Long-term culture of hepatocytes from human adults

A long-term primary human hepatocyte culture retraining liver-specific func tions is important and essential for basic research and for the future deve lopment of hepatocyte-based applications. We established a normal hepatocyt e culture system from excess normal tissues obtained from adult liver cance r patients who received partial liver resection, Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to coll agen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix a nd increased in size and numbers with time of culture and eventually grew i nto spheroids of variable sizes. After being transferred to collagen gel, t he cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to prolifera te. Cellular functions specific for hepatocytes were analyzed using immunob lot assay for proteins specifically secreted by the liver cells on differen t days of culture. The cells secreted albumin, transferrin and alpha-fetopr otein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a longterm culture of hepatocytes from human adults, wh ich will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infec tious hepatitis, pharmacology, pharmacokinetics, and toxicology.