Purification and Differentiation of Human Adipose-Derived Stem Cells through Silk Screen/PLGA Hybrid Membranes by Membrane Filtration Method

• Main Authors: Li-Yu Chen, 陳俐伃
• Other Authors: Akon Higuchi
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/50498965500078257344

碩士 === 國立中央大學 === 化學工程與材料工程學系 === 101 === Human adipose-derived stem cells (hADSC) represent one of the most promising cell candidates in the field of regenerative medicine, it can easily be extracted in large amount compared to bone marrow stem cells and also exhibits a higher proliferation rate in the medium. Furthermore, hADSCs also have a high differentiation capability into mesoderm (muscle, bone, and cartilage) and ectoderm (nerves and epidermal). The cultivation of adipose tissue cells generates hADSCs with contamination of several other types of cells. Therefore, it is necessary to purify hADSCs before using hADSCs for clinical applications. Purification of hADSCs via the cell culture process requires 5-12 days by using the conventional culture method. In this study, we developed a membrane filtration method to purify hADSCs using novel membranes where the operation time is less than 30 min to purify hADSCs from adipose tissue solution. We prepared silk screen/PLGA hybrid membranes (scaffolds) by a freeze drying method where silk screens (170 mesh size) are natural and readily biodegradable protein fibers to be used for reinforcement of the membranes. PLGA has biodegradability and biocompatibility to be used to generate a sponge pore morphology of the membranes. The average pore size of the silk screen/PLGA hybrid membranes was measured to be 3.5-7µm from scanning electron microscopy. After the preparation of home-made silk screen/PLGA hybrid membranes, we investigated the purification of hADSCs from adipose tissue solution (adipose tissue-derived stromal vascular fraction [SVF]) having different cell density by the membrane filtration method and evaluated the purification efficiency of hADSCs. The mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 expressed by hADSCs were less than 10% in the adipose tissue solution (SVF), whereas the MSC markers in the permeate solution were found to be 30-50%, indicating hADSCs were concentrated after permeation through the silk screen/PLGA hybrid membranes, when 9 ml of the adipose tissue solution having 1×106 cells/ml was permeated through the membranes. The MSC markers of the cells after 12 days of culture of the adipose tissue solution (hADSCs purified by the conventional culture method) were found to be 60-80%. The efficiency of hADSC purification in the permeation solution through the silk screen/PLGA hybrid membranes analyzed by MSC markers depended on the cell density of the adipose tissue solution. Currently 1×106 cells/ml was the optimal cell density compared to 2×106 cells/ml or 4×106 cells/ml when 9 ml of adipose tissue solution was used. It was demonstrated that more than two fold higher osteogenic gene expression, Alizarin red staining, and von Kossa staining was observed in the permeate solution compared to the adipose tissue solution (SVF) when the cells were cultured in osteogenic induction medium for 28 days. Therefore, the hADSCs were purified in the permeation solution and demonstrated a superior capacity for osteogenic differentiation than the cells in the adipose tissue solution (SVF). The polyurethane foaming membranes having pore size of 11 µm could not purify hADSCs in the permeate solution. This result indicates that the pore size and membrane material are important factors to purify the hADSCs by the membrane filtration method. It is concluded that the hADSCs can be easily isolated through the permeation through the silk screen/PLGA hybrid membranes, whereas non-hADSCs are blocked by the sieving effect of the membrane pore size and/or adhered on the membranes.