title: Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
abstract: 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.
authors: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
authors: Laura
authors: L
authors: Broutier
authors: Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.
authors: Amanda
authors: A
authors: Andersson-Rolf
authors: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
authors: Christopher J
authors: 0000-0002-5294-1270
authors: CJ
authors: Hindley
authors: Foundation Hubrecht Organoid Technology (HUB), Utrecht, the Netherlands.
authors: Sylvia F
authors: SF
authors: Boj
authors: Hubrecht Institute-KNAW, University Medical Centre Utrecht, Utrecht, the Netherlands.
authors: Hans
authors: H
authors: Clevers
authors: Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Genetics, University of Cambridge, Cambridge, UK.
authors: Bon-Kyoung
authors: BK
authors: Koo
authors: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.|Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors: Meritxell
authors: 0000-0002-1545-5265
authors: M
authors: Huch
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D000066673:Cell Self Renewal; D005260:Female; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D008099:Liver / Q000166:cytology*; D008297:Male; D051379:Mice; D009940:Organoids / Q000166:cytology* / Q000378:metabolism; D010179:Pancreas / Q000166:cytology*