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

Nature Protocols volume 14, pages 1884–1925 (2019)Cite this article

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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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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.

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Data availability

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.

References

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    PubMed  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

Download references

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

Author notes

  1. These authors contributed equally: Olivia C. Tysoe, Alexander W. Justin, Teresa Brevini.

  2. These authors jointly supervised this work: Kourosh Saeb-Parsy, Athina E. Markaki, Ludovic Vallier and Fotios Sampaziotis.

Authors and Affiliations

  1. Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge, UK

    Olivia C. Tysoe, Teresa Brevini, Si Emma Chen, Hajer Zedira, Ludovic Vallier & Fotios Sampaziotis

  2. Department of Surgery, University of Cambridge, Cambridge, UK

    Olivia C. Tysoe, Krishnaa T. Mahbubani, Kourosh Saeb-Parsy, Ludovic Vallier & Fotios Sampaziotis

  3. Department of Engineering, University of Cambridge, Cambridge, UK

    Alexander W. Justin & Athina E. Markaki

  4. Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milan, Italy

    Teresa Brevini

  5. Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

    Anna K. Frank & Espen Melum

  6. Research Institute of Internal Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

    Anna K. Frank & Espen Melum

  7. Department of Medicine III, University Hospital Aachen, Aachen, Germany

    Anna K. Frank

  8. Section for Gastroenterology, Department of Transplantation Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital Rikshospitalet, Oslo, Norway

    Espen Melum

  9. Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

    Espen Melum

  10. Department of Hepatology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK

    Fotios Sampaziotis

  11. Department of Medicine, University of Cambridge, Cambridge, UK

    Fotios Sampaziotis

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Contributions

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.

Corresponding authors

Correspondence to Ludovic Vallier or Fotios Sampaziotis.

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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.

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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

Supplementary Figure 1

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

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.

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.

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

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.

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

Supplementary Information

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

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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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