (20)(21)(22), to our knowledge, this is the first description of the use of microcarriers, without coating with mouse fibroblasts or Matrigel™, that successfully scaled-up human embryonic stem cell production in a stirred culture system. The cultivation method presented here will facilitate expansion of hES cells needed for cell therapy, research and industrial applications.…”

Section: Introductionmentioning

confidence: 99%

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Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Fernandes

Marinho

Sartore

et al. 2009

Braz J Med Biol Res

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106

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Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10 8 karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder-and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Fernandes

Marinho

Sartore

et al. 2009

Braz J Med Biol Res

Self Cite

106

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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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“…In general, bone tissue engineering consists of three basic elements, which is cells, growth factors and bone scaffolds [7,8]. As a framework for bone tissue regeneration, scaffold materials directly affect the biological properties of seed cells, such as its survival, migration, proliferation and metabolism.…”

Section: Function Of Bone Scaffoldmentioning

confidence: 99%

Application of 3D printing technology in bone tissue engineering

Wang

Wei

et al. 2018

Bio-des. Manuf.

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Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Cited by 145 publications

References 33 publications

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

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

Application of 3D printing technology in bone tissue engineering

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