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

生物人工肝长期冷冻保存临床应用的研究。

基本信息

批准号：
09557093
负责人：
MATSUSHITA Michiaki
金额：
$ 8.19万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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

1.肝细胞球体快速形成的实验研究建立了肝细胞球体的快速形成和大规模制备方法，作为生物人工肝的生物反应器。通过使用不含EGTA和Ca 2+的灌注缓冲液制备分离的大鼠肝细胞。体外旋转培养6 h，约80%的肝细胞形成肝细胞球状体。在使用猪肝细胞的初步研究中，与大鼠肝细胞相比，10倍浓缩的猪肝细胞在旋转培养中在6小时内形成肝细胞球状体。大鼠肝细胞球体的冷冻保存：我们检查了大鼠肝细胞球体的冷冻保存。将肝细胞球状体在室温下悬浮于冷冻保护剂培养基（L-15 + 10%（v/v）FCS + 10%（v/v）DMSO）中20分钟。将混悬液在-5.5℃下过冷，并在结冰后保持15分钟。以1℃/min的速度冷冻至-40℃，在-80℃下保存7 ~ 10 d ...更多信息是的。将冻存的肝细胞球状体在37℃水浴中快速解冻，并对冷冻保护剂培养基进行逐步稀释。通过相差显微镜观察，检测LDL滞留量、尿素合成和白蛋白生成。形态学检查显示，肝细胞旋转培养后形成圆形球状体，表面有一些气泡，这些气泡在冷冻保护剂中浸泡期间仍然存在。解冻后肝细胞球体仍保持圆形，但大泡被破坏。解冻后乳酸脱氢酶的保留量约为新鲜球体的27%，并不断下降。尿素合成量约为新鲜植株的50%，培养24小时后降至18%。冻存后白蛋白的生成量明显减少，为新鲜肝细胞的1/200，冻存后肝细胞球状体出现不可逆性死亡。我们推测水泡的破坏导致了球状体的致命损伤。但冻存肝细胞球体可用于短期解毒。我们必须建立一个更复杂的方法来制作肝细胞球体。肝细胞球的培养条件肝细胞球的尿素合成在培养的早期有所下降，但在胶原凝胶培养条件下可长期维持高水平。少