E.H. Chestnut1, M.T. Le1, B. Wei1, P. Li1, W. Zhang1, B. Ekser1 1Indiana University School Of Medicine, Transplant Surgery, Indianapolis, IN, USA
Background: Developing clinically relevant cellular models for studying cholangiopathies has been challenging. CHOL organoids offer the promise to recapitulate the structure and functionality of in vivo biliary trees. Previously, our lab has developed methods for isolating and culturing cholangiocytes (CHOL) from explanted human liver tissue. To enhance their expansion capability, the isolated CHOLs were immortalized using lentiviral vectors expressing human papillomavirus (HPV) E6/E7. However, it remains unclear whether the immortalization process alters other cellular properties. This study aims to determine whether immortalized CHOLs maintain the capability to self-organize into bile duct-like structures ex vivo, thus providing insight into their potential for modeling cholangiopathies and biliary tree development.
Methods: Human cholangiocytes were isolated from explanted human liver tissue (Figure 1A). 5,000 human primary or immortalized CHOL were seeded into a 24-well plate at 50ml/well in a mixture of Matrigel and culturing media supplemented with EGF, DKK1, R-Spondin1, and dexamethasone (Figure 1B). At 10 days of growth, the diameter and number of organoids from each condition were quantified. The expression of the key markers for CHOLs such as CK19, EpCAM, SCT, SR, CFTR, JAG1, and RS1 were measured by qPCR. Rhodamine123 transport assay was performed to further assess the functionality of the CHOL organoids.
Results: Fluorescent microscopy analysis confirmed that the cholangiocyte organoids generated with HPV E6/E7 immortalized CHOLs maintain the expression of cholangiocyte-specific cell markers CK19 and CK7 (Figure 1D), as well as maintain p-glycoprotein function. However, organoids derived from immortalized CHOLs showed a significant reduction in both diameter (12.68 ± 2.4 vs 20.61 ± 2.8, p<0.01) and number per well (17.3 ± 4.9 vs 27.0 ± 3.2, p<0.02) compared to the organoids derived from primary CHOL. qPCR analysis revealed elevated expression of progenitor cell marker EpCAM and reduced expression of the mature CHOL marker CK19 in immortalized cells derived organoids (Figure 1C), suggesting that immortalization impaired the differentiation of CHOLs under 3D culture conditions.
Conclusion: Although establishing immortalized CHOL cell lines from normal and diseased human livers provides effective cellular models for studying cholangiopathies, caution should be exercised when using these cells for studies that require fully functional, mature CHOL behavior or complex 3D organization. Future work should focus on developing alternative immortalization methods that better preserve the functional characteristics and differentiation potential of primary cholangiocytes in 3D culture systems.