Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (44): 8253-8256.doi: 10.3969/j.issn.1673-8225.2010.44.021

Previous Articles     Next Articles

Separation of rat hepatocytes using modified collagenase perfusion via the abdominal aorta

Yu Song-lin1, Han Bao-san1, Zhang Rui2, Du Zhi-yong1, Wu Xu-bo1, Wu Wei1, Wang Jia-xiang1, Huang Fang1, Li Hong-wei1,  Shen Bo-yong1, Peng Cheng-hong1   

  1. 1 Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Department of General Surgery, Center of Organ Transplantation, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai  200025, China; 2 Department of Surgical Oncology, First Affiliated Hospital of Inner Mongolia Medical College, Hohhot  010020, Inner Mongolia Autonomous Region, China　
  • Online:2010-10-29 Published:2010-10-29
  • Contact: Peng Cheng-hong, Chief physician, Professor, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Department of General Surgery, Center of Organ Transplantation, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China chhpeng@hotmail.com
  • About author:Yu Song-lin☆, Studying for doctorate, Attending physician, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Department of General Surgery, Center of Organ Transplantation, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China ysl1997@sina.com
  • Supported by:

    the National "Eleventh Five-Year" Hi-tech Research and Development Program - "863" Project, No. 2008AA02Z417*; the Key Project of the National Natural Science Foundation of China, No. 20434030*; the National Natural Science Foundation of China, No. 30772105*, 20074031*; the Natural Science Fund Project of Shanghai Science and Technology Commission, No. 07ZR14076*

PDF

951

Abstract:

BACKGROUND: Good separation technique is the basis of harvesting hepatocytes with high viability. At present, two-step collagenase perfusion method via portal vein is extensively used in the world. However, this method has some problems, such as high dose of collagenase, complicated operation, long process and high requirement for the instrument.
OBJECTIVE: To investigate a simple effective method of separation and primary culture of rat hepatocytes. 
METHODS: A total of 10 Sprague Dawley rats were obtained to harvest hepatocyte by modified perfusion method through the abdominal aorta. Hepatocyte tests were repeated ten times to observe results of each index and to compare with published literatures. SD rats served as hepatocyte donors using Ⅳ type collagenase perfusion via the abdominal aorta. Liver hepatic portal structures, the upper and inferior vena cava of the donors were closed to retain collagenase digestion to obtain hepatocytes. Following filtration through 200-mesh and 300-mesh sieves, the suspension was transferred to a centrifuge tube, and then purified hepatocytes at 1 000, 500, 300 r/min centrifugation about respectively (each 3 minutes). Trypan blue staining was utilized to measure cell viability. The purity and morphology of hepatocytes were observed under the inverted microscope.
RESULTS AND CONCLUSION: Hepatocytes obtained by collagenase digestion were pure, intact and have a very good viability. The modified collagenase perfusion through the abdominal aorta was a better method for isolation of hepatocytes.

CLC Number: