US 7,351,584 B2Granted

Hepatocyte bioreactor system for long term culture of functional hepatocyte spheroids

AssigneeIn Vitro Technologies, Inc.

InventorsPaul M. Silber, Nancy L. Cowger, Ana P. Miller, Lanika A. Brown, Albert P. Li

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Abstract

A rotating wall vessel is used as a culture vessel and bioreactor for the cultivation of hepatocytes in the form of spheroids to generate a culture with many properties of the intact liver. These properties include enzyme activity comparable to fresh cells and long-term maintenance of viability and cellular function for periods on the order of months. The cultures may be used to produce hepatocyte products, evaluate metabolism of an agent, propagate Hepatitis C virus and test agents as inhibitors of this virus. Thus, the culture system disclosed herein makes long term functional cultivation of human hepatocytes feasible.

Claims11

Dependent claims

A method for cultivation of mammalian hepatocytes in a viable functional state, which comprises the steps of:

(a) 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;
(b) transferring said spheroids into a rotating wall vessel (RVW) culture chamber where said spheroids remain substantially in suspension during subsequent culture with rotation;
and
(c) 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:
(i) producing and secreting normal hepatocyte products;
and
(ii) 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.

5 more elements

Dependencies are inferred from the claim text.