微囊包载体肝细胞培养方式对肝细胞功能及活性的影响

The function and viability of hepatic cells in ACA microcarrier

目的:探讨采用海藻酸钠/壳聚糖/海藻酸钠(ACA)微囊包裹微载体的肝细胞培养方式对肝细胞功能及活性的影响。方法:设立微囊包载体组和普通单层细胞培养组;以微载体Cytodex3进行肝细胞(HL-7702)培养;选择合适时机进行ACA微囊化包裹。收集两组第1～6天的培养液,测定其中白蛋白及乳酸脱氢酶(LDH)含量;并用Calcein-AM/PI双染细胞,在荧光显微镜下观察细胞的荧光强度变化,了解细胞活性的改变。结果:观察培养6d,微囊包载体组培养液中的白蛋白含量至第4天达到峰值,之后缓慢下降;而普通单层培养组培养液中白蛋白含量至第2天达到峰值,之后迅速下降。微囊包载体组培养液的LDH含量明显低于单层培养组(P<0.01)。微囊包载体组中活细胞的绿色荧光强度逐渐减弱,至培养第6天,显示黄绿色荧光;而单层培养组中每天有大量失活细胞悬浮于培养液中,至培养第6天,贴壁细胞数量仅占培养前的27%。结论:微囊包载体肝细胞培养方式中前期,微载体细胞培养可在较短的时间及较小的空间内实现细胞扩增;外周的微囊机构对肝细胞营养物质的供给及合成产物的排出无影响,但可阻挡大分子免疫球蛋白;两者结合有利于维持肝细胞的白蛋白分泌功能及细胞活性,是一种兼具高密度培养及免疫屏障功能的细胞培养方式,在生物人工肝中有一定的可行性。

Objective To observe the function and viability of the hepatocyes in microcarrier encapsuled by alginate/chitosan/alginate.Methods Two groups were designed according to different styles of cell culture.The 1st group consisted of using the ACA-microcarrier and the other group belonged to the monolayer culture group.Hepatocytes of the HL-7702 cell-line were cultured in the microcarrier cytodex-3 and then encapsulated by ACA.The monolayer culture group had the same number of HL-7702 hepatocytas which were cultured in the 12 holes plate.We collected the media of both groups from day 1 to day 6.The albumin and LDH were measured.The cell viability was observed by using Calcein AM/PI to stain the cells in the microcapsules.Results The albumin level in the ACA-microcarrier group stepped up and reached the climax at day 4 and than declined slowly.In the monolayer culture group,the albumin level reached the climax at day 2 and declined lower till day 6.The LDH level in the ACA-microcarrier group was lower than that of the monolayer culture group（P0.01）.After stained by Calcein-AM/PI,the living cells showed green and the dead cells showed red under the fluorescence microscope.The viability of the cells in the ACA-microcarrier group disappeared slowly.The cells in the ACA-microcarrier group showed faint green at day 6.The cells in the monolayer culture group died more quiekly.There were only 27% living cells remaining at day 6.Conclusions Hepatocytes cultured on the Cytodex 3 generally used to proliferate within a short time and in a small space.The microcapsule membrane permits the nutrient substances entering into the cells and prevents IgG attacking the cells.Both the support of the Cytodex 3 and the protection of the microcapsule help maintain the hepatocytic function and viability.Cells in microcarrier encapsuled by ACA is a new style of hepatic cells culture,which has a potential value to be used in the bioartificial liver system.