Human hepatocytes are being used in a broad range of in vitro studies including drug metabolism, drug-drug interaction, and cytotoxicity. These results are then correlated with in vivo data to help predict the pharmacokinetics and undesirable side effects of NCEs. While human data is critical for the development of an NCE, animal studies are still required by regulatory agencies. To reduce and refine these animal in vivo studies, in vitro animal studies have been encouraged.
Cryopreserved hepatocytes, including cryo-plateable hepatocytes, from various clinically relevant animals are readily available. As in human cells, cryopreserved animal cells can be used to analyze metabolism and induction potential. One study compared the use of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes with fresh and cryopreserved human hepatocytes for in vitro induction studies. Previous studies demonstrated the successful application of suspension cells in metabolism assays. However, these induction studies required plated cells.
Researchers plated fresh cynomolgus monkey hepatocytes (FCMH), cryopreserved cynomolgus monkey hepatocytes (CCMH), and fresh human hepatocytes (FHH) on collagen-coated 48-well plates. Following standard induction protocol, five inducers were added at the concentrations noted: ß-naphthoflavone (BNF, 33 µM), 3-methylcholanthrene (3-MC, 8 µM), rifampicin (RIF, 25 µM), phenobarbital (PB, 2 mM), or omeprazole (OMEP, 50 µM) in InVitroGRO™ HI medium. A vehicle control dosing solution (0.1% DMSO or 1% ACN) was also added. At the end of the five days in culture, all plates had hepatocyte monolayers of 70% or greater confluence (Figure 2).
After the induction period, hepatocytes were evaluated for 7-ethoxyresorufin O-deethylase (CYP1A) and testosterone 6b-hydroxylase (CYP3A) enzyme activities. CYP1A was induced greater than twofold in CMH, CCMH, and FHH by BNF, 3- and OMEP. RIF did not induce CYP1A activity in any of the hepatocyte monolayers (Figure 3). CYP3A was induced greater than twofold by RIF and PB in all hepatocyte monolayers, while CYP3A activity was not induced by 3-MC or BNF. OMEP significantly induced CYP3A in FHH but did not induce in CCMH or FCMH.
No significant differences were observed in treated versus control samples or among the FCMH, CCMH, and FHH monolayers, therefore demonstrating the utility of this cryopreserved nonhuman primate hepatocyte model. It was concluded that cryo-plateable CCMH offer a reliable and convenient alternative to freshly plated cynomolgus monkey or human hepatocytes. Greater availability of cryo-plateable CCMH provides a valuable in vitro model that avoids the burdensome requirements of obtaining fresh monkey tissue and preparing fresh hepatocytes. The use of appropriate inducers is also stressed given the difference between human and cynomolgus responses to OME and RIF—preferred inducers for human—illustrating interspecies variability.
Cryopreserved hepatocytes perform comparably to freshly isolated hepatocytes allowing for similar uses like metabolic stability. When handled appropriately, cryo-plateable products are equally effective for induction and transport studies, providing researchers with greater convenience, larger lot sizes, fully characterized activities, and a shelf life of five years or more. Given these advantages, the benefits of using cryopreserved suspended and plateable hepatocytes well exceed those of fresh products.