A study of long term cryopreservation of a bio-artificial liver for clinical applications.

1.肝细胞球体快速形成的实验研究我们建立了快速形成和规模化生产肝细胞球体的方法，作为生物人工肝的生物反应器。用不含EGTA和Ca~(2+)的灌流缓冲液分离大鼠肝细胞。在旋转培养中，约80%的分离肝细胞在6h内形成肝细胞球形。在猪肝细胞的初步研究中，猪肝细胞与大鼠肝细胞相比，在6小时内形成了肝细胞球体。2.大鼠肝细胞球体的冷冻保存。将肝细胞球体置于冷冻保护剂(L-15+10%(v/v)胎牛血清+10%(v/v)二甲基亚砜)中室温悬浮20min。悬浮液在-5.5℃下过冷，结冰后保持15分钟。冰冻速率为1℃/℃直到-40℃，肝细胞球体在-80…下保存7~10dmin更多的钱。冷冻保存的肝细胞球体在37℃水浴中快速解冻，并进行冷冻保护剂的逐步稀释。通过相差显微镜观察、低密度脂蛋白滞留、尿素合成和白蛋白生成等指标进行检测。形态观察显示，旋转培养后的肝细胞球体呈圆形，表面有气泡，这些气泡在冷冻保护剂中浸泡过程中仍然存在。解冻后肝细胞球体仍保持圆形，但这些小泡被破坏。解冻后乳酸脱氢酶的保留量约为新鲜球体的27%，并持续下降。尿素合成量约为新鲜尿素的50%，培养24小时后降至18%。超低温保存可显著降低白蛋白的产量，约为新鲜白蛋白的1/200。我们推测，气泡的破坏在椭球体中造成了致命的侮辱。但在解毒主题中，冷冻保存的肝细胞球体持续时间较短。3.肝细胞球体的培养条件肝细胞球体的尿素合成在培养早期呈下降趋势，但在胶原胶培养条件下可长期维持较高水平。较少

1.An experimental study of rapid formation of hepatocyte spheroids.We established methods of a rapid formation and a large scale production of hepatocyte spheroids as a bioreactor of bio-artificial liver. Isolated rat hepatocytes prepared by using perfusion buffers excluding EGTA and Ca2+. About 80% of isolated hepatocytes formed hepatocyte spheroids in 6 hours in rotation culture. In preliminary studies using pig hepatocytes, ten times concentrated pig hepatocytes compared with rat hepatocytes formed hepatocyte spheroids in 6 hours in rotation culture.2..Cryopreservation of rat hepatocyte spheroids.We examined cryopreservation of rat hepatocytes spheroids. Hepatocyte spheroids were suspended in cryoprotectant medium(L-15 + 10%(v/v) FCS + 10%(v/v) DMSO) for 20 minutes at room temperature. The suspension was supercooled at -5.5℃ and maintained for 15 minutes after ice formation. A freezing ratio of 1℃/min was applied until -40℃ and hepatocyte spheroids were stored at -80℃ for 7 to 10 da … More ys. Cryopreserved hepatocyte spheroids were rapidly thawed in a 37℃ water bath and stepwise dilutions of cryoprotectant medium were performed. Phasecontrast microscopic observations, an amount of LDL retention, urea synthesis and albumin productions were examined. Morphological examinations revealed hepatocyte spheroids forming a round shape after rotation culture with some blebs on the surface, and these blebs remained during submerging in cryoprotectant medium. Hepatocyte spheroids maintained the round shape after thawing, but those blebs were destroyed. The amount of LDH retention was about 27% of fresh spheroids after thawing and declined continuously. Urea synthesis was about 50% of fresh ones and declined to 18% after 24 hours in culture. Albumin productions were significantly impaired by cryopreservation, the amounts were 1/200 compared with fresh ones.In this study, an unreversal death of hepatocyte spheroids occurred by cryopreservation. We speculated the destruction of blebs caused lethal insults in spheroids. But cryopreserved hepatocyte spheroids were available for short duration in themes of detoxification. We have to establish a more sophisticated method to make hepatocyte spheroids.3..Culture conditions of hepatocyte spheroidsUrea synthesis of hepatocyte spheroids declined in a early period in culture, but can be maintained high levels for a long duration in the collagen gel culture conditions. Less