脂肪幹細胞可分化為許多種細胞，如脂肪細胞、成骨母細胞、軟骨細胞等間葉細胞，也因如此、脂肪幹細胞在組織再生工程學上被視為一種極具潛力的細胞來源。現今的幹細胞分離技術中，膜分離技術乃是一種相當簡便、快速、易保持細胞活性的方法，此項研究意圖針對小鼠脂肪幹細胞的初代細胞溶液做出進一步的膜純化技術。我們準備了具有11微米孔徑簡單結構的尼龍過濾膜及12 微米複雜結構的聚氨酯膜進行過濾。此外，我們也準備了兩種過濾方法來進行膜純化法，分別是槽式過濾及傾注式過濾，不同點在於槽式過濾中存在一適當壓力使細胞通過過濾膜，而傾注式過濾則有一開孔以宣洩壓力，不會推擠細胞通過過濾膜。在初代細胞溶液通過過濾膜後稱為過濾液，而在相反方向注入培養基以取得未能通過過濾膜的細胞的溶液則稱為回收液。在此項研究中使用流式細胞儀及分化實驗來比較不同過濾條件下其過濾液及回收液中脂肪幹細胞之純度。其結果顯示，11微米的尼龍過濾膜無法有效分離脂肪幹細胞，即使增加至五層尼龍過濾膜亦無法有效分離脂肪幹細胞。而12微米孔徑的聚氨酯膜在搭配傾注式過濾法的條件下，其回收液可取得最高純度的脂肪幹細胞溶液。但在經過一代的增殖培養後，其脂肪幹細胞之表面抗體的表現量及分化比率並無顯著差異。 另一研究則討論細胞外間質對人類脂肪細胞之影響，有許多研究顯示細胞外間質如膠原蛋白會對幹細胞之分化、增殖能力產生影響，且幹細胞於繼代後可能失去其多能性。故此研究將三種典型之細胞外間質塗佈於培養盤上，並將第三代的人類脂肪幹細胞培養於其上，以QRT-PCR及分化實驗測試其多能性基因的表現量及分化能力。結果顯示於膠原蛋白塗佈條件下培養之脂肪幹細胞具有較強之多能性基因表現，且同樣具有較強之脂肪細胞分化能力，此兩項檢測應可得到同樣的結論，膠原蛋白可保持較多的人類脂肪幹細胞之多能性。 Adipose tissue-derived stromal cells (ADSCs) can be induced into several mesenchymal cell lineages such as adipocytes, osteoblasts, and chondrocytes, and other germ layer cell lineages. Therefore, ADSCs are considered as a promising stem cell source for tissue engineering repair. Purification and isolation of specific mesenchymal stem cells are necessary to obtain adequate stem cells for use in clinical applications. The membrane filtration method is a good candidate for the purification of ADSCs because it is simple, inexpensive and because it is easy to maintain sterility during the filtration process. In this study, we developed a novel method to purify primary mouse ADSCs (mADSCs) by the membrane filtration method for rapid use. We investigated two filtration methods to purify mADSC, (i.e., batch-type and perfusion-type). Main differences between these two filtration methods are cell flow direction to the membranes. Nylon mesh filters having 11 μm of pore size and polyurethane foaming (PU) membranes having 12 μm of pore size were used as the membranes for the filtration method. After filtration of adipose-tissue digestion solution, the recovery solution was permeated through the membranes to recovery the adhered cells on the membranes. The cells in permeate and recovery solutions were analyzed by flow cytometry from ADSC surface marker (CD73 and CD90) and Nile-red staining. The differentiation capability of cells separated by the membrane filtration method was also evaluated to confirm the enriched mADSCs. The results indicate that the Nylon mesh filter of 11 μm can not effectively separate mADSCs because of the simple membrane structure, even though we increased sheet numbers of Nylon mesh filters. We found that cells separated through PU membranes by the perfusion method showed twice higher MSCs surface marker expression and differentiation capability. It is concluded that the recovery solution by perfusion-type with polyurethane foaming membranes is the best condition to purify mADSC from mouse adipose tissue by the membrane filtration method. The micro-environment of stem cells plays an important role on gene expression and differentiation. Therefore, the effect of interaction between human ADSCs (hADSCs) and extracellular matrix (ECM)-coated dishes on the expression and maintenance of pluripotent genes (Oct-4 and Sox-2) and differentiation ability was investigated from qRT-PCR analysis and differentiation experiments. The decrease of pluripotent gene expressions was found in hADSCs from passage 2 to passage 3, which were cultured on TCPS. It seems that the pluripotant gene expression of hADSCs is gradually losing during culture. The hADSCs cultured on collagen-coated dishes showed the highest expression of Oct-4, followed by that on gelatin and fibronectin, while TCPS was the lowest at passage 3. Sox-2 expression of hADSCs also showed similar tendency to the Oct-4 expression. In this point, Collagen-coated dishes should be the best dishes for the culture of hADSCs keeping pluripotent genes. The result of differentiation of hADSCs cultured on ECM-coated dishes also showed the same tendency to qRT-PCR result. It is concluded that hADSCs cultured on collagen-coating dishes could keep higher pluripotency and higher differentiation ability than TCPS and other ECM-coating dishes in this research.
