生物人工肝用C3A细胞在零下非结冰时的保存

Subzero nonfreezing storage of C3A hepatocytes for use in bioartificial liver support systems

目的:探讨零下非结冰保存肝细胞的效果及其与细胞凋亡的关系.方法:UW液保存的C3A细胞悬液分为3组:-0.8℃组(零下非结冰组),0℃组(0℃非结冰组),4℃组(对照组).低温保存24、48及72h后,采用流式细胞术分别测定细胞存活率及凋亡率,同时测定LDH、乳酸释放,细胞内ATP含量、尿素合成功能及白蛋白分泌功能,同时观察细胞形态.结果:零下非结冰组较0℃非结冰组及对照组明显提高了低温保存72h的C3A细胞的存活率(86.49%±2.80%vs81.50%±2.83%,77.83%±3.40%,均P<0.05),降低了细胞凋亡率(1.26%±0.84%vs5.34%±1.20%,9.16%±1.99%,均P<0.05);明显抑制了低温保存72h乳酸以及LDH释放(乳酸:10.38μg/106cells±1.40μg/106cellsvs12.02μg/106cells±1.64μg/106cells,17.41μg/106cells±2.40μg/106cells;LDH:80.10U/L±11.10U/Lvs120.04U/L±14.32U/L,148.98U/L±15.37U/L,均P<0.05);更好地维持了低温保存72hC3A细胞内ATP含量、尿素合成功能、及白蛋白分泌功能(均P<0.05).形态上,零下非结冰组保存细胞死亡比例少,细胞接触良好,未见对照组及0℃非结冰组细胞膜周围出现"毛刺"样改变及细胞内的"空泡样"改变.结论:在零下非结冰条件下保存肝细胞,建立一个"血库样"(readytouse)肝细胞库,可以有效促进生物人工肝的发展.

AIM：To investigate whether subzero nonfreezing storage （-0.8°C） is superior to conventional cold storage in preservation of C3A hepatocytes for use in bioartificial liver support systems.METHODS：C3A hepatocytes suspended in University of Wisconsin （UW） solution were divided into three groups：subzero nonfreezing group （-0.8°C）,zero nonfreezing group （0°C） and control group （4°C）.After 24,48 and 72 hours of hypothermic storage,cell viability and apoptosis were detected by flow cytometry;intracellular adenosine triphosphate （ATP） content,lactate dehydrogenase （LDH） release,lactic acid production,urea synthesis and albumin secretion were determined;and cell morphological changes were observed.RESULTS：Compared to the zero nonfreezing group and the control group,after 72 hours of hypothermic storage,the percentage of viable C3A hepatocytes was significantly higher （86.49% ± 2.80% vs 81.50% ± 2.83% and 77.83% ± 3.40%,respectively;both P 0.05）,and cell apoptosis rate was significantly lower （1.26% ± 0.84% vs 5.34% ± 1.20% and 9.16% ± 1.99%,respectively;both P 0.05） in the subzero nonfreezing group.Lactic acid and LDH production was more significantly suppressed （lactic acid：10.38 μg/10 6 cells ± 1.40 μg/10 6 cells vs 12.02 μg/10 6 cells ± 1.64 μg/10 6 cells and 17.41 μg/10 6 cells ± 2.40 μg/10 6 cells;LDH：80.10 U/L ± 11.10 U/L vs 120.04 U/L ± 14.32 U/L and 148.98 U/L ± 15.37 U/L,respectively;all P 0.05）,and the ability of hepatocytes to synthesize urea and secrete albumin was better maintained in the subzero nonfreezing group （both P 0.05）.Moreover,cells in the subzero nonfreezing storage group had lower death rate and better cellular morphology.A burr-like structure around the cell membrane and an intracellular vacuole-like structure were found in cells in the zero nonfreezing group and the control group,but not in the subzero nonfreezing group.CONCLUSION：Subzero nonfreezing storage （-0.8 °C） of hepatocytes to construct a ＂ready-touse＂ hepatocyte bank like the ＂blood bank＂ will efficiently promote the development of bioartificial liver support systems.