| Background: It has been proved that biological artificial liver support system(BALSS)with porcine hepatocytes has good liver support function in the treatment of dog and patients with acute hepatic failure . A large sum of porcine hepatocytes with enough function is the key point of success of treatment. Establishment of method of large-scale cryopreservation of porcine hepatocytes can reduce preparation period. The only method of large-scale cryopreservation of porcine hepatocytes was established by Watanabe, but it cost high and complex. Our job is the elongation of previous study and the aim of which is to establish a bank of porcine hepatocytes. We cryopresrved Chinese experimental miniswine hepatocytes with kinds of methods.In the circumstances of in vitro culture and BALSS culture, We established method of large-scale cryopreservation of porcine hepatocytes by contrast of the viability, morphology and function of rewarming hepatocytes with fresh hepatocytes. Methods: 1. Porcine hepatocytes were isolated with enzymetic digestion method and divided into three groups randomly: Fresh hepatocytes group, fresh hepatocytes were cultured directly; The other two were cryopreserved groups with Gradually freezing method and programmed freezing method. The hepatocytes were rewarmed 1 month later and DMSO were eluted first, then hepatocytes were cultured in flask with type- I collagen and lidocah, NH4Cl fructose were added into NSAL nutrious solution . Culture solutionsamples were obtained timely to measure the concentration of lidocaine, glucose, urea nitrogen and albumin.The hepatocytes's microstructure and ultrastructure were observed so that the best freezing method could be determined.2. Porcine hepatocytes were isolated with enzymetic digestion method and divided into fresh hepatocytes group and cryopreserved hepatocytes group. Fresh hepatocytes were cultured in BALSS for 6 hours directly and cryopreserved hepatocytes firstly were conserved in -196 C Nitrogen tank and then cultured in BALSS after being rewarmed 1 month later. Porcine hepatocytes circulated in the exterior space and interior space was connected with peristaltic pump and triangle flask via medical silica tube. The whole device was a closed loop. Triangle flask contained 250ml DMEM solution with lidocaine, NH4C1 and fructose. The hepatocytes' viability, morphology was detected at 0, 2, 4, 6hours and the nutrition samples were obtained to determine the concentration of albumin, urea nitrogen , glucose and lidocaine .Living state of cryopreserved hepatocytes in BALSS was determined by contrast with fresh hepatocytes and cryopreserved hepatocytes. Results: 1. Viability of fresh porcine hepatocytes was 81%+3.6%, While Gradually freezing group had a viability of 76.3%+1.9% and programmed freezing group's viability was 72.4%+1.5%. There was no significant difference of albumin and lidocaine concentration among three groups (P>0.05) . There was no significant difference of the synthesis function of urea nitrogen and glucose between gradually freezing group and fresh hepatocyte group and they both superior to programmed group. The ultrastructure of fresh hepatocytes was basically normal and partial membrane had little pores.After 30 minutes' culture,the structure of membrane was restored. While the ultrastructure of cryopreserved hepatocytes were integrated and mitochondrias were lightly swollen, After 30 minutes'cculture, microvilli structure emerged in parts of hepatocytes, organelles were intact and two cryopreserved groups had no significant differences.2. The viability of fresh porcine hepatocytes changed little during the first 4 circulating hours while cryopreserved hepatocytes changed dramatically. The concentration of total protein , albumin , urea nitrogen and glucose increased with time and there had no significant difference at the same circulating time point( P>0.05). They both could transformated lidocaine and the concentration of which droped gradually. The broken fresh porcine hepatocytes increased aft... |
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