Optimization of isolation and culture of primary liver cancer parenchyma cells and cancer-associated fibroblasts

Objective To isolate and culture human primary liver cancer (PLC) parenchyma cells and cancer-associated fibroblasts (CAF) and to investigate the biological characteristics. Methods Fresh PLC tissues were collected from 15 patients with PLC who underwent surgical resection in the First Affiliated Hospital of Xi'an Jiaotong University from September 2015 to October 2016. Among them, 12 cases were male and 3 female, aged 28-64 years with a median of 45 years old. 13 cases were diagnosed with hepatocellular carcinoma and 2 cases of cholangiocarcinoma. The informed consents of all patients were obtained and the local ethical committee approval was received. Primary human hepatoma cells and CAF were isolated and cultured with collagenase digestion. The morphological characteristics of PLC parenchyma cells and CAF were observed under inverted microscope. The cell viability was assessed by CCK-8 assay. The expression levels of PLC cell marker alpha-fetoprotein (AFP) and CAF marker Vimentin were measured by immunofluorescence staining. Results The success rate of primary PLC parenchymal cells culture was 2/15, and 11/15 for CAF culture, all of which were hepatocellular carcinoma. The PLC parenchymal cells were epithelioid cells, which were distributed in a paving stone pattern. CAF was in long fusiform or polygonal shape, distributed in a fish swarm manner. PLC cells and CAF grew and proliferated well in the logarithmic growth phase. The proliferation ability of CAF was slightly stronger than that of PLC cells. Positive AFP was observed in the cytoplasm of primary PLC cells by immunofluorescence staining and positive Vimentin protein expression was observed in CAF. Conclusions Human primary PLC parenchyma cells and CAF with high proliferation can be successfully obtained through collagenase digestion. Key words: Carcinoma, hepatocellular; Fibroblasts; Culture techniques