Continuous harvest system for expansion of human ES and iPS cells cultured on biomaterials immobilized with thermoresponsive nanosegments
| Autor: | Jia-Sin Yang, 楊佳馨 |
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| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 Human pluripotent stem cells (hPSCs) are an attractive prospect for regenerative medicine and tissue engineering. Typical stem cell cultivation methods are still based on batch-type culture, which is laborious and expensive. In this study, It is developed that the partial detachment stem cell culture system by using thermoresponsive nanosegments, a polymer having low critical solution temperature (LCST), which were coated on the surface of cell culture dishes for continuous stem cell harvest. This method enables cell aggregates or cell sheets to be obtained in culture medium without applying an enzymatic digestion. The themoresponsive nanobrush surfaces are composed by three copolymers having polystyrene, which are (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm, (b) biocompatible polyethylene glycol methacrylate (PEGMA) and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin) could be conjugated via carboxylic acid of PAA. P[St-NIPAAm] (poly[styrene-co-N-isopropylacrylamide]), and P[St-PEGMA] (poly[styrene-co-polyethylene glycol methacrylate]) were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization. In this study, I successfully cultured human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) on the thermoresponsive surface and continuously harvested for seven and ten cycles respectively. The hESCs and hiPSCs have high pluripotency and differentiation ability by immunostaining analysis, embryoid body (EB) formation and teratoma formation after seven cycles. In addition, over 60% of hESCs and hiPSCs could be detached from the surface after 30 minutes of low temperature incubation (7-9°C). This continuous culture system prevents hESCs and hiPSCs from enzymatic digestion damages and allows cells maintain their pluripotency on the surface. The continuous harvest of stem cells should downsize the equipment requirements for stem cell culture and simplify the culture process. Moreover, This cultivation method also can be scaled up cell numbers by shifting 2D to a novel 3D culture system, which will be a great benefit to its clinical application in regenerative medicine. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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