Subzero Nonfreezing Storage Of The Immortalized Hepatocytes Used By Bioartificial Liver Support System

The mortality of liver failure caused by multiple risk factors is as high as 80%, and the original treatment and the general support treatment do a little effect. Liver transplantation is currently considered to be the most effective treatment for acute or chronic liver failure. However, the donor liver for transplantation is deficient and the liver transplantation have many disadvantages such as complex surgery, lifelong immunosuppressive therapy, a great number of expenditure and so on. At last, many patients die while waiting for the donor liver. The development of bioatificial liver support system (BLASS) has brought up a new avenue for the treatment of liver failure. On the one hand, some patients with acute liver failure are reversible, and the liver function is recovery by the compensatory hyperplasia of the remaining normal liver cells with short-term effective BLASS supporting; on the other hand, if acute or chronic liver failure is irreversible, the patients could win the valuable time for the final liver transplantation undergoing the BLASS supporting. In conclusion, the BLASS construct a bridge between the liver failure and liver transplantation.The hepatocytes in vitro used by the BLASS should achieve the following three requirements:①Quality requirements:The hepatocytes should-have the major functions such as toxins clearing, biological transformation, albumin secretion, urea synthesis and so on.②Quantity requirements:in order to achieve the clinical supporting, we need at leALT a great number of liver cells at 109 magnitudes.③ Rapid clinical application:The patients with liver failure caused by various reasons are often in critical condition, so a large number of liver cells in vitro for BLASS must be obtained within a short time. Therefore, exploring a reliable cryopreservation method and creating a ready to use "cell bank" which have a large number of high-quality available hepatocytes is the key point of BLASS, and the"cells bank" which restrict the clinical widely application is the bottleneck of BLASS.Temperature is key factor in cold storage. The lower the temperature is, the lower the cells' metabolic activity and the longer retention time are on the whole. Subzero nonfreezing temperature (SZNFT) is a range from 0℃to the freezing point of a solution. On the one hand, SZNFT could avoid the cryopreservation injury such as "ice formation" and"the lethal osmolality"; On the other hand, SZNFT is different from the conventional hypothermic temperature(4℃). Therefore, SZNFT is an ideal way for hepatocytes cold storage. At first, SZNFT is used for donor liver preservation, and the preservation time is extended. However, the donor liver preservation is mixed with the ischemia reperfusion injury, which can not fully reflect the effect of hepatocytes preservation. Nowadays, the effect of the immortalized hepatocytes stored at a SZNFT is still not clear. Therefore, compared with the conventional low temperature (4℃and 0℃), the effect of the hepatocytes stored in UW solution at-0.8℃is investigated in the first part of this article.The hepatocytes used by bioatificial liver support system have not yet been satisfactorily resolved. The C3A is a kind of immortalized tumor derived hepatocyte with good proliferate activity and the specific functions such as the ALB secretion, urea synthesis and glycogen synthesis. The L02 is a kind of immortalized normal hepatocyte with stable proliferate activity and the specific functions such as amino clearance and albumin secretion. The function changes of the C3A and the L02 hepatocytes stored at a SZNFT are still unclear. We would evaluate the biological features of C3A and L-02 hepatocytes and theirs application proneness undergoing subzero nonfreezing storage in University of Wisconsin (UW) solution for use in bioartificial liver support system in the second part.The University of Wisconsin solution is now considered the standard solution for donor liver preservation. With the development of BLASS, more and more hepatocytes are needed. UW solution has also been used for hepatocytes short term preservation and the effect is obvious. However, UW solution is very expensive and some ingredients are hard to obtain. The Celsior solution and Histidine-tryptophan-Ketoglutarate (HTK) solutions which are relatively inexpensive are also used for donor liver preservation, but the differences of the hepatocytes respectively stored in UW solution, Celsior solution and HTK solution at a SZNFT have not been reported. Therefore, compared with the Celsior solution and HTK solution, the effect of the L02 hepatocytes stored in UW solution 72h at-0.8℃and the possible mechanism are investigated in the third part of this article.Besides of cell necrosis, many research indicat that cell apoptosis is another important way of cryopreservation death. However, the specific mechanism of cell apoptosis caused by cold storage is still not clear. A major apoptotic-signaling pathway involves the release of cytochrome C (cyt C) from the mitochondrial intermembrane space into the cytoplasm in response to apoptotic stimulation. Current models implicate the mitochondrial permeability transion pore (MPTP) as a key player in cyt C release. Integral to the maintenance of the MPTP are members of the Bcl-2 protein family. Bax, a known pro-apoptotic protein, located on the outer mitochondrial membrane, causing the MPTP opening and the cyt C release. Bcl-2, an anti-apoptotic protein located on the mitochondrial membrane, binding to the Bax and blocking the cyt C release. Therefore, the Bcl-2/Bax ratio can be used as a predictive marker of apoptotic index. However, cell apoptosis caused by cold preservation and the relationship with the Bcl-2/Bax mRNA and protein expression of hepatocytes which are stored at a subzero nonfreezing temperature are still unclear. Therefore, our research respectively store L02 hepatocytes at a subzero nonfreezing temperature(-0.8℃) and conventional hypothermic temperature(4℃and 0℃). At 1ALT, we investigate cell apoptosis caused by cold preservation and the relationship with the Bcl-2/Bax mRNA and protein expression, which would promote our comprehension of possible apoptosis mechanism. ObjectiveThe L02 immortalized hepatocytes were cultured in vitro and the suspensions were stored in UW solutions. After 24h,48h,and 72h cold storage, we investigate the effect and the relationship with cells apoptosis of Subzero nonfreezing storage (-0.8℃) compared with conventional hypothermic storage (4℃and 0℃) by using L02 hepatocytes for bioartificial liver support system.Methods1. Cell Culture and Treatment:The L02 hepatocytes were removed from the liquid nitrogen, and the cells were rapidly put in 37℃water bath. Then the cells were transferred into the super clean bench until the cells were completely dissolved.①According to the strict aseptic method, the cells were inoculated to the 25cm culture bottle. The medium was used DMEM/F-12 nutrient medium with 10% FBS,20ng/ml EGF, 10mg/L insulin,10kU/Lpenicillin, andlOg/L streptomycin.②the culture bottles were put into the carbon dioxide incubator, and the culture conditions were maintained at 37℃,5% CO2,100% Humidity.③The cells were observed by inverted microscope (CK2 model, Olympus), and cell culture medium (5ml) was replaced every 24h until the bottle walls were covered with hepatocytes.④The culture bottles were taken out of carbon dioxide incubator and the culture medium were abolished. The cell monolayer was detached by addition of lml of 0.25% trypsin for 1-3min at 37℃. Once the cell cytoplasm retracted and the cell gap increased, the 0.25% trypsin was abolished and the cells digestion was inhibited.⑤About 2ml culture medium was added to cell monolayer and the cell monolayers were prepared into cell suspension by the repeated pipetting with a straw wall.⑥The same batch of the L02 cells were mixed together. Taking a drop of the cell suspension to the CBC board and calculating the cell density. At 1ALT, the cell concentration was finally adjusted to 1×109/L and then filled to the freezing tube (2mL). 2. Cell Cold Storage and Treatment:①The packing tubes were centrifuged (1000r/min,2min), and the culture medium with 10%FBS was replaced with 2ml UW solution and the centrifuged cells and UW solution were mixed together. Then the freezing tubes were respectively divided into the following three groups:Subzero nonfreezing group (-0.8℃), zero nonfreezing group (0℃) and control group (4℃), and every group had 18 samples.②After 24h,48h and 72h cryopreservation,6 samples in each group were removed from the hypothermic storage. By the rapid thawing method, the samples were placed in 37℃bath and shocked for 1-2min.③The thawing samples were centrifuged (1000r/min,2min), and the UW solution was replaced with the 2ml culture medium. The centrifuged cells and the culture medium were mixed together, and then were put into the carbon dioxide incubator for 30min which was maintained at 37℃,5% CO2,100% Humidity.3. The Testing Component:The testing contents of the rewarming cells after cold storage were summarized as follows:(1)About lml hepatocytes suspensions were inoculated to the 6-well culture plates and every well had lml culture medium with 4mmol/L Ammonium Chloride, and the wells were put into the carbon dioxide incubator, and the culture conditions were maintained for 24h at 37℃,5% CO2,100% Humidity.①the cell suspensions were centrifuged (2000r/min,20min), and the ALT, LDH release and the urea in the cell-free supernate were determined by automatic biochemical analyzer.②According to the introduction of the Human albumin ELISA kit, The albumin in the cell-free supernate was measured by automatic microplate reader.③The cell suspensions from the adherent cells were prepared by HE staining, and the cell morphology was observed. (2)According to the introduction of the ATP kit, about 0.5 ml L02 hepatocytes suspensions were used for the test of the ATP content. According to the introduction of the lactic acid kit,the lactic acid production in the cell-free supernate was determined by the Spectrophotometer. (3)About 0.5ml cell suspensions were centrifuged (1000r/min, 2min). According to the introduction of the AnnexinV-FITC apoptosis kit, the centrifuged cells were tested by the Flow Cytometry and the cell viability rate and cell apoptosis rate were calculated.4. The cooling curve and the freezing point of different solutions were determined as follows:about 1ml distilled water, UW solution or cell suspensions were respectively placed in the freezing tube (2mL), and the smart thermometer was inserted into the solution (Not connecting with the wall). The tubes were put into the program controlled incubator at-10℃, and the temperature was recorded every minute. Then the temperatures were plotted as time-temperature curve [temperatures were taken from 10℃to (-10)℃].Results1. The freezing point and SZNFT of different solutions were different as follows: The freezing point of the distilled water was 0℃; The freezing point of the UW solution was -1℃, and the SZNFT was from 0℃to-1℃; The freezing point of L02 suspensions was-1℃, and the SZNFT was from 0℃to-1℃.2. The viability rate of the fresh cells was 86.95%±0.58%, and the cell viability rate descended with the prolonged cold storage (P<0.001). After 24h cold storage, there were no differences among the three groups; After 48h cold storage, compared with the control group (4℃), the difference of the cell viability rate in Subzero nonfreezing group(-0.8℃) began to appear. Compared with the fresh cells, the viability rate in different groups apparently descended after 72 h of hypothermic storage, but the percentage of viable L02 hepatocytes was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance (P<0.001).3. The cell apoptosis rate of the fresh cells was 1.73%±0.20%, and the cell apoptosis rate increased with the prolonged cold storage (P<0.001). After 24h,48h cold storage, there were no differences among the three groups;Compared with the fresh cells, the cell apoptosis rate in different groups apparently increased after 72 hours of hypothermic storage, but the percentage of cell apoptosis hepatocytes was significantly lower in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance (P=0.017).4. The ALT and LDH release of the fresh cells were respectively 3.83U/L±0.48U/L and 49.52U/L±5.57U/L, and the ALT and LDH release increased with the prolonged cold storage(BothP<0.001). After 24h cold storage, there were no differences among the three groups about the ALT and LDH release; After 48h cold storage, compared with the control group (4℃), the differences of the ALT and LDH release in Subzero nonfreezing group(-0.8℃) began to appear. Compared with the fresh cells, the ALT and LDH release in different groups apparently increased after 72h of hypothermic storage, but the ALT and LDH release were significantly lower in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(Both P≤0.001).5. The urea synthesis of the fresh cells was 2.63mmol/L±0.46mmol/L, and the cell urea synthesis decreased with the prolonged cold storage(P<0.001). After 24h cold storage, there were no differences among the three groups; After 48h cold storage, compared with the control group (4℃), the differences of urea synthesis in Subzero nonfreezing group(-0.8℃) began to appear. Compared with the fresh cells, the urea synthesis in different groups apparently decreased after 72 hours of hypothermic storage, but the urea synthesis of the hepatocytes was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(P=0.001).6. The albumin secretion of the fresh cells was 11.90mg/L±0.39mg/L, and the albumin secretion decreased with the prolonged cold storage(P<0.001). After 24h cold storage, there were no differences among the three groups; After 48h cold storage, compared with the control group (4℃), the difference of albumin secretion in Subzero nonfreezing group(-0.8℃) began to appear; Compared with the fresh cells, the albumin secretion in different groups apparently decreased after 72 hours of hypothermic storage, but the albumin secrete of the hepatocytes was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(P=0.003).7. The lactic acid production of the fresh cells was 3.48ug/10^6cells±0.81 ug/10^6cells, and the lactic acid production increased with the prolonged cold storage(P<0.001).After 24h cold storage, compared with the control group (4℃), the difference of the lactic acid production in Subzero nonfreezing group(-0.8℃) began to appear. Compared with the fresh cells, the lactic acid production in different groups apparently increased after 48h,72h of hypothermic storage, but the lactic acid production was significantly lower in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(P<0.001).8. The ATP Contents of the fresh cells was11.29ug/10^6cells±1.66ug/10^6cells, and the ATP Contents decreased with the prolonged cold storage(P<0.001). After 24h cold storage, compared with the control group (4℃), the differences of the ATP Contents in Subzero nonfreezing group(-0.8℃) began to appear. Compared with the fresh cells, the ATP Contents in different groups apparently decreased after 48h,72h of hypothermic storage, but the ATP Contents was significantly lower in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(.P<0.001).9. The fresh cells had integral membrane and had a distinguishable cell nucleus and cell cytoplasm, and the death rate was lower; After 72h cold storage,there were sentus-like structures found around the cell membrane and vacuole-like micro-structures found in those cells preserved both in zero nonfreezing group(0℃) and control group (4℃), which were not observed in Subzero nonfreezing group(-0.8℃).Conclusion1. Subzero nonfreezing storage (-0.8℃) of the hepatocytes stored in UW solution could pride better cell viability and the hepatocytes function, and lower cell apoptosis.2. The cell viability rate of the hepatocytes stored at a Subzero nonfreezing temperature for 72h was about 70%, and Subzero nonfreezing was an effective way to store the hepatocytes need by the BLASS for a short-term time.3. The cell viability rate of the hepatocytes stored at a Subzero nonfreezing temperature was significantly improved by reducing the apoptosis rate.4. Subzero nonfreezing storage(-0.8℃) of hepatocytes and constructing a "ready to use" cell bank which likes the "blood bank" would efficiently promote the development of the BLASS.The second part The biological features comparison of C3A and L02 hepatocytes stored at a SZNFT in UW solutionObjectiveTo evaluate the biological features of C3A and L-02 hepatocytes and theirs application proneness undergoing subzero nonfreezing storage in University of Wisconsin (UW) solution used by the bioartificial liver support systems in the second part.MethodThe specific methods had been shown in the first part.Results1. The viability rate of the C3A and L02 hepatocytes descended with the prolonged cold storage(P<0.001).After 24h,48h and 72h cold storage, the viability rate of the C3A hepatocytes was higher than that of the L02 hepatocytes with statistical significance (all P<0.001).2. The apoptosis rate of the C3A and L02 hepatocytes increased with the prolonged cold storage (P<0.001). After 24h,48h and 72h cold storage, the apoptosis rate of the C3A hepatocytes was lower than that of the L02 hepatocytes with statistical significance (all P≤0.005).3. The ALT release of the C3A and L02 hepatocytes increased with the prolonged cold storage (P<0.001). After 24h,48h and 72h cold storage, the ALT release of the C3A hepatocytes was lower than that of the L02 hepatocytes with statistical significance (all P≤0.011).4. The LDH release of the C3A and L02 hepatocytes increased with the prolonged cold storage (P<0.001). After 24h,48h and 72h cold storage, the ALT release of the LDH hepatocytes was lower than that of the L02 hepatocytes with statistical significance (all P≤0.001).5. The urea synthesis of the C3A and L02 hepatocytes decreased with the prolonged cold storage (P<0.001). After 24h,48h and 72h cold storage, the urea synthesis of the C3A hepatocytes was lower than that of the L02 hepatocytes with statistical significance (all P≤0.002).6. The albumin secretion of the C3A and L02 hepatocytes decreased with the prolonged cold storage (P<0.001). After 24h,48h and 72h cold storage, the albumin secretion of the C3A hepatocytes was higher than that of the L02 hepatocytes with statistical significance (all P<0.001).Conclusion1. The cell viability, apoptosis rate, ALT and LDH release, and the albumin secretion of the C3A hepatocytes were better than the L02 hepatocytes after cold storage.2. The urea synthesis of the L02 hepatocyte was better than the C3A hepatocyte after cold storage.3. The L02 hepatocyte was more probably suitable for BALSS for the treatment of liver failure with hepatic encephalopathy4. The C3A hepatocyte was more probably suitable for BALSS for the treatment of liver failure with low albuminoidal. The third part To compare the effect of hepatocytes respectively stored in UW, Celsior and HTK solution at a SZNFTObjectiveThe immortalized L02 hepatocytes were cultured in vitro, and the suspensions were respectively stored in UW solution, Celsior solution and HTK solution. After 72h cold storage, we compared the effect and the possible reasons of L02 hepatocytes respectively stored in UW solution, Celsior solution and HTK solution at a SZNFT (-0.8℃).Methods1. Cell Culture and Treatment:The L02 hepatocytes were removed from the liquid nitrogen, and the cells were rapidly put in 37℃water bath. Then the cells were transferred into the super clean bench until the cells were completely dissolved.①According to the strict aseptic method, the cells were inoculated to the 25cm culture bottle. The medium was used DMEM/F-12 nutrient medium with 10% FBS,20ng/ml EGF, 10mg/L insulin,10kU/Lpenicillin, andlOg/L streptomycin.②The culture bottles were put into the carbon dioxide incubator, and the culture conditions were maintained at 37℃,5% CO2,100% Humidity.③The cells were observed by inverted microscope (CK2 model, Olympus), and cell culture medium (5ml) was replaced every 24h until the bottle walls were covered with hepatocytes.2. Cell Cold Storage:①The culture medium with 10%FBS was respectively replaced with 5ml UW solution, Celsior solution and HTK solution, and then were clod stored at-0.8℃and each group had 6 samples.②After 72h cryopreservation,6 samples in each group were removed. The UW solution, Celsior solution and HTK solution were replaced with culture medium (5ml). By the rapid thawing method, the samples were placed in the carbon dioxide incubator for 30min which was maintained at 37℃,5% CO2,100% Humidity.3. The cell suspensions were got as follows:①The culture bottles were taken out of carbon dioxide incubator and the culture medium were abolished. The cell monolayers were detached by addition of 1ml of 0.25% trypsin for 1-3min at 37℃Once the cell cytoplasm retracted and the cell gap increased, the 0.25% trypsin was abolished and the cells digestion was inhibited.②About 2ml culture medium was added to cell monolayer and the cell monolayers were prepared into cell suspension by the repeated pipetting with a straw wall.③Taking a drop of the cell suspension to the CBC board and calculating the cell density. At 1ALT, the cell concentration was finally adjusted to 1×109/L.4.The testing contents of the rewarming cells after cold storage were summarized as follows:(1)About 1ml hepatocytes suspensions were inoculated to the 6-well culture plates and every well had 1ml culture medium with 4mmol/L Ammonium Chloride. The wells were put into the carbon dioxide incubator, and the culture conditions were maintained for 24h at 37℃,5% CO2,100% Humidity.①the cell suspensions were centrifuged (2000r/min,20min), and the ALT, LDH release and the urea in the supernate were determined by automatic biochemical analyzer.②According to the introduction of the Human albumin ELISA kit, The albumin secrete in the cell-free supernate was measured by automatic microplate reader. (2)About 1ml cell suspensions were centrifuged (1000r/min,2min). According to the introduction of the Live/Dead kit, the centrifuged cells were tested by the Flow Cytometry and the cell viability rate and cell dead rate were calculated.Results1. The viability rate of the fresh cells was 89.33%±2.64%, and the cell death rate was 10.08%±2.56%. Compared with the fresh cells, the cell viability rate descended after 72h of hypothermic storage. The percent of the viable cells stored in UW solution was higher than that in the Celsior solution and HTK solution (P<0.001).Compared with the fresh cells, the cell dead rate increased after cold storage for 72h, but the percent of dead cells stored in UW solution was lower than that in the Celsior solution and HTK solution (P<0.001).2. The ALT release of the fresh cells was 3.93 U/L±1.04U/L, and the LDH release of the fresh cells was 44.00 U/L±5.72U/L. Compared with the fresh cells, the ALT and LDH release increased after 72 hours of hypothermic storage, but the ALT and LDH release of the cells stored in UW solution were lower than that in the HTK solution(BothP≤0.010). There were no differences about the ALT release between the UW solution and the Celsior solution (P=0.070). However, the LDH release was adverse between the UW solution and the Celsior solution (P=0.004).3. The urea synthesis of the fresh cells was 2.60mmol/L±0.56mmol/L,and the albumin secretion of the fresh cells was 12.21mg/L±1.68mg/L. Compared with the fresh cells, the ability of urea synthesis and albumin secretion decreased after hypothermic storage for 72 h. The urea synthesize of the cells stored in UW solution was higher than that in the HTK solution (P=0.002). However, there were no differences about the urea synthesis between the UW solution and the Celsior solution (P=0.358). The albumin secretion of the cells stored in UW solution was lower than that in the Celsior solution and the HTK solution (P=0.015), and there were no differences about the albumin secretion between the Celsior solution and the HTK solution (P=0.618).Conclusion1. The cell viability of the hepatocytes stored in Subzero nonfreezing UW solution (-0.8℃) was higher than that in the Celsior solution and the HTK solution, and the HTK solution has the lowest cell viability.2. There was no difference between the UW solution and the Celsior solution about urea synthesis of L02 hepatocytes, which were better than the HTK solution.3. There was no difference between the HTK solution and the Celsior solution about the albumin secretion of L02 hepatocytes, which were lower than the UW solution.4. The time of the hepatocytes stored in UW solution at a SZNFT was better no more than 72h, and sometimes the UW solution could be replaced with the Celsior solution.The fourth part To investigate the mechanisms of the cell apoptosis caused by cold storage at a SZNFTObjectiveThe immortalized L02 hepatocytes were cultured in vitro, and the suspensions were respectively stored at a subzero nonfreezing temperature(-0.8℃) and the conventional temperature(4℃and 0℃).Compared with the conventional temperature(4℃and 0℃), we investigated the differences of cell apoptosis caused by cold preservation and the relationship with the Bcl-2/Bax mRNA and protein expression of the L02 hepatocytes at a SZNFT(-0.8℃).Methods1. The specific methods had been shown in the third part.2. The testing contents of the rewarming cells after cold storage were summarized as follows:(1)About lml hepatocytes suspension were centrifuged (2000r/min,20min), and the ALT, LDH releas in the cell-free supernate were determined by automatic biochemical analyzer. According to the introduction of the AnnexinV-FITC apoptosis kit, the centrifuged cells were tested by the Flow Cytometry, and the cell viability rate and cell apoptosis rate were calculated. (2) About lml hepatocytes suspensions were centrifuged (1000r/min,2min), and the precipitated hepatocytes were used for Real-time quantitative PCR (Eppendorf realplex4, Germany). Total cells RNA were extracted by using MagExtraor-RNA kit (NPK201, TOYOBO). Real-time quantitative PCR analysis was performed according to the manufacturer's instructions (RNA-direct SYBR Green Real-time PCR kit, QRT201, TOYOBO). The target genes were Bcl-2 and Bax, and theβ-actin was applied as an internal control. (3)About 1ml hepatocytes suspension were centrifuged (1000r/min,2min), and the precipitated hepatocytes were used for Western Blotting. Total Cell extracted from cultured hepatocyte was obtained according to the manufacturer's instructions of the protein extraction kit. The target protein was Bcl-2 and Bax, and theβ-actin was applied as an internal control.Results1. The viability rate of the fresh cells was 87.35%±1.79%, and the apoptosis rate was 1.73%±0.26%. Compared with the fresh cells, the viability rate in different groups apparently descended after 72 hours of hypothermic storage, but the percentage of viable L02 hepatocytes was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance(P<0.001); However, the apoptosis rate was totally antipodal with statistical significance (P=0.019).2. The ALT release of the fresh cells was 3.78U/L±0.48U/L, and the LDH release was 39.48U/L±5.73U/L. Compared with the fresh cells, The ALT and LDH release in different groups apparently ascended after 72 hours of hypothermic storage, but both the ALT and the LDH release were significantly lower in Subzero nonfreezing group(-0.8℃) than that in control group (4℃) with statistical significance (bothP≤0.001). There were no differences about the ALT release between Subzero nonfreezing group(-0.8℃) and the zero nonfreezing group (0℃) (P=0.088). However, the LDH release was adverse between Subzero nonfreezing group(-0.8℃) and the zero nonfreezing group (0℃) (P=0.002).3. The total cells RNA were extracted by using MagExtraor-RNA kit (NPK201, TOYOBO), and the manipulate program was strictly performed according to the manufacturer's instructions. Then, the ratio of A260/A280 of the extracted total RNA was measured by spectrophotometer, and the normal ratio were from 1.8 to 2.0; the 1.2% agarose electrophoresis of total cells RNA in every group showed three clear RNA bands:28S,18S, and 5S. Therefore, the total extracted RNA had a good quality and could be used for Real-time-PCR assay.4. Theβ-actin was applied as internal control, and the real-time quantitative Bcl-2 and Bax mRNA expression of hepatocytes in different groups including the fresh cells were done. The Bcl-2 and Bax mRNA expression of the fresh cells was on a basic level. The Bcl-2 mRNA expression in was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and the control group (4℃) with statistical significance (P<0.001), and there was no difference between the zero nonfreezing group (0℃) and the control group (4℃) (P=0.387).However, the Bax mRNA expression was totally antipodal (P<0.001), and there was no difference between the zero nonfreezing group and the control group (4℃) (P=0.432). Compared with Subzero nonfreezing group, the ratio of Bcl-2/Bax were significantly lower in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance (P<0.001).5. The P-actin was applied as internal control, and the Bcl-2/Bax protein expression of hepatocytes in different groups including the fresh cells were done. The Bcl-2 and Bax protein expression of the fresh cells was on a basic level. The Bcl-2 protein expression was significantly higher in Subzero nonfreezing group(-0.8℃) than that in the zero nonfreezing group (0℃) and the control group (4℃) with statistical significance (P<0.001), and there was no difference between the zero nonfreezing group (0℃) and the control group (4℃) (P=0.300).The Bax protein expression was totally antipodal (P=0.006). Compared with Subzero nonfreezing group, the ratio of Bcl-2/Bax were significantly lower in the zero nonfreezing group (0℃) and control group (4℃) with statistical significance (P<0.001).Conclusion1. Compared with conventional temperature(0℃and 4℃), hepatocytes stored at a subzero nonfreezing temperature(-0.8℃) had the lower cell apoptosis rate.2. The mitochondrial associated apoptosis pathway played an important role in the hepatocytes cold stored. 3. Subzero nonfreezing storage of hepatocytes having lower apoptosis might be related with the changes of the Bcl-2/Bax ratio.