A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.

Seven promutagens belonging to two chemical classes--polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene, dimethyl benz[a]anthracene, 3-methylcholanthrene, fluoranthene) and alkyl nitrosamines (dimethyl, diethyl, and dibutyl nitrosamine)--were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254-induced rat liver homogenate (S9) in the CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Our novel findings are (1) the inclusion of CaCl2 in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for structurally similar promutagens. The inclusion of CaCl2 in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.

Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli beta-galactosidase gene (beta-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-1 antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.

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