Comparison of methods for isolating primary hepatocytes from mini pigs.
Successful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N-acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24-hour rocker culture in serum-free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×10(9) cells in the NB 8+NAC group, compared to (9.5±2.1)×10(9) cells in the NB 8 group (P<.01) and (9.1±1.1) ×10(9) cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Yi
Y
Li
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.|Division of Transplant Surgery, Department of Surgery, Mayo Clinic, Rochester, MN, USA.
Yujia
Y
Wang
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Qiong
Q
Wu
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Li
L
Li
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Yujun
Y
Shi
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.|Department of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Hong
H
Bu
Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu, Sichuan, China. baoji@scu.edu.cn.|Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China. baoji@scu.edu.cn.
Ji
J
Bao
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D019164:Liver, Artificial; D013552:Swine; D013556:Swine, Miniature; D014183:Transplantation, Heterologous / Q000379:methods
bioartificial liver; cell isolation; hepatocyte transplantation; porcine hepatocyte; spheroid culture