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Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation


Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

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Figure 1: Schematic representation of organoid isolation, culture and characterization.
Figure 2: Examples of isolated ducts and growing organoids.
Figure 3: Differentiation of liver organoids.
Figure 4: Scheme summarizing the relevant steps in the organoid transduction (left) and transfection (right) protocols.
Figure 5: Transfection and transduction of liver organoids.


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L.B. is supported by an EMBO postdoctoral fellowship (EMBO ALTF 794-2014). C.J.H. is supported by a Cambridge Stem Cell Institute Seed Fund award and the Herchel Smith Fund. B.-K.K. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society. M.H. is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z). A.A.-R. is funded by an MRC PhD fellowship. We gratefully acknowledge the kind gift of 293T-HA-Rspo1-Fc cells from Calvin Kuo, Stanford University.

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Authors and Affiliations



M.H. and S.F.B. developed the culture system for liver and pancreas. B.-K.K. developed the genetic manipulation of organoid cultures. H.C. supervised the development of organoid cultures and their genetic manipulation. L.B., A.A.-R. and C.J.H. prepared figures. L.B., A.A.-R., C.J.H., S.F.B. and M.H. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Bon-Kyoung Koo or Meritxell Huch.

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The authors declare no competing financial interests.

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Broutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11, 1724–1743 (2016).

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