MORPHOLOGY AND FUNCTIONALITY OF PORCINE HEPATOCYTE SPHEROIDS

Introduction: It was assumed tha succesful long term culture of porcine hepatocyte spheroids require the culture continuosly in suspension in order to achieve the best functionality. Our working hypothesis is that after obtaining a tissue level of organization pig hepatocytes could maintain metabolic functions independently of the methods of culture. Objective: To demonstrate metabolic functionality on porcine hepatocyte spheroids characterized by electron microscopy. Methods: The cells were isolated and cultured in spinner flasks, feeded with oxygenated enriched E-Williams medium. Metabolism of diazepam and production of urea were used as markers of detoxification. The metabolism of diazepam was measured through the decrease of its concentration and the increase of its metabolites temazepam and oxazepam. Results: The spheroids formed compact structures, with relatively uniform size and shape, and smooth surface. They were evaluated by TEM, where viable cells were seen inside the spheroids, with preserved organelles and structures corresponding to bile canaliculi and desmosome junctions. Diazepam metabolites were present during the whole period of study (6 days), with a peak at day 1 (temazepam: 18,37 +/- 10,79 μg/ml, oxazepam: 0,11 +/-0,07 μg/ml), in agree with the maximum decrease of diazepam. Urea was also detected during the 6 days period (954.55 +/-238.36 μg/mg protein). Conclusion: This work suggest that when hepatocytes raise the level of a tissue with tight junctions, canaliculus and other structures, maintain metabolic abilities.