Translating human embryonic stem cells from 2-dimensional to 3-dimensional cultures in a defined medium on laminin- and vitronectin-coated surfaces.
While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Boon Chin
BC
Heng
Jian
J
Li
Allen Kuan-Liang
AK
Chen
Shaul
S
Reuveny
Simon M
SM
Cool
William R
WR
Birch
Steve Kah-Weng
SK
Oh
D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000502:physiology* / Q000637:transplantation; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D059785:Karyotype; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D009374:Neoplasms, Experimental / Q000473:pathology; D013499:Surface Properties; D013724:Teratoma / Q000473:pathology; D015854:Up-Regulation; D019096:Vitronectin / Q000737:chemistry / Q000378:metabolism*
D000943:Antigens, Differentiation; D016895:Culture Media, Serum-Free; D055767:Immobilized Proteins; D007797:Laminin; D019096:Vitronectin