Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

Johanna F Dekkers, Esmée J van Vliet, Norman Sachs, Jennifer M Rosenbluth, Oded Kopper, Heggert G Rebel, Ellen J Wehrens, Carol Piani, Jane E Visvader, Carla S Verissimo, Sylvia F Boj, Joan S Brugge, Hans Clevers, Anne C Rios

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgaveArtikelWetenschappelijkpeer review

Samenvatting

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

Originele taal-2Engels
Pagina's (van-tot)1936-1965
Aantal pagina's30
TijdschriftNature Protocols
Volume16
Nummer van het tijdschrift4
DOI's
StatusGepubliceerd - apr. 2021

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