Effect of the silk protein sericin on cryopreserved rat islets

Background/purpose Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS...

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Veröffentlicht in:Journal of hepato-biliary-pancreatic sciences 2012-07, Vol.19 (4), p.354-360
Hauptverfasser: Ohnishi, Kenji, Murakami, Makoto, Morikawa, Mitsuhiro, Yamaguchi, Akio
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container_title Journal of hepato-biliary-pancreatic sciences
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creator Ohnishi, Kenji
Murakami, Makoto
Morikawa, Mitsuhiro
Yamaguchi, Akio
description Background/purpose Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori . Methods Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3 days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation. Results There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS. Conclusion The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.
doi_str_mv 10.1007/s00534-011-0415-4
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Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori . Methods Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3 days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation. Results There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS. 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Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori . Methods Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3 days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. 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Murakami, Makoto ; Morikawa, Mitsuhiro ; Yamaguchi, Akio</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5756-888aa131faeabf526dd8ed4c7d772e2bddf4d10a2410b6d4027034338af4d0e93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Abdominal Surgery</topic><topic>Animals</topic><topic>Cell culture</topic><topic>Cryopreservation - methods</topic><topic>Culture Media, Serum-Free - chemistry</topic><topic>Gastroenterology</topic><topic>Hepatology</topic><topic>Insulin</topic><topic>Insulin - metabolism</topic><topic>Insulin Secretion</topic><topic>islet cryopreservation</topic><topic>islet transplantation</topic><topic>Islets of Langerhans</topic><topic>Male</topic><topic>Medicine</topic><topic>Medicine &amp; Public Health</topic><topic>Organ Culture Techniques</topic><topic>Organ Preservation - methods</topic><topic>Original Article</topic><topic>Pancreas</topic><topic>Rats</topic><topic>Rats, Inbred Lew</topic><topic>Rodents</topic><topic>sericin</topic><topic>Sericins - therapeutic use</topic><topic>serum-free freezing medium</topic><topic>Surgical Oncology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ohnishi, Kenji</creatorcontrib><creatorcontrib>Murakami, Makoto</creatorcontrib><creatorcontrib>Morikawa, Mitsuhiro</creatorcontrib><creatorcontrib>Yamaguchi, Akio</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Health &amp; 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The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation. Results There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS. Conclusion The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.</abstract><cop>Japan</cop><pub>Blackwell Publishing Ltd</pub><pmid>21678022</pmid><doi>10.1007/s00534-011-0415-4</doi><tpages>7</tpages></addata></record>
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subjects Abdominal Surgery
Animals
Cell culture
Cryopreservation - methods
Culture Media, Serum-Free - chemistry
Gastroenterology
Hepatology
Insulin
Insulin - metabolism
Insulin Secretion
islet cryopreservation
islet transplantation
Islets of Langerhans
Male
Medicine
Medicine & Public Health
Organ Culture Techniques
Organ Preservation - methods
Original Article
Pancreas
Rats
Rats, Inbred Lew
Rodents
sericin
Sericins - therapeutic use
serum-free freezing medium
Surgical Oncology
title Effect of the silk protein sericin on cryopreserved rat islets
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