混成膜法從脂肪組織中分離高純度脂肪誘導幹細胞之研究;A hybrid-membrane migration method to isolate high-purity adipose-derived stem cells from fat tissue

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题名:	混成膜法從脂肪組織中分離高純度脂肪誘導幹細胞之研究;A hybrid-membrane migration method to isolate high-purity adipose-derived stem cells from fat tissue
作者:	王靖棠;WANG,CHING-TANG
贡献者:	化學工程與材料工程學系
关键词:	脂肪誘導幹細胞;純化;多能性;adipose-derived stem cells;purification;pluripotency
日期:	2015-07-27
上传时间:	2015-09-23 10:47:33 (UTC+8)
出版者:	國立中央大學
摘要:	對於再生醫療而言，人類脂肪誘導幹細胞(ADSCs)是許多富有前景的細胞來源之一，因為它們不只能以大量且以最少的小型手術流程取得，例如:抽脂手術。不像人類胚胎幹細胞(hESCs)，需破壞胚胎，成體幹細胞並不會有道德上的爭議。對人類胚胎幹細胞(hESCs)和人類誘導多能性幹細胞(hiPSCs)而言，培養成體幹細胞只需相對低的花費。此外，人類胚胎幹細胞和人類誘導多能性幹細胞的無異種培養非常困難，且(或者)需要花費相當龐大的資金，因此人類脂肪誘導幹細胞比起人類胚胎幹細胞和人類脂肪誘導幹細胞被視為更有潛力的幹細胞來源。但是，人類脂肪誘導幹細胞的使用還是有相當的侷限性，因為比起人類胚胎幹細胞和人類誘導多能性幹細胞，它們的多能性和分化能力非常低，因此如何維持或增加人類脂肪誘導幹細胞的多能性變得更加重要。在這個研究中，脂肪組織被膠原蛋白酶消化，緊接著進行離心，隨後我們可以得到間質血管部分(SVF)。間質血管部分的細胞不但由多種細胞組成且雜亂，造成各式各樣的基因型態，且這些細胞具有非常高的多能性。雖然人類脂肪誘導幹細胞可以藉由培養在細胞培養盤上去純化，但是他們的多能性隨著時間顯著的下降。在此研究，為了改善人類脂肪誘導幹細胞的臨床應用，相對於傳統的培養方法，將花費更少時間建立混成薄膜法。除此之外，在此研究中，從間質血管部分細胞溶液中純化的人類脂肪誘導幹細胞，具有極高的純度和多能性。間質血管部分細胞溶液被過濾於8m到25m孔徑的薄膜且這些薄膜被培養在細胞培養液中15到18天，從10% PLGA/silk sscreen 混成薄膜中爬出的細胞包含了極高的表現間葉幹細胞表面抗原細胞的百分比，且這些細胞較於經過傳統培養法培養的細胞，表現了更高的多能性基因(Oct4, Sox2, Klf4, Nanog)。值得注意的是，在10% PLGA/silk screen 混成薄膜過濾的permeate 和recovery 溶液中，間質血管部分中細胞的多能性可以被維持，甚至加強。此外，烷鍊磷酸酶活性(ALP)在換誘導骨分化培養液後的14天後被分析，Alizarin red staining, von Kossa staining則是在換誘導骨分化培養液後的28天被檢測。由此可證被10% PLGA/silk screen 混成薄膜過濾之permeate和recovery 溶液中的細胞，較於primary 細胞和經由傳統培養法培養的細胞，具有較高的骨分化能力。;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.
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