A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes.
Primary human hepatocytes are widely used for metabolic stability evaluations. However, there are limited data directly comparing phase I and phase II drug-metabolizing enzymes in fresh and cryopreserved hepatocytes prepared from the same human donor liver. We evaluated the metabolic competency of human hepatocytes prepared from seven donor tissues before and after cryopreservation. Temporal-dependent enzyme activity in suspension and matched adherent cultures of primary human hepatocytes was also assessed. Cryopreservation of hepatocytes resulted in statistically significant increases in activities of CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A but not CYP2C8, CYP2C19, FMO, UGT, and SULT, relative to fresh hepatocytes. In suspension cultures of hepatocytes, enzyme stabilities were as follows: UGT<CYP3A<CYP1A2<CYP2D6<CYP2C9<SULT. CYP1A2 and CYP3A enzyme stability was significantly greater in plated cells relative to suspension with mean enzyme inactivation time values of 2.69 ± 0.39 and 1.62 ± 0.09 h in suspension and 21.3 ± 2.1 and 28.8 ± 20.4 h in culture, respectively. These data demonstrate that cryopreservation is not detrimental to primary human hepatocytes enzyme activities, indicate time-dependent changes in metabolic activity in both suspension and adherent cultures, and support the utility of adherent cultures of cryopreserved hepatocytes for prediction of metabolic clearance for low-clearance drugs.
ADME/TOX Division of Life Technologies, Durham, North Carolina 27703, USA. Cornelia.smith@lifetech.com
Cornelia M
CM
Smith
Christina K
CK
Nolan
Manda A
MA
Edwards
Jean B
JB
Hatfield
Todd W
TW
Stewart
Stephen S
SS
Ferguson
Edward L
EL
Lecluyse
Jasminder
J
Sahi
D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008657:Metabolic Clearance Rate; D050216:Metabolic Detoxication, Phase I / Q000502:physiology*; D050217:Metabolic Detoxication, Phase II / Q000502:physiology*; D013535:Suspensions / Q000378:metabolism
D013535:Suspensions; D003577:Cytochrome P-450 Enzyme System