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 100&mgr;m. 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.
Gao
Yi
Y
Hu
Huan-Zhang
HZ
Chen
Ke
K
Yang
Ji-Zhen
JZ