Molecular Design of Nanosegments on Cell Culture Biomaterials for Proliferation of Human Embryonic Stem Cells

• Main Authors: Yeh-Chia Tseng, 曾也家
• Other Authors: Akon Higuchi
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/4hq4mv

碩士 === 國立中央大學 === 化學工程與材料工程學系 === 106 === Stem cells are an attractive prospect for regenerative medicine and tissue engineering. The dishes coated with Matrigel, Synthemax II, Laminin 511, Laminin 521, and Cellstart are typically used for the cell culture substrates for human embryonic stem cells (hESCs). These coating materials were based on extracellular matrix (ECM), which were secreted from cells. Our previous study showed that hESCs can be cultured on polyvinylalcohol-co-itaconic acid (PVA-IA) hydrogels conjugated with higher concentration of oligovitronectin (>500 μg/mL) as hESC culture substrates. In order to solve the reason of the higher concentration usage of oligovitronectin conjugated on PVA-IA hydrogels, we designed another PVA-IA hydrogels conjugated with oligovitronectin for hESCs culture substrates using crosslinker of PEG-AEAC (Poly(ethylene glycol)-2-aminoethyl ether acetic acid) and we evaluated hESC attachment and pluripotency cultured on these hydrogels. The PVA-IA hydrogels were prepared by coating aqueous PVA-IA solution on the tissue culture polystyrene (TCPS) dishes and were crosslinked with glutaraldehyde to control hydrogel stiffness. Twice activation was performed to conjugate oligovitronectin on PVA-IA hydrogels via PEG-AEAC crosslinker by the peptide bonding reaction between carboxylic group and amino group. We investigated the effect of long crosslinking agent of PEG-AEAC on the culture of hESCs on PVA-IA hydrogels grafted with oligovitronectin via PEG-AEAC. We have also tried same reaction but with different length of PEG-AEAC to evaluate the length effect on the culture of hESCs in the dishes. After the long term culture of hESCs on the PVA-IA hydrogels (more than 10 passages), we evaluated pluripotency and differentiation ability of hESCs. Moreover, we design several oligopeptide sequence with different length of joint segment to investigate which oligopeptide-grafted hydrogel molecular design with optimal length of joint segments supported the proliferation of hESCs while hESCs maintained their pluripotency and differentiation ability into cardiomyocytes under xeno-free cell culture conditions. In addition, we designed the small molecules of oligopeptides for hESC culture, which could be coated directly to the TCPS dishes in order to decrease the concentration of these small molecules of oligopeptides used and also to provide a much more convenient way to prepare hESC culture dishes.