Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

Abstract

in Wiley InterScience (www.interscience.wiley.com).As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. V

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…However, methodologies such as preservation on microcarriers might provide the advantages of freezing adherent cells at higher densities that are not possible on flat surfaces. This is what has been described by Nie et al, who used Cytodex 3 microcarriers to cryopreserve adherent hESCs (Nie et al, 2009). These microcarriers consisted of a thin layer of denatured collagen covalently coupled to a matrix of cross-linked dextran.…”

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

“…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.…”

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

“…An attractive approach for scaling up production is to move cell culture from 2D to 3D (9,17), and accordingly several 3D suspension systems have been probed for hPSCs production: cell aggregates (18)(19)(20)(21), cells on microcarriers (22,23), and cells in alginate microencapsulates (24) (SI Appendix, Table S1). Although these approaches have some attractive aspects, they also highlight significant challenges for 3D hPSC culture (9) (SI Appendix, Table S1) including the following: (i) the use of components from human or animal tissue (e.g., Matrigel, serum, and/ or albumin), which limit reproducibility and/or scalability, pose risks for pathogen and immunogen transfer (18)(19)(20)(21)(22)(23)(24), and are thus problematic for good manufacturing practice (GMP) cell production (25); (ii) substantial cell agglomeration that can in some cases lead to differentiation and/or death (22,23); (iii) shear forces in agitated cultures that can compromise cell viability (18)(19)(20)(21)(22)(23); (iv) limited cell expansion rates and cell yields per volume (18)(19)(20)(21)(22)(23)(24); and (v) unclear potential for long-term serial expansion. As an example, in a recent culture of hPSCs within alginate hydrogel microspheres in mouse embryonic fibroblast conditioned medium, 5% of the encapsulated single hPSCs remained viable after 7 d, and an ∼10-to 20-fold expansion to a peak density of 3 × 10 6 cells per mL occurred after 20 d (24).…”