Establishment of Human Myofibroblast Like Cell Line Immortalized by Transfection of SV40 Largw T Gene

Kim, Chung Yong; Lee, Hyo Suk; Jun, Hyung Sik; Woo, Gwang Hoon; Yoon, Jung Hwan; Lee, June Sung

Abstract

BACKGROUND/AIMS: Myofibroblast like cell (MFLC) plays an important role in hepatic fibrogenesis. A continuously proliferating human MFLC line would be valuable in studying human hepatic fibrogenesis. The present study was attempted to establish immortalized human liver MFLC line.
METHODS
Fetal liver cells were transfected with SV40 large T gene. The transfected cells were selected and those colonies with morphologic characteristics of MFLCs were further cloned by limiting dilution method. Growth characteristics of subcultured cells were investigated. Indirect immuno-fluorescece studies were carried out to confirm the expression of SV40 large T antigen, to analyze the expression pattern of intermediate filaments, and to detect smooth muscle alpha-actin. Moreover, the activity to store vitamin A was analyzed.
RESULTS
Subcultured cells were spindle in shape and aggregated to form a typical hills-and-valleys structure in an overconfluent culture condition. They were stained positively for SV40 large T antigen. Doubling time of the cells was approximately 36 hours. Their growth rates were accelerated by increasing serum concentration in the media and they did not form colonies in soft agar assay. They were stained to vimentin and smooth muscle alpha-actin but not to cytokeratin and factor-VIII related antigen. These cells were also able to take up and store vitamin A.
CONCLUSIONS
We established an immortalized human MFLC line derived from the hepatic stellate cells.