March 15, 2009 (Vol. 29, No. 6)

For Metabolic Studies, Frozen Trumps Fresh for Most Uses

In vitro drug-metabolism data has been increasingly utilized in understanding and predicting pharmacokinetics in vivo. Traditionally, predictive studies have been conducted in animal models. Because extrapolation from animal to human is difficult due to species differences, attaining human in vitro data is critical for accurate human in vivo correlation.

This data is often obtained using human hepatocytes isolated from donated livers. The limitations of freshly isolated hepatocytes for studies like induction severely restrict the researcher’s choices for timing, demographics, and retesting.  Cryopreservation of these cells permits expanded investigation while retaining clinically significant activity. By removing constraints associated with tissue availability, cryopreservation allows for further testing of activity and genomic characterizations, retesting from the same donor, and scheduling at the researcher’s discretion.

Furthermore, some cryopreserved hepatocyte preparations retain the fresh hepatocytes’ ability to attach to a collagen I coated matrix (cryo-plateable), extending their use for long-term culturing applications like induction. Both human and animal cryopreserved hepatocytes offer another benefit as well: utilizing the entire liver produces larger lot sizes and, in the case of animals, reduce the number and frequency of animals sacrificed. Thus, cryo-plateable hepatocytes present an ideal system by offering the dual benefits of freshly isolated plateability and the convenience of cryopreservation.

Fresh vs. Frozen

In today’s competitive environment, time and predictability are essential. Promising drugs may save or improve the lives of patients. In addition, the financial implications of being the first to file a promising new chemical entity (NCE) cannot be underestimated.

In the race to succeed, fresh hepatocytes present a number of challenges for the researcher. Freshly isolated hepatocytes may not be readily available due to reliance on liver donation. This unpredictability may slow the testing of NCEs and may require staff to remain on call for a liver.

Liver resects from biopsies may improve availability but are potentially dangerous as they do not include full serology reporting. In contrast, fully characterized cryopreserved hepatocytes, like those from Celsis In Vitro Technologies, can be immediately procured in large inventories and stored at the laboratory, controlling the scheduling of studies and accelerating time to market. For example, standard induction studies typically may be completed 50% faster  (Figure 1).

Cryopreserved hepatocytes may be pre-characterized for enzyme activities, genotypes, and for applications like induction or transport, representing a second major advantage over fresh products. In fact, cryopreserved products may be obtained to meet specific donor requirements while maintaining high levels of viability, confluence, or activity. Large inventories and a minimum five-year shelf life allow researchers to repeat tests at future dates with no difference in product performance. Likewise, research being conducted at multiple sites can draw from the same product lot for consistent, simultaneous research worldwide.

Cryopreserved hepatocytes, however, are sensitive to storage conditions, thawing and plating procedures, and culture maintenance. To mitigate any storage damage, cryopreserved hepatocytes must be stored at less than -150°C. Cryo-plateable and cryo-suspension hepatocytes must be thawed according to the manufacturer’s instructions using appropriate medium. Cryo-plateable hepatocytes also must be seeded uniformly at the optimal cell density, which can affect their plating efficiency. When treated properly, cryo-plateable hepatocytes can be used as if they were freshly isolated hepatocytes.

The use of cryopreserved hepatocytes is recognized as equivalent to using freshly isolated hepatocytes. Acknowledging this trend, the U.S. FDA drafted a Guidance for Industry in 2006 advising that cryopreserved tools are a viable and acceptable alternative to fresh research products, especially in metabolism, toxicity, and drug-drug interaction testing. Many laboratories have demonstrated cryopreserved equivalence to freshly isolated hepatocytes.

As such, several large pharmaceutical companies have adopted cryopreserved hepatocytes as their primary source for stability, transport, and induction studies. As researchers realize the benefits of cryopreserved hepatocytes that can be selected, reserved, and scheduled in advance, an expanding range of applications will be employed like high-throughput screening  and high-content screening. 

Figure 1. Viability of hepatocyte monolayers as determined by MTT reduction.

Comparative Efficacy

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.

Figure 2. Induction of CYP1A activity as measured by ethoxyresorufin O-deethylation and expressed as percent of vehicle control.


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.

Figure 3.

Ji Young Lee, Ph.D. ([email protected]), and Timothy A. Moeller ([email protected]) are scientific advisors at Celsis In Vitro Technologies. Web:

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