Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

AIM:To develop a culture mode providing durable biomaterials with high yields and activities used in bioartificial liver.METHODS:Hepatocytes were isolated from a whole pig liver by Seglen s method of orthotopic perfusion with collagenase. In culture on microcarriers, primary porcine hepatocytes were inoculated at a concentration of 5center dot10(7)/mL into the static culture systems containing 2g/L Cytodex-3, then supplemented with 100mL/L fetal calf serum (FCS) or 100mL/L porcine portal vein serum (PPVS) respectively. In spheroidal aggregate culture hepatocytes were inoculated into 100mL siliconized flasks at a concentration of 5.0center dot10(6)/mL.RESULTS:In culture on microcarriers hepatocytes tended to aggregate on Cytodex-3 obviously after being inoculated. Typical multi-cellular aggregated spheroids could be found in the two systems 24h-48h after hepatocytes were cultured. The morphological charact-eristics and synthetic functions were maintained for 5wk in FCS culture system and 8wk in PPVS culture system. In spheroidal aggregate culture about 80%-90% isolated hepatocytes became aggregated spheroids 24h after cultured in suspension and mean diameter of the spheroids was 100m. The relationship among the hepatocytes resembled that in the liver in vivo. Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytes cultured on monolayers.CONCLUSION:As high-yields and high-activity modes of culture on microcarriers or in spheroidal aggregate culture with portal vein serum are promising to provide biomaterials for bioartificial liver (BAL) efficiently.