A Hybrid-Membrane Migration Method to Isolate Adipose-Derived Stem Cells from Fat Tissues Through Membranes Coated with Extracellular Matrices

• Main Authors: Yu-Chun Lee, 李雨駿
• Other Authors: Akon Higuchi
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/5m95dy

碩士 === 國立中央大學 === 化學工程與材料工程學系 === 107 === Human adipose-derived stem cells, hADSCs, can be obtained by isolation from fat tissue, which is currently a more practical source of stem cells than human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Currently, several clinical trials use hADSCs, whereas only a few clinical trials have been performed using hiPSCs and hESCs. However, hADSCs are known to show heterogeneous characteristics and contain different pluripotency and differentiation abilities. Therefore, it is expected that the stem cell characteristics, pluripotency, and differentiation abilities should be different for hADSCs isolated by different isolation methods. hADSCs are typically isolated by cell culture of stromal vascular fraction (SVF, primary hADSC solution) where the SVF solution can be obtained by collagenase digestion of fat tissues followed by centrifugation. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. It is innovated that the membrane migration method through Nylon mesh filter purifies hADSCs from a fat tissue solution with extremely high purity and pluripotency in my laboratory. A primary fat-tissue solution was permeated through the porous membranes (e.g., Nylon mesh) with a pore size from 8 to 25 μm, and the membranes were incubated in cell culture medium for 15-18 days. In this study, I developrd a new membrane migration method using Nylon mesh membranes having optimal pore sizes, 11 and 20 μm, and PLGA/silk membranes where optimal extracellular matrix (ECM) was coated on the membranes, which could purify hADSCs. The isolated hADSCs are expected to have high pluripotency and high differentiation ability into chondrocytes, osteoblasts and adipocytes. hADSCs were isolated from adipose tissue by the membrane migration method where different membranes were used, e.g., (a) Nylon mesh and PLGA (poly (lactic-co-glycolic acid))/silk screen membrane, (b) Nylon mesh and PLGA/silk screen membrane coated with collagen type I, (c) Nylon mesh and PLGA/silk screen membrane coated with human recombinant-vitronectin, (d) Nylon mesh and PLGA/silk screen membrane coated with human fibronectin. Collagen type I is xeno-containing materials, whereas another extracellular matrices (ECMs) were xeno-free materials. The hADSCs that migrated from the membranes kept an extremely high percentage (e.g., >98%) expression of mesenchymal stem cell markers (CD44, CD73, and CD90) and showed almost one order of magnitude higher expression of some pluripotency genes (Oct4, Sox2, and Nanog) compared with cells isolated using the conventional culture method.