| Summary: | 碩士 === 國立中央大學 === 化學工程與材料工程學系 === 103 === Human adipose-derived stem cells (hADSCs) are one of the promising cell sources in regenerative medicine because they can be obtained in abundant quantity and harvested by minimally invasive procedure such as liposuction. Unlike embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), adult stem cells do not generate the ethical concerns that accompany hESCs and are cultivated in relatively low cost. Furthermore, the xeno-free culture of hESCs and hiPSCs is difficult to achieve and/or are extremely costly. Therefore, hADSCs are considered to be a more attractive source of stem cells than hESCs and hiPSCs. However, hADSCs are limited for application because of their low pluripotency and differentiation ability compared with hESCs and hiPSCs. It is a critical issue how to maintain or increase the pluripotency of hADSCs. The adipose tissue was treated with collagenase to digest, followed by centrifugation in this study. Subsequently, stromal vascular fraction (SVF) was obtained. Cells in SVF were not only heterogeneous and contain many different types of cells leading to various genotypes but also held high pluripotency. Although hADSCs could be purified by culturing on tissue culture dishes, the pluripotency significantly decreased with time. In order to improve the clinical application of hADSCs, the hybrid membrane method that takes shorter time to purify hADSCs (i.e. less than 30 min) than conventional culture method (i.e. 5-12 days) has developed in this study where hADSCs are purified from SVF cell solution with extremely high purity and pluripotency. A SVF cell solution was permeated through the porous membranes with a pore size from 8 m to 25 m, and membranes were incubated in cell culture medium for 15-18 days. The hADSCs migrated from 10% PLGA/silk screens hybrid membranes contain an extremely high percentage (e.g. >95%) of cells positive for mesenchymal stem cell markers and express higher pluripotent genes (Oct4, Sox2, Klf4 and Nanog) than the cells after culturing by conventional culture method. It is noteworthy that the pluripotency of cells in SVF could be kept or even enhanced in permeate and recovery solution filtered by 10% PLGA/silk screen hybrid membranes. Besides, the alkaline phosphatase activity (ALP) was analyzed after 14 days. Alizarin red staining, von Kossa staining were observed after 28 days. It was indicated that cells in permeate and recovery solution purified by10% PLGA/silk screen hybrid membranes had higher ability to differentiate into osteoblasts compared with primary cells and cells after culturing by conventional culture method.
|
|---|
