Translating Human Embryonic Stem Cells from 2-Dimensional to 3-Dimensional Cultures in a Defined Medium on Laminin- and Vitronectin-Coated Surfaces
| Autor: | Allen Chen, Shaul Reuveny, Jian Li, Steve Oh, Boon Chin Heng, Simon M. Cool, William R. Birch |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Surface Properties
Karyotype Cell Culture Techniques Mice SCID Culture Media Serum-Free Mice chemistry.chemical_compound Adsorption Laminin Cell Adhesion Bioreactor Animals Humans Vitronectin Cells Cultured Embryonic Stem Cells biology business.industry Teratoma Microcarrier Cell Differentiation Neoplasms Experimental Cell Biology Hematology Antigens Differentiation Embryonic stem cell Up-Regulation Biotechnology Chemically defined medium Immobilized Proteins chemistry biology.protein Biophysics Polystyrene business Developmental Biology |
| Zdroj: | Stem Cells and Development. 21:1701-1715 |
| ISSN: | 1557-8534 1547-3287 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|