平滑肌细胞在旋转生物反应器内的微载体培养与快速扩增

为获取足量数量活性良好的膀胱平滑肌种子细胞,本实验探索在旋转生物反应器内应用微载体技术快速扩增膀胱平滑肌细胞的方法。将培养的第二代新西兰兔膀胱不滑肌细胞应用Cytodex-3微载体在旋转生物反应器（RCCS）内进行动态培养,观测平滑肌细胞的生长情况和细胞代谢率,进行a-肌动蛋白免疫组化染色,并与常规方法培养平滑肌细胞进行比较。结果显示膀胱平滑肌细胞在Cytodex-3微载体上生长迅速、细胞代谢率高、生长倍增时间缩短。在培养第九天,细胞数量可达最初接种的23倍。免疫组化显示平滑肌细胞活性良好。结果表明利用微载体细胞培养技术可简便快速地在体外扩增平滑肌细胞,可为构建组织工程化人造膀胱提供大量活性良好的平滑肌细胞。

In vitro amplification of smooth muscle cell of rabbit bladder with micro-carriers in rotary cell culture system

It's of great importance in bladder tissue engineering to find a proper way for acquiring well differentiated smooth muscle cells in large scale in a short time. This experiment was conducted for this purpose by culturing the second passage smooth muscle cells of New Zealand rabbits bladder with micro carriers in rotary cell culture system(RCCS). Smooth muscles were mechanically isolated from the bladders and cells were disassociated by type I collagenase and cultured in flasks and in RCCS respectively. Kinetic growth of the smooth muscle cells on cytodex 3 micro carriers were recorded and cell products in culture medium were measured. Immuno cyto chemical analysis against alpha smooth muscle actin was also performed both in micro carriers amplified and flasks augmented smooth muscle cells. Result shows that the bladder smooth muscle cells attached rapidly to the surface of cytodex 3 micro carriers. In comparison with the cells statically cultured in flasks, a quicker growth and a higher metabolization rate of these cells were observed. Cells cultured in RCCS increased 23 times in number on the ninth day and were stained positively for anti alpha smooth muscle actin. This study demonstrates that smooth muscle cells can be expanded up to more than 20 folds within a short time when cultured with micro carriers in RCCS and it will be of much advantages for reconstruction of bladder in tissue engineering.