組織工程主要的三大要素包括有細胞、支架和生長因子。其中幹細胞於組織工程上的應用，成為近年來被廣泛研究的對象，主要是幹細胞具備高度的分化能力。然而如何在體外大量培養幹細胞，並維持幹細胞的特性，最終誘導為所需的組織，是組織工程實際應用非常重要的課題。
近年來科學家從脂肪組織中可以分離大量的幹細胞，而人類脂肪幹細胞的優點包括:來源取得容易及安全、細胞量多、分化能力佳且不具道德爭議等問題。由於傳統單層培養會受限於培養面積的大小不易取得大量的細胞。使用三維懸浮培養可以克服單層培養受限於細胞貼附面積的影響，可獲得大量的細胞數對組織工程的應用有正面的幫助，然而幹細胞在懸浮培養下，是否仍具備高度分化之能力尚不清楚，。本研究目的探討懸浮培養對於人類脂肪幹細胞增生與分化之影響，利用不同於傳統的單層培養方式，期望能培養大量的細胞，可日後提供組織工程細胞培養參考之應用。
本實驗利用Ultra-Low Attachment Surface Plates (ULASP) 培養細胞，讓細胞懸浮於培養液中，形成懸浮培養系統。利用此方法培養人類脂肪幹細胞，觀察不同細胞密度於懸浮培養下細胞增生的表現。並且探討懸浮培養的人類脂肪幹細胞，經由骨化、軟骨化和脂化等不同的培養基誘導後，細胞不同分化能力的表現。
實驗結果顯示懸浮培養能維持人類脂肪幹細胞的存活，細胞具有細胞聚集 (Cell aggregation) 的特性，進而形成細胞團塊。以不同的細胞密度培養下，細胞形成的團塊平均直徑範圍皆在50~200 μm間。細胞密度的增加有助於增加細胞團塊的形成以及維持團塊的數量。從組織化學染色觀察發現懸浮培養的人類脂肪幹細胞能維持正常的分化能力，經誘導能分化為骨母細胞、軟骨細胞和脂肪細胞等。進一步由Real-Time PCR結果中得知經懸浮培養及誘導骨化後，骨分化基因的表現較貼附培養的細胞有更明顯的表現，更能有效促進人類脂肪幹細胞的分化。
另外為了增進細胞間快速的聚集，使提高細胞的生存，藉由添加天然高分子包括5%的膠原蛋白 (Collagen)溶液與0.33%的幾丁聚醣 (Chitosan) 溶液於懸浮培養系統，評估可以幫助細胞間聚集與存活的變化。初步結果發現添加天然高分子能增加細胞之間的聚集，使細胞團塊的數量增加，並且增加細胞貼附因子Integrin β3的表現量。
最後，由本實驗結果發現懸浮培養系統能夠維持人類脂肪幹細胞的存活與分化之能力，於培養液中添加天然高分子也許能改善懸浮培養的條件，可日後作為組織工程的細胞培養參考之應用。

Cell is considered the most important component for 3 major tissue engineering factors, cell, scaffold and stimulation. In recent years, stem cells became popular cell source for tissue engineering application because of high self renew capacity, good proliferation ability and multiple linkage differentiation quality. It is an important issue to efficiently multiple the stem cell number in vitro with proper desired differential capacity for practical tissue engineering application.
Recently, adipose tissue has been discovered to provide a large number of adult stem cells. Therefore, adipose-derived stem cell (hADSC) has become popular choice for stem cell source for its easy accessibility, abundant supply, highly differential ability and less ethical concern. Due to the limit surface area by regular 2-D culture dish, the cell number for monolayer culture is restricted. A more efficient approach to multiple stem cell number would be a great benefit for stem cell application in tissue engineering. Comparing to conventional 2-D dish culture, suspension culture allows cells to aggregate into 3-D block which can accommodate much more cells in low-attachment surface plate. However, the differentiation ability of cells, especially stem cells, is still a major concern. The purpose of this study was to observe the effect of suspension culture on the proliferation and differentiation of human adipose-derived stem cells. We anticipated this culture environment could be applied for cell culture for tissue engineering.
We cultured hADSC in the suspension culture, which was the Ultra-Low Attachment Surface Plates (ULASP) condition. Effect of different cell densities on the proliferation of hADSC was investigated, and differentiation ability of hADSC was evaluated by application of induction medium of osteogensis, chondrogensis and adipogensis in suspension culture.
Our results showed that hADSC could maintain the cell viability in suspension culture system and form several cell aggregates. The diameter of aggregates were in 50~200 μm in suspension culture, although cell densities were different. However, increasing cell density could promote the formation of cell aggregates. In addition, hADSC could maintain the ability of the differentiation in suspension culture and could be effectively induced into multilineage. The results of histomorphometric analysis displayed that hADSCs were indeed induced into adipocytes, osteoblasts and chondrocytes in suspension culture. Further, Q-PCR assay discussed the expression of osteogenic genes. The results exhibited that the expression of osteogenic genes were significantly increased in suspension culture. Suspension culture was more efficiency in hADSC differentiation than the monolayer culture.
We also studied the influence of the addition natural biomaterials, including collagen and chitosan into suspension culture on the formation of cell aggregation. The results showed greatly increase in cell aggregates by adding collagen or chitosan. The intercellular molecules integrin β3 were significantly increased by adding biomaterials.
We conclude that suspension culture can maintain cell viability and contribute to the effective differentiation of hADSC, and offer an better approach of cell culture for tissue engineering application.

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