The Experimental Study Of A New Hypothermic Storage Solution Of Hepatocytes Used For Bioarticicial Liver Support System

Background:The date of our liver transplantation is more than 700 cases per annum, and the survival rate is more than 50% per annum, then the longest survival time is more than six years. However, our organ supplies for serious hepatitis patient are destitute. So, many patients have no time of liver transplantation and lost his chance of survivi- ng.Meanwhile, effective donor resources failed to capitalize on. Therefore, the cellul- ar level of new treatments such as Bioartificial Liver or hepatocellular transplantation has obtained research and development. Hepatocellular transplantation and Bioarti- ficial Liver which have got favourable effect in a lot of animal experiments and clini- cal experiments become a promising way to treat hepatopathy. Actually, as the great applications of Hepatocellular transplantation and Bioartificial Liver in clinical, the demand for hepatocytes will be going to go up.Then, the situation of donor shortage will be same as the organ transplantation. If there is a practical and reliable technolo- gy of the low temperature storage and an establishment of a hepatocytes library, crit- ical patients with hepatopathy should be provided plenty of high vigour and functional hepatocytes.Then, Hepatocellular transplantation and Bioartificial Liver technology can be promoted everywhere. Since the 1970s, the experiment study of preservation in many kinds of organ and tissue from basic theories, animal experiment to clinical application has achieved great progress. As the pathogenesis of injury of cell and tissue in organ preservation has inquired more deeply, organs of the effective pres- ervation time were significantly prolonged. The development and application of organ preservation such as University of Wiseonsin (UW) solution, Celsiorsolution (CS) and HTK solution promote the development of clinical organ transplantation. Our recent ten years in organ transplants work has been developing rapidly, and the pathogenesis of injury of cell and tissue in organ preservation and the research in organ preserve solution have made certain breakthrough. However, due to rely on preservation solution imports, high costs and difficult to obtain, to a certain extent, our organ transplants development will be restrained.Design features of the composition of hypothermia hepatocytes preservation solution used in bioartificial liver support system.Based on the analysis of University of Wisconsin (UW) and Bretschneider solution (HTK), the advantages of organ preservation solution both at home and abroad were drawed, and, the principle of preservation solution composistion was obeyed. Belzer made an effective hypothermia organ preservation according to the organ under hypothermia condition of all kinds of pathological physical features and put forward that it must satisfy the following six aspects:①abate cell swelling;②avoid cell acidification;③avoid enlarging the space between cells;④lighten reperfusion injury;⑤supply material to synthesize energy substance;⑥maintain the internal environment of the cells.Keeping with the recent advancement of organ preservation field,improving the above two advanced preservation solution composition and reducing the cost,the compositions were repeatedly proved by facts. The hypothermia hepatocytes preservation solution used in bioartificial liver support system was abundant in source and cheap in price. Other than the former preservation solution,the compositions which were chiefly of disaccharide,glycans, polypeptide metalion, phosphate,et al were certain.Because of no need to add the other Heterologous Antigens and dimethyl sulfoxide which has a toxic effect on cells,the risk of contaminated with unknown virus and the antigen-antibody reaction were avoided.So,cells can be used to bioartificial liver support system after hypothermia preservation without special processing and then will be well satisfied with ready-to-use deman of the materials used in bioartificial liver support system.The fundamental ingredient of the hypothermia hepatocytes preservation solution used in bioartificial liver support system are able to maintain hepatocytes activity and electrolytes cell physiological level under hypothermia condition.The compositions of this preservation solution maily act on lightening cell acidification, preventing release of hepatocytes lipid peroxidation products, elevating the cellular energy, abate cell swelling.The formula desing absorbed the advancement of University of Wisconsin (UW) and Bretschneider solution (HTK) and also had its own traits.1. With a strong buffer action capacity.2. Choose the impermeability protective agent-trehalose which plays an important role in stabilizing hepatocytes membrane and protein structure and abates cell swelling.3. Added ectogenesis adenosine triphosphate and magnesium chloride as the energy rich phosphate compound substrate so as to elevate cellular energy through ATP-Mg2+.4. Adopted reduced glutathione andα-lipoic acid as Antioxidant System and Antiapoptotic System so as to relieve reperfusion injury and prevent release of hepatocytes lipid peroxidation products.5. Inducted Matrine as the active ingredient in Chinese medicine.Used its antioxidant to enhance protection.6. HES were used to raise the colloid osmotic Pressure and ease the hepatocytes edema.Object:This experimental purposes of research is aimed at finding out the limitation of existing preservation solution, manufacturing a new hypothermia hepatocytes preservation solution used in Biological Artificial Liver support system which is in possession of intellectual property and with low costs,then,the preservation effect of C3A cell which is used in Biological Artificial Liver support system and the principle of hepatocytes preservation will be explored to compare with UW solution. Finally, The results of the experiments are willing to prolong the period of hepatocytes preservation and provide experimental basis for clinical application.Methods:1. Adopted normative praeparatum standardization, in accordance with prescription to prepare the hypothermia hepatocytes preservation solution used in bioartificial liver support system. Regulated the pH of 7.2 to 7.4 and regulated the osmotic pressure of 305±10mmol/L.2.Experiment group:Ⅰgroup:called control group;Ⅱgroup:Ringer lactate solution(RL)group;Ⅲgroup:New hypothermia hepatocytes preservation solution used in Biological Artificial Liver support system(NBS) group; IVgroup:University of Wisconsin (UW) group.3. Hypothermia preservation and resuscitation of hepatocytes:For hypothermic experiments, cells were trypsinized and concentration to 3×105 cells/mL and then seeded onto 6 hole cell culture flask for 48h to allow adherence..The medium was replaced with prechilled RL, NBS and UW solution, respectively, and air-tight sealed with parafilm.Then the cells were preserved by refrigerating at 4℃for indicated time. Cells grown in medium at 37℃were used for negative control. For heat preconditioning, the plate was then moved to regular incubator at 37℃. Then the supernatant was taken to detect.4. Research content and testing methods:After the cells were preserved by refrigerating at 4℃for indicated time, viability of hepatocytes, morphological changes, liver enzymology (ALT; AST), MDA level in hepatocytes, Alb level of hepatocytes after resuscitation and hepatocytes apoptosis rate were detected.5.Statistical analysis:Date processed by SPSS software.Date are presented as mean±SD. Statistical analysis was performed using factorial analysis and ANOVA analysis;p values lower than 0.05 were considered to be significant. Result:1.To characterize the viability of cultured hepatocytes in cold preservatives, we ex-amined the cellular morphology of hepatocytes preserved at 4℃in different pres-ervation solution:The cells that were hypothermically preserved in different pr-eservation solution turned round in shape in varying degrees such as swollen and vi-sible vacuole-like microstructures and so on.There were various degrees of degener-ation and necrosis in those cells preserved in RL for 24 h. In contrast, NBS and UW solution preserved better membrane integrity at respective time points.2.The survival rate of hepatocytes was assayed after cryopreservation:The-re was no significant difference between preserved in NBS and UW for 24h or 48h in the su- rvival rate of hepatocytes.However,the survival rate of hepatocytes preversed in NBS solution at 4℃for 72h was lower than that in UW group. There were vari-ous degrees of degeneration and necrosis in those cells preserved in RL for 24 h and its survival rate of hepatocytes significantly decreased.3. Liver Enzymology changes:ALT and AST were raised in both NBS and UW group after preserved at 4℃.There presented a tremendous rise peak after pres-erved at 4℃for 24h.Then, the peak significantly decreased after the culture medium was renewed. The hepatocytes cultured in DMEM completed culture medium conta-ining 10% FCS continuously after anabiosis.The cell viability and general growth co- ndition in UW group are both better than the NBS group for 24h,48h and 72h.4. Alb level of hepatocytes after resuscitation:There was no significant dif-ference between preserved in NBS and UW for 24h or 48h in the Alb level of hepat-ocytes after resuscitation. However, the Alb level of hepatocytes after resuscitation in NBS group was lower than that in UW group for 72h.5. The analysis of hepatic apoptosis:Apoptosis was determined by acridine orange/PI (AO/PI) double staining.There were abundance hepatic apoptosis in RL group after cryopreservation for 24h. Late apoptotic and necrotic cells presented in RL group for 48h and 72h. The hepatic apoptosis phenomena also can be seen both in NBS and UW group for 24h and 48h. However, there was no significant difference between NBS and UW group at the same time. Conclusion:As a bioartificial liver cells with the effects of cold preservation solution, the NBS preservation solution and UW solution play a similar role in improving the survival rate of hepatocytes and reduce hepatocytes injury, protect the hepatocytes function and prevent hepatocytes apoptosis in cryopreservation.