title: Expansion of human embryonic stem cells on cellulose microcarriers.
abstract: This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrix-coated two-dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three-dimensional suspension culture using Matrigel-coated cellulose microcarriers. hESCs from a tissue culture plate can be seeded directly onto the microcarriers; hESC microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100-ml spinner flask is also described. Long-term culture of hESC on the microcarriers maintains typical pluripotent markers (OCT-4, Tra-1-60, and SSEA-4) and stable karyotype. Spontaneous differentiations of microcarrier-maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells.
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Centros, Singapore.
authors.forename: Allen K
authors.initials: AK
authors.lastname: Chen
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.forename: Andre B H
authors.initials: AB
authors.lastname: Choo
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.forename: Steve K W
authors.initials: SK
authors.lastname: Oh
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002482:Cellulose / Q000378:metabolism* / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D008858:Microscopy, Phase-Contrast; D008863:Microspheres; D011509:Proteoglycans / Q000494:pharmacology
chemical_list: D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D002482:Cellulose; D003094:Collagen