Culture of Intrahepatic Bile Duct Cells Isolated from Normal Rat Liver

1989年，我们研制了大鼠离体肝灌流仪，它主要由胶原酶溶液再循环部分和充氧部分组成。使用该系统，可获得95%以上的肝细胞存活率。用等温离心法或等温离心法+免疫亲和法分离肝内胆管细胞，单独等温离心法可获得细胞悬液，其中50-60%为胆管细胞。为了获得适合培养的细胞，我们试图通过在灌流液中省略胰酶来获得成团的细胞。这些细胞在10%FCS/William‘s培养液中培养。我们还将它们与含有IV型胶原和层粘连蛋白的塑料、胶原或Matrigel进行电镀，以观察基质对细胞附着的影响。细胞在Matrigel上的粘附率最高(10.5%)。他们至少活了6天。在培养过程中，胆管细胞没有增殖或形成对…的附着特征。下一步我们尝试将免疫亲和力应用于胆管细胞的分离方法，以提高胆管细胞的纯度。抗胆管细胞的单抗来自梅奥诊所的医学教授N.F.LaRusso医学博士。用该抗体可将纯度提高到70%以上。此外，我们要求布朗大学的R.Faris医学博士使用他的单抗来对抗从胆总管结扎大鼠分离的增殖的胆管细胞。该抗体的纯度可达80%以上。我们正在努力确定长期培养的培养条件，使细胞增殖。到目前为止，我们还没有值得公布的数据。如果我们成功地长期培养形成紧密连接的单层胆管细胞，我们认为我们应该发表结果。但从我们的经验来看，由于胆管细胞增殖活性低，可能很难获得纯培养的胆管细胞。为了克服这一问题，应该考虑新的方法，如用SV-40转染细胞。较少

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 … More 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