Attachment and growth of human embryonic stem cells on microcarriers.
The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.
Phillips
Blaine Wesley
BW
Institute of Medical Biology, 8A Biomedical Grove, #06-06, Singapore 138648, Singapore.
Horne
Rachel
R
Lay
Tan Suat
TS
Rust
William Lathrop
WL
Teck
Tan Thong
TT
Crook
Jeremy Micah
JM
D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D023822:Tissue Engineering / Q000379:methods*