Developing Primary Culture from Human Cholangiocarcinoma

Abstract

Background: Our goal is to isolate and expand tumor cells in culture and identify the spectrum of malignant attributes. Methods: Fresh tumor tissue from Grade 3, Stage IA, intrahepatic CCA was minced and subjected to enzymatic digestion for 75 minutes at 37°C. Digested contents were filtered through a 40 μm filter and the cells in the filtrate after washing were plated in 6 well collagen coated dishes using Williams E. Media supplemented with 2% FBS and several growth factors. One batch of cells was immortalized by pBABE hTERT retroviral vector. Results: Within 2-3 days, epithelial cells started propagating and cells grew up to 7 passages (P) over the course of two months both with (CCA-telo) or without (CCA) telomerase immortalization. Using qPCR analysis in P6 CCA/CCA-tel lines, expression profiles of liver cell specific markers like HP, ALB, APOA2, SERPINA1, and AFP were found to be very low,compared to HCC line HepG2 but exression of these was similar to lung adenocarcinoma A549 cell line. CCA line showed good telomerase expression however lower than CCA-telo line with exogenous telomerase induction. Conclusions: Cells isolated from human CCA show good viability and propagated for up to 7 passages. Though cells exhibited typical epithelial and mesenchymal morphology, the cells did not show liver epithelial markers. CCA line appears to be spontaneously immortalized, independent of exogenous hTERT induction. The gene expression profile of malignant tissue, if retained by primary culture cells, could facilitate the development and testing of novel molecular targets for cholangiocarcinoma.