Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes
Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials o...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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SAGE Publishing
2001-01-01
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| Series: | Cell Transplantation |
| Online Access: | https://doi.org/10.3727/000000001783987043 |
| _version_ | 1818961526479912960 |
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| author | Tao Fu Danqing Guo Xuemei Huang Maurice R. G. O'gorman Lijun Huang Susan E. Crawford Humberto E. Soriano M.D. |
| author_facet | Tao Fu Danqing Guo Xuemei Huang Maurice R. G. O'gorman Lijun Huang Susan E. Crawford Humberto E. Soriano M.D. |
| author_sort | Tao Fu |
| collection | DOAJ |
| description | Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes. |
| first_indexed | 2024-12-20T12:14:50Z |
| format | Article |
| id | doaj.art-4a8b8b0a13934cee80deda62b3fe68e7 |
| institution | Directory Open Access Journal |
| issn | 0963-6897 1555-3892 |
| language | English |
| last_indexed | 2024-12-20T12:14:50Z |
| publishDate | 2001-01-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Cell Transplantation |
| spelling | doaj.art-4a8b8b0a13934cee80deda62b3fe68e72022-12-21T19:41:09ZengSAGE PublishingCell Transplantation0963-68971555-38922001-01-011010.3727/000000001783987043Apoptosis Occurs in Isolated and Banked Primary Mouse HepatocytesTao Fu0Danqing Guo1Xuemei Huang2Maurice R. G. O'gorman3Lijun Huang4Susan E. Crawford5Humberto E. Soriano M.D.6Department of Pediatrics, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Pediatrics, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Pathology, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Immunology, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Pathology, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Pathology, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Department of Pediatrics, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.https://doi.org/10.3727/000000001783987043 |
| spellingShingle | Tao Fu Danqing Guo Xuemei Huang Maurice R. G. O'gorman Lijun Huang Susan E. Crawford Humberto E. Soriano M.D. Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes Cell Transplantation |
| title | Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes |
| title_full | Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes |
| title_fullStr | Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes |
| title_full_unstemmed | Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes |
| title_short | Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes |
| title_sort | apoptosis occurs in isolated and banked primary mouse hepatocytes |
| url | https://doi.org/10.3727/000000001783987043 |
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