Tissue-like Culture Of Hepatocytes For Application In Bioartificial Liver

Bioartificial liver (BAL) is the most important issue in the field of studying artificial liver supporting system (ALSS). The development of BAL technique could benefit from evaluating hollow fiber bioreactors, together with developing new methods of hepatocyte culture. In this paper, we investigated diffusion in mammalian cell culture by gel entrapment within hollow fibers, invent a new method of culturing hepatocyte spheroids, and set up a small BAL bioreactor by entrapment of rat hepatocyte spheroids inside hollow fibers. This paper mainly include the following three parts:1. Evaluation of diffusion in gel entrapment cell culture within hollow fibersEffective diffusion coefficients of glucose in collagen gels were firstly determined by lag time analysis in a diffusion cell. It was found that the gel contractions by entrapment of viable hepatocytes caused a significant reduction on effective diffusion coefficient of glucose. Then the cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Either hepatocytes or KB cells entrapped in hollow fibers with molecular weight cut off (MWCO) of 30 kDa didn't survive. And urea was secreted about 50 % more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 kDa.2. Rat hepatocyte spheroids cultureIt was found that 48 h after incubation hepatocytes formed spheroids spontaneously within hollow fibers with an efficiency of about 50 % by adding type I collagen solution into intracellular culture medium. With the change of collagen concentration in the medium, spheroids could be spherical or cylindrical. Scanning electronmicroscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies showed a resemblance between cells in the spheroids and in the liver in vivo. Urea genesis and albumin synthesis by spheroids were more efficient than that by gel entrapped dispersed hepatocytes.3. The development and evaluation of small BAL reactor utilizing hepatocyte spheroidsA small BAL utilizing cylindrical hepatocyte spheroids was developed. Spheroids entrapped within hollow fibers maintained viability and liver specific functions for at least 5 days. Assessment of urea genesis and albumin synthesis within this BAL indicated much higher activity than a dispersed hepatocyte entrapment BAL respectively.