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


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.

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Fig. 1: Schematic overview of the protocol describing human breast organoid derivation, culturing, genetic manipulation and xenotransplantation.
Fig. 2: Organoid derivation, culturing and passaging.
Fig. 3: Genetic manipulation of breast organoids.
Fig. 4: Estrogen pellet implantation and organoid xenotransplantation.
Fig. 5: Characterization of BC organoid cultures.
Fig. 6: Characteristics of breast organoid cultures derived from histologically normal breast tissues.

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All data described in this protocol are included in the article figures or were published previously in the supporting primary research papers (, and


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We are very grateful for the technical support of the Princess Máxima Center for Pediatric Oncology, the Hubrecht Institute, the Foundation Hubrecht Organoid Technology (HUB), the Dana-Farber Cancer Institute and the Walter and Eliza Hall Institute of Medical Research for generation of the described methods and the HUB for managing the breast organoid biobank. This work was financially supported by the Princess Máxima Center for Pediatric Oncology. J.F.D. was supported by a Marie Curie Global Fellowship and a VENI grant from the Dutch Organization for Scientific Research. J.M.R. and J.S.B. were supported by an R35 grant (CA242428) from the National Cancer Institute and acknowledge the support of the Susan G. Komen Breast Cancer Foundation. J.M.R. was supported by the American Cancer Society. J.E.V. was supported by the Australian National Health and Medical Research Council. A.C.R. was supported by a Starting Grant 2019 (project 804412) from the European Research Council.

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Authors and Affiliations



J.F.D., E.J.V., N.S., J.M.R., O.K., H.G.R. and C.P. performed experiments and developed the protocol. J.F.D., E.J.V., N.S., J.M.R., H.G.R., C.S.V. and S.F.B. generated figures. J.F.D. and E.J.V. conceptualized and wrote the manuscript. H.G.R., E.J.W., C.S.V. and S.F.B. co-wrote the manuscript. J.E.V., C.S.V., S.F.B., J.S.B., H.C. and A.C.R. supervised the work.

Corresponding author

Correspondence to Johanna F. Dekkers.

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Competing interests

J.F.D., N.S., S.F.B., H.C. and A.C.R. are inventors on patents related to the organoid technology (WO201309812-A3/WO2016083612-Al/P309013GB).

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key references using this protocol:

Sachs, N. et al. Cell 172, E10 (2018):

Rosenbluth, J. M. et al. Nat. Commun. 11, 1171 (2020):

Dekkers, J. F. et al. J. Natl Cancer Inst. 112, djz196 (2019):

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Dekkers, J.F., van Vliet, E.J., Sachs, N. et al. Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids. Nat Protoc 16, 1936–1965 (2021).

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