Abstract:

Objective To study the morphological changes and the function of long-term cultured primary rat hepatocytes in a sandwich configuration. Methods Hepatocyte were isolated by a two-step collagenase perfusion based on a procedure described by Seglen and modified by us. Hepatocyte viability was determined by trypan blue exclusion test. Primary rat hepatocytes were cultured in a sandwich configuration. The morphological changes were observed under a inverted microscope. The concentration of albumin, BUN and lactate dehydrogenase(LDH) in the collected supernatant of different cultured periods were examined with single collagen gel configuration as control. Results Routinely, 2×10⁸～3×10⁸ cells were obtained per liver, with viability ranging from 90% to 96%[(93±3)%]. By the third day the function of the albumin secretion and urea synthesis recovered dramatically . The functions of albumin secretion and urea synthesis of the hepatocytes cultured in sandwich configuration maintained high level for 14 d. Heptocytes cultured in sandwich configuration formed hepatic cords, and bile canaliculi-like structures appeared, its polygonal morphology lasted for more than 28 d. Conclusion Sandwich configuration more closely mimics the normal histological sturcture of the liver, hepatocytes cultured in this configuration may maintain its morphology and function for a long period, it is not only used for the study of basic science, but also serves as the basis of a bioreactor and hepatocyte transplantation for the treatment of liver failure.

Key words: sandwich, hepatocyte, primary culture