S Yoshida1, Y Yamagata2, K Murayama3, K Watanabe4, T Imura4, Y Igarashi4, A Inagaki4, K Fujimori1, K Ohashi5, N Ohuchi1, S Satomi1, M Goto6. 1. Division of Advanced Surgical Science and Technology, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan. 2. New Industry Creation Hatchery Center, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan; Department of Applied Biological Chemistry, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan. 3. Division of Biomedical Measurements and Diagnostics, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan. 4. New Industry Creation Hatchery Center, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan. 5. Department of Surgery, Nara Medical University, Kashihara, Japan. 6. Division of Advanced Surgical Science and Technology, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan; New Industry Creation Hatchery Center, Graduate School of Medical Engineering, Tohoku University, Sendai, Japan. Electronic address: gotokichi@aol.com.
Abstract
OBJECTIVE: We previously demonstrated that collagenase H (ColH) plays a crucial role in rat islet isolation, whereas collagenase G (ColG) plays only a supporting role. We also showed that collagen III appears to be one of the key targets of ColH based on a mass spectrometry analysis. In the present study, we investigated whether our novel findings in an islet isolation model are universally applicable for other types of cell isolation, such as a hepatocyte isolation, with the use of enzyme blends of recombinant collagenases. METHODS: As the first step, the expression of one of the main matrix components, collagen III, on rat pancreatic and hepatic tissues was assessed with the use of immunohistochemical staining. ColG and ColH were expressed in recombinant E. coli carrying expression plasmids for each collagenase. Then the efficiency of the collagenase subtype on rat hepatocyte isolation was evaluated in terms of cell yield with the use of thermolysin combined with either ColG or ColH (n = 3, respectively). RESULTS: The expression of collagen III on rat hepatic tissues was dramatically lower than that of rat pancreatic tissues. In the rat hepatocyte isolation, a substantial amount of hepatocytes (0.81 ± 0.11 × 10(6)) were obtained in the ColG group, whereas almost no hepatocytes were retrieved in the ColH group, indicating that the influence of the collagenase subtypes in rat hepatocyte isolation are completely opposite to that observed in rat islet isolation. CONCLUSIONS: Considering that the expression of collagen III on hepatic tissues was relatively low and that almost no hepatocytes were retrieved when ColH and thermolysin were used, the present study supports our novel finding that collagen III appears to be one of the key targets of ColH in hepatocyte isolation. Therefore, the semiquantification of collagen III on the target tissues not only may positively contribute to efficient islet isolation, but also may affect other types of cell isolation by optimizing the ColH amount.
OBJECTIVE: We previously demonstrated that collagenase H (ColH) plays a crucial role in rat islet isolation, whereas collagenase G (ColG) plays only a supporting role. We also showed that collagen III appears to be one of the key targets of ColH based on a mass spectrometry analysis. In the present study, we investigated whether our novel findings in an islet isolation model are universally applicable for other types of cell isolation, such as a hepatocyte isolation, with the use of enzyme blends of recombinant collagenases. METHODS: As the first step, the expression of one of the main matrix components, collagen III, on ratpancreatic and hepatic tissues was assessed with the use of immunohistochemical staining. ColG and ColH were expressed in recombinant E. coli carrying expression plasmids for each collagenase. Then the efficiency of the collagenase subtype on rat hepatocyte isolation was evaluated in terms of cell yield with the use of thermolysin combined with either ColG or ColH (n = 3, respectively). RESULTS: The expression of collagen III on rat hepatic tissues was dramatically lower than that of ratpancreatic tissues. In the rat hepatocyte isolation, a substantial amount of hepatocytes (0.81 ± 0.11 × 10(6)) were obtained in the ColG group, whereas almost no hepatocytes were retrieved in the ColH group, indicating that the influence of the collagenase subtypes in rat hepatocyte isolation are completely opposite to that observed in rat islet isolation. CONCLUSIONS: Considering that the expression of collagen III on hepatic tissues was relatively low and that almost no hepatocytes were retrieved when ColH and thermolysin were used, the present study supports our novel finding that collagen III appears to be one of the key targets of ColH in hepatocyte isolation. Therefore, the semiquantification of collagen III on the target tissues not only may positively contribute to efficient islet isolation, but also may affect other types of cell isolation by optimizing the ColH amount.