Spheroid and monolayer cultures of male albino rat hepatocytes: a comparative study of in-vitro and ultrastructural features

ABSTRACT

This study was designed to compare the morphology and function of rat hepatocytes cultured as spheroids and as conventional monolayers for Ten days. Hepatocytes were isolated from liver of 15 male albino rats [8-10 weeks old] and cultured on the basal William's E medium. For spheroidal cultures, the tissue culture plates were coated with 2.5% pHEMA, whereas, for the conventional mnonolayers, the tissue culture plates were coated with rat tail collagen. Hepatocyte integrity and morphology were assessed by inverted phase-contrast and electron microscopies. Hepatocyte function was measured by the albumin secretion of the cells in the culture medium. The study revealed that hepatocytes cultured as spheroids retained a more in vivo-like morphology including their polyhedral/round shape together with normal ultrastructural features despite minimal nuclear injury of some cells and reduction in glycogen and lipid deposits. Hepatocytes in conventional monolayers appeared flattened and exhibited a spindle-like appearance. Moreover, marked clumping of nuclear chromatin, nucleolar segregation, lysis of the mitochondrial cristae, dilatation of smooth endoplasmic reticulum, depletion of glycogen and lipid stores with loss of intactness of the cell membranes were signs of prominent cellular injury in hepatocytes cultured as monolayers. In addition to maintaining a more in vivo-like morphology, rat hepatocytes cultured as spheroids retained more capacity for secreting albumin than those in conventional monolayers. These observations could suggest that spheroidal cultures allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. The improved morphology and function of hepatocyte cultured as spheroids may provide a more appropriate in-vitro model for hepatocyte research work, especially when long-term culture is crucial