Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation
Andersson Rolf, Amanda
Boj, Sylvia F
Nature Publishing Group
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Broutier, L., Andersson Rolf, A., Hindley, C., Boj, S. F., Clevers, H., Koo, B., & Huch Ortega, M. (2016). Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nature Protocols, 11 1724-1743. https://doi.org/10.1038/nprot.2016.097
Adult somatic tissues have proven difficult to expand in vitro, largely due to 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 describe 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 whilst retaining their tissue-of-origin commitment, genetic stability and differentiation potential 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 perform genetic manipulation experiments and requires basic scientific training to perform.
organoid, liver, pancreas, genetic manipulation, stem cell culture
LB is supported by an EMBO Postdoctoral fellowship (EMBO ALTF 794-2014). CH is supported by a Cambridge Stem Cell Institute Seed Fund award and the Herchel Smith Fund. BK is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society. MH is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z).
WELLCOME TRUST (104151/Z/14/Z)
External DOI: https://doi.org/10.1038/nprot.2016.097
This record's URL: https://www.repository.cam.ac.uk/handle/1810/256520