772 The influence of the synergistic effect of neutral protease derived from Clostridium histolyticum and clostripain on the hepatocyte isolation
Wednesday November 18, 2015 from 11:00 to 12:30
Room 111-112

Satoru Yoshida, Japan

graduate student

Division of Advanced Surgical Science and Technology

Tohoku University Graduate School of Medicine


The influence of the synergistic effect of neutral protease derived from Clostridium histolyticum and clostripain on the hepatocyte isolation

Satoru Yoshida1, Youhei Yamagata2,3, Kazutaka Murayama4, Kimiko Watanabe2, Takehiro Imura2, Yasuhiro Igarashi2, Akiko Inagaki2, Shigehito Miyagi1, Kazuo Ohashi5, Noriaki Ohuchi1, Susumu Satomi1, Masafumi Goto1,2.

1Division of Advanced Surgical Science and Technology, Tohoku University School of Medicine, Sendai, Japan; 2New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; 3Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan; 4Graduate school of Biomedical Engineering, Tohoku University, Sendai, Japan; 5Laboratory of Drug Development and Science, Graduate School of Pharmaceutical Sciences , Osaka University, Osaka, Japan

Background: We recently reported that clostripain (CP) has a strong synergistic effect with the neutral protease derived from Clostridium histolyticum (ChNP), but not with thermolysin (TL), and using CP/ChNP in combination with collagenase derived from the same bacteria (Clostridium histolyticum) could dramatically increase the efficiency of pancreatic islet isolation. Considering that the tissue dissociation enzymes used for islet isolation have thus far been effectively used for hepatocyte isolation, it may be speculated that a combination of CP and ChNP would exert similar biological synergy for hepatocyte isolation. Therefore, we examined the synergistic effect of CP and ChNP for the efficiency of rat hepatocyte isolation using highly purified recombinant collagenases of each subtype (collagenase G (ColG) and collagenase H (ColH) provided by Meiji Seika Pharma Co., Ltd.), highly purified recombinant CP, highly purified recombinant ChNP, and/or TL.
Methods: Rat livers were digested using TL combined with ColG and ColH (TL group), or ChNP and CP combined with ColG and ColH (ChNP + CP group) (n=11, 6, respectively). The amount of ChNP (0.5 mg) was also adjusted to contain the same amount of protein as TL. The efficiency of rat hepatocyte isolation was evaluated in terms of cell yield and viability using trypan blue exclusion. Furthermore, the plating efficiency and deoxyribonucleic acid (DNA) quantitation of the cultured hepatocytes were measured as in vitro evaluation of the hepatocyte function.
Results: There were no significant differences in the yield or viability of isolated rat hepatocytes between TL and ChNP + CP groups. In addition, no beneficial effects were observed on the plating efficiency or DNA amount by introducing ChNP + CP in the rat hepatocyte isolation.
Conclusions: We recently reported that one of the main mechanisms underlying the beneficial effects of CP + ChNP on islet isolation is that the digestion procedure is effectively facilitated by chymotrypsin, without direct damage to the islets by trypsin. The present finding that no beneficial effects on the efficiency of hepatocyte isolation were observed by introducing CP + ChNP were most likely due to absence of endogenous proteases such as trypsin and chymotrypsin on the liver tissues. The present data strongly support our previous finding that protecting the islets from the damage caused by trypsin could be an essential reason why CP + ChNP was extraordinarily effective in pancreatic islet isolation. No additional enzyme components other than collagenases and neutral proteases seemed to be needed for hepatocyte isolation.

Kozue Imura. Megumi Goto.

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