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

1.肝细胞球状体快速形成的实验研究。我们建立了快速形成和大规模生产肝细胞球体作为生物人工肝的生物反应器的方法。用不含EGTA和Ca2+的灌注缓冲液制备的分离大鼠肝细胞。约80%分离的肝细胞在旋转培养6小时内形成肝细胞球状体。在猪肝细胞的初步研究中，与大鼠肝细胞相比，10倍浓度的猪肝细胞在6小时的旋转培养中形成了肝细胞球体。大鼠肝细胞球体的低温保存。我们研究了大鼠肝细胞球状体的冷冻保存。在室温下，肝细胞球体悬浮在低温保护剂(L-15 + 10%（v/v) FCS + 10%(v/v) DMSO）中20分钟。悬浮液在-5.5℃过冷，结冰后保持15分钟。1℃/min冷冻至-40℃，肝细胞球体在-80℃保存7 ~ 10 da。冷冻保存的肝细胞球体在37℃水浴中快速解冻，并逐步稀释冷冻保护剂培养基。相衬显微镜观察，LDL保留量，尿素合成和白蛋白产生进行了检查。形态学检查显示，肝细胞球体在旋转培养后形成圆形，表面有一些水泡，这些水泡在冷冻保护剂中浸泡后仍然存在。肝细胞球体在解冻后仍保持圆形，但水泡被破坏。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。尿素合成率约为新鲜的50%，培养24 h后降至18%。冻存后白蛋白产量明显下降，为新鲜贮藏的1/200。在本研究中，肝球细胞通过低温保存发生非逆转性死亡。我们推测气泡的破坏会对球体造成致命的伤害。但在解毒的主题中，冷冻保存的肝细胞球体可用于短时间。我们必须建立一种更复杂的方法来制造肝细胞球状体。肝球细胞的培养条件：肝球细胞的合成在培养初期下降，但在胶原凝胶培养条件下可以长时间保持高水平。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