Cryopreservation, hepatocytes

Cryopreserved human hepatocytes Cryopreserved human hepatocytes are extremely useful for the evaluation of drug metabolism, but in general cannot be cultured due to their impaired cell-attachment. There are preparations of cryopreserved human hepatocytes with high plating efficiency and therefore can be cultured for induction studies. It has been shown that hepatocytes cultured after cryopreserva-tion are responsive to CYP1A and 3A inducers, but they have a significantly lower basal (uninduced) levels of these enzymes. Cryopreserved human hepatocytes therefore represent a more convenient experimental model than freshly isolated human hepatocytes for enzyme induction studies (Roymans etal. 2005). 

Shitara, Y., Lu, C., Li, A. P., Y. Kato, Y., Suzuki, H., Ito, K., Itoh, T., Sugiyama, Y., Cryopreserved human hepatocytes as a tool for the prediction of in vivo transport and transporter-mediated drug-drug interactions, Abstract of International Conference on Drug Interaction, Hamamatsu, October 21-23, 1999, p. 87. 

Lau, Y.Y. et al. 2002. Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. Drug Met. Disp. 30 1446. 

Li, A. P. (1999) Overview hepatocytes and cryopreservation — a personal historical perspective. Chemico-Biological Interactions, 121 (1), 1—5. 

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. 

Digoxin uptake into rat hver shces showed a temperature-dependent component, compatible with the involvement of carrier-mediated uptake mechanisms. Quinine markedly inhibited the uptake of digoxin, in contrast to its diastereomer quinidine, which only slightly inhibited the digoxin uptake in rat liver slices. This stereoselective inhibition is in line with results obtained in isolated rat hepatocytes and isolated perfused rat hvers [90,91]. These results were also found after cryopreservation of the slices, indicating that carrier-specific phenomena can be studied after cryopreservation [92]. 

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). 

Westerink, W.M.A. and Schoonen, W.G.E.J. (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology In Vitro, 21, 1581-1591. 

Demetriou et al. [25] described a capillary hollow fiber membrane based bioreactor in which microcarrier-attached hepatocytes are placed in the extracapillary space on the exterior surface of the capillary hollow fiber membranes as shown in Fig. 1. Recent experimental studies with this device have demonstrated its efficacy in animal models. By using cryopreserved microcarrier-attached hepatocytes this system offers the convenience of being readily available when needed. 

Primary human hepatocytes in particular have not yet proven to be compatible with cryopreservation (Utesch et al., 1992), therefore studies must be performed when the tissue becomes available. Characterization of the enzyme composition of the tissue derived from an individual must be conducted in parallel with characterization of the unknown xenobiotic. This can lead to the devotion of considerable experimental effort to studies which, in the end, do not meet quality control criteria. Despite these limitations, human hepatocytes are uniquely suited for studies of cytochrome P450 regulation and also provide the only current system which maintains a balanced and physiological ratio of cofactors and individual Phase I and Phase II enzymes. 

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

Shitara Y, Li AP, Kato Y, et al. Function of uptake transporters for taurocholate and estradiol 17b - D-glucuronide in cryopreserved human hepatocytes. Dmg Metab Pharmacokinet 2003 18 33 41. 

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... 

Zhao P, Kunze KL, Lee CA. Evaluation of time-dependent inactivation of CYP3A in cryopreserved human hepatocytes. Drug Metab Dispos 2005 33(6) 853-861. 

Niro, R., Byers, J., Fournier, R., and Bachmann, K., Application of a convective-dispersion model to predict in vivo hepatic clearance from in vitro measurements utilizing cryopreserved human hepatocytes, Current Drug Metabolism, Vol. 4, No. 5, 2003, pp. 357-369. 

Lee SH, Slattery JT (1997) Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes. Drug Metab Dispos 25 1354-1358 Li AP (1999) Cryopreserved human hepatocytes characterization of DME activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug-drug interaction potential. Chem Biol Interact 121 17-35... 

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. 

Hepatocytes - both freshly prepared or cryopreserved - are commercially available e.g. Gen test, IVT, Xenotech or prepared in-house. Isolation of the animal hepatocytes follows after a two step collagenase perfusion of the liver via the vena portae in situ (Seglen 1976) or via several blunt-end cannulae inserted into vessels available on the cut surface of pieces of human liver obtained from resection. Liver cells are gently scraped out into suspension buffer and washed twice to three times by centrifugation to remove cell fragments and non-vital cells. Hepatocytes are used immediately or cryopreserved for further use. 

CRITICAL ASSESSMENT OF THE METHOD Human hepatocytes (fresh or cryopreserved) are now commercially available e.g. from BD/Gentest, In Vitro Technology or Xenotech. However, the quality, stability and availability of the commercial preparation remain questionable (Mandan 2002). Isolation (and cultivation) of hepatocytes is still time- and labintensive and needs to be optimized for livers of every different animal species (De Graaf 2002). Metabolism studies in hepatocytes might be a good compromise between perfused livers and subcellular fractions such as microsomes, since the complete cellular machinery is available. Nevertheless, some pitfalls have to be taken in account ... 

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, Van Looveren C, Leone A et al. (2004) Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes. Biochem Pharmacol 67 427 137... 

Hepatotoxicity—enzyme release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide (MTT) assay on fresh or cryopreserved (human) hepatocytes Endocrine disruption—yeast and mammalian reporter gene assay (oestrogenicity and adrogenicity)... 

Table 5-5. Viability and plateability (ability of hepatocytes to be cultured as monolayer cultures) of the various lots of cryopreserved human hepatocytes3...

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