Thawed human hepatocytes in primary culture
Identifieur interne : 000157 ( France/Analysis ); précédent : 000156; suivant : 000158Thawed human hepatocytes in primary culture
Auteurs : M. Dou [France] ; G. De Sousa [France] ; B. Lacarelle [France] ; M. Placidi [France] ; P. Lechene De La Porte [France] ; M. Domingo [France] ; H. Lafont [France] ; R. Rahmani [France]Source :
- Cryobiology [ 0011-2240 ] ; 1992.
Abstract
In drag metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (−1.9 °C/min for 18 min and −30 °C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50–65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.
Url:
DOI: 10.1016/0011-2240(92)90048-7
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Istex, to step Corpus: 000621
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- to stream Istex, to step Checkpoint: 003601
- to stream Main, to step Merge: 004028
- to stream Main, to step Curation: 003E97
- to stream Main, to step Exploration: 003E97
- to stream France, to step Extraction: 000157
Links to Exploration step
ISTEX:4885FA155D807A74B0D49152F49CDB2197A40480Le document en format XML
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In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (−1.9 °C/min for 18 min and −30 °C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50–65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Provence-Alpes-Côte d'Azur</li></region><settlement><li>Marseille</li></settlement></list><tree><country name="France"><region name="Provence-Alpes-Côte d'Azur"><name sortKey="Dou, M" sort="Dou, M" uniqKey="Dou M" first="M." last="Dou">M. Dou</name></region><name sortKey="De La Porte, P Lechene" sort="De La Porte, P Lechene" uniqKey="De La Porte P" first="P. Lechene" last="De La Porte">P. Lechene De La Porte</name><name sortKey="De Sousa, G" sort="De Sousa, G" uniqKey="De Sousa G" first="G." last="De Sousa">G. De Sousa</name><name sortKey="Domingo, M" sort="Domingo, M" uniqKey="Domingo M" first="M." last="Domingo">M. Domingo</name><name sortKey="Lacarelle, B" sort="Lacarelle, B" uniqKey="Lacarelle B" first="B." last="Lacarelle">B. Lacarelle</name><name sortKey="Lafont, H" sort="Lafont, H" uniqKey="Lafont H" first="H." last="Lafont">H. Lafont</name><name sortKey="Placidi, M" sort="Placidi, M" uniqKey="Placidi M" first="M." last="Placidi">M. Placidi</name><name sortKey="Rahmani, R" sort="Rahmani, R" uniqKey="Rahmani R" first="R." last="Rahmani">R. Rahmani</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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