Formation and Maintenance of Rat Hepatocyte Spheroids on Lactose-Silk Fibroin Conjugates in Primary Cultures

We examined the formation and maintenance of spherical multicellular aggregates (spheroids) of rat primary hepatocytes on dishes coated with lactose-silk fibroin conjugates bearing galactose residues (Lac-CY-SF) as substrate materials. Rat hepatocytes that had attached onto the conjugate-coated dishes were subsequently cultured in a medium supplemented with epidermal growth factor (EGF) and insulin. After the rat hepatocytes extended flat on the conjugate-coated dishes in 2 days of culture, these hepatocytes formed spheroids about 100 to 300 μm in diameter at day 4. After 6 days of culture the spheroids were maintained without any obvious change in size on the conjugate-coated dishes. However, the detachment of the spheroids from the conjugate-coated dishes began from day 8, and the spheroids clearly shrank at day 10. These results suggested that hepatocyte spheroid formation was induced on the Lac-CY-SF conjugate-coated dishes in the presence of EGF and insulin, but the conjugate-coated dishes were not capable of keeping the spheroids for more than 5 days.