Cryopreserved hepatocytes

Shen, J., Cheng,Y., Sun, C., Ge, X., Subramanyam, B. and Tseng, J. L., A Systematic Approach in Metabolic Stability Studies Using Cryopreserved Hepatocyte, American Society for Mass Spectrometry 2002 Conference Abstract, Orlando, FL, USA, 2002. 

XenoTech offers a selection of services for drug metabobsm-related research including liver and pulmonary microsomes and S9, cryopreserved hepatocytes from human and six other relevant species, antibodies directed against CYP enzymes, recombinant CYPs, and bDNA probe sets (322). 

Liver Uptake Blood Parenchymal cells Isolated, cultured cryopreserved hepatocytes, sinusoidal membrane vesicles, transporter expressions system... 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

An attractive strategy for predicting the clinical significance of irreversible inhibition is to use human hepatocytes wherein the natural turnover of enzymes might be preserved and in vivo cellular concentrations of inhibitors and metabolites would be achieved. Zhao et al. demonstrated time-dependent inactivation of CYP3A in cryopreserved hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and TAO (126). Except for TAO, significant differences in inactivation efficiency potency between hepatocytes and HLMs were... 

Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as metabolizing system in mutagenicity assays (Hengstler 2000), providing qualitative metabolic information and quantitative pharmacokinetic parameters from key animal species and human at the early stage of drug discovery and drug development. 

CRITICAL ASSESSMENT OF THE METHOD Using primary cultures of hepatocytes the retention of transporter expression and activity needs to be guaranteed strictly speaking after each preparation. Cryopreserved hepatocytes from one preparation could be a powerful alternative for industrial applications in the future, because transporter expression and activity could only be characterized once. Houle et al. (2003) compared the transporter activities in cropreserved and freshly isolated hepatocytes and found no significant difference in the transport rates of 14C-taurocholate, 3H-estrone sulfate and 3H-estradiol-17 3-D-glucuronide. 

Roymans D, Annaert P, Van Houdt J, et al. Expression and induction potential of cytochromes P450 in human cryopreserved hepatocytes. Drug Metab Disp. 2005 33 1004-1016. 

Li AP, Gorycki PD, Hengstier JG, Kedderis GL, Koebe HG, Rahmani R, de Sousas G, Silva JM, Skett P. Present statns of the application of cryopreserved hepatocytes in the evaluation of xenobiotics consensns of an international expert panel. Chem Biol Interact 1999 121 117-123. 

Martignoni M, Monshouwer M, de Kanter R, Pezzetta D, Moscone A, Gross P. Phase I and phase II metabolic activities are retained in liver slices from mouse, rat, dog, monkey and human after cryopreservation. Toxicol In Vitro 2004 18 121-128. McGinnity DF, Soars MG, Urbanowicz RA, Riley RJ. Evaluation of fresh and cryopreserved hepatocytes as in vitro drug metabolism tools for the prediction of metabolic clearance. Drug Metab Dispos 2004 32 1247-1253. 

Cryopreservation of hepatocytes allows their storage and transport rather than immediate use after isolation. However there is an associated loss of phenotype with this technique, making cryopreserved hepatocytes less physiologically relevant than primary human hepatocytes (Terry et al., 2006). 

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