BACKGROUND: Cryopreserved hepatocytes are extensively used in drug industry today. However, comparing with cryopreserved human hepatocytes the single animal hepatocytes particularly rodent hepatocytes are limited on small size of batch regarding what is needed for setting up large scale study. In addition, animal hepatocytes are less resistant to isolation and cryopreservation process, with a significant alteration both at viability and plateability. Hence, the aim of the present study was designed to (i) to prepare freshly pooled hepatocytes from several small animals for making large size batch of pooled and cryopreserved rodent hepatocytes; (ii) to validate the pooled and cryopreserved rodent hepatocytes by comparing with fresh and cryopreserved hepatocytes from single animal. (iii) to characterize the post-thawing cell quality and pre-qualification of cryopreserved cells
STUDY DESIGN AND METHODS: For producing large size batch, rat and mouse hepatocyte were isolated from several animals and pooled and then cryopreserved by using an optimized process in BPI. The post-thaw viability, yield and plateability as well as the functionality of cryopreserved hepatocytes were checked and compared with both fresh and small size batch of cryopreserved hepatocyte from single animal donor. The pooled cryopreserved hepatocytes were also pre-qualified according to application including prediction of metabolic clearance, evaluation of CYP induction and hepatocyte transporter uptake assays.
RESULTS: We have developed an optimized technique for preparing and freezing of large size lot of pooled hepatocytes from multiple animal donors like rat and mice. They retain their fresh hepatocytes ability to attach to a collagen I coated matrix (post-thaw plateability), thereby permitting their use for long-term plated assay, such as induction and sandwich cultures. The comparison study shows that metabolism activity is comparable between the fresh and pooled cryopreserved hepatocytes. As well, a good lot-to-lot reproducibility was observed. Furthermore, some pre-qualified applications like induction or transport on cryopreserved pooled cells shown an acceptable inducibility of cytochrome P450 and efflux activity with sandwich-cultured hepatocytes.
Conclusions: Our pooled cryopreserved hepatocytes from multi-animal donors and multi-animal species represent a good alternative for use of freshly isolated hepatocytes for drug studies.