人類脂肪幹細胞於生醫材料塗佈細胞外間質之純化及分化;Isolation and differentiation of human adipose-derived stem cells cultured on biomaterials Immobilized with ECM

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Title:	人類脂肪幹細胞於生醫材料塗佈細胞外間質之純化及分化;Isolation and differentiation of human adipose-derived stem cells cultured on biomaterials Immobilized with ECM
Authors:	古埝如 Ku, Nien-Ju
Contributors:	國立中央大學
Keywords:	幹細胞;人類脂肪幹細胞;細胞外間質;幹細胞培養;人類血小板裂解液體;細胞分化能力;mesenchymal stem cells;human adipose-derived stem cells;extracellular matri
Date:	2018-08-23
Issue Date:	2018-08-31 13:07:46 (UTC+8)
Abstract:	人類脂肪幹細胞(hADSCs)具有不均一的特性，此特性可從其多樣的基因型及分化能力中表現。依不同純化方式所分離出的人類脂肪幹細胞會具有不同的純度及多樣性。細胞其生長的微環境於人類脂肪幹細胞之基因表現及分化能力中，扮演著重要的角色。於先前的研究，我們發展了(一)培養於特定材料，例如聚苯乙烯細胞培養盤(簡稱培養盤、TCPS)之培養法，以及(二)利用通過網狀尼龍膜過濾器之膜遷移法。1在此研究中，我們正在發展新的人類脂肪幹細胞純化方式，期望利用優化後的純化方法所獲得之脂肪幹細胞能具有較高多能性，以及細胞分化能力，如：軟骨細胞、成骨細胞及脂肪細胞之分化。我們將從脂肪組織中萃取出的人類脂肪幹細胞藉由培養方法培養在不同的基材上，如：(一)聚苯乙烯細胞培養盤、(二)培養盤塗佈基質膠(Matrigel)、(三)培養盤塗佈第一型膠原蛋白(Collagen type I)、(四)培養盤塗佈Synthemax? II (市售合成生醫材料，以寡玻璃粘連蛋白為主)、(五)培養盤塗佈人類重組玻璃粘連蛋白(human recombinant Vitronectin)，以及(六) 培養盤塗佈人類纖維連接蛋白(Fibronectin)。基質膠及第一型膠原蛋白為異種來源的材料，其餘細胞外間質之來源則為人類(無異種材料)。在上述的材料中，培養在基質膠塗佈之培養盤上的人類脂肪幹細胞具有最高純度，最高之基因表現，以及最高的分化能力。儘管培養在塗佈無異種細胞外間質之培養盤的人類脂肪幹細胞，其多能性及分化能力相對低。然而，在無異種材料中，我們發現脂肪幹細胞培養在塗佈人類重組玻璃粘連細胞及Synthemax? II的培養盤上，其結果表現最佳。未來若希望將人類脂肪幹細胞使用在臨床應用上，發展出恰當的無異種培養環境是必須的。此外，目前我們正在發展一新的培養液，利用人類血小板裂解液取代小牛血清作為養分添加劑以優化人類脂肪幹細胞之純度，並建立一更完全的無異種培養環境。 ;Human adipose-derived stem cells (hADSCs) exhibit heterogeneous characteristics, indicating various genotypes and differentiation abilities. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. Microenvironment plays an important role on gene expression and differentiation of hADSCs. Previously, we developed purification of hADSCs from adipose (fat) tissue by (a) the culture method on specific culture materials and (b) the membrane migration method through Nylon mesh filter.1 In this study, we developed a new hADSC line from adipose tissue by culturing on the optimal cell culture condition, which can purify hADSCs having high pluripotency and high differentiation ability into chondrocytes, osteoblasts and adipocytes. We isolated hADSCs from adipose tissue by the culture method where different substrates were used; (a) tissue culture polystyrene (TCPS) dishes and TCPS dishes coated with Matrigel (b), collagen type I (c), Synthemax II (oligo-vitronectin based substrate) (d), human recombinant-vitronectin (e), or human fibronectin (f) where Matrigel and collagen type I are xeno-contained materials and another extracellular matrices (ECMs) are xeno-free materials. Among these substrates, hADSCs cultured on Matrigel-coated dishes presented the highest purity of hADSCs, the highest pluripotent gene expression, and the highest differentiation abilities. Despite hADSCs cultured on TCPS dishes coated with xeno-free ECMs showed lower pluripotency and differentiation ability compared to those on Matrigel-coated TCPS dishes. However, TCPS dishes coated with human recombinant-vitronectin and Synthemax II were found to be the best dishes for hADSCs isolation and culture among the TCPS dishes coated with xeno-free ECMs. It should be necessary to develop optimal xeno-free culture dishes and conditions of hADSCs for clinical application of hADSCs in future. Furthermore, we are now developing a new culture medium by replacing the fetal bovine serum (FBS) with human platelet lysate (hPL) as the supplement to improve the purity of hADSCs and build up more complete xeno-free culture conditions of hADSCs for clinical applications.
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