Research articles
ScienceAsia 51 (2023): 1-8 |doi:
10.2306/scienceasia1513-1874.2023.059
Optimization of in vitro cell culture conditions suitable for the
cholangiocarcinoma stem cell study
Krittiyabhorn Kongtanawanicha, Pornlada Likasitwatanakulb,c, Methichit Wattanapanitchd,
Siwanon Jirawatnotaia,e,f,g,*
ABSTRACT: 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
f
luorescence 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
CCACSCpercentagesinboth2-Dand3-Dsettings. Notably, 3-Dculture markedly increased CSCpercentages 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.
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| a |
Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 Thailand |
| b |
Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University 10700 Thailand |
| c |
Department of Medicine, University of Minnesota, Minnesota 55414 USA |
| d |
Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700 Thailand |
| e |
Siriraj Center of Research Excellence for Precision Medicine and Systems Pharmacology, Faculty of Medicine Siriraj
Hospital, Mahidol University, Bangkok 10700 Thailand |
| f |
Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto
860-8555 Japan |
| g |
Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000 Thailand |
* Corresponding author, E-mail: siwanon.jir@mahidol.ac.th
Received 28 Oct 2024, Accepted 0 0000
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