利用3D細胞培養系統維持脂肪幹細胞分化特性

Abstract

近年來人們開始利用細胞進行組織工程相關研究，但是不足的細胞數量和來源常是阻礙實驗進行的原因之一，一般常用的細胞培養方法為在培養皿上的2D細胞擴增，此種方法操作容易，但可能會造成細胞的型態改變或因酵素作用破壞細胞外間質；為了改善此種情況，另有3D細胞培養方法，包含了利用旋轉培養瓶培養於微載體上培養細胞，微載體有高比表面積的優點，而此種培養環境也和生理環境較相似。 本研究希望利用BioLevitator TM此種3D培養系統，建立適合不同細胞(3T3纖維母細胞、Hela cell癌症細胞、BAEC內皮細胞、hASC人類脂肪幹細胞)生長的培養平台，比較這幾種細胞在2D/3D培養環境下的生長狀況，利用螢光染色我們可以知道這些細胞在3D環境下的細胞型態，由live/dead染劑也可知道當細胞長滿於微載體上時仍有高存活率。 hASC脂肪幹細胞因具分化能力，有相較於其他幹細胞更為方便的萃取方法和充足的來源等優點，為一在組織工程和在生醫學領域上新興的幹細胞來源，因此我們針對hASC比較其在2D/3D培養環境下的分化能力，將細胞先以骨分化和脂肪分化的培養液培養後，利用RT-PCR和染色量化的方式比較，雖然在基因表現上兩種培養方式對分化能力沒有顯著差異，但透過染色分化可得到在分化第21天時，3D培養後骨分化和脂肪分化的hASC都有較佳的分化表現。 我們希望能利用此一系統培養後的具有較佳分化能力的hASC，直接用於組織工程或在生醫學相關的研究上，或將此種細胞注射於組織受損的部位，以期達到幫助治療恢復的效果。

With the emergence and promising evidence of tissue engineering in recent years, researchers have utilized stem cells for drug delivery, tissue repair and regenerative medicine related studies. However, limited amount of cell sources, especially stem cells is one of the challenges in studies of drug delivery tissue repair and regenerative medicine. Traditional 2D cell cultures have been widely used for in vitro cell proliferation and cell-based assays. Though easy to operate, they may have some drawbacks such as: unnatural flat-surface culture condition, cell phenotype changes…etc. 3D cultures have been viewed as a system which better mimic the physiological environment and improve cell culture condition at the same time. Microcarrier-bioreactor culture systems are one of the promising 3D culture methods for cell amplification while maintaining the phenotype of gene expression. Here, we wanted to establish a 3D culture system for different cell types (3T3 cell, fibroblast; Hela cell, cancer cell; BAEC, endothelial cell; hASC, stem cell) using BioLevitatorTM as the bioreactor for microcarrier-based culture. Fluorescence staining was used for cell morphology observation on microcarrier during culture period, multilayers of cell proliferation could be seen in 3T3 cell and Hela cell; BAEC showed a single layer formation over the surface of the microcarrier; hASC prefers to grow in gaps between microcarriers. Live/dead staining indicated high viability of all the cell types as cell reach confluence over microcarrier. Then we used adipogenic and osteogenic differential medium induction for hASCs previously cultured in 2D or 3D culture environment, and compared the differential potential via RT-PCR and staining quantification. Though no significant difference showed in gene expression, staining quantification indicated that cells cultured in 3D condition might have better differential potential at day 21 in either adipogenic or osteogenic lineage.