Mechanism of cryopreserved porcine hepatocyte xenotransplantation in treating acute hepatic failure

Abstract

AIM: To study the effect of hypothermia on structure of membrane proteins of pig hepatocytes and the mechanism of transplantation of cryopreserved porcine hepatocytes in treating acute hepatic failure in Wistar rats.

METHODS: Isolated porcine hepatocytes were cryopreserved in liquid nitrogen for 150 d and then transplanted to the rat abdominal cavity. Three days later, 80% of rat liver by weight was resected. Conformation of membrane proteins of the porcine hepatocytes, liver function and weight of the remaining liver in the rats were measured.

RESULTS: Viability of the cryopreserved hepatocytes was 78%. Light and electron microscopy showed that the hepatocytes were morphologically intact. The cells were rich in glycogen and glucose-6-phosphatase. The α-helix content in the membrane proteins of fresh cells was 48%. After freezing the cells, the content was increased to 59.5%. Survival rate of rats in the transplanted group was 16/17, significantly higher than 3/9 in the control group (P = 0.0022). However, there were no significant differences in the weight of the remaining liver or indices of liver function between the two groups.

CONCLUSION: Transplant of frozen porcine hepatocytes is effective in treating acute hepatic failure. The mechanism is suggested to be a result of multiple effects. Hypothermia could increase α-helix content in the cellular membrane proteins, which might be related to the report that hypothermia was able to reduce the immunogenicity of the transplanted tissues and cells.

Key Words: Liver failure, acute, Cryopreservation, Liver/cytology, Transplantation, Heterologous, Membrane proteins

INTRODUCTION

Hepatocyte transplantation has been applied to the treatment of acute hepatic failure (AHF) and congenital disturbed hepatic diseases[1]. It was shown that after long-term cryopreservation of hepatocytes, the shape and functions of hepatocytes could be maintained[2] and AHF could be treated effectively[3]. The immunogenicity of tissues and cells decreased obviously after being frozen[4], but the mechanism is not known completely. In this study, we explored the effect of hypothermia on antigen structure of pig hepatocellular membranes and the mechanism of transplantation of cryopreserved porcine hepatocytes in treating AHF in Wistar rats.

MATERIALS AND METHODS

Preparation of pig hepatocytes and histologic examination

A Chinese experimental minipig[5] weighing 7 kg was used as the donor. After gradient cooling, the isolated porcine hepatocytes[6] were preserved in liquid nitrogen (-196 °C) for 150 d[3]. Before transplantation, the cell viability was measured and the hepatocytes were observed under light and electron microscope. HE staining and histochemical staining for glycogen were used in light microscopic observations. The percentage of cells containing glucose-6-phosphatase (G6Pase; ++ - ++++) was counted. Fresh and cryopreserved hepatocytes of the pig were cultured routinely for 48 h.

Preparation of hepatocellular membrane proteins and conformational measurement

Porcine hepatocytes were washed with a solution containing 0.05 M boric acid, 0.15 mol/L NaCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂ and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) (pH 7.2). The cells were centrifuged at 450 g for 5 min and then lysed with 100 volumes of 0.02 mol/L boric acid, 0.2 mmol/L EDTA and 0.1 mmol/L PMSF (pH 10.2). Whole cells were removed by centrifugation at 12000 g for 30 min. Cell membranes were then suspended in PBS (pH 7.4), layered on 35% (w/v) sucrose in PBS and centrifuged at 7000 g for 30 min. The material at the sucrose-PBS interface was membrane proteins[7]. Their conformations were calculated from their circular dichroism spectra.

Animal groups and pig hepatocyte transplantation

Wistar rats weighing 200-250 g were used, and 80% of the liver by weight was resected to induce AHF. The AHF rats usually died within 7 d after the operation[3]. The experimental animals were divided into two parts. The first part was used to observe the survival rate and living situation during the survival for one month after 80% hepatectomy. This part was further divided into two groups: transplant group (I_t, 17 rats) and control group (I_c, 9 rats). The second part was sacrificed on the 2^nd or 7^th d following AHF to measure the liver function and the weight of the remaining liver in the AHF rats, and to do histological examination. This part was divided into two groups too: transplant group (II_t, 37 rats) and control group (II_c, 27 rats). Fifteen normal rats provided comparative standards of the liver weight and function. 4 × 10⁷ cryopreserved hepatocytes in 2 mL were injected into the abdominal cavity of the rats in the two transplant groups before and after 80% hepatectomy[6]. Normal saline was injected into the rats of the two control groups.

Biochemical values of liver function

Biochemical values of liver function included serum concentrations of albumin (ALB), total bilirubin (TBr) and direct bilirubin (DBr).

Statistical analysis

Data were processed by variance analysis and q-test.

RESULTS

The viability of fresh pig hepatocytes was 84%, and that of cryopreserved cells was 78%. Light microscopy showed that the frozen hepatocytes had a spherical shape and were rich in glycogen and G6Pase. The hepatocytes containing G6Pase (++ - ++++) accounted for 97% of total liver cells. There was no apparent difference between the cryopreserved cells and fresh ones under an electron microscope. The cells grew very well in culture and there was no difference either.

As regards to the conformation of membrane proteins, the α-helical structure accounted for 48% in fresh cells, and 59.5% in frozen cells.

The survival rate of group I_t was 16/17, which was significantly higher than that of group I_c (3/9) (P = 0.0022, exact probability). The AHF rats showed low consciousness and appetite, but the situation in the transplant group was a little better than that in the control group. Twelve rats of group II_t and 16 of group II_c died before sacrifice. Varying degrees of light yellow ascites were found by autopsy in the dead rats. The remaining livers of 10 out of 13 sacrificed rats in group II_t and of 6 out of 8 sacrificed rats in group II_c were weighed 2 d after the 80% hepatectomy. The same procedures were performed in another set of rats and in 10 normal rats 7 d after the 80% hepatectomy. The weights of remaining livers in groups II_t and II_c were 6.34 ± 0.88 g (n = 10) and 6.22 ± 0.95 g (n = 6), respectively, 2 d after the 80% hepatectomy, and 8.80 ± 1.02 g (n = 10) and 8.22 ± 0.50g (n = 3), respectively, 7 d after the 80% hepatectomy. There was no significant difference between groups II_t and II_c (P > 0.05), but the weights of livers at 7 d in both groups were significantly lower than that of the normal rats (13.27 ± 1.99 g, n = 10) (P < 0.01). Histological examination revealed that the structure of the remaining livers was normal. The results of the hepatic function are showed in the Table 1.

Table 1 Assay of biochemical values of sera after liver failure (x^-± s).

	Group IIt	Group IIc	Normal rats
2 d after liver failure
ALB (g/L)	23.62 ± 3.69 (13)	24.13 ± 3.56 (8)	33.87 ± 2.36b (15)
TBr (μmol/L)	20.30 ± 13.23 (13)	24.00 ± 17.22 (8)	2.29 ± 1.61b (15)
DBr (μmol/L)	12.89 ± 9.12 (13)	15.61 ± 13.63 (8)	0.40 ± 0.39b (15)
7 d after liver failure
ALB (g/L)	30.00 ± 4.22 (12)	26.00 ± 3.00 (3)	33.87 ± 2.36b (15)
TBr (μmol/L)	2.97 ± 1.02 (11)	2.90 ± 0.36 (3)	2.29 ± 1.61 (15)
DBr (μmol/L)	1.14 ± 0.49 (11)	0.80 ± 0.56 (3)	0.40 ± 0.39 (15)

Note: group IIt : Transplant group in the part two; group IIc: Control group in the part two. Numbers in parentheses are numbers of rats.

^bP < 0.01, group IIt vs group IIc, and group IIc vs normal rats. ALB: Albumin. TBr: Total bilirubin. DBr: Direct bilirubin.

DISCUSSION

The survival rate (16/17) of the AHF rats after the transplantation was significantly higher than that of group I_c (3/9), and the living situation of the former during the survival was apparently superior to that of the latter. We found that after the 30^th d of cryopreservation in liquid nitrogen, the viability of hepatocytes no longer decreased in subsequent freezing up to 180 d, and the appearance and function of the hepatocytes remained well[3]. In view of the above, it is possible to cure AHF using pig hepatocytes cryopreserved for 150 d.

The transplanted cells will be destroyed by immunological rejection. To enable the grafted porcine hepatocytes to play the therapeutic role in the body of a xenogeneic animal, we adopted the transplanting method before and after the 80% hepatectomy, respectively. Moreover, as hypothermia is able to reduce the immunogenicity of cells[4], and thus delay or abate the rejection, the hepatocytes can gain more time to recover their functions. The conformational investigation of membrane proteins revealed that the content of α-helical structure increased from 48% to 59.5% after freezing. This result was possibly responsible for the report that hypothermia was able to reduce the immunogenicity of cells. The conformational change of membrane proteins might subsequently affect the characteristics of their functioning domains.

The possible mechanism of treatment of AHF with hepatocyte transplantation was thought to be that the transplanted hepatocytes produce hepatocyte growth factor which promotes liver regeneration and recovery of liver function, and the cells act as a substitution for the AHF liver. From our experiment, ALB concentrations of groups II_t and II_c 2 d after the 80% hepatectomy were significantly lower than that of the normal rats. TBr and DBr increased rapidly, but there was no significant difference between groups II_t and II_c , neither was there any significant difference in the weight of remaining livers between the two groups 2 and 7 d after the 80% hepatectomy. However, the survival rate of the transplant group was significantly higher than that of the control group. The results suggest that the mechanism of hepatocyte transplantation in treating AHF may not only be the effects of the substitution for the liver and the hepatocyte growth factor, but also be the effect of other unknown factors. It may be a result of multiple effects.