TY - JOUR
T1 - The influence of collagen III expression on the efficiency of cell isolation with the use of collagenase H
AU - Yoshida, S.
AU - Yamagata, Y.
AU - Murayama, K.
AU - Watanabe, Kimiko
AU - Imura, T.
AU - Igarashi, Yasuhiro
AU - Inagaki, A.
AU - Fujimori, Keisei
AU - Ohashi, K.
AU - Ohuchi, Noriaki
AU - Satomi, Susumu
AU - Goto, M.
N1 - Funding Information:
The authors thank Kozue Imura and Megumi Goto for their excellent technical assistance and Meiji Seika Pharma Co for the supply of the recombinant collagenases. The authors also acknowledge the support of the Biomedical Research Core of Tohoku University, Graduate School of Medicine, and Tohoku Advanced Medical Research and Incubation Center, and the grant for “Coordination, Support and Training Program for Translational Research” from the Ministry of Education, Culture, Sports, Science, and Technology.
Funding Information:
The authors received partial financial support from Meiji Seika Pharma Co .
Funding Information:
Supported by a grant for “Coordination, Support, and Training Program for Translational Research” from the Ministry of Education, Culture, Sports, Science, and Technology .
PY - 2014
Y1 - 2014
N2 - 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 × 106) 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.
AB - 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 × 106) 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.
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U2 - 10.1016/j.transproceed.2014.06.007
DO - 10.1016/j.transproceed.2014.06.007
M3 - Article
C2 - 25131077
AN - SCOPUS:84906088377
SN - 0041-1345
VL - 46
SP - 1942
EP - 1944
JO - Transplantation Proceedings
JF - Transplantation Proceedings
IS - 6
ER -