ALBUMIN SYNTHESIS FROM ISOLATED RAT HEPATOCYTES

A222 Aims: alternative approaches to get over the limit of shortage of donors for liver transplantation may be the use of hepatocytes for cell transplantation or for the bioartificial devices. The aim was to evalute if isolated rat hepatocytes are able to maintain synthetic and metabolic functions when seeded on homologous acellular matrix (HAM). Methods: 1.6×105/cm2 of viable hepatocytes detected by using an antialbumin rat antibody were seeded on 60 HAM, on 60 collagen coated wells and on 60 uncoated wells. Albumin concentration (commercial colorimetric assay), albumin mRNA expression (PCR analysis), urea nitrogen concentration (commercial enzymatic assay) were detected on rat hepatocytes culture 24 hours after seeding and then daily till day 9 to evaluate synthetic and metabolic functions. RNA was extracted from hepatocytes using a total RNA extraction kit. The RNA concentration was measured by absorption at 260nm. The primers were designed to amplify a region of the rat serum albumin. In parallel, negative PCR controls (no DNA) were performed. PCR products were analysed on agarose gel. Hepatocytes cultures were examined by scanning electron microscopy (SEM). Results: cells stained positive with the antialbumin rat antibody were 95% of the isolated cells. The presence of albumin and urea nitrogen concentration was detected in all the 9 days of the study in the three supports, but albumin mRNA, expression of neo-synthetized albumin, was detected for the 9 days of the study in hepatocytes seeded on HAM only. Albumin mRNA was detected day 1-3-5 in hepatocytes seeded on collagen coated wells and day 1 on uncoated wells. At SEM examination hepatocytes seeded on HAM were organized on clusters, well anchored to HAM and morphologically stable. Conclusions: rat hepatocytes maintain synthetic and metabolic functions for more than one week expecially when seeded on homologous acellular matrix.