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Cryopreservation of assay-ready hepatocyte monolayers by chemically-induced ice nucleation : preservation of hepatic function and hepatotoxicity screening capabilities
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Tomás, Ruben M. F., Dallmann, Robert, Congdon, Thomas R. and Gibson, Matthew I. (2024) Cryopreservation of assay-ready hepatocyte monolayers by chemically-induced ice nucleation : preservation of hepatic function and hepatotoxicity screening capabilities. Biomaterials Science, 11 (23). pp. 7639-7654. doi:10.1039/d3bm01046e ISSN 2047-4849.
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Official URL: https://doi.org/10.1039/d3bm01046e
Abstract
Cell culture plays a critical role in biomedical discovery and drug development. Primary hepatocytes and hepatocyte-derived cell lines are especially important cellular models for drug discovery and development. To enable high-throughput screening and ensure consistent cell phenotypes, there is a need for practical and efficient cryopreservation methods for hepatocyte-derived cell lines and primary hepatocytes in an assay-ready format. Cryopreservation of cells as adherent monolayers in 96-well plates presents unique challenges due to low volumes being susceptible to supercooling, leading to low recovery and well-to-well variation. Primary cell cryopreservation is also particularly challenging due to the loss of cell viability and function. In this study, we demonstrate the use of soluble ice nucleator materials (IN) to cryopreserve a hepatic-derived cell line (HepG2) and primary mouse hepatocytes, as adherent monolayers. HepG2 cell recovery was near 100% and ∼75% of primary hepatocytes were recovered 24 hours post-thaw compared to just 10% and 50% with standard 10% DMSO, respectively. Post-thaw assessment showed that cryopreserved HepG2 cells retain membrane integrity, metabolic activity, proliferative capacity and differentiated hepatic functions including urea secretion, cytochrome P450 levels and lipid droplet accumulation. Cryopreserved primary hepatocytes exhibited reduced hepatic functions compared to fresh hepatocytes, but functional levels were similar to commercial suspension-cryopreserved hepatocytes, with the added benefit of being stored in an assay-ready format. In addition, normal cuboidal morphology and minimal membrane damage were observed 24 hours post-thaw. Cryopreserved HepG2 and mouse hepatocytes treated with a panel of pharmaceutically active compounds produced near-identical dose–response curves and EC50 values compared to fresh hepatocytes, confirming the utility of cryopreserved bankable cells in drug metabolism and hepatotoxicity studies. Cryopreserved adherent HepG2 cells and primary hepatocytes in 96 well plates can significantly reduce the time and resource burden associated with routine cell culture and increases the efficiency and productivity of high-throughput drug screening assays.
Item Type: | Journal Article | ||||||||||||||||||
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Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School | ||||||||||||||||||
SWORD Depositor: | Library Publications Router | ||||||||||||||||||
Library of Congress Subject Headings (LCSH): | Cryopreservation of organs, tissues, etc. , Cells -- Cryopreservation , Cell culture, Liver cells , Ice nuclei | ||||||||||||||||||
Journal or Publication Title: | Biomaterials Science | ||||||||||||||||||
Publisher: | The Royal Society of Chemistry | ||||||||||||||||||
ISSN: | 2047-4849 | ||||||||||||||||||
Official Date: | 7 December 2024 | ||||||||||||||||||
Dates: |
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Volume: | 11 | ||||||||||||||||||
Number: | 23 | ||||||||||||||||||
Page Range: | pp. 7639-7654 | ||||||||||||||||||
DOI: | 10.1039/d3bm01046e | ||||||||||||||||||
Status: | Peer Reviewed | ||||||||||||||||||
Publication Status: | Published | ||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||||||||||||
Date of first compliant deposit: | 17 October 2023 | ||||||||||||||||||
Date of first compliant Open Access: | 18 October 2023 | ||||||||||||||||||
RIOXX Funder/Project Grant: |
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