|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1990 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1989 : ¥1,100,000 (Direct Cost : ¥1,100,000)
In 1989, we made isolated rat liver perfusion apparatus consisting mainly of two parts : a re-circulation part for collagenase solution and a part for oxygenizing the solution. Using this system, we could get more than 95% viability of isolated liver cells. Intrahepatic bile duct cells were isolated with either isopycnic centrifugation or isopycnic centrifugation plus immunoaffinity.
With isopycnic centrifugation alone, we could get cell suspension in which 50-60% were bile duct cells. To get cells suitable for culturing, we have tried to get cells as clumps by ommiting trypsin in perfusion solution. These cells were cultured in 10% FCS/William's medium. We also plated them onto plastic, collagen or Matrigel containing type IV collagen and laminin to see the effect of matrix on cell attachment. The cells were found attached most effectively (10.5%) onto Matrigel. They were alive for at least 6 days. During culture, bile duct cells did not proliferate or form attachment characteristic fo
r epithelial cells.
The next step we have tried was to apply immunoaffinity to isolation method to increase the purity of bile duct cells. The monoclonal antibody against bile duct cells was gifted from N. F. LaRusso, M. D., a professor of Medicine at Mayo Clinic. Using this antibody, the purity was increased up to 70%. Furthermore, we asked R. Faris, M. D. at Brown University to use his monoclonal antibody which was raised against proliferated bile duct cells isolated from common bile duct ligated rats. With this antibody, the purity became above 80%. We are now trying to determine the culture conditions for long-term culture and making the cells proliferate.
Up to now we have not had data worthwhile to be published. If we succeed long-term culture of the bile duct cells which form monolayer with tight junctions, we think we should publish the results. But from our experiences, it may be difficult to get pure cultured bile duct cells because of their low proliferative activity. To overcome this problem, new methods, such as transfection of the cells with SV-40, should be considered. Less