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標題: Evaluation of the alginate culture system for chondrogenesis of human adipose tissue-derived stromal cells and assessment of the rotational speed for the novel bioreactor vessel
作者: Lee, Ying-Ying-Yi
關鍵字: human adipose tissue derived stromal cell;人類脂肪幹細胞;alginate gel;chondrogenesis;bioreactor;褐藻膠;軟骨新生;生物反應器
出版社: 化學工程學系
In view of that the three-dimensional (3-D) structure was necessary for chondrogenesis, we fabricated alginate beads and pads to be the cell carriers. Human adipose tissue derived stromal cell (ADSC) were embedded in the gel to study the effects of inducers, culture model and components of extracellular matrix on chondrogenesis. Transforming growth factor-beta 3 (TGF-β3) was found to be a better inducer for chondrogenesis of ADSC than TGF-β1. Cell proliferation was limited in divalent cations crosslinked alginate beads; however, matrix synthesis and deposition were benefited from the 3-D environment. Addition of hyaluronic acid in alginate gel increased type II collagen and GAG contents, while addition of chondroitin sulfate promoted cell proliferation and up-regulated the aggrecan gene expression. In the aspect of bioreactor, better results of cell number, scaffold dry weight, mechanical strength and total GAG contents were obtained from dynamic culture than static culture. At the same time, dynamic culture condition stimulated the cells to secrete NO, which could reduce collagen synthesis. Large amount of GAG was released to media upon the flush arising from the rotation. Based on the data of cell proliferation, matrix accumulation and mechanical strength, the optimal rotational speed for the novel bioreactor vessel was smaller than 25 rpm. This study provided a carrier system for chondrogenesis and established suitable parameters for the bioreactor.

本研究使用脂肪來源之人類成體幹細胞,並考量三維培養環境對軟骨新生之必要性,配合褐藻膠製作之載體,用以探討分化劑、培養模式及基質成份對其體外軟骨發生(chondrogenesis)能力之影響。由基因表現、細胞數及GAG含量分析,本研究發現TGF-β3對ADSC於軟骨發生有較TGF-β1佳的誘導效果。使用褐藻膠載體進行培養,雖然會使細胞的增殖受限,但對於基質的合成及累積有所助益;在褐藻膠中添加透明質酸可提高第二型膠原蛋白及GAG的含量,添加硫酸軟骨素則可促進細胞增生及蛋白聚醣基因表現。在生物反應器方面,在動態環境下,細胞數、支架乾重、機械強度、總GAG含量均較靜態培養時佳,但動態環境亦刺激細胞分泌NO,降低膠原蛋白的合成,且流場的沖刷使得大量GAG釋放至培養液中;綜合細胞生長、基質分泌及機械強度之表現,該旋轉式生物反應器的較適轉速應小於25 rpm。本研究提供了一個可以促進分化的載體系統,及適當的生物反應器條件。
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