- 學位論文
以溫感性材料接枝微載體在生物反應器中進行放大華通氏膠間質幹細胞的研究
Temperature-responsive microcarriers with poly(N-isopropylacrylamide) grafted for wharton's jelly mesenchymal stromal cell expansion in functionally-closed process bioreactor
摘要
間質幹細胞為多功能性幹細胞，因為其俱有良好的分化能力及免疫調節的特性對於發展組織修復 (tissue repair)、組織再生 (tissue regeneration)及細胞治療 （cell therapy）是非常好的細胞來源。所以細胞需求量日趨增加，但是目前最大的挑戰為細胞數不足，現今仍然沒有一個細胞培養系統能夠穩定且有效率的培養到所需細胞數且要維持其型態（phenotype）。 本研究利用溫感性中孔洞明膠微載體作為間質幹細胞的細胞載體，並在生物反應器內進行細胞增生的研究。由於明膠有良好的生物相容性及細胞貼附能力好，常被用於細胞載體的材料。本實驗所使用過程功能封閉式生物反應器。利用灌流的方式使細胞生長在液體流動的環境，不僅能充足的提供細胞所需養分還能帶走細胞代謝物，由於過程利用蠕動幫補來進行液體流動的動力，所以不需要更換細胞培養基減少細胞污染機會。在細胞獲取方面，為了避免利用胰蛋白酶對細胞造成的傷害，本研究接枝溫感型材料PIPAAm在微載體上，細胞的獲得僅需要將溫度降低到低於LCST，即可已不傷害細胞的方式來獲取細胞，以增加細胞放大的成功機會與使用方便性。 從結果顯示，進行FTIR與ESCA可證實PIPAAm有成功接枝在微載體上。利用WST-1與Live/Dead staining可以得到細胞不論在靜態或生物反應器內培養，均可以穩定的在溫感性微載體上增生，且在生物反應器的速率大於在靜態培養的生長速率。並利用SEM與 Actin/Hoechst 染色，可以看得細胞可以貼附在整顆微載體，達到增加細胞培養密度的效果，且細胞呈現圓球狀。在細胞獲取方面，有接枝PIPAAm利用降低溫度獲取細胞可以達到很高的收穫效率。將靜態或生物反應器內培養七天後，分別利用RT-PCR與流式細胞儀，均可以證實細胞沒有因為在此系統多天培養而分化，並且維持間質幹細胞的幹性。 所以利用溫感性微載體並在生物反應器放大細胞，不但可以用最有效率且最方便的方式培養大量細胞，並且在收穫細胞方面，僅需要將溫度降低而不需要蛋白質水解酶的處理，不僅不會傷害細胞且使可以細胞獲取步驟操作更加便利。可見將溫感性微載體放入生物反應器後，將可以穩定的培養出大量且健康的細胞對於組織工程及細胞治療都是很有潛力培養系統。
並列摘要
Mesenchymal stem cells (MSC) are a multipotent stromal cell. MSC can differentiate into a variety of call types and immunologic properties. It is a good source for tissue engineering, regenerative medicine and cell therapy. Therefore, increasing demand of cell number. But the most serious challenge is lack of cell number, it doesn’t have a capable of stable and efficient cell culture system to expand the enough cell and maintain cell phenotype. In this study, thermosensitive gelatin microcarriers was used as cell carrier mesenchymal stem cells. Because gelatin has good biocompatibility and cell adhesion. Functionally-closed process bioreactor system used for expanding cells. This is perfusion system, cells culture in a circulating liquid environment, not only sufficient to provide the necessary nutrients, but also take out cell metabolism. It is no need to replace the cell culture medium to reduce the opportunities of cell contamination. Traditional process ues the proteolytic enzyme to harvest cells, but it needs degradate the cell surface membrane protein. In order to avoid the trypsinization, grafting temperature-sensitive materials poly(N-isopropylacrylamide) (PIPAAm) on microcarriers was developed in this study. The cells harvest only need to reduce the temperature to 20 °C for 30 mins. It was chemically characterized by FTIR and ESCA, the result showed that PIPAAm was successfully grafted onto gelatin microcarrier. The result of proliferation rate showed that MSC cultured in both static and bioreactor systems, cell proliferation rate at bioreactor was higher than the static culture. For the result of SEM and Actin/Hoechst staining, the photographs showed that cell could attach and proliferate onto complete microcarrier, achieving the highest cell culture density. After 7-day culture in both static and bioreactor, the result showed that cells maintain their phenotype. Therefore, the thermosensitive microcarrier was used for expanding MSC in bioreactor, not only it can achieve more efficient and convenient system to culture a large number of cells, but also without repeated trypsinization. Thermosensitive microcarrier in bioreactor is a promising culture system to culture a lot of health cell for tissue engineering and cell therapy.