长期体外培养条件下海藻酸钙-多聚赖氨酸-海藻酸钙微囊化牛肾上腺嗜铬细胞的变化

Changes in alginate-polylysine-alginate microencapsulation of bovine chromaffin cells after a long period in vitro culture

目的:了解在体外长期培养的条件下,海藻酸钙-多聚赖氨酸-海藻酸钙微囊化牛肾上腺嗜铬细胞的形态及其细胞活率的变化。方法:实验于2003-09/2004-05在解放军总医院老年医学研究所细胞生物学室完成。①材料:细胞为牛肾上腺嗜铬细胞;海藻酸钠和多聚赖氨酸由SIGMA公司提供;DMEM-H培养液由GIBCO公司提供;加强型小牛血清由HYCLONE公司提供。②方法:用海藻酸钙-多聚赖氨酸-海藻酸钙微胶囊包裹牛肾上腺嗜铬细胞,以含有体积分数为0.1的小牛血清的DMEM-H培养基培养于CO2孵箱中,定期换液。③观察指标:在光学显微镜下观察微囊及囊内细胞的形态;以锥虫蓝染色法计数囊内细胞的数量和存活率,观察时间为8个月以上。结果:①微囊包裹后1d时,光镜下见微囊呈圆形,囊膜完整,表面光滑;囊内为单个细胞,均匀散在分布。体外培养35d时,微囊仍呈圆形,囊膜完整,表面光滑;囊内细胞聚集成体积较小的团块。体外培养240d时,微囊形态无明显变化;囊内大部分细胞已聚集成多个体积较大的团块,少数微囊内的细胞聚集成一个大的细胞团。②随着培养时间的延长,囊内细胞数量逐渐减少,从最初的(54±10)个/囊减少到培养243d时的(35±7)个/囊,但差异无显著性意义(P>0.05);囊内细胞的活率无明显改变,开始为95%,到培养243d时仍有93%的细胞存活。结论:在体外培养条件下,海藻酸钙-多聚赖氨酸-海藻酸钙微囊化牛肾上腺嗜铬细胞可较长时间地保持良好状态。

AIM： To investigate the variation of the form and the motility rate of the microencapsulated bovine chromaffin cells （APA-BCC） with alginate-polylysine-alginate after being cultivated in vitro for a long period. METHODS： The experiment was accomplished in Cell Biology Laboratory, Institute of Gerontology and Geriatrics, General Hospital of Chinese PLA between September 2003 and May 2004.（1）Materials： BCC; Alginate and polylysine were provided by SIGMA company; DMEM-H solution was offered by GIBCO company; Cosmic calf serum was supplied by HYCLONE company. （2）Methods： APA-BCC was cultivated in CO2 incubator with DMEM-H solutions containing 0.1 volume fraction of calf serum, and the solutions were exchanged periodically.（3）Evaluations： The form of APA microcapsules and microencapsulated cells were observed under optical microscope. The motility rate of microencapsulated cells was recorded with trypan blue dye for more than 8 months. RESULTS： （1）One day after being microencapsulated, it was observed under the optical microscope that APA microcapsules presented spherical and the member was smooth, the single cells in the microcapsule were well and diffusely distributed. After being cultivated in vitro for 240 days, the form of APA-BCC had no marked change but the most cells had aggregated to form lots of clumping with relatively large volume, while few cells aggregated to a cell mass.（2）With the elongation of cultivating time, the number of the cells in the microcapsule decreased, there was no statistics difference between the beginning and 243 days of culture [（54±10）, （35±7） cells/microcapsule, P 〉 0.05]. The motility rate of microencapsulated cells showed no remarkable changes （95%, 93%）. CONCLUSION： The APA-BCC can keep a good status for a relatively long period during in vitro culture.