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

Fernandes, Aline Marie; Marinho, Paulo A.; Sartore, Rafaela C.; Paulsen, Bruna S.; Mariante, Rafael M.; Castilho, Leda R.; Rehen, Stevens K.

doi:10.1590/s0100-879x2009000600007

Cited by 108 publications

(54 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Nie et al 28 and Lock and Tzanakakis 29 achieved lower maximum cell concentrations (1.1 Â 10 6 and 2 Â 10 6 cells/mL, respectively), even though in both studies commercial collagencoated microcarriers were treated with Matrigel prior to cell inoculation. Fernandes et al 30 were the only authors to report human ES cell growth on commercial microcarriers without any further coating, but the maximum human ES cell density achieved was 1.5 Â 10 6 cells/mL, as compared to $8 Â 10 6 cells/mL achieved with murine ES cells in the present study.…”

Section: Discussioncontrasting

confidence: 58%

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

Marinho

Fernandes

Cruz

et al. 2009

Biotechnology Progress

View full text Add to dashboard Cite

The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers.

show abstract

Section: Discussioncontrasting

confidence: 58%

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

Marinho

Fernandes

Cruz

et al. 2009

Biotechnology Progress

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

“…hPSCs have been successfully expanded and differentiated to definitive endoderm, cardiomyocytes, and neural progenitor cells 6,8,9 in stirred-suspension microcarrier vessels. Despite success in cultivating hPSCs in microcarrier SSBs, the beads utilized in most studies are coated with animal-derived matrices such as Matrigel 6,[9][10][11] or collagen 12 barring the applicability of this culture method from clinical settings. Similarly, the proposed use of rodent and human feeder cells for coating microcarriers 10,13 raises issues with the downstream separation of multiple cell types and beads in addition to the expression of nonhuman immunogens by hPSC derivatives.…”

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

View full text Add to dashboard Cite

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.

show abstract

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

Cited by 108 publications

References 41 publications

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

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

Contact Info

Product

Resources

About