A modified methodology for human hepatic stellate cells isolation

Objective To establish and optimize human hepatic stellate cells (hu-HSCs) isolation method for primary culture and research use. Methods Human hepatic tissues obtained from operation were perfused with collagenase and EGTA solution. Hepatocytes in the digested liver suspension were removed by low speed centrifugation. hu-HSCs were further separated from other nonparenchymal cells in the supernatant by Nycodenz density gradient centrifugation. The viability of the harvested hu-HSCs was determined by Trypan blue exclusion staining. The purity of hu-HSCs was identified by oil red O staining and immunofluorescence staining for both crSMA and desmin. Results The yield rate of hu-HSCs was 2 to 3 × 10⁶gram of hepatic tissue and the purity was 98% , which was higher than previous isolation method containing pronase and DNAase digestion steps. Conclusions The modified isolation method of primary hu-HSCs is simple and more cost-effective.