Spheroid Cultures of Human Hepatoblastoma Cells (HuH-6 Line) and Their Application for Cytotoxicity Assay of Alcohols

Spheroid cultures of human hepatoblastoma cells (HuH-6 line) were established by rotating 3 x 10(6) cells/3 ml culture medium in 25-ml Erlenmeyer flasks on a gyratory shaker. The size of the spheroids rapidly increased until 4 days of culture, and thereafter their size gradually increased until 8 days of culture. A considerable amount of lactate dehydrogenase (LDH) was detected in the culture medium at 24h after seeding because of cell damage by subculturing, but thereafter the amount released was small, indicating that the spheroids were in healthy condition. Albumin production, one of the differentiated functions of hepatocytes, was higher in spheroid cultures than in monolayer cultures. Using this spheroid culture model, the cytotoxic effects of alcohols on HuH-6 cells were studied by measuring the activity of LDH released in the medium from damaged cells. The results indicate that the increasing order of toxicity of the alcohols was as follows: methanol