Hepatic stellate cells play a cardinal role in the development of liver fibrosis. Quiescent hepatic stellate cells isolated from normal liver are activated by plating on a plastic culture dish. Therefore, a culture method that maintains hepatic stellate cells in a quiescent state is required for studies of fibrosis. We attempted to deactivate human hepatic stellate cells by culturing on VECELL® culture inserts (Preset VECELL). VECELL is a cell culture scaffold consisting of an expanded polytetrafluoroethylene mesh that is coated with collagen type I. Cryopreserved human hepatic stellate cells and LI90 cells, which is a cell line established from an outgrowth of a human hepatic mesenchymal tumor, were cultured on Preset VECELL. The expression of activation markers a-SMA and COL1A was decreased by VECELL cultivation. In addition, actin filaments (markers of activated hepatic stellate cells) were not detected in LI90 cells on VECELL inserts. These results suggest that human hepatic stellate cells were deactivated by VECELL cultivation, which could provide a model system for the analysis of deactivated human hepatic stellate cells. Thus, Preset VECELL will be a useful in vitro tool for the clarification of underlying mechanisms and the development of drugs to treat liver fibrosis. This study will contribute to provide alternative methods to animal tests that have been mainly carried out in studies of hepatic stellate cell, liver fibrosis, and liver cirrhosis.