Protocol | Published:

Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue

Abstract

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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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

References

  1. 1.

    Park, S. M. The crucial role of cholangiocytes in cholangiopathies. Gut Liver 6, 295–304 (2012).

  2. 2.

    Lazaridis, K. N. & LaRusso, N. F. The cholangiopathies. Mayo Clin. Proc. 90, 791–800 (2015).

  3. 3.

    Murray, K. F. & Carithers, R. L. AASLD practice guidelines: evaluation of the patient for liver transplantation. Hepatology 41, 1407–1432 (2005).

  4. 4.

    Sampaziotis, F. et al. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids. Nat. Med. 23, 954–963 (2017).

  5. 5.

    Sampaziotis, F. et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat. Biotechnol. 33, 845–852 (2015).

  6. 6.

    Sampaziotis, F. et al. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells. Nat. Protoc. 12, 814–827 (2017).

  7. 7.

    Lugli, N. et al. R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders. EMBO Rep. 17, 769–779 (2016).

  8. 8.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

  9. 9.

    Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247 (2013).

  10. 10.

    Kessler, M. et al. The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun. 6, 8989 (2015).

  11. 11.

    Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015).

  12. 12.

    Karthaus, W. R. et al. Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159, 163–175 (2014).

  13. 13.

    Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).

  14. 14.

    Koo, B.-K. & Clevers, H. Stem cells marked by the R-spondin receptor LGR5. Gastroenterology 147, 289–302 (2014).

  15. 15.

    Glinka, A. et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357 (1998).

  16. 16.

    Fedi, P. et al. Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J. Biol. Chem. 274, 19465–19472 (1999).

  17. 17.

    Bafico, A., Liu, G., Yaniv, A., Gazit, A. & Aaronson, S. A. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/arrow. Nat. Cell Biol. 3, 683 (2001).

  18. 18.

    Cui, S., Capecci, L. M. & Matthews, R. P. Disruption of planar cell polarity activity leads to developmental biliary defects. Dev. Biol. 351, 229–241 (2011).

  19. 19.

    Strazzabosco, M. & Fabris, L. Development of the bile ducts: essentials for the clinical hepatologist. J. Hepatol. 56, 1159–1170 (2012).

  20. 20.

    Place, E. S., George, J. H., Williams, C. K. & Stevens, M. M. Synthetic polymer scaffolds for tissue engineering. Chem. Soc. Rev. 38, 1139–1151 (2009).

  21. 21.

    Dhandayuthapani, B., Yoshida, Y., Maekawa, T. & Kumar, D. S. Polymeric scaffolds in tissue engineering application: a review. Int. J. Polym. Sci. 2011, 290602 (2011).

  22. 22.

    O’Brien, F. J. Biomaterials & scaffolds for tissue engineering. Mater. Today 14, 88–95 (2011).

  23. 23.

    Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47 (2005).

  24. 24.

    Cheung, H.-Y., Lau, K.-T., Lu, T.-P. & Hui, D. A critical review on polymer-based bio-engineered materials for scaffold development. Compos. Part B Eng. 38, 291–300 (2007).

  25. 25.

    Kehoe, S., Zhang, X. F. & Boyd, D. FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy. Injury 43, 553–557 (2012).

  26. 26.

    Frisch, S. & Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124, 619–626 (1994).

  27. 27.

    Zeugolis, D. I., Paul, R. G. & Attenburrow, G. Engineering extruded collagen fibers for biomedical applications. J. Appl. Polym. Sci. 108, 2886–2894 (2008).

  28. 28.

    Abou Neel, E. A., Cheema, U., Knowles, J. C., Brown, R. A. & Nazhat, S. N. Use of multiple unconfined compression for control of collagen gel scaffold density and mechanical properties. Soft Matter 2, 986–992 (2006).

  29. 29.

    Landi, F. et al. Endoscopic treatment of anastomotic biliary stricture after adult deceased donor liver transplantation with multiple plastic stents versus self-expandable metal stents: a systematic review and meta-analysis. Transpl. Int. 31, 131–151 (2018).

  30. 30.

    Seehofer, D., Eurich, D., Veltzke-Schlieker, W. & Neuhaus, P. Biliary complications after liver transplantation: old problems and new challenges. Am. J. Transpl. 13, 253–265 (2013).

  31. 31.

    Cellon. BioFELT scaffolds from BMS. Cellon http://www.cellon.lu/biofelt-scaffold.html (2019).

  32. 32.

    Justin, A. W., Saeb-Parsy, K., Markaki, A. E., Vallier, L. & Sampaziotis, F. Advances in the generation of bioengineered bile ducts. Biochim. Biophys. Acta Mol. Basis Dis. 1864, 1532–1538 (2018).

  33. 33.

    Zhang, L. & Hui, L. Bile ducts regenerated. Nature 547, 171 (2017).

  34. 34.

    Thomas, H. Bioengineering the common bile duct. Nat. Rev. Gastroenterol. Hepatol. 14, 504 (2017).

  35. 35.

    Günther, C., Brevini, T., Sampaziotis, F. & Neurath, M. F. What gastroenterologists and hepatologists should know about organoids in 2019. Dig. Liver Dis. https://doi.org/10.1016/j.dld.2019.02.020 (2019).

  36. 36.

    Huch, M. et al. Unlimited in vitro expansion of adult bi‐potent pancreas progenitors through the Lgr5/R‐spondin axis. EMBO J. 32, 2708–2721 (2013).

  37. 37.

    Cho, W. K., Mennone, A. & Boyer, J. L. Isolation of functional polarized bile duct units from mouse liver. Am. J. Physiol. Gastrointest. Liver Physiol. 280, G241–G246 (2001).

  38. 38.

    Demetris, A. J. et al. Isolation and primary cultures of human intrahepatic bile ductular epithelium. In Vitro Cell. Dev. Biol. 24, 464–470 (1988).

  39. 39.

    Auth, M. K. et al. Morphogenesis of primary human biliary epithelial cells: induction in high‐density culture or by coculture with autologous human hepatocytes. Hepatology 33, 519–529 (2001).

  40. 40.

    Ogawa, M. et al. Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat. Biotechnol. 33, 853 (2015).

  41. 41.

    De Assuncao, T. M. et al. Development and characterization of human induced pluripotent stem cell-derived cholangiocytes. Lab. Investig. 95, 684–696 (2015).

  42. 42.

    Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560 (2016).

  43. 43.

    Petrowsky, H. & Clavien, P.-A. in Transplantation of the Liver 3rd edn. (eds Busuttil, R. W. & Klintmalm, G. B. G.) 582–599 (W.B. Saunders, 2015).

  44. 44.

    Badylak, S. F. The extracellular matrix as a scaffold for tissue reconstruction. Semin. Cell Dev. Biol. 13, 377–383 (2002).

  45. 45.

    Dong, C. & Lv, Y. Application of collagen scaffold in tissue engineering: recent advances and new perspectives. Polymers 8, 42 (2016).

  46. 46.

    Glowacki, J. & Mizuno, S. Collagen scaffolds for tissue engineering. Biopolymers 89, 338–344 (2008).

  47. 47.

    Drury, J. L. & Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Polymers 24, 4337–4351 (2003).

  48. 48.

    Brown, R. A., Wiseman, M., Chuo, C.-B., Cheema, U. & Nazhat, S. N. Ultrarapid engineering of biomimetic materials and tissues: fabrication of nano- and microstructures by plastic compression. Adv. Funct. Mater. 15, 1762–1770 (2005).

  49. 49.

    Gieseck, R. L. 3rd et al. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One 9, e86372 (2014).

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Acknowledgements

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.

Author information

O.C.T.: manuscript writing and editing, coordination of study, execution of experiments and data acquisition, validation of the CO culture protocol, design and production of figures, final approval of the manuscript. A.W.J.: manuscript writing and editing, design, concept, development and validation of the collagen densification protocol, final approval of the manuscript. T.B.: manuscript writing and editing, collection of data, validation of the CO culture, collagen densification and scaffold seeding protocols. S.E.C.: production of schematics for Figs. 1, 2, 3, 4, 5, 7, 8, 9 and 10 and validation of the collagen densification protocol. K.T.M.: execution of experiments and data acquisition. A.K.F.: development and validation of the CO and ERCP brushing collection protocols. H.Z.: validation of CO culture and data acquisition. E.M.: critical revision of the manuscript, validation of the CO protocols. K.S.-P.: design and concept of the study, development of the protocol, critical revision and final approval of the manuscript. A.E.M.: design and concept of the collagen densification protocol, critical revision and final approval of the manuscript. L.V.: design and concept of the study, critical revision and final approval of the manuscript. F.S.: design and concept of the study, development and validation of the protocol, manuscript writing and editing, critical revision and final approval of the manuscript. O.C.T., A.W.J., and T.B. contributed equally to this work.

Competing interests

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.

Correspondence to Ludovic Vallier or Fotios Sampaziotis.

Integrated supplementary information

  1. Supplementary Figure 1

    Flowchart showing recommended order of primary tissue processing for CO line derivation.

  2. Supplementary Figure 2 Derivation of intrahepatic organoids through EpCAM sorting.

    (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.

  3. Supplementary Figure 3 Enlarged images of COs before and after organoid breaking.

    Enlarged images of COs before and after organoid breaking (from Fig. 5). Enlarged brightfield images of Fig. 5b, image 1 and 5b, image 5. Scale bars, 100 μm.

  4. Supplementary Figure 4 Representative images of COs.

    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

  5. Supplementary Figure 5 Derivation of a CO line from low cell numbers.

    (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.

  6. Supplementary Figure 6 Gating strategy for flow cytometric analyses.

    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.

Supplementary information

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    Supplementary Figs. 1–6 and Supplementary Tables 1 and 2

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  3. Supplementary Software 1 and 2

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Fig. 1: Flowchart of key steps in the generation of bioengineered biliary tissue.
Fig. 2: Derivation of ECOs from extrahepatic biliary tissue.
Fig. 3: Derivation of ECOs through ERCP brushings.
Fig. 4: Derivation of intrahepatic organoids.
Fig. 5: Passaging of COs.
Fig. 6: Characterization of COs.
Fig. 7: Generation of densified collagen sheets.
Fig. 8: Generation of densified collagen tubes.
Fig. 9: Seeding of flat densified collagen or PGA scaffolds.
Fig. 10: Seeding of densified collagen tubular scaffolds.
Fig. 11: Characterization of bioengineered biliary tissue.
Supplementary Figure 1
Supplementary Figure 2: Derivation of intrahepatic organoids through EpCAM sorting.
Supplementary Figure 3: Enlarged images of COs before and after organoid breaking.
Supplementary Figure 4: Representative images of COs.
Supplementary Figure 5: Derivation of a CO line from low cell numbers.
Supplementary Figure 6: Gating strategy for flow cytometric analyses.

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