Cold Storage of Rat Hepatocyte Spheroids
Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
SAGE Publishing
2014-07-01
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| Series: | Cell Transplantation |
| Online Access: | https://doi.org/10.3727/096368913X664847 |
| _version_ | 1818958356630470656 |
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| author | Hongling Liu Yue Yu Jaime Glorioso Shennen Mao Brian Rodysil Bruce P. Amiot Piero Rinaldo Scott L. Nyberg M.D., Ph.D. |
| author_facet | Hongling Liu Yue Yu Jaime Glorioso Shennen Mao Brian Rodysil Bruce P. Amiot Piero Rinaldo Scott L. Nyberg M.D., Ph.D. |
| author_sort | Hongling Liu |
| collection | DOAJ |
| description | Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids. |
| first_indexed | 2024-12-20T11:24:27Z |
| format | Article |
| id | doaj.art-c86f5debbb40432a913fe834309f2dc7 |
| institution | Directory Open Access Journal |
| issn | 0963-6897 1555-3892 |
| language | English |
| last_indexed | 2024-12-20T11:24:27Z |
| publishDate | 2014-07-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Cell Transplantation |
| spelling | doaj.art-c86f5debbb40432a913fe834309f2dc72022-12-21T19:42:25ZengSAGE PublishingCell Transplantation0963-68971555-38922014-07-012310.3727/096368913X664847Cold Storage of Rat Hepatocyte SpheroidsHongling Liu0Yue Yu1Jaime Glorioso2Shennen Mao3Brian Rodysil4Bruce P. Amiot5Piero Rinaldo6Scott L. Nyberg M.D., Ph.D.7Liver Failure Diagnosis and Treatment Center, 302 Military Hospital, Beijing, P.R. ChinaLiver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, P.R. ChinaDivision of Experimental Surgery, Mayo Clinic College of Medicine, Rochester, MN, USADivision of Experimental Surgery, Mayo Clinic College of Medicine, Rochester, MN, USADivision of Experimental Surgery, Mayo Clinic College of Medicine, Rochester, MN, USABrami Biomedical, Inc., Minneapolis, MN, USADepartment of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USADivision of Experimental Surgery, Mayo Clinic College of Medicine, Rochester, MN, USACell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 μM cyclosporin A (CsA); SFM + 1 mM Def + 1 μM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.https://doi.org/10.3727/096368913X664847 |
| spellingShingle | Hongling Liu Yue Yu Jaime Glorioso Shennen Mao Brian Rodysil Bruce P. Amiot Piero Rinaldo Scott L. Nyberg M.D., Ph.D. Cold Storage of Rat Hepatocyte Spheroids Cell Transplantation |
| title | Cold Storage of Rat Hepatocyte Spheroids |
| title_full | Cold Storage of Rat Hepatocyte Spheroids |
| title_fullStr | Cold Storage of Rat Hepatocyte Spheroids |
| title_full_unstemmed | Cold Storage of Rat Hepatocyte Spheroids |
| title_short | Cold Storage of Rat Hepatocyte Spheroids |
| title_sort | cold storage of rat hepatocyte spheroids |
| url | https://doi.org/10.3727/096368913X664847 |
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