Efficient expansion of clinical-grade human fibroblasts on microcarriers: cells suitable for ex vivo expansion of clinical-grade hESCs.
Human embryonic stem cells hold considerable potential for cell-based treatments of a variety of degenerative diseases, including diabetes, ischemic heart failure, and Parkinson's disease. However, advancing research to provide clinical-grade product requires scale-up to therapeutic quantities of stem cells and their differentiated progeny. Most human embryonic stem cell culture platforms require direct support by a fibroblast feeder layer or indirect support using fibroblast conditioned medium. Accordingly, large numbers of clinically compliant fibroblasts will be requisite for stem cell production. Published platforms for feeder production are insufficient for stem cell scale-up, being costly to operate and requiring considerable effort to prepare, maintain and harvest. Here we describe the expansion of cGMP-grade, FDA-approved human foreskin fibroblasts using cGMP-grade reagents and polystyrene-based cationic trimethyl ammonium-coated microcarriers in spinner flasks. Fibroblasts attach rapidly to the microcarriers (T(1/2)=75 min), and expand with a maximum doubling time of 22.5h. Importantly, microcarrier-expanded fibroblasts and their conditioned medium support pluripotent stem cell growth through >5 passages, enabling extended self-renewal and expansion while retaining full differentiation potential. In summary, the method described is an economical and cGMP-compliant means of producing human fibroblast cells in support of cGMP human embryonic stem cell culture.
Phillips
Blaine Wesley
BW
Institute of Medical Biology, 8A Biomedical Grove, #05-15 Immunos, Singapore 138648, Singapore. blaine.phillips@imb.a-star.edu.sg
Lim
Ricky Yee Meng
RY
Tan
Thong Teck
TT
Rust
William Lathrop
WL
Crook
Jeremy Micah
JM
D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction