Experimental study on isolation and cultivation of primary rat hepatocytes

Objective:To investigate the feasibility of isolation and cultivation of primary rat hepatocytes by collagenase perfusion through hepatic portal vein.Methods:10 Sprague-Dawley rats as liver cell donor were used to obtain hepatocytes by Seglen in situ two-step collagenase perfusion through hepatic portal vein.Following filtration through 200-mesh sieves,the suspension was transferred to a centrifuge tube,and then purified hepatocytes at 200 r/min centrifugation for three times(3~5 min each time).Trypan blue staining was utilized to measure cell viability.Hepatocytes were cultured on the plates covered with rat tail collagen.The morphology of hepatocytes was observed under the inverted microscope.The isolated liver cells were identified by Periodic acid-Schiff reaction(PAS).The proliferation of the hepatocytes was detected by MTT.Results:The yield of the hepatocytes was(1.8±0.14)×108/mL from each rat.The viability of harvested primary hepatocytes were above 85%.Numerous glycogenosomes were found in cultured hepatocytes and revealed redness by PAS staining.The MTT results showed that the proliferation ability of the cultured hepatocytes reached peak on 8 d cultivation,but the viability of the hepatocytes began to decline on 9 d.Conclusion:Obtaining liver cells from the rat liver by collagen perfusion through hepatic portal vein is a simple and effective method.