| The life of patients with the end-stage liver illness is indispensable to support the bioartificial liver(BAL). A typical BAL is generally composed of hepatocytes, biology reactor and plasm perfusion system, and hepatocytes act as the core. At present, most of the hepatocytes for HBAL come from the animal derived primary culture cells, liver stem cell, immortalized hepatocytes, gene-engineered hepatocytes and human derived hepatocytes. While the virus contamination was an unresolved problem for animal derived hepatocytes, liver stem cell can not be used for HBAL right now for it studies only begins recently; gene-engineered hepatocytes are not only an technologically difficult but there are not known suitable genes now. Human hepatocytes are an ideal cell source for BAL, because they have no concern of animal derived virus infection and with satisfactory biological function, such as the production of albumin, hepatocyte regeneration factors, blood clotting factors and so on. Immortalized human hepatocytes are also a potentical cell source of BAL, because they are easy obtain, morphologically identical, functionally stabilized, as well as the same biological products the human liver cells, for example the albumin.A BAL system needs 200 gram hepatocytes for operation. In most clinically used, bAL's human hepatocytes, should account for 15% or the levelof 109. To solve the problem of hepatocytes shortage are usually cultured continuously or seed in some carrier, are laborious, easy pollution, highly expensive, as well as low cell viability and inadguate function, cryopreservation of hepatocytes is an important method to solve the problem of hepatocytes shortage. Although step freezing and rapid thaw are worldwidely used for hepaocyte cryopreservation, There are no consent on cryoprotective agent(CPA) dosage, cryopreservation solution ingredients and preservation time. CPA is the core of hepatocytes cryopreservation, and among them, DMSO is a widely used CPA that can lower cell freezing point , reduce cell freezed crystal, reduce cell damage by free radicals, stabilze membrane permenbility to electrolyte, drug, toxic substances and metabolizes matter. However, DMSO itself possesses serious toxicity, which leads to protein denaturalization by interacting with ion groups of protein. It is important to find some other CPA to reduce the dosage or toxicity of DMSO, and to improve hepatocyte function. Matrine, an alkaloids, is a main ingredient of quinoilizidine. Matrine can reduce hepatocyte damage by eliminating free ridicals and protecting cell membrance. Matrine can cure some liver diseases in clinical. Therefore,it is interested to know Whether can matrine itself or plus DMSO protect hepatocytes. Here we conducted experiments to identify the factors influence the function of human fetal hepatocytes and immortalized L-02 hepatocytes preserved in liquid nitrogen .Part I : the influence of Matrine on the thawed human fetal hepatocytespreserved in liquid nitrogenObjective To find out the influence of Matrine on the thawed human fetal hepatocytes preserved in liquid nitrogen and establish a suitable method for the cryopreservation of hepatocytes.Methods Human fetal hepatocytes were harvested by two-step collagenaseperfusion, and then stored in a liquid nitrogen for one month with five different cryoprotectants (only 10%DMSO, 5%DMS0+ 0.2mg/L Matrine, 5%DMSO+2mg/L Matrine, 5%DMSO+ 6mg/L Matrine ,and 5%DMSO+ lOmg/L Matrine). One month later, the cells were thawed rapidly and its viability, morphology and cell function were measured.Results The human fetal hepatocytes, preserved in different cryoprotectants, showed the different viability, morphological manifestation and cell function. Among the five methods, a combination of 5%DMSO with 6mg/L Matrine was the best. After thawing, the viability was 88% and flash adhereing was 82%, cell function in the group of cells was better cryopreserved with 5%DMSO+ 6ug/ml Matrine than that in others groups(P<0.05).Conclusions First, the human hepatocytes were preserved at -196 degree C with Matrine, with different dosages of Matrine,different preservation effect on human hepatocytes obtained,the 6mg/L Matrine dosage was the best; Second, Matrine has synergisic effects with DMSO,the dosage of DMSO can be reduced in the preservation solution.Part II :The influence of Matrine on the thawed immortalized L-02 hepatocytespreserved in liquid nitrogenObjective To find out the influence of combined use of Matrine ^ DMSO with Detrax-40 on the thawed immortalized L-02 hepatocytes preserved in liquid nitrogen and establish a suitable method for the cryopreservation of hepatocytes.Methods The immortalized L-02 hepatocytes were cultured as usual, Harvested by trypsin digestion and stored in a liquid nitrogen for one month with six different cryoprotectants (only 10%DMSO, 5%DMSO+ 5%Detrax-40, 5%DMSO+ 5%Detrax-40+0.2mg/LMatrine, 5%DMSO+5%Detrax-40+2mg/LMatrine,5%DMSO+5%Detrax-40+6mg/LMatrine,5%DMSO+5%Detrax-40+10mg/LMatrine). After one month, the cells were thawed rapidly and its viability, morphology and cell function were measured.Results The immortalized L-02 hepatocytes, preserved in different cryoprotectants, showed the different viability, flask addhering, morphological manifestation and cell function. Among the six methods, the group of only DMSO showed the worst effect while the group of DMSO+ Detrax-40+6 rag/L Matrine had the best results. After thawing, the viability of immortalized L-02 hepatocytes was 90% and flask addhering efficiency was 83%, These parameters including cell function were more favorable in the immortalized L-02 hepatocytes cryopreserved with the method of the combination use of DMSO+ Detrax-40+ 6 mg/ L Matrine than with that of others(P<0.05).Conclusions First, the immortalized L-02 hepatocytes were preserved at -196 °C with Matrine, with different dosages of Matrine,different preservation effect on immortalized L-02 hepatocytes obtained,the 6mg/L Matrine dosage was the best; Second, Matrine has plus effects with DMSO -. Detrax-40,combination of 5%DMSO+ 5%Detrax-40+6 mg/L Matrine was a good cryoprotectant and can improve the preservative quality of immortalized L-02 hepatocytes significantly. |
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