Cell culturing method and medium for producing proliferated, normal, differentiated human liver cells

摘要

The present invention provides improvements in a method for producing an expanded non-transformed cell culture of human liver cells comprising the steps of: (1) preparing partially purified, minced human liver tissue, (2) concentrating the resulting cells and tissue pieces, (3) resuspending the concentrated tissue cells and pieces in a growth medium, (4) culturing the resuspended cells in the growth medium for a time and under conditions to effect sustained cell division, and (5) passaging the cultured human liver cells periodically to expand the culture. The growth medium comprises a combination of a basal medium and ingredients to provide a medium in which the cultured human liver cells are selectively proliferated without being transformed, providing an expanded culture of proliferated, functionally differentiated human liver cells that is substantially free of fibroblast, macrophage and capillary endothelial cells, the improvement comprising the steps of harvesting cells of the expanded culture at a selected PDL preferably 5, providing a high density cell suspension of such proliferated human liver cells, and incubating such high density cell suspension in a calm-down medium to induce a mitotically quiescent state and, using a culture procedure which encourages aggregation, making the cells adhere tightly to form a three-dimensional cell organisation typical of the organ of origin, thereby forming organoids. Methods of gel-embedding the single cells, aggregates and organoids produced by the method; the growth medium and calm-down medium; various different methods for use of the normal, proliferated human liver cells produced by the method, are also provided.