Patent ID: 7575859

Claim:
A method for testing an agent for its activity as an inhibitor of Hepatitis C Virus (HCV) replication or propagation in hepatocytes, comprising: (a) preparing cultured hepatocytes by (i) culturing a single cell suspension of mammalian hepatocytes in a culture medium for a period of between about 12 and about 168 hours in a culture vessel having a flat surface under conditions that permit aggregation of hepatocytes into spheroids which comprise viable hepatocytes; (ii) transferring said spheroids into a rotating wall vessel (RVW) culture chamber where said spheroids remain substantially in suspension during subsequent culture with rotation; (iii) incubating said suspended spheroids under conditions of vessel rotation such that said spheroids remain substantially in suspension; wherein suspended spheroids remain viable and capable of: (1) producing and secreting normal hepatocyte products; and (2) metabolizing drugs or toxins via enzymes of the cytochrome P450 system, which drugs or toxins are ones that are normally metabolized by hepatocytes in vivo for more than 30 days; (b) inoculating the cultured hepatocytes with an inoculum of Hepatitis C Virus (HCV) to prepare virus-infected cultures and permitting the virus to infect, replicate in and be released from the hepatocytes; (c) before, during or after step (b), adding to the prepared cultures the agent being tested for inhibitory activity; (d) to a parallel hepatocyte culture or set of cultures of hepatocytes infected with HCV as in steps (a) and (b), adding negative control agent that does not inhibit HCV replication or propagation (e) harvesting the medium of the cultures of (c) and (d); and, (f) measuring the amount or titer of HCV present in the harvested medium, wherein a lower amount or titer of virus in the cultures of (c) compared to the cultures of (d) is indicative that the agent is an inhibitor.