Optimization of in vitro cell culture conditions suitable for the cholangiocarcinoma stem cell study

Currently, cancer stem cells (CSCs) are extensively studied due to their roles in tumorigenesis, metastasis, and drug resistance.However, the low quantity, and its transient nature present a challenge for their study.Therefore, a culture system supporting high CSC quantity and flexible for high-throughput drug testing is needed.In this study, we used cholangiocarcinoma (CCA) cell lines with a CSC-specific live-cell biosensor system, which expresses green fluorescence protein (GFP) in CSCs, to study factors/conditions affecting CCA stem-like cell quantity in culture.We investigated culture media, seeding density, and compared 2-dimension (2-D) vs 3-dimensional (3-D) culture methods.The effects of anti-CCA drug dosages on CSC numbers were also studied.By comparing three media recipes, a defined serum-free CCA medium was selected for its high CSC yields.We found seeding density and time significantly influence CCA CSC percentages in both 2-D and 3-D settings.Notably, 3-D culture markedly increased CSC percentages compared to 2-D, supporting its application in CSC studies.In 3-D culture, a higher CSC index correlated with higher anti-CCA drug doses, suggesting loss of differentiation as a prominent characteristic under standard therapy.Thus, we report culture conditions influencing CCA CSC quantity, useful for CSC study and anti-CSC drug discovery in CCA.