Growth versus function in the three-dimensional culture of single and aggregated hepatocytes within collagen gels

Hepatocytes require cell-cell and cell-substrate interactions to survive and function. Furthermore, cell proliferation and differentiated function are often reciprocally related activities, both of which are highly regulated in the liver microenvironment. To mimic the physiological organization of hepatocytes into multicellular plates, small hepatocyte aggregates containing an average of 4-6 cells were formed. The aggregates were suspended within three-dimensional collagen gels and compared to both single hepatocytes (suspended within collagen gels) and hepatocyte monolayers (on tissue culture polystyrene) over 6 and 10 days in culture. The comparison of aggregates with single cells represents a novel in vitro system for the study of hepatocyte cell-cell interactions. All hepatocyte-populated gels displayed almost identical patterns of cell behavior: hyperplastic or hypertrophic growth (indicated by DNA or protein concentration) coincided with the decline of phenotypic function (assessed by uric acid and albumin production), while functional recovery often accompanied the cessation of growth. However, aggregate cultures maintained an average of 2-fold higher concentrations of functional parameters than singlet cultures when cells were not involved in growth activities. Much higher concentrations of all cell parameters were sustained by gel-dispersed hepatocytes than by hepatocyte monolayers.