Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.
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The authors declare that the main data supporting this study are available within the article. Additional data are available from the corresponding authors upon request.
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We acknowledge the Cambridge Biorepository for Translational Medicine for the provision of human tissue used in the study and thank N. Georgakopoulos and B. Bareham for their roles in collecting and distributing this tissue. We also thank W.G. Bernard and L. Gambardella for their help and advice with regard to the fixation and staining of tubular bioengineered biliary constructs. O.C.T. was supported by an MRC-Sackler Doctoral Training Partnership. F.S. was supported by an Addenbrooke’s Charitable Trust Grant, an Academy of Medical Sciences Clinical Lecturer Starter Grant (SGL019/1071) and an NIHR Clinical Lectureship. A.W.J. and A.E.M. acknowledge support from EPSRC (EP/R511675/1 and EP/N509620/1) and the Isaac Newton Trust. T.B. was supported by an EASL PhD Juan Rodes Studentship. The L.V. lab is funded by the ERC Proof of Concept grant Relieve-Chol, by the ERC advanced grant New-Chol, the Cambridge University Hospitals National Institute for Health Research Biomedical Research Centre and the core support grant from the Wellcome Trust and Medical Research Council of the Wellcome–Medical Research Council Cambridge Stem Cell Institute. A.W.J., F.S., L.V., A.E.M. and K.S.-P. gratefully acknowledge support from the Rosetrees Trust (REAG/240 and NMZG/233). A.K.F. and E.M. were supported by funds from the Norwegian PSC Research Center.
L.V. is a founder and shareholder of DefiniGEN. L.V., F.S. and K.S.-P. are founders and shareholders of Bilitech. The remaining authors declare no competing interests.
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Key references using this protocol
Sampaziotis, F. et al. Nat. Med. 23, 954–963 (2017): https://doi.org/10.1038/nm.4360
Sampaziotis, F. et al. Nat. Biotechnol. 33, 845–852 (2015): https://doi.org/10.1038/nbt.3275
Sampaziotis, F. et al. Nat. Protoc. 12, 814–827 (2017): https://doi.org/10.1038/nprot.2017.011
Integrated supplementary information
Flowchart showing recommended order of primary tissue processing for CO line derivation.
(a) Schematic representation of the optional EpCAM+ sorting step (procedure steps D III-DXXVI) for the derivation of intrahepatic COs. (b) Representative brightfield images of key EpCAM+ sorting steps. Numbers correspond to schematic stages in (a). 3: Liver tissue before enzymatic dissociation. 4: Liver tissue after enzymatic dissociation demonstrating release of cells in the medium and remnants of the extracellular matrix. 6: Single-cell suspension after filtration and before EpCAM+ sorting. Scale bars, 100 μm. (c) Representative brightfield image of an organoid derived from a single EpCAM+ cell, 48 hours after plating. Scale bar, 50 µm.
Additional characterisation and troubleshooting of CO lines. (a) Representative brightfield images of healthy CO lines derived from all tissue types: 1: bile duct (BD), gallbladder (GB), Endoscopic Retrograde Cholangio-Pancreatography (ERCP), liver biopsy (biopsy) and EpCAM+ sorted cells (EpCAM). Scale bars- 200 μm. (b) Representative brightfield images of CO lines showing typical CO culture issues contrasted with optimal CO lines. Scale bars, 200 μm
(a) Representative brightfield images of a CBD CO line derived from ~3.0 ×103 viable cells/well (total: ~2.0 ×104 viable cells). D0- D12: days after plating. P1: passage 1. (b) Graph illustrating cell number over time for the CBD line derived in (a) demonstrating appropriate expansion.
Representative flow cytometry plots showing gating strategy for all flow cytometric analyses. (a) Exclusion of debris. (b) Exclusion of doublets. (c) Secondary-only control to exclude negative population. (d) Representative CK19+/CK7+ population. A minimum of 2 × 104 gated events were used for analysis.
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Tysoe, O.C., Justin, A.W., Brevini, T. et al. Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue. Nat Protoc 14, 1884–1925 (2019). https://doi.org/10.1038/s41596-019-0168-0
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