NL2027546B1 - Method of producing an organoid - Google Patents

Abstract

The present application provides a method for producing a breast organoid comprising: (a) isolating breast cells from breast milk; and (b) culturing the breast cells in a culture medium and under conditions suitable to form a breast organoid. A breast organoids obtainable by a method of the invention and their uses in research are provided and in the formation of assembloids are also provided.

Description

METHOD OF PRODUCING AN ORGANOID FIELD OF THE INVENTION

[0001] This invention relates to breast organoids, method for production thereof, and uses of the organoid as various research tools.

BACKGROUND OF THE INVENTION

[0002] Breast cancer (BC) remains the most widely diagnosed and second deadliest malignancy in women. BC comprises multiple subtypes that arise through the sequential accumulation of mutations in resident epithelial cells and result in tumours with complex genomic and biological heterogeneity. Standard therapeutic approaches are currently based on clinical and pathologic features, complemented by the expression status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2}, and are thereby inadequately tailored to individual patients. An extensive understanding of the processes underlying normal breast development, carcinogenesis and disease progression is necessary to devise novel personalized therapies, but hampered by a lack of suitable and widely available in vitro models.

[0003] There are various breast tissue models available in the prior art which are used to study breast tissue health and disease. For example, such models include breast cancer cell lines (2D), immortalized cell lines (2D), tumouroids (3D), spheroids (3D), breast organoids derived from healthy or tumour breast epithelial cells and mouse PDX models.

[0004] Organoid technology, the long-term propagation of human adult stem cells in three- dimensional (3D) cultures, has provided unprecedented and transformative ways to study healthy organ development and tumourigenesis in vitro.

[0005] State of the art methods to grow breast organoids use the culture media first described in Sachs, Norman, et al. "A living biobank of breast cancer organoids captures disease heterogeneity." Cell 172.1-2 (2018): 373-386.

[0006] In the prior art, breast organoids derived from breast tissue are often generated from tissue obtained after surgical resection of the breast tumour. These resections include normal tissue in addition to the tumour, which can be used as a source of healthy breast organoids. However, contamination with tumour cells often occurs and cannot be fully excluded.

[0007] Thus, there is a need in the prior art for improved in vitro models that recapitulate the complex organisation of healthy and tumour human breast tissue, to better enable investigation of human breast development and function in health and disease. The mammary tissue organoids and methods for their production described in the prior art, particularly healthy mammary organoids, are inefficient as they have low growth rate and limited long-term passaging.

BRIEF SUMMARY OF THE INVENTION

[0008] The inventors have surprisingly found that breast organoids may be generated from cells found in breast milk. This is particularly advantageous as there is increased certainty of capturing healthy epithelial cells form breast milk, thereby generating a healthy breast organoid that avoids contamination with tumour cells.

[0009] Breast milk as a source of cells for organoid generation has additional advantages.

Breast milk is an easily accessible source material compared to tissue resection, and the ethical and regulatory requirements for obtaining breast milk are relatively straightforward. Breast organoid generation from breast milk allows co-culturing with autologous cells from the same sample, for example immune cells such as macrophages, such that the organoid more closely mimics in vivo conditions.

[0010] The inventors have also surprisingly found a culture medium that is particularly efficient at growing breast organoids from breast milk. The breast organoids developed using the culture medium show improved outgrowth and long-term maintenance of the organoid through more passages than observed with breast organoids in the prior art.

[0011] In accordance with a first aspect the present invention there is provided a method for producing a breast organoid comprising: (a) isolating breast cells from breast milk; and (b) culturing the breast cells in a culture medium and under conditions suitable to form a breast organoid.

[0012] In certain embodiments, breast milk is obtained from a mammalian subject. In certain embodiments, breast milk is obtained from a human subject.

[0013] At least one advantage of using breast milk as a source for breast cells is the absence of tumorous cells. This allows for the formation of organoids that consist of healthy cells. In certain embodiments. organoids cultured from breast cells isolated from breast milk do not comprise tumorous or cancerous cells. Such organoids may be useful as models of healthy breast tissue and may be used as controls in a number of different methods and experiments such as toxicity assays. For example, testing the toxicity and/or specificity of chemotherapeutic agents against non-cancerous or tumorous cells.

[0014] In certain embodiments, the isolated breast cells are stem cells. In certain embodiments, the isolated breast cells are epithelial stem cells.

[0015] In certain embodiments, the culture medium comprises a Wnt agonist. In certain embodiments, Wnt agonists increase the efficiency of organoid establishment.

[0016] In certain embodiments, the Wnt agonist is a surrogate wnt, chir or wnt3a.

[0017] In certain embodiments, the Wnt agonist is provided by Wnt conditioned media at a concentration of 5 to 50%yv/v of the final culture medium volume. In certain embodiments, the Wnt agonist is provided by Wnt conditioned media at a concentration of 10 to 30%v/v of the final culture medium volume.

[0018] In certain embodiments, the culture medium comprises the wnt agonist at concentration of 0.01 to 2 nM. In certain embodiments, the culture medium comprises the wnt agonist at concentration of 0.05 to 1 nM. In certain embodiments, the culture medium comprises the wnt agonist at concentration of 0.2 nM.

[0019] In certain embodiments, the Wnt agonist is provided by Wnt conditioned media at a concentration of 20% v/v of the final culture medium volume.

[0020] In certain embodiments, the Wnt agonist is wnt3a.

[0021] In certain embodiments, the culture medium further comprises one or more of at least one Lgr5 agonist, at least one BMP inhibitor, at least one ROCK inhibitor, at least one ErbB3/4 ligand, at least one FGFR2b ligand, at least one TGF-beta inhibitor, at least one p38 inhibitor, at least one receptor tyrosine kinase ligand, B27 supplement plus Vitamin A, nicotinamide, at least one antimicrobial agent, and/or N-Acetylcysteine.

[0022] In certain embodiments, the culture medium further comprises one or more of at least one Lgr5 agonist, at least one BMP inhibitor, at least one ROCK inhibitor, at least one ErbB3/4 ligand, at least one FGFR2b ligand, at least one TGF-beta inhibitor, at least one receptor tyrosine kinase ligand, B27 supplement plus Vitamin A, nicotinamide, at least one antimicrobial agent, and/or N-Acetylcysteine.

[0023] In certain embodiments, the culture medium further comprises at least one Lgr5 agonist.

[0024] In certain embodiments, the culture medium further comprises at least one BMP inhibitor.

[0025] In certain embodiments, the culture medium further comprises at least one ROCK inhibitor.

[0026] In certain embodiments, the culture medium further comprises at least one ErbB3/4 ligand.

[0027] In certain embodiments, the culture medium further comprises at least one FGFR2b ligand.

[0028] In certain embodiments, the culture medium further comprises at least one TGF-beta inhibitor.

[0029] In certain embodiments, the culture medium further comprises at least one receptor tyrosine kinase ligand.

[0030] In certain embodiments, the culture medium further comprises B27 supplement plus Vitamin A.

[0031] In certain embodiments, the culture medium further comprises hydrocortisone and/or forskolin. In certain embodiments, the culture medium further comprises hydrocortisone. In certain embodiments, the culture medium further comprises forskolin. In certain embodiments, the culture medium further comprises hydrocortisone and forskolin.

[0032] In certain embodiments, the culture medium further comprises nicotinamide.

[0033] In certain embodiments, the culture medium further comprises N-Acetylcysteine.

[0034] In certain embodiments, the culture medium further comprises at least one antimicrobial agent.

[0035] In certain embodiments, the culture medium comprises the LgrS agonist at a concentration of 50 to 1000 ng/ml. In certain embodiments, the culture medium comprises the Lgr5 agonist at a concentration of 100 to 500 ng/ml. In certain embodiments, the culture medium comprises the Lgr5 agonist at a concentration of 250 ng/ml.

[0036] In certain embodiments, the culture medium comprises 1 to 50% v/v Lgr5 agonist conditioned media. In certain embodiments, the culture medium comprises 2 to 20% v/v Lgr5 agonist conditioned media. In certain embodiments, the culture medium comprises 10% v/v Lgr5 agonist conditioned media.

[0037] In certain embodiments, the culture medium comprises 1 to 50%v/v BMP inhibitor conditioned media. In certain embodiments, the culture medium comprises 2 to 20%v/v BMP inhibitor conditioned media. In certain embodiments, the culture medium comprises 10% v/v BMP inhibitor conditioned media.

[0038] In certain embodiments, the culture medium comprises the BMP inhibitor at a concentration of 10 to 500 ng/ml. In certain embodiments, the culture medium comprises the BMP inhibitor at a concentration of 50 to 250 ng/ml. In certain embodiments, the culture medium comprises the BMP inhibitor at a concentration of 100 ng/ml.

[0039] In certain embodiments, the culture medium comprises 1 to 20% v/v of 50 times concentrated B27 supplement plus Vitamin A. In certain embodiments, the culture medium comprises 1 to 5% v/v of 50 times concentrated B27 supplement plus Vitamin A. In certain embodiments, the culture medium comprises 2% v/v of 50 times concentrated B27 supplement plus Vitamin A.

[0040] In certain embodiments, the culture medium comprises 1 to 100 mM nicotinamide. In certain embodiments, the culture medium comprises 1 to 50 mM nicotinamide. In certain 5 embodiments, the culture medium comprises 10 mM nicotinamide.

[0041] In certain embodiments, the culture medium comprise 0.1 to 15 mM N-Acetylcysteine. In certain embodiments, the culture medium comprise 0.5 to 2 mM N-Acetylcysteine. In certain embodiments, the culture medium comprise 1.25 mM N-Acetylcysteine.

[0042] In certain embodiments, the culture medium comprise 0.1 to 5 pg/ml hydrocortisone. In certain embodiments, the culture medium comprise 0.1 to 1 pg/ml hydrocortisone. In certain embodiments, the culture medium comprise 0.5 ug/ml hydrocortisone.

[0043] In certain embodiments, the culture medium comprise 50 to 500 nM B-estradiol. In certain embodiments, the culture medium comprise 50 to 150 nM B-estradiol. In certain embodiments, the culture medium comprise 100 nM B-estradiol.

[0044] In certain embodiments, the culture medium comprise 1 to 50 uM forskolin. In certain embodiments, the culture medium comprise 5 to 15 uM forskolin. In certain embodiments, the culture medium comprise 10 uM forskolin.

[0045] In certain embodiments, the culture medium comprise 1 to 50 uM ROCK inhibitor. In certain embodiments, the culture medium comprise 1 to 10 pM ROCK inhibitor. In certain embodiments, the culture medium comprise 5 uM ROCK inhibitor.

[0046] In certain embodiments, the culture medium comprise 5 to 100 ng/ml FGFR2b ligand. In certain embodiments, the culture medium comprise 5 to 30 ng/ml FGFR2b ligand. In certain embodiments, the culture medium comprise 20 ng/ml FGFR2b ligand.

[0047] In certain embodiments, the culture medium comprise 0.1 to 5 uM TGF-beta inhibitor. In certain embodiments, the culture medium comprise 0.1 to 1 uM TGF-beta inhibitor. In certain embodiments, the culture medium comprise 0.5 uM TGF-beta inhibitor.

[0048] In certain embodiments, the culture medium comprise 1 to 50 ng/ml receptor tyrosine kinase ligand. In certain embodiments, the culture medium comprise 1 to 10 ng/ml receptor tyrosine kinase ligand. In certain embodiments, the culture medium comprise 5 ng/ml receptor tyrosine kinase ligand.

[0049] In certain embodiments, the culture medium comprise at least one antimicrobial agent at a concentration of 1 to 100 pg/ml. In certain embodiments, comprise at least one antimicrobial agent at a concentration of 5 to 50 ug/ml. In certain embodiments, comprise at least one antimicrobial agent at a concentration of 20 ug/ml.

[0050] In certain embodiments, the culture medium comprise 1 to 50 nM ErbB3/4 ligand. In certain embodiments, the culture medium comprise 1 to 10 nM ErbB3/4 ligand. In certain embodiments, the culture medium comprise 5 nM ErbB3/4 ligand

[0051] In certain embodiments: (i) the at least one Lgr5 agonist comprises R-spondin1; (ii) the at least one BMP inhibitor comprises Noggin; (iii) the at least one ROCK inhibitor comprises Y-27632; (iv) the at least one ErbB3/4 ligand comprises heregulin B1; (v) the at least one FGFR2b ligand comprises FGF-10; (vi) the at least one TGF-beta inhibitor comprises A83-01; (vii) the at least one p38 inhibitor comprises SB202190; and (viii) the at least one receptor tyrosine kinase ligand comprises EGF.

[0052] In certain embodiments, the culture medium comprises 1 to 50% v/ R-spondin1 conditioned media. In certain embodiments, the culture medium comprises 2 to 20% v/v R- spondin1 conditioned media. In certain embodiments, the culture medium comprises 10% v/v R- spondin1 conditioned media.

[0053] In certain embodiments, the culture medium comprises the R-spondin1 at a concentration of 50 to 1000 ng/ml. In certain embodiments, the culture medium comprises the R- spondin1 at a concentration of 100 to 500 ng/ml. In certain embodiments, the culture medium comprises the R-spondin1 at a concentration of 250 ng/ml.

[0054] In certain embodiments, the culture medium comprises 1 to 50%v/v Noggin conditioned media. In certain embodiments, the culture medium comprises 2 to 20%v/v Noggin conditioned media of the final volume. In certain embodiments, the culture medium comprises 10% v/v Noggin conditioned media.

[0055] In certain embodiments, the culture medium comprises Noggin at a concentration of 10 to 500 ng/ml. In certain embodiments, the culture medium comprises Noggin at a concentration of 50 to 250 ng/ml. In certain embodiments, the culture medium comprises Noggin at a concentration of 100 ng/ml.

[0056] In certain embodiments, the culture medium comprises 1 to 20% v/v of 50 times concentrated B27 supplement plus Vitamin A. In certain embodiments, the culture medium comprises 1 to 5% v/v of 50 times concentrated B27 supplement plus Vitamin A. In certain embodiments, the culture medium comprises 2% v/v of 50 times concentrated B27 supplement plus Vitamin A.

[0057] In certain embodiments, the culture medium comprises 1 to 100 mM nicotinamide. In certain embodiments, the culture medium comprises 1 to 50 mM nicotinamide. In certain embodiments, the culture medium comprises 10 mM nicotinamide.

[0058] In certain embodiments, the culture medium comprise 0.1 to 15 mM N-Acetylcysteine. In certain embodiments, the culture medium comprise 0.5 to 2 mM N-Acetylcysteine. In certain embodiments, the culture medium comprise 1.25 mM N-Acetylcysteine.

[0059] In certain embodiments, the culture medium comprise 0.1 to 5 ug/ml hydrocortisone. In certain embodiments, the culture medium comprise 0.1 to 1 pg/ml hydrocortisone. In certain embodiments, the culture medium comprise 0.5 ug/ml hydrocortisone.

[0060] In certain embodiments, the culture medium comprise 50 to 500 nM B-estradiol. In certain embodiments, the culture medium comprise 50 to 150 nM B-estradiol. In certain embodiments, the culture medium comprise 100 nM B-estradiol.

[0061] In certain embodiments, the culture medium comprise 1 to 50 uM forskolin. In certain embodiments, the culture medium comprise 5 to 15 uM forskolin. In certain embodiments, the culture medium comprise 10 pM forskolin.

[0062] In certain embodiments, the culture medium comprise 1 to 50 uM Y-27632. In certain embodiments, the culture medium comprise 1 to 10 pM Y-27632. In certain embodiments, the culture medium comprise 5 HM Y-27632.

[0063] In certain embodiments, the culture medium comprise 5 to 100 ng/ml FGF-10. In certain embodiments, the culture medium comprise 5 to 30 ng/ml FGF-10. In certain embodiments, the culture medium comprise 20 ng/ml FGF-10.

[0064] In certain embodiments, the culture medium comprise 0.1 to 5 pM A83-01. In certain embodiments, the culture medium comprise 0.1 to 1 uM A83-01.In certain embodiments, the culture medium comprise 0.5 uM A83-01.

[0065] In certain embodiments, the culture medium comprise 1 to 50 ng/ml EGF. In certain embodiments, the culture medium comprise 1 to 10 ng/ml EGF. In certain embodiments, the culture medium comprise 5 ng/ml EGF.

[0066] In certain embodiments, the culture medium comprise 1 to 50 nM Heregulin B1. In certain embodiments, the culture medium comprise 1 to 10 nM Heregulin B1. In certain embodiments, the culture medium comprise 5 nM Heregulin B1.

[0067] In certain embodiments, the culture medium primocin at a concentration of 1 to 100 g/ml. In certain embodiments, comprise primocin at a concentration of 5 to 50 ug/ml. In certain embodiments, comprise primocin at a concentration of 20 pg/ml.

[0068] In certain embodiments, the culture medium comprises Wnt3a, R-spondin1, Noggin, B27 plus Vitamin A, nicotinamide, N-Acetylcysteine, hydrocortisone, B-estradiol, forskolin, Y-27632, heregulin B1, FGF-10, A83-01, primocin, and EGF.

[0069] In certain embodiments, the culture medium consists of Wnt3a, R-spondin1, Noggin, B27 plus Vitamin A, nicotinamide, N-Acetylcysteine, hydrocortisone, B-estradiol, forskolin, Y-27632, heregulin B1, FGF-10, A83-01, primocin and EGF.

[0070] In certain embodiments, the culture medium comprises: (i) 20% v/v Wnt3a conditioned media; (ii) 10% v/v R-spondin1 conditioned media; (iii) 10% v/v Noggin conditioned media of the final volume; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 pM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

[0071] In certain embodiments, the culture medium consists of: (i) 20%v/v Wnt3a conditioned media; (ii) 10% v/v R-spondin1 conditioned media; (iii) 10% v/v Noggin conditioned media; (iv) 2% 50 times concentrated B27 supplement plus Vitamin A;

(v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 pM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 pM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

[0072] In certain embodiments, the culture medium comprises: (i) 0.2nM Wnt agonist; (ii) 250 ng/ml Rspondin1; (iii) 100 ng/ml Noggin; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 pM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

[0073] In certain embodiments, the culture medium consists of:

(i) 0.2nM Wnt agonist; (ii) 250 ng/ml Rspondin1; (iii) 100 ng/ml Noggin; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 pM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

[0074] In certain embodiments, the ROCK inhibitor is removed from the culture medium 2 to 3 days after organoid establishment, passaging or thawing. In certain embodiments, the Y-27632 is removed from the culture medium 2 to 3 days after organoid establishment, passaging or thawing.

[0075] In certain embodiments, prior to culturing the breast cells, the breast cells are suspended in a matrix and are seeded onto a surface.

[0076] In certain embodiments, the matrix comprises one or more of laminin, collagen IV, entactin and heparan sulfate proteoglycan, preferably wherein the matrix is basement membrane extract (BME).

[0077] In certain embodiments, the matrix is a BME.

[0078] In certain embodiments, culturing the breast stem cells comprises refreshing the culture medium at least twice a week. In certain embodiments, culturing the breast stem cells comprises refreshing the culture medium at least every 2 to 3 days.

[0079] In certain embodiments, culturing the breast stem cells comprises passaging the cells. In certain embodiments, culturing the breast stem cells comprises passaging the organoids formed from the breast stem cells.

[0080] In certain embodiments, the breast stem cells and/or breast organoids are passaged at a ratio of 1:2 to 1:10, In certain embodiments, the breast stem cells and/or breast organoids are passaged at a ratio of 1:2 to 1:6. In certain embodiments, the breast stem cells and/or breast organoids are passaged at a ratio of 1:2.

[0081] In certain embodiments, the breast stem cells and/or breast organoids are passaged every 7 to 21 days. In certain embodiments, the breast stem cells and/or breast organoids are passaged every 7 to 14 days.

[0082] In certain embodiments, the breast stem cells and/or breast organoids are passaged at least 4 times, at least 6 times, at least 10 times, at least 15 times. In certain embodiments, the breast stem cells and/or breast organoids are passaged at least 6 times. In certain embodiments, the breast stem cells and/or breast organoids are passaged at least 10 times. In certain embodiments, the breast stem cells and/or breast organoids are passaged at least 15 times. In certain embodiments, the breast stem cells and/or breast organoids are passaged more than 10 times. In certain embodiments, the breast stem cells and/or breast organoids are passaged more than 15 times.

[0083] According to a second aspect of the invention there is provided, a breast organoid obtainable by the method according to the first aspect, wherein the organoid withstands more than 4 passages, more than 10 passages and/or is maintained for at least 4 to 10 weeks.

[0084] In certain embodiments, the organoid withstands more than 4 passages. In certain embodiments, the organoid withstands more than 6 passages. In certain embodiments, the organoid withstands more than 10 passages. In certain embodiments, the organoid withstands more than 15 passages.

[0085] In certain embodiments, the organoid is maintained for at least 4 weeks. In certain embodiments, the organoid is maintained for at least 8 weeks. In certain embodiments, the organoid is maintained for at least 10 weeks. In certain embodiments, the organoid is maintained for at least 12 weeks. In certain embodiments, the organoid is maintained for at least 15 weeks. In certain embodiments, the organoid is maintained for more than 10 weeks. In certain embodiments, the organoid is maintained for more than 15 weeks.

[0086] In certain embodiments, the breast organoid comprises: polarized progenitor luminal cells; matured luminal cells; and basal cells.

[0087] In certain embodiments, the breast organoid comprises:

an inner compartment of polarized progenitor luminal cells and matured luminal cells and an outer of network of basal cells.

[0088] In certain embodiments, the breast organoid comprises a spherical shape.

[0089] According to a third aspect of the invention there is provided a breast organoid according to the second aspect, for use as a medicament.

[0090] According to a fourth aspect of the invention there is provided, use of the breast organoid as described herein for any one or more of: (i) drug discovery; (ii) toxicity assay; (iii) research of nutrient and drug metabolism; (iv) cancer research; (v) research of tissue embryology, cell lineages, or differentiation pathways; (vi) research of breast milk production and composition; and/or (vii) recombinant breast milk production.

[0091] In certain embodiments, the breast organoid is for use in drug discovery. A breast organoid formed from healthy cells (for example from breast milk) may be used as control for testing the specificity or toxicity of compounds. In certain embodiments, wherein the organoid is formed from tumorous cells, the organoid may be used to test the efficacy of compounds such as anti-cancer agents and/or chemotherapeutics.

[00982] In certain embodiments, the breast organoid is for use in toxicity assays. In certain embodiments, wherein the organoid is formed from healthy cells (for example from breast milk) the organoid may be used as a control for testing the specificity or toxicity of compounds.

[0093] In certain embodiments, the breast organoid is for use in cancer research. In certain embodiments, the breast organoid may be subjected to genetic manipulation in order to determine the effects of mutations and formation of cancerous cells.

[0094] In certain embodiments, the breast organoid is for use in research of tissue embryology, cell lineages, or differentiation pathways. Organoids formed from healthy or tumorous cells can be studied in order to provide information and insight into the normal functioning of mammary or breast organs as well as to study disease mechanisms and outcomes.

[0095] In certain embodiments, the breast organoid is for use in research of breast milk production and composition.

[0096] In certain embodiments, the breast organoid is for use in recombinant breast milk production.

[0097] According to a fifth aspect of the invention there is provided a method for producing an assembloid, comprising: (a) isolating breast cells from breast milk; (b) isolating at least one second cell type or cell line from a tissue or breast milk; and (c) combining the cells from step (a), and step (b); (d) culturing the combined cells of step (c) in a culture medium and under conditions suitable to form an organoid comprising the cells of step (a) and step (b).

[0098] In certain embodiments, the combined cells are cultured according to the method of the first aspect.

[0099] In certain embodiments, the assembloid is a hybrid organoid. That is to say that the assembloid comprises two or more cell types or cell lines. In certain embodiments, the assembloid comprises two or more cell types. As used herein cell type refers to healthy and un- healthy cell types such as cancerous or tumorous cells. In certain embodiments, both cell types are breast cells. In certain embodiments, both cell types are breast epithelial stem cells.

[00100] In certain embodiments, the assembloid may be formed from 2, 3, 4, 5 or more cell types orcell lines. That is to say the assembloid may be a hybrid organoid formed by fusing 2, 3, 4, 5, or more cell types or cell lines.

[00101] In certain embodiments, the cells of step (a) and step (b) are cultured in a culture medium as described herein.

[00102] In certain embodiments, the cells of step (a) are autologous cells to the cells of step (b). In certain embodiments, the cells of the step (a) and step (b) are obtained from the same subject.

[00103] In certain embodiments, the cells of step (b) comprise non-autologous cells to the cells of step (a). In certain embodiments, the cells of the step (a) and the cells of the step (b) are obtained from the different subjects.

[00104] In certain embodiments, the cells of step (b) comprise cells derived from a tumour tissue.

[00105] In certain embodiments, the cells of step (b) are isolated from tumour tissue. In certain embodiments, the cells of step (a) are derived from breast milk breast cells.

[00106] In certain embodiments, the method for producing an assembloid comprises a step of producing a first organoid from the cells of step (a) and producing at least one further organoid from the at least one second cell type or cell line prior to combining the cells.

[00107] In certain embodiments, the organoid formed from the cells of step (a) and/or the at least one further organoid formed from the at least one second cell type or cell line are produced according to the method of the first aspect.

[00108] In certain embodiments, step (c} comprises combining the organoid formed from the cells of step (a) and the at least one further organoid formed from the cells of step (b).

[00109] In certain embodiments, step (d) comprises incubating the organoid formed from the cells of step (a) and the least one further organoid formed from the at least one second cell type or cell line under conditions suitable to fuse the first organoid and the least one further organoid to form an assembloid.

[00110] In certain embodiments, the assembloid may be formed from 2, 3, 4, 5 or more organoids. That is to say the assembloid may be a hybrid organoid formed by fusing 2, 3, 4, 5, or more organoids.

[00111] In certain embodiments, each organoid of the assembloid is formed from different cell lines or types. As used herein cell line refers to morphologically or phenotypically distinct cell forms within a species. In certain embodiments, the further organoid is derived from breast cells, immune cells, ovarian cells or any other cell line or type. In certain embodiments, the further organoid is derived from any one or more of epithelial cells, barrier cells, hormone-secreting cells, neurons, sensory transducer cells, extracellular matrix cells, contractile cells, blood cells, immune cells, nurse cells, and/or intestinal cells.

[00112] In certain embodiments, the further organoid is cultured in a culture medium as described herein.

[00113] In certain embodiments, the further organoid comprises autologous cells to the cells of the first organoid. In certain embodiments, the cells of the first organoid and further organoid are obtained from the same subject.

[00114] In certain embodiments, the further organoid comprises non-autologous cells to the cells of the first organoid. In certain embodiments, the cells of the first organoid and further organoid are obtained from the different subjects.

[00115] In certain embodiments, the further organoid comprises cells derived from a tumour tissue.

[00116] In certain embodiments, the further organoid is derived from tumour tissue. In certain embodiments, the first organoid is derived from breast milk breast cells.

[00117] Assembloids may be for use according to the third and fourth aspects.

[00118] In certain embodiments, assembloids are for use as a medicament.

[00119] In certain embodiments, assembloids are for use in any one or more of; (i) drug discovery; (ii) toxicity assay; (iii) research of nutrient and drug metabolism; (iv) cancer research; (v) research of tissue embryology, cell lineages, or differentiation pathways; (vi) research of breast milk production and composition; and/or (vii) recombinant breast milk production.

[00120] According to a further aspect of the invention there is provided, use of a culture medium for producing a breast organoid derived from breast cells isolated from breast milk, wherein the culture medium is as described herein.

[00121] In certain embodiments, the organoid is an organoid according to the first aspect of the invention. In certain embodiments, the organoid may be an assembloid according to the fifth aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[00122] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[00123] Figure 1. Schematic overview of the protocol describing human breast organoid derivation, culturing, genetic manipulation, and xenotransplantation. Organoids are established from resections of normal breast or tumour tissue, followed by organoid maintenance or freezing for long-term storage. Genetic manipulation by lipofectamine-based transfection, electroporation- based transfection, or lentiviral transduction is described, as well as (clonal) organoid selection. Orthotopic injection can be performed to grow organoid-derived tumours in vivo, with the stated time indicating time until first signs of tumour formation. Corresponding steps of the protocol and their timing are indicated in yellow boxes.

[00124] Figure 2. Organoid derivation, culturing, and passaging. (a) Schematic overview of the organoid derivation process. Biopsies are sliced into 0.5 - 1 mm? pieces, digested with collagenase, and plated in BME. (b) Representative piece of healthy breast tissue. Scale bar 1 cm. (c) Representative images of the absence of a pellet (left), a pellet containing residual BME (middle), and a clean pellet (right), after organoid washing. (d) Representative images of BME drops in a 12-well plate. Scale bar 5 mm. (e) Brightfield images of BC organoids (Sample 36 of Table 1) grown in either Type 1 or Type 2 expansion medium (EM), demonstrating increased organoid outgrowth in Type 2 compared to Type 1 EM. P = passage number, d = number of days since last split. Scale bar 400 um. (f) Representative images of BC organoid cultures passaged as single cells (left) or fragments of 5-20 cells (right). Scale bars 100 um. (g) Representative brightfield images of BC organoids seeded too sparsely (left), too densely (middle), or at the proper density (right). Scale bars 100 um. (h) Representative image of an organoid culture with clear presence of cell debris. Scale bar 100 um. (i) Representative images of confluent organoid wells of solid (left) and grape-like (right) cultures. Scale bars 100 um. Numbers in f — i refer to sample numbers in Table 1.

[00125] Figure 3. Genetic manipulation of breast organoids. (a) Schematic overview of genetic manipulation of breast organoids. Dissociated organoids (1) can be incubated with a Lipofectamine 2000-based transfection mix (2a), electroporated (2b), or incubated with high-titer lentivirus (2c), and plated in BME (3). (b) Representative fluorescent images of normal breast organoids 7 days in culture after transduction with lentivirus expressing fluorescent reporters and single guide (sg)RNAs or Cas9, as indicated. Scale bar 500 um. (€) Representative brightfield images of normal breast organoids (control) or normal breast organoids knocked out for £53 and PTEN by CRISPR/Cas9 and treated with Nutlin-3a (10 uM) for 7 days. Scale bar, 200 um. (d,e) Representative images of a single organoid (d) or multipe organoids (e) (sample 20 of Table 1) that were genetically edited to stably express H2B::mNeonGreen and a puromycin resistane gene after 14 days of puromycin selection. Scale bars 100 um (d) and 1000 um (e).

[00126] Figure 4. Estrogen pellet implantation and organoid xenotransplantation. (a) Subcutaneous estrogen pellet implantation. (b) Shaving of the injection site (i) and orthotopic injection of organoids into the mammary fat pad (ii). (c) Representative image of a tumour grown from orthotopically injected breast cancer organoids. Scale bar 5 mm.

[00127] Figure 5. Characterization of BC organoid cultures. (a) Representative brightfield images of different morphologies of breast cancer (BC) organoids. Scale bars, as indicated in the bottom right corner of the first image, 100 um. (b) Donut chart depicting the distribution of the major morphologies of 31 BC organoid cultures. (¢) Donut chart showing the distribution of BC subtypes in 155 tissue samples and 95 related organoid cultures. (d) Stacked bar chart demonstrating the success rate of organoid establishment, grouped by receptor expression status. ‘Yes’ indicates successful establishment (> 5 in vitro passages), ‘No’ indicates unsuccessful establishment. (e) Stacked bar charts showing the percentage of organoid cultures with positive (green) or negative (pink) receptor status, as compared to the original tissue receptor status. (f) Comparative immunohistochemical images of breast cancer tissue and derived organoids. Shown are representative images of receptor positive (pos) and receptor negative (neg) samples. Scale bar, as indicated in the top left corner of the first image, 100 um. ER = estrogen receptor, PR = progesterone receptor and HER2 = human epidermal growth factor receptor 2. In panel a and d, numbers 15, 18, 16, 27, 22 and 19 refer to sample numbers in Table 1, and 35T, 68T, 86T and 72T refer to sample numbers as published in Sachs, N. et al. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 172, 373-386.e10 (2018).

[00128] Figure 6. Characteristics of breast organoid cultures derived from histologically normal breast tissues. (a) Diverse structure types can be seen in a single normal breast organoid culture at passage 5. (b) Representative examples of organoid cultures showing structures of the mature luminal-type (acinar structures with a large lumen), luminal progenitor-type (smaller structures with a small inner lumen that can exhibit budding), and basal/stem cell-type (larger structure types that can exhibit budding or branching). (c}) Fluorescent confocal image of a representative branching organoid at passage 3 of culture labelled for DAPI (white) and F-actin (red). (d) Fluorescent confocal image of a luminal structure at passage 5 of culture labelled for DAPI (white) and F-actin (red). (e) CyTOF analysis using a breast-specific antibody panel demonstrates the presence of all of the major mammary epithelial cell types in a single culture. Heatmap showing normalized protein expression levels for the indicated markers (x-axis) for single cells in a representative organoid culture (y-axis). (f) The indicated medium components were omitted from Type 1 expansion medium and breast organoid cultures were generated. The distribution of mammary lineages as measured by CyTOF is shown in the donut plots (upper panel), and representative brightfield images of the cultures are shown (bottom panel}. Basal: LP = luminal progenitor, ML = mature luminal. Other = cells falling outside of the CD49f and EpCAM gating parameters, and can thus not be defined as basal, LP or ML. In all panels: organoids were maintained in Type 1 expansion medium from which Y-27632 was removed 2 — 3 days after organoid establishment, passaging, or thawing; scale bars 100 um (a-c) and 20 pm (d).

[00129] Figure 7. Establishment and characterization of organoids derived from human milk. (A) Organoid formation efficiency of two milk donors in different medium conditions. Organoids were cultured in ‘Type 1" medium, ‘Type 2’ medium, or ‘Type 2’ medium without Wnt3A. (B) Growth rate of organoids established from tissue (blue; left hand column; n = 12 organoid cultures) compared to organoids established from milk (red; right hand column; n = 4 organoid cultures). (C) Bright-field images of morphologies observed in milk-derived organoid cultures. Scale bar 50 um. (D) 3D imaging of milk-derived organoids (left) and 2D slice (right). Organoids were stained for DAPI (grey), E-cadherin (green), Cytokeratin 5 (magenta) and Cytokeratin 8/18 (blue). Scale bars 20 um. (E) 3D imaging and 2D slice of milk-derived organoids virally transduced to express mNeonGreen (green; exterior structures) and mScarlett-1 (magenta; interior structures). Scale bars 10 um. (F) 3D live cell imaging of tumour organoids (top) and milk-derived organoids (bottom) cultured with engineered T-cells over 10 hours. Scale bars 100 um. (G) 3D live cell images of an assembloid consisting of a milk-derived organoid (yellow; white (top) arrows) fused to a tumour organoid (yellow and green; green (bottom) arrows) at the indicated time points of incubation with the chemotherapy pactitaxel, showing that the tumour part dies (increase in red (dead) signal) while the normal part remains alive. Scale bars 20 pm.

DETAILED DESCRIPTION

[00130] In one embodiment the present invention is based on the use of an expansion medium, comparable to that developed for long-term ovarian cancer organoid cultures previously. The expansion medium is surprisingly good at maintaining outgrowth and maintenance of healthy and tumour breast organoids derived from breast milk.

[00131] The term organoid is used to refer to self-organized three-dimensional tissue cultures derived from stem cells. Organoids may include artificial, in vitro three-dimensional structures made to mimic or resemble the functional and/or histological structure of an organ or portion thereof, such as a mammary or breast organ.

[00132] In comparison to commonly used breast cancer cell lines or breast cancer spheroids, breast cancer organoids can be grown long-term, while recapitulating the complex genetic and phenotypic heterogeneity that is characteristic of breast cancer. The organoids of the present invention allow the extended culture of all mammary lineages, in contrast to the normal breast organoids in the prior art. Generally, establishing a breast organoid culture, from cell isolation until the first passage, typically takes 7-21 days. To genetically manipulate an organoid culture and generate a selected clonal line at passage 1, typically takes a 14-21 days. To expand an organoid culture for transplantation usually requires more than 4 weeks. The present invention provides breast organoids, both healthy and tumour organoids which may be passaged for more than 4 weeks. In certain embodiments, an organoid of the invention may be passaged for 4 to 10 weeks, preferably 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks. More preferably the organoid of the invention may be passaged for more than 10 weeks.

[00133] The proliferation rate of breast organoids is relatively slow, compared to intestinal organoids for example. The splitting ratio can vary from 1:6 weekly to 1:2 weekly, biweekly, or even less frequently (Table 1).

[00134] The methods of the invention and breast organoids obtainable thereby recapitulate the original patient tumour in terms of histopathology, hormone receptor expression and HER2 status, as well as copy number variations and sequence changes, thereby capturing the full heterogeneity of breast cancer cells. Furthermore, healthy breast organoids of the present invention contain basal/stem cells, luminal progenitors (LPs) and mature luminal (ML) cells.

[00135] The methods and organoids of the invention may be used to study the development and function of the human mammary gland. For understanding de novo oncogenesis, normal breast organoids can be subjected to CRISPR/Cas9 editing to efficiently knock out tumour suppressor genes, or organoids from individuals with a hereditary cancer predisposition syndrome, such as BRCA1/BRCAZ2 mutations, can be used.

[00136] The methods and organoids of the present invention may be used to assess the efficacy of novel drugs, both in vitro, as well as upon engraftment in vivo. These organoid xenograft models can also be applied to visually study tumour growth and cancer cell behaviour in vivo through the introduction of a mammary imaging window. Due to the retained genetic and histological features of the original patient tumour, breast cancer organoids can be used as a clinical tool to aid personalized medicine, by assessing drug responses in a patient-specific manner.

[00137] The methods and organoids of the present invention may be used to provide an injection- based method for estrogen pellet implantation and breast cancer organoid xenotransplantation. This circumvents the need for complex surgical procedures.

[00138] The methods and organoids of the present invention may recapitulate or mimic phenotypic features of in vivo mammary epithelium. The organoids of the invention may have a structure including an inner compartment of polarized progenitor cells and matured luminal cells and an outer network of myoepithelial cells. The term myoepithelial cells may be interchangeably used with the term basal cells, Thus, the organoids of the invention may have a structure including an inner compartment of polarized progenitor cells and matured luminal cells and an outer of network basal cells. Therefore, the methods of the invention may provide a breast tissue organoid that is a mimetic of in vivo breast tissue.

[00139] The methods and organoids of the present invention may be used for a variety of therapeutic and medical methods.

[00140] For example, the organoids of the invention may be used for researching tissue embryology, cell lineages, or differentiation pathways by providing a model system that can be studied and interrogated.

[00141] The methods and organoids of the present invention may be used for recombinant production of breast milk. For example, organoids may be stimulated with prolactin in order to induce production of breast milk.

[00142] Recombinant breast milk production also allows the use of the organoids of the invention to investigate the components in breast milk and the mechanism of production in vitro.

[00143] Organoids that have been induced to recombinantly produce breast milk may also be used to research the effects of different compounds and nutrients on the production and composition of breast milk produced.

[00144] Organoids of the invention may be genetically modified or manipulated. Methods to genetically modify organoids of the invention include transfection, for example by lipofection, electroporation, and/or transduction, for example by lentivirus transduction.

[00145] Organoids of the invention may be selected for specific properties. Selection may be done by competitive selection methods. For example, by the use of targeted toxins or markers that will kill, destroy or label organoids that do or do not have desired properties such specific genes or genetic mutations.

[00146] In certain embodiments breast organoids of the invention may be for use as a medicament. For example, for use in methods of treating diseases such as cancer.

[00147] Breast organoids of the invention may be provided in a composition, such as pharmaceutical composition. A pharmaceutically composition may include one or more pharmaceutically acceptable carriers, excipients and/or additional compounds.

[00148] The methods and culture medium disclosed herein may be used for the production of breast organoids from breast cells derived from breast tissue.

[00149] Isolation of breast cells from breast tissue can be achieved by taking a resection of tissue from a subject. The resection material may be homogenised by any known methods, for example by cutting or shearing the tissue. The tissue may then be washed and digested. For example, by enzymatic digestion using enzymes such collagenase. Once homogenised and/or digested, the tissue may be further washed and/or filtered in order to remove unwanted cell types, dead cells and/or cellular debris. The isolated breast cells are epithelial stem cells,

[00150] Tissue used to isolate cells may be healthy tissue or tumour tissue. “Tumour tissue” includes tissue of a solid tumour, a semi-solid tumour, a primary tumour, a metastatic tumour, and the like. As used herein, “tumour tissue” not only includes a tissue made up exclusively of cancer cells, but also a tissue that includes cancer cells and one or more additional cell types, including but not limited to, immune cells (e.g., tumour associated macrophages (TAMs)) associated with (e.g., infiltrated within) the tissue. “Cancer cell” may be used interchangeably herein with “tumour cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of tumour tissue.

[00151] As used herein, “healthy tissue” is a relative terminology, generally referring to tumour- free tissue at the level of the statistical evaluation, and according to some embodiments, may refer to cancer-free tissue, or breast cancer free tissue.

[00152] A subject may refer to any animal that has breast cells, such as any mammal. In certain embodiments, the subject is a human.

[00153] In certain embodiments, the methods of the invention provide for the production of breast organoids from breast cells derived from breast milk. Breast cells may be derived from breast milk by isolating breast cells from breast milk. Without being bound by theory, the use of cells from breast milk provides cells that are healthy. That is to say that cells obtained or derived from breast milk do not include, for example, cancerous or tumorous cells.

[00154] Breast milk may be obtained from any subject that is capable of producing breast milk, for example any mammal. In certain embodiments, the breast milk is obtained from a human.

[00155] “Breast milk" refers to fluid produced by a mammary gland and expressed or exuded by the breast. Breast milk includes all lactation products, including, but not limited to, colostrum, whole milk and skim milk taken at any time before or after birth. As used herein, " breast milk " typically refers to human whole milk. "Whole milk" means breast milk that has not had fat removed therefrom.

[00156] Breast milk may contain functionally distinct bioactive components that are involved in remodelling of the immune system of neonates and infants. Cells of eukaryotic origin (i.e, excluding probiotic bacteria) found in breast milk may include two main groups: blood-derived and breast-derived cells. Both groups may include progenitor and/or stem cells.

[00157] The main cell lines found in breast milk may be CK18+ luminal epithelial cells and betacasein-positive lactocytes that synthesize milk proteins. Human milk may also include luminal and/or myoepithelial cells. The epithelial component of breast milk may include not only mature epithelial cells, but also their precursors and stem cells such as epithelial stem cells.

[00158] Breast milk stem cells may be able to differentiate into cells of all three germ layers (ectodermal germ layer, mesodermal germ layer, and/or endodermal germ layer) and the level of pluripotency may be comparable with that of human embryonic stem cells. Breast milk stem cells may express one or more markers. For example one or more of, Alpha36 Integrin (CD49f), p63, Cytokeratin 5 (CKS), CD34, CD44, CD90, CD271, CD146, SSEA4, TRA-1-80, TRA-1-81, OCT4, Sox2 TRA-60-1, Nanog, and/or KLF4.

[00159] Isolation of breast cells from breast milk may be achieved by centrifugation of breast milk. Centrifugation of breast milk may provide a pellet that includes breast cells. For example, breast epithelial stem cells.

[00160] Isolated breast cells, derived from tissue samples or breast milk may be cultured under conditions in order to form an organoid.

[00161] For example, the breast cells may be suspended in on 3-dimensional support such as a matrix. For example, the support may be a basement membrane extract. Base membrane extracts (BME) are a gel-like substance that polymerizes at temperatures above 10 °C, and can be used to mimic the extracellular matrix and provide support for 3D organoids. BMEs include a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumour. The extract may provide a natural extracellular matrix hydrogel that polymerizes at 37°C to form a reconstituted basement membrane. As used herein, the term basement membrane refers to continuous sheets of specialized extracellular matrix that forms an interface between for example endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma and plays a role in tissue organization by influencing cell adhesion, migration, proliferation, and differentiation. The major components of BME may include laminin, collagen IV, entactin, and heparan sulfate proteoglycans. Examples of BMEs include Cultrex® RGF BME Type 2, and Cultrex® Basement Membrane Extract. Matrigel™ can be used as alternative for BME.

[00162] Once cells are suspended in a support, the cells and supported may be deposited onto a surface. For example, the cells and support may be deposited or seeded into the well of culture plate. The number of cells that may be deposited onto a surface may range from 500,000 cells up to 1,000,000 cells or more.

[00163] Once deposited the cells may be contacted with a culture medium and incubated or cultured in the presence of the culture medium in order to allow for expansion of cells and formation of organoids.

[00164] The cells, and organoids formed therefrom may be cultured for at least one week. The cells and organoids formed therefrom may be cultured and maintained for 10 weeks or more.

[00165] The culture medium may be refreshed. For example, the culture medium may be refreshed at least twice a week. In certain embodiments, the culture medium may be refreshed every 2 to 3 days or more.

[00166] The cells and organoids formed therefrom may be passaged. The terms passaging, substituting or splitting may be used interchangeably throughout to refer to methods of removing medium and transfer of cells from a previous culture into fresh culture medium, in order to help enable the further propagation of cells or organoids.

[00167] The cells or organoids formed therefrom may be passaged at a ratio of 1:2 to 1:10. In certain embodiments, the cells or organoids formed therefrom may be passaged at a ratio of 1:2 to 1:6. Preferably the cells or organoids formed therefrom may be passaged at a ratio of 1:2.

[00168] In certain embodiments, an organoid of the invention may be passaged for 4 to 10 weeks, preferably 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks. More preferably the organoid of the invention may be passaged for more than 10 weeks.

[00169] In order to passage cells or organoids formed therefrom any known method may be used. For example, cells or organoids may be detached from their support using any suitable method. For example, cells or organoids may be detached using shake-off, scarping and/or enzymatic dissociation. Examples of enzymatic dissociation may involve the use of trypsin, collagenase, dispase and/or other enzymes or enzyme mixtures which are commercially available, for example Gibco™ TrypLE™ and TrypLE Express™ (Thermo Fisher).

[00170] The use of a culture medium according to the invention may allow for an improved number of passaging events and therefore may provide organoids with a longer maintenance period or lifetime. The terms culture medium, expansion medium and growth medium are used herein interchangeably.

[00171] The culture medium of the invention may include a Wnt agonist. The Wnt signalling pathway is a conserved pathway that regulates aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans. The Wnt signalling pathways include a group of signal transduction pathways made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. Three Wnt signalling pathways have been characterized: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. All three Wnt signalling pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which passes the biological signal to the protein Dishevelled inside the cell. Without being bound by theory, the canonical Wnt pathway may lead to regulation of gene transcription, the noncanonical planar cell polarity pathway may regulate the cytoskeleton that is responsible for the shape of the cell, and the noncanonical Wnt/calcium pathway regulates calcium inside the cell. Wnt signalling pathway has been demonstrated to play a role in a variety of diseases, including cancer (such as breast and prostate cancers), glioblastoma, and/or type II diabetes.

[00172] Wnt agonists may include surrogate wnt, chir and/or wnt3a.

[00173] The wnt agonist may be provided by including in the culture medium a composition that has been conditioned to include a wnt agonist. Such a composition may be referred to as wnt agonist conditioned medium. In certain embodiments, the wnt agonist may be provided by a recombinant wnt agonist.

[00174] In certain embodiments, culture mediums of the invention comprise at least 5%v/v wnt agonist conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 10%v/v wnt agonist conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 20%v/v wnt agonist conditioned medium. In certain embodiments,

culture mediums of the invention consist of 20%v/v wnt agonist conditioned medium. In certain embodiments, culture mediums of the invention consist of 20%v/v wnt3A conditioned medium.

[00175] The culture mediums of the invention also include one or more of: at least one Lgr5 agonist such as R-spondin1; at least one BMP inhibitor such as Noggin; at least one ROCK inhibitor such as Y-27632; at least one ErbB3/4 ligand such as heregulin B1; at least one FGFR2b ligand such as FGF-7 and/or FGF-10; at least one TGF-beta inhibitor such as A83-01; at least one p38 inhibitor such as SB202190; the at least one antimicrobial agent such as primocin; and/or at least one receptor tyrosine kinase ligand such as EGF.

[00176] ROCK inhibitors refer to Rho kinase inhibitors such as compounds that target rho kinase.

[00177] In certain embodiments, the Rock inhibitor may be included in the culture medium at a concentration of 5uM. In certain embodiments, the ROCK inhibitor may be removed from the culture medium 2 to 3 days after organoids have been formed. In certain embodiments, the ROCK inhibitor may be removed from the culture medium after passaging. In certain embodiments, the ROCK inhibitor may be removed from the culture medium after thawing. In certain embodiments, the ROCK inhibitor may be provided by a recombinant ROCK inhibitor.

[00178] ErbB3/4 ligands refer to Receptor tyrosine-protein kinase erbB-3 ligands. ErbB3/4 is also known as HER3 (human epidermal growth factor receptor 3), and is a membrane bound protein that in humans is encoded by the ERBB3 gene. Ligand binding causes a change in conformation that allows for dimerization, phosphorylation, and activation of signal transduction.

[00179] ErbB3 is a member of the epidermal growth factor receptor (EGFR/ERBB) family of receptor tyrosine kinases. The kinase-impaired ErbB3 is known to form active heterodimers with other members of the ErbB family, most notably the ligand binding-impaired ErbB2.

[00180] In certain embodiments, the ROCK inhibitor may be included in the culture medium at a concentration of 5nM.

[00181] FGFR2b ligands refers to fibroblast growth factor receptor 2.

[00182] FGF-7 refers to Fibroblast growth factor 7. FGF-10 refers to Fibroblast growth factor 10. FGF-10. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumour growth and invasion. They exhibit mitogenic activity for keratinizing epidermal cells, but essentially no activity for fibroblasts, which is similar to the biological activity of FGF7. Studies of the mouse homolog of suggested that this gene is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of lim bud formation. The FGF-10 gene is also implicated to be a primary factor in the process of wound healing.

[00183] In certain embodiments, of the culture mediums of the invention do not include FGF-7.

[00184] In certain embodiments, of the culture mediums of the invention FGF-10 may be included at a concentration of 20ng/ml.

[00185] TGF-beta inhibitors refer to inhibitors of Transforming growth factor beta (TGF-beta). TGF-beta promotes or inhibits tumourigenesis depending on the concurrent gene mutations and tissue microenvironment present through the small mothers against decapentaplegic (Smad) and non-Smad pathways.

[00186] In certain embodiments, the culture mediums of the invention TGF-beta inhibitors may be included at a concentration of 0.5uM.

[00187] p38 inhibitor refers to inhibitors of p38 mitogen-activated protein kinase. p38 are mitogen-activated protein kinases (MAPKs) that are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell differentiation, apoptosis and autophagy.

[00188] In certain embodiments, the culture mediums of the invention p38 inhibitors may be included at a concentration of 1M.

[00189] In certain embodiments, the culture mediums of the invention do not include p38 inhibitors.

[00190] Receptor tyrosine kinase ligands refers to ligands of receptor tyrosine kinases such as epidermal growth factor receptor family, fibroblast growth factor receptor (FGFR) family, vascular endothelial growth factor receptor (VEGFR) family, RET receptor family, Eph receptor family, and discoidin domain receptor (DDR) family.

[00191] EGF refers to Epidermal growth Factor. EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture.

[00192] In certain embodiments, of the culture mediums of the invention EGF may be included at a concentration of 5ng/ml.

[00193] In certain embodiments, culture mediums of the invention include noggin. Noggin (also known as NOG) is a protein involved with the development of many different body tissues such as nerve tissues, bones and muscles. In human development, noggin is encoded by the NOG gene. The amino acid sequence of noggin is highly homologous to that of rats, mice and Xenopus.

Noggin is a signalling molecule that plays a role in promoting somite patterning in the developing embryo. It's released from the notochord and regulates bone morphogenic protein (BMP4) during development. The secreted polypeptide noggin binds and inactivates members of the transforming growth factor-beta (TGF-beta) superfamily signalling proteins, such as bone morphogenetic protein-4 (BMP4). By diffusing through extracellular matrices more efficiently than other members of the TGF-beta superfamily, noggin is believed to have a role in creating morphogenic gradients. Noggin also appears to have pleiotropic effects in both early development and later stages.

[00194] The noggin may be provided by including in the culture medium a composition that has been conditioned to include noggin. Such a composition may be referred to as noggin conditioned medium. In certain embodiments, the noggin may be provided by a recombinant noggin.

[00195] In certain embodiments, culture mediums of the invention comprise at least 2%v/v noggin conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 5%v/v noggin conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 10%v/v noggin conditioned medium. In certain embodiments, culture mediums of the invention consist of 10%v/v noggin conditioned medium.

[00196] In certain embodiments, culture mediums of the invention include R-spondin1. R- Spondin-1 (Rspo-1) belongs to the (Rspo) family of Wnt modulators. The family includes four structurally related secreted ligands (Rspo 1-4), all containing the furin-like and thrombospondin structural domains. Rspo-1 is expressed in certain areas of the developing central nervous system, as well as in the adrenal glands, ovary, testis, thyroid, and trachea. Rspo can interact with the Frizzled/LRP6 receptor complex in a manner that stimulates the Wnt/B-catenin signalling pathway. Recombinant Human R-Spondin-1 is a 26.7 kDa protein consisting of 243 amino acid residues. Due to glycosylation, R-Spondin-1 may migrate at an apparent molecular weight of approximately 40.0 kDa by SDS-PAGE analysis under reducing conditions.

[00197] The R-spondin1 may be provided by including in the culture medium a composition that has been conditioned to include R-spondin1. Such a composition may be referred to as R- spondin1 conditioned medium. In certain embodiments, the R-spondin1 may be provided by a recombinant Rspo protein such as recombinant R-spondin1, R-spondin2, and/or R-spondin3.

[00198] In certain embodiments, culture mediums of the invention comprise at least 2%v/v R- spondin1 conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 5%v/v R-spondin1 conditioned medium. In certain embodiments, culture mediums of the invention comprise at least 10%v/v R-spondin1 conditioned medium. In certain embodiments, culture mediums of the invention consist of 10%v/v R-spondin1 conditioned medium.

[00199] In certain embodiments, culture mediums of the invention include forskolin. Forskolin refers to a labdane diterpenoid isolated from the Indian Coleus plant. It has a role as a plant metabolite, an anti-HIV agent, a protein kinase A agonist, an adenylate cyclase agonist, an antihypertensive agent and a platelet aggregation inhibitor. It is a labdane diterpenoid, an acetate ester, an organic heterotricyclic compound, a triol, a cyclic ketone and a tertiary alpha-hydroxy ketone.

[00200] In certain embodiments, culture mediums of the invention include hydrocortisone. Hydrocortisone, or cortisol, is a glucocorticoid secreted by the adrenal cortex.

[00201] In certain embodiments, the culture media includes B27 supplement plus Vitamin A. B27 plus Vitamin A refers to compositions that include B27 and vitamin A. B27 supplement may comprise one or more of catalase, reduced glutathione, insulin, superoxide dismutase, Holo- Transferin, T3, L-carnitine, Ethanolamine, D+-galactose, Putrescine, Sodium selenite, Corticosterone, Linoleic acid, Linolenic acid, Progesterone, Retinol acetate, DL-alpha tocopherol (vitamin E), DL-alpha tocopherolacetate, Oleic acid, Pipecolic acid, and/or Biotin.

[00202] In certain embodiments, compositions may be serum free. B27 supplement plus vitamin may be provided at 50x concentration. Therefore, a final working concentration of 1x may be used. In certain embodiments, the culture media includes 2%v/v B27 plus vitamin A supplement. That is to say in a final volume of 200ml, 4ml of 50x concentrated B27 plus vitamin a supplement would be included in the culture medium. In certain embodiments, the culture media comprises 1x concentrated B27 supplement plus vitamin A.

[00203] Examples of B27 supplement plus Vitamin A include, B-27™ Plus Supplement (50X) (available from ThermoFisher under catalogue number A3582801).

[00204] In certain embodiments, the culture medium includes B-estradiol. B-estradiol is a major estrogen secreted by the premenopausal ovary. Estrogens direct the development of the female phenotype in embryogenesis and during puberty by regulating gene transcription and, thus, protein synthesis.

[00205] In certain embodiments, the culture medium includes N-acetylcysteine. In certain embodiments, the culture medium includes N-acetyl-L-cysteine. N-acetylcysteine is an antioxidant and mucolytic agent. It may increase cellular pools of free radical scavengers. It has been reported to prevent apoptosis in neuronal cells but induce apoptosis in smooth muscle cells and may serve as a substrate for microsomal glutathione transferase.

[00206] In certain embodiments, the culture medium includes nicotinamide. Nicotinamide is a water-soluble form of vitamin B3 or niacin. Nicotinamide is an amide derivative of vitamin B3 and a PARP inhibitor.

[00207] In certain embodiments, the culture medium includes at least one antimicrobial agent. As used herein, "antimicrobial agent" refers to a compound or substance having antibacterial properties. include antibiotics (also termed antibacterial) and anti-fungal, anti-viral, and anti- parasitic agents. Also encompassed in the terms "antimicrobial" and "antimicrobial agents" are antimicrobial antibodies {e.g., antibodies that bind to and directly kill organisms or enhance their clearance during infection), antimicrobial peptides, phages, phage lysins (e.g., bacteriophage endolysins, which are phage- encoded peptidoglycan hydrolases able to cause lysis of cells such as bacteria), anti-virulence compounds (e.g., anti-toxins that interfere with bacterial disease progression by binding to target proteins produced during infection or anti-adhesins that interfere with bacteria binding to tissue), and other alternative class or non-standard agents developed as therapeutic agents for treating infections caused by one or more microbial organisms.

[00208] In In certain embodiments, the culture medium includes 20 pg/ml of an antimicrobial agent.

[00209] In In certain embodiments, the antimicrobial agent is primocin.

[00210] As used herein, the term Type 2 medium (as defined in Table 2) is used to refer to a certain embodiment of culture mediums of the invention. As used herein, Type 1 medium (as defined in Table 2) is used to refer to standard organoid culture or expansion medium. Type 2 medium as used as described herein, for culturing of breast organoids has surprisingly be found to allow for increased throughput of organoid formation and increased passaging.

[00211] In certain embodiments, culture mediums of the invention consist of Type 2 medium (as defined in Table 2).

[00212] Methods of the invention and the organoids formed therefrom may be used to form hybrid organoids. The term hybrid organoids refers to an organoid formed from two or more individual cell types or cell lines or from two or more organoids formed from two or more individual cell types or cell lines which are fused. Hybrid organoids may be referred to as assembloids.

[00213] In certain embodiments, assembloids are formed by combining a cells of first cell type or cell line and cells of second cell type or cell line and forming an organoid therefrom according to the methods of the invention.

[00214] In certain embodiments, cells of first cell type or cell line are first formed into a first organoid and the cells of second cell type or cell line first formed into a second or further organoid and the two organoids are incubated or cultured together according to the methods described herein to form a fused organoid.

[00215] In certain embodiments, assembloids are formed by combing a first organoid, such as an organoid of the invention with at least one second organoid. The second organoid may be formed by a method as described herein or by other suitable methods. The two organoids are then incubated or cultured together in order to allow the two organoids to fuse to each other. Upon fusion a hybrid organoid is formed.

[00216] In certain embodiments, the cell types or lines or the two organoids formed therefrom are isolated or formed from two different tissue types. For example one cell type or line or organoid may be derived from healthy breast tissue cells or breast milk and the second cell type or line or organoid is derived from cancerous, tumourous or unhealthy cells.

[00217] In certain embodiments, the second cell type or cell line or the organoids formed therefrom are isolated or formed from one or more of breast cells, immune cells, ovarian cells, epithelial cells, barrier cells, hormone-secreting cells, neurons, sensory transducer cells, extracellular matrix cells, contractile cells, blood cells, immune cells, nurse cells, and/or intestinal cells.

[00218] In certain embodiments, the second cell type or cell line or the organoids formed therefrom are isolated or formed from one or more of CK18+ luminal epithelial cells, betacasein- positive lactocytes luminal cells, myoepithelial cells, and/or epithelial stem cells.

[00219] Hybrid organoids or assembloids may be formed by fusing more than two organoids.

For example, 3, 4, or more organoids. Each organoid may be derived from a different cell type, tissue type or cell class. That is to say a hybrid organoid or assembloid may be include 1, 2, 3, 4, 5, 10 or more different cell types.

[00220] Assembloids may be used for assessment of drug or therapy toxicity. For example, by determining the different effects of a drug or therapy on cells of the first organoid and cells of the second organoid.

[00221] It will be understood that the methods described herein may be applied to the production of breast organoids derived from tissue. For example, organoids formed from cells derived from a breast tissue sample and not isolated from breast milk.

EXAMPLES Example 1

[00222] The inventors have investigated morphology, growth rate and passaging conditions for 45 organoid cultures encompassing normal, primary tumour, as well as metastatic cultures as a reference guide for organoid maintenance, taking into account inter-culture variation (Table 1).

The organoid cultures that were investigated are available at Huburect Organoid Technology, Yalelaan 62, 3584 CM Utrecht (www.huborganoids.nl). The deposit information for each organoid investigated is described in Table 1.

[00223] Fig 1 details step-by-step protocol outlines the derivation, manipulation, and xenotransplantation of human breast organoids. Organoid derivation

[00224] An organoid culture may be established as described in Sachs, N. et al. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 172, 373-386.e10 (2018).

[00225] Patient resection material is cut into small pieces, washed thoroughly and digested with collagenase. After additional washing and filtration, the digested tissue is plated in basement membrane extract (BME) and supplemented with expansion medium (Fig. 2a, b). BME, a gel-like substance that polymerizes at temperatures above 10 °C, is used to mimic the extracellular matrix and provide support for 3D organoids (Fig. 2c,d). Matrigel can be used as alternative for BME with comparable performance (see Reagents section). Organoid derivation is most optimal for tissues that contain a minimal amount of fat and necrotic tissue. Expansion medium

[00226] The preparation of an expansion medium has been previously described in Sachs, N. et al A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 172, 373-

386.e10 (2018). This expansion medium is referred to as ‘Type 1’ in Table 2. A second, alternative, expansion medium was also investigated, which is comparable to that previously developed for long-term ovarian cancer organoid cultures. This is referred to as ‘Type 2’ in Table

2. This expansion medium improves the growth properties of some organoid cultures (Fig. 2e; Table 1). The components used in each medium are described in Table 2. We advise to grow newly established organoid cultures in both Type 1 and Type 2 expansion medium, to define the most optimal medium type per donor. The inventors used home-made R-spondin-1, Noggin and Whnt3a conditioned media, for which detailed production protocols are available, for example, Cattaneo, C. M. et al. Tumour organoid—T-cell coculture systems. Nat. Protoc. 15, 15-39 (2020), Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347-358 (2016). Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474-85 (2015) and Broutier, L. 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). Organoid maintenance

[00227] The split ratio, passage interval and method of organoid dissociation significantly differs between donors and was optimized for each newly established organoid culture (Table 1). Single cell organoid passaging by trypsinization (step 43A, Fig. 2f) or via organoid fragments by mechanical disruption, referred to as shearing, (step 43B, Fig. 2f) was conducted. Fig. 2g-1 provides guidance for culturing at appropriate organoid densities, how to minimize cell death and how to recognize a confluent well of organoids together with procedures and conditions for long- term storage and recovery after cryopreservation. At least 6 cryovials for each newly established organoid culture at an early passage (< passage 5) were cryopreserved. Genetic manipulation

[00228] Three different methods to genetically modify breast organoids were conducted, including transfection by Lipofectamine 2000, electroporation, or stable transduction using lentivirus (Fig. 3a-c). Control transfection, or transduction vectors with a fluorescent label, to assess efficiency of the procedure was used (Fig. 3b,d and e). Organoid selection and clonal cultures

[00229] Genetically modified organoids can be selected by antibiotic addition if a resistance gene has been introduced, or by addition or withdrawal of other medium components. For instance, addition of Nutlin-3a will kill all cells expressing wild-type P53 and can be used to select P53- mutated organoids (Fig. 3c). In addition, withdrawal of EGF can select for cells overexpressing KRAS Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43-47 (2015). Preferably, the optimal concentration per selection agent and organoid culture, which is often the lowest concentration that kills 100% of untreated organoids was calculated. Selection and propagation of individual organoids can be used to generate a clonal culture (Fig. 3d, e). Non-manipulated organoids are used as a control. Although sub-cloning for each organoid culture can be assessed, the use of organoid cultures that efficiently expand after passaging from single cells give a better success rate. Xenotransplantation

[00230] A large quantity of organoids is necessary for engraftment (steps 102-113), usually varying between 0.25x10°-1x10° cells per injected site in the form of intact organoids for a better engraftment rate compared to single cells. These numbers require optimization per organoid culture. Organoid cultures with a fast growth rate in vitro tend to engraft better in vivo. We describe surgery-free estrogen pellet implantation (steps 114-121; Fig. 4a) prior to orthotopic organoid injection (steps 122-132; Fig. 4b) to provide an exogenous source of estrogen that facilitates tumour engraftment and growth (Fig. 4¢). Organoid injection was carried out by injecting 30 pl transplantation medium with 5%Trypan Blue, followed by dissection to confirm localization in the mammary fad pad. Reagents

[00231] Bovine serum albumin (BSA), modified fraction V (Sigma-Aldrich, cat. no. A9418)

[00232] BSA, fatty acid free (Sigma Aldrich, cat. no. A6003)

[00233] Cell recovery solution (Corning, cat. no. 354253)

[00234] Collagenase Type II (Thermo Fisher Scientific, cat. no. 17101-015)

[00235] Cultrex RGF BME, Type 2 (R&D systems, cat. no. 3533-005-02); Matrigel (e.g. BD Biosciences, cat. no. 356231) can be used as an alternative with equal performance.

[00236] Ethanol, 70% (vol/vol) (Klinipath, cat. no. 4070-9010)

[00237] Formalin, 4% solution (e.g. Sigma Aldrich, cat. no. 1.00496)

[00238] DPBS (Dulbecco’s phosphate-buffered saline, no calcium, no magnesium, 1x; Thermo Fisher Scientific, cat. no. 14190-144)

[00239] Recovery cell culture freezing medium (Thermo Fisher Scientific, cat. no. 12648-010)

[00240] Trypan Blue solution (Sigma Aldrich, cat. no. T8154)

[00241] TrypLE Express (1x, Thermo Fisher Scientific, cat. no. 12605-010)

[00242] Fetal bovine serum (FBS), heat-inactivated (Thermo Fisher, cat. no. 10500064)

[00243] Dimethyl sulfoxide (DMSO; Sigma Aldrich, cat. no. D2650).

[00244] Reagents for Organoid Culture

[00245] A83-01 (Tocris Bioscience, cat. no. 2939)

[00246] Advanced DMEM/F12 (adDMEM/F12; Thermo Fisher Scientific, cat. no. 12634-028)

[00247] B27 Supplement (50%, Thermo Fisher Scientific, cat. no. 17504-44)

[00248] B-estradiol (Sigma-Aldrich, cat. no. E2257)

[00249] DMEM GlutaMAX (Thermo Fisher Scientific, cat. no. 31966-047)

[00250] Forskolin (Sigma-Aldrich, cat. no. F6886)

[00251] GlutaMAX (100%, Thermo Fisher Scientific, cat. no. 35050-061)

[00252] HEPES 1M (100%, Thermo Fisher Scientific, cat. no. 15630-080)

[00253] Heregulin 31 (Peprotech, cat. no. AF-100-03)

[00254] Hydrocortisone (Sigma-Aldrich, cat. no. H-0888)

[00255] N-acetyl-L-cysteine (Sigma-Aldrich, cat. no. A9165-59)

[00256] Nicotinamide (Sigma-Aldrich, cat. no. NO636)

[00257] Noggin-conditioned medium, produced by a Noggin-producing cell line, which can be obtained from the laboratory of Prof. H. Clevers (Hubrecht Institute, The Netherlands).

Preparation instructions can be found in box 2 of Cattaneo, C. M. et al. Tumour organoid-T-cell coculture systems. Nat. Protoc. 15, 15-39 (2020). Commercially available Noggin (e.g. U-Protein Express BV, cat. no. N002, at a final concentration of 1-2% (vol/vol) or recombinant Noggin (Peprotech, cat. no. 120-10C, at a final concentration of 100 ng/ml) can be used as alternatives.

[00258] Nutlin-3a (Merck, cat. no. SML0580)

[00259] Penicillin-Streptomycin (Thermo Fisher Scientific, cat. no. 15140-122)

[00260] Primocin (Invivogen, cat. no. Ant-pm-1)

[00261] Recombinant human epidermal growth factor (EGF) (Peprotech, cat. no. AF-100-15)

[00262] Recombinant human fibroblast growth factor (FGF)-7 (Peprotech, cat. no. AF-100-19)

[00263] Recombinant human FGF-10 (Peprotech, cat. no. AF-100-26)

[00264] Red blood cell lysis buffer (Sigma Aldrich, cat. no. 11814389001)

[00265] R-spondin-1-conditioned medium, produced by a R-spondin-1-producing cell line, which can be obtained from the laboratory of Prof. C. Kuo (Stanford University, USA). Preparation instructions can be found in box 2 of Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347-358 (2016), Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474-85 (2015) and Broutier, L. 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). Commercially available recombinant R-spondin-3 (e.g. R&D Systems, cat. no. 3500-RS/CF, at a final concentration of 250 ng/ml) can be used as an alternative.

[00266] SB 202190 (Sigma-Aldrich, cat. no. S7067)

[00267] Wnt3a-conditioned medium, produced by an L Wnt3A-producing cell line, which can be obtained from the laboratory of Prof. H. Clevers (Hubrecht Institute, The Netherlands). Preparation instructions can be found in box 2 of Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474-85 (2015) and Broutier, L. 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). Commercially available Wnt surrogate (U-Protein Express BV, cat. no. NOO1, at a final concentration of 0.2 nM) can be used as an alternative.

[00268] Y-27632 dihydrochloride (ROCK inhibitor; Abmole Bioscience, cat. no. M1817) Genetic Manipulation

[00269] Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668-019)

[00270] Opti-MEM, reduced serum medium (Thermo Fisher Scientific, cat. no. 11058-021) Xenotransplantation

[00271] 17B-estradiol pellet, 0.36 mg/pellet, 60-day release {Innovative Research of America, cat. no. SE-121)

[00272] Isoflurane (100% (wt/wt); inhalation anesthetic (e.g. Isoflutek; Laboratorios Karizoo) Reagent Setup Collagenase Type II

[00273] To prepare a 20 mg/ml stock solution, dissolve 1 gram of powder in 50 ml of adDMEM/F 12+++ (RT) and filter through a 0.22 um filter. Store 1 ml aliquots, protected from light, at -20 °C for up to 3 months. DMEM with 0.1% (wt/vol) BSA (D-BSA)

[00274] Supplement 500 ml of DMEM GlutaMAX with 5 ml penicillin-streptomycin (1% vol/vol) and 5 ml 10% BSA (fatty acid free, wt/vol in DPBS) solution (final concentration: 0.1% BSA). Store at 4 °C for up to 4 weeks. adDMEM/F12+++

[00275] Supplement 500 ml of Advanced DMEM/F12 with 5 ml penicillin-streptomycin (1% vol/vol), 5 ml GlutaMAX (1% vol/vol), and 5 ml HEPES (1% vol/vol). Store at 4 °C for up to 6 months. DPBS with 0.1% (wt/vol) BSA (DPBS-B)

[00276] Used for diluting specific growth factors. Dissolve 0.1 g BSA (Modified Fraction V) per 100 ml DPBS (RT) and filter sterilize over a 0,22 um filter. Store 1 ml aliquots at -20 °C. Type 1 Expansion medium

[00277] The medium components with their end concentrations are listed in Table 2. To make 200 ml of Type 1 expansion medium, add to 153 ml of adDMEM/F12+++: 20 ml of R-spondin-1- conditioned medium, 20 ml of Noggin-conditioned medium, 4 ml of 50x B27 supplement, 2 ml of nicotinamide (1 M in DPBS), 500 pl N-acetyl-L-cysteine (500 mM in HzO), 400 pl Primocin (1 mg/ml), 10 ul Y-27632 (100 mM in DMSO), 100 ul Heregulin $1 (10 uM in DPBS-B), 100 pl human FGF-7 (10 pg/ml in DPBS-B), 100 pl human FGF-10 (40 pg/ml 0.1% DPBS-B), 20 pl A83-01 (5 mM in DMSO), 2 ul human EGF (500 ug/ml 0.1% DPBS-B) and 6.8 ul SB202190 (30 mM in DMSO). This medium can be stored for 1 week at 4 °C. Type 2 Expansion medium

[00278] The medium components with their end concentrations are listed in Table 2. To make 200 ml of Type 2 expansion medium, add to 112 ml of adDMEM/F12+++: 40 ml of Wnt3a- conditioned medium, 20 ml of R-spondin-1-conditioned medium, 20 ml of Noggin-conditioned medium, 4 ml of 50x B27 supplement, 2 ml of nicotinamide (1 M in DPBS), 500 pl N-acetyl-L- cysteine (500 mM in H20), 400 ul Hydrocortisone (250 ug/ml), 200 pl B-estradiol (100 uM), 200 pl Forskolin (10 mM in DMSO), 400 pl Primocin (1 mg/ml), 10 pl Y-27632 (100 mM in DMSO), 100 pl Heregulin B1 (10 uM in DPBS-B), 100 pl human FGF-10 (40 pg/ml in DPBS-B), 20 pl A83- 01 (5 mM in DMSO) and 2.0 pl human EGF (500 pg/ml 0.1% DPBS-B). This medium can be stored for 1 week at 4 °C. Wnt3a-conditioned medium

[00279] The procedure of Wnt3a-conditioned medium production has been described in box 1 of Fuji, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474-85 (2015) and Broutier, L. 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). Wnt3a-conditioned medium can be stored at 4 °C for 6 months.

[00280] Growth medium: DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin and 300ug/ml Zeocin. Store at 4 °C for up to one week.

[00281] Harvest medium: DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin. Store at 4 °C for up to one month. Step 1 Plate 1.5-2x106 cells of the L-Wnt3a cell line into a T150 flask in 35ml of growth medium, prewarmed to 37 °C. Step 2 Expand cells in growth medium by passaging when cells are at ~75% confluence. Passage by removing medium and incubating in 3ml TrypLE, pre-warmed to 37 °C. Incubate for 2-3min until all cells are in suspension, then add ~5ml of growth medium to the flask. Pool cells and re-seed 1.5%106 cells per T150 flask in 35ml of growth medium as in step 1. Step 3 When there are 20-30 T150 flasks each at ~75% confluence, passage cells as in step 2 but reseed 500cm plates with 4.5x1086 cells per plate in 100ml of growth medium. Step 4 When plates are ~70% confluent then change growth medium to 100ml harvest medium per plate. Incubate in this medium for 1 week. Step 5 Remove medium into 50ml tubes. Centrifuge at 500g for 5min to remove cells. Step 6 Filter medium using 500ml filter cups. Mix and aliquot into 25ml aliquots. R-spondin-1-conditioned medium

[00282] The procedure of R-spondin-1-conditioned medium production has been described in box 1 of Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat.

Protoc. 11, 347-358 (2016), Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474-85 (2015) and Broutier, L. 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).

R-spondin-1-conditioned medium can be stored at 4 °C for 6 months.

[00283] Growth medium: DMEM supplemented with 10% FBS and 150ug/ml Zeocin. Store at 4 °C for up to one week.

[00284] Harvest medium: Advanced DMEM/F12 supplemented with 1% Penicillin/Streptomycin, 1% Glutamax, and HEPES 10mM. Store at 4 °C for up to one month.

Step 1 Plate 1.5-2x106 cells of the L-Wnt3a (Wnt3a conditioned medium) or 293T-HA- Rspol-Fc (Rspol conditioned medium) cell lines into a T150 flask in 35ml of growth medium, pre- warmed to 37 °C.

Step 2 Expand cells in growth medium by passaging when cells are at ~75% confluence. Passage by removing medium and incubating in 3ml TrypLE, pre-warmed to 37 °C. Incubate for 2-3min until all cells are in suspension, then add ~5ml of growth medium to the flask. Pool cells and re-seed 1.5x106 cells per T150 flask in 35ml of growth medium as in step 1.

Step 3 When there are 20-30 T150 flasks each at ~75% confluence, change growth medium to 35ml harvest medium per flask. Incubate in this medium for 1 week.

Step 4 Remove medium into 50ml tubes. Centrifuge at 500g for 5min to remove cells. Step 5 Filter medium using 500ml filter cups. Mix and aliquot into 5ml aliquots. Noggin-conditioned medium

[00285] The procedure of Noggin-conditioned medium production has been described in box 2 of Cattaneo, C. M. et al. Tumour organoid-T-cell coculture systems. Nat. Protoc. 15, 15-39 (2020). Noggin-conditioned medium can be stored at 4 °C for 6 months.

A83-01

[00286] To prepare a 5 mM stock solution (10,000x%), dissolve 10 mg powder in 5 ml DMSO (RT). Store 50 pl aliquots at -20 °C for up to 1 year.

B-estradiol

[00287] To make a 100 pM stock solution (1000x%), dissolve 1 mg powder in 1 ml 100% EtOH (RT). Add 35.7 ml adDMEM/F12+++ and filter sterilize over a 0.22 um filter. Store 100 pl aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Forskolin

[00288] To prepare a 10 mM stock solution (1000x), dissolve 50 mg of powder in 12.2 ml DMSO (RT). Store 100 pl aliquots at -20 °C for up to 1 year. Nicotinamide

[00289] To prepare a 1M stock solution (100x), dissolve 6 g of powder in 50 ml of DPBS (RT) and filter sterilize over a 0.22 um filter. Store 1 and 5 ml aliquots at -20 °C for up to 1 year. N-acetyl-L-cysteine

[00290] To prepare a 500 mM stock solution (400x), dissolve 815 mg powder in 10 ml H2O (RT) and filter sterilize over a 0.22 pm filter. Store 500 pl aliquots at -20 °C for up to 1 year. Nutlin-3a.

[00291] To prepare a 1 mM stock solution, dissolve 5 mg powder in 860 ul DMSO (RT). Store 50 ul aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Heregulin 31

[00292] To prepare a 10 pM stock solution (2000x), dissolve 100 ug powder in 1333 yl DPBS (RT). Store 100 pl aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Human EGF

[00293] To prepare a 500 pg/ml stock solution (100,000x}, dissolve 500 ug powder in 1 ml DPBS- B (RT). Store 100 ul aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Human FGF7

[00294] To prepare a 100 ug/ml master stock solution (20,000x), dissolve 100 ug powder in 1 ml DPBS-B (RT). Store 100 ul aliquots at -80 °C for up to 1 year. To prepare a 10 ug/ml stock solution (2000x), dilute the master stock 10 times in DPBS-B (add 100 pl master stock to 900 ul DPBS- B). Store 100 pl aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Human FGF10

[00295] To prepare a 40 ug/ml stock solution (2000x), dissolve 100 ug powder in 2.5 ml DPBS- B (RT). Store 100 pul aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. Hydrocortisone

[00296] To make a 250 ug/ml stock solution (500x}, dissolve 10 mg powder in 500 pl 100% EtOH (RT). Add 39.5 ml adDMEM/F12+++ (RT) and filter sterilize over a 0.22 um filter. Store 200 pl aliquots at -20 °C for up to 1 year. Prevent freeze-thaw cycles. SB 202190

[00297] To prepare a 30 mM stock solution (30,000x}, dissolve 5 mg powder in 500 yl DMSO (RT). Store 10 pl aliquots at -20 °C for up to 1 year. Y-27632 dihydrochloride (ROCK inhibitor)

[00298] To prepare a 100 mM stock solution (20,000x), dissolve 10 mg powder in 312 ul DMSO (RT). Store 100 pl aliquots at -20 °C for up to 1 year.

[00299] In general, 4 °C-stored expansion medium can be used at any moment for organoid propagation with good performance within a week from preparation. However, for organoid establishment from tissue, after genetic manipulation, or when passaging single organoids it is preferable to use expansion medium as fresh as possible for the most optimal recovery of single cells.

Transplantation medium

[00300] Prepare while harvesting organoids for transplantation. Account for 40 pl transplantation buffer per injection, plus at least 10% extra. Mix equal volumes of ice-cold Type 1 or Type 2 medium (donor-dependent) and BME, avoiding bubbles. Store on ice until organoid pellet is ready for resuspension.

Equipment

[00301] 1.5-ml Safe-lock tubes (Eppendorf, cat. no. 0030120086)

[00302] 2.0-ml Safe-lock tubes (Eppendorf, cat. no. 0030120094)

[00303] 2.0-ml Cryovials (Greiner Bio-One, cat. no. 122263)

[00304] Biosafety cabinet

[00305] -80 °C Freezer (e.g. Eppendorf CryoCube F740hi, cat. no. 740320011)

[00306] CoolCell™ LX Freezing Container (Corning®, cat. no. CLS432001)

[00307] Counting chamber (e.g. Bürker-Türk, Merck, cat. no. BR719520)

[00308] Micro-dissecting forceps (Merck, cat. no. F3767)

[00309] Cell culture dishes, 60 x 15 mm (Greiner Bio-One, cat. no. 664160)

[00310] Cell strainer, 70 um (Greiner, cat. no. 542070)

[00311] Cell strainer, 100 um (Greiner, cat. no. 542000)

[00312] Centrifuge for 15/50 ml Falcon tubes (e.g. Eppendorf, cat. no. 5804R)

[00313] CO: incubator, 37 °C and 5% CO: (e.g. Thermo Fisher Scientific, model no. 4141)

[00314] Disposable scalpels (Swann-Morton, code 0501)

[00315] Electroporation Chambers (2mm), Cuvettes Plus (Fisher Scientific, cat. no. BTX620)

[00316] Falcon tubes, 15 ml, conical bottom (Greiner Bio-One, cat. no. 188271)

[00317] Falcon tubes, 50 ml, conical bottom (Greiner Bio-One, cat. nos. 227261)

[00318] Gene electroporator (NEPA GENE, model no. NEPA21 Super Electroporator)

[00319] Heated Incubator Oven (e.g. Panasonic, model no. MIR-H163-PE)

[00320] Petri dish, glass, 100 x 20 mm (VMR, cat. no. 391-0579)

[00321] Inverted brightfield microscope, objectives 5x, 10x, 20x (e.g. Leica, model no. DMi1)

[00322] Microcentrifuge (e.g. Eppendorf, cat. no. 5424R)

[00323] Millex-GS Syringe Filter Unit, 0.22 um (Merck, cat. no. SLGS033SS)

[00324] Multi-well suspension plates 12, 24, 48-well (Greiner Bio-One, cat. nos. 665102, 662102, 677102)

[00325] Orbital shaker (e.g. Panasonic, model no. MIR-S100-PE)

[00326] Pipetman L, P20L, P200L, P1000L (Gilson, cat. nos. FA10003M, FA10005M, FA10006M)

[00327] Pipette tips with filter (e.g. Gilson Pipetman, Diamond tips)

[00328] Serological pipettes 5, 10, 25 ml (Greiner Bio-One, cat. nos. 606180, 607160, 760160) Equipment for Xenotransplantation

[00329] MicroFine* Insulin Syringes, 29G (Becton Dickinson, cat. no. 324825)

[00330] Precision Trochar, 10 Gauge (Innovative Research of America, cat. no. MP-182)

[00331] Trimmer, Exacta GT416 (Aesculap, cat. no. AEX415) Equipment Setup For organoid electroporation, NEPA21 electroporator was set to the following settings:

Poring pulse Transfer pulse Voltage 200 V 20V Pulse length 5ms 50 ms Pulse interval 50 ms 50 ms Number of pulses 2 5 Decay rate 10% 40% Polarity + +/- Mice

[00332] Female NSG (NOD.Cg-Prkdcs® I[2rg'"1Wi! /SzJ) mice, between 6-8 weeks of age. Human Material

[00333] Human material; this protocol can be applied to human resection or biopsy material of normal, precancerous, cancerous, and metastatic tissue. Fresh material is the preferred starting material, but tissue can be kept in adDMEM/F12+++ with 100 pg/ml Primocin at 4 °C for up to 72 h before starting the procedure. For the use of human material, approval of the relevant national and institutional regulatory bodies is necessary, and informed consent should be obtained from all donors. Occasional profiling of the organoid cultures with a single nucleotide polymorphism (SNP) array through commercial resources available (e.g. Thermo Fisher Scientific, cat. no. 4475394, following the company's specific instructions of sample preparation) to rule out cross- contamination with a different culture, and to regularly test for mycoplasma contamination.

PROCEDURE Establishment of organoids from breast tissue resections (TIMING: 4.5-6.5h)

[00334] This section describes the isolation of epithelial cells from breast tissue resections for the establishment of organoid cultures (Fig. 2a). When pipetting disrupted tissue, always pre-wet the 5 or 10 ml sterile serological pipettes with D-BSA medium for preventing tissue sticking to the plastic (Steps 12, 13, 19, 21, 24, 32). When receiving resected material from two different locations (i.e. normal and tumour), start processing the normal tissue and while digesting, process the tumour tissue. The derivation success of normal organoids depends on the viability of the stem cells in the normal tissue, which are usually present in lower numbers as compared to cancer (stem) cells in tumour tissue. Normal tissue should thus be processed as soon as possible upon arrival. Resected tissue should be placed in a Falcon tube containing 14 ml of adDMEM/F12+++ with Primocin (100 pg/ml) and be transferred to the lab as soon as possible for isolation.

[00335] As the derivation process is performed with primary human material, which could potentially carry human pathogens, all steps should be performed wearing appropriate Personal Protective Equipment (PPE) and while working in a biosafety cabinet, and with the tissue in closed tubes or petri dishes when checking it under the microscope. Working in a biosafety cabinet also reduces the risk of contaminating the tissue and isolated organoids with microorganisms.

[00336] Store Falcon tube containing tissue on wet ice in case it takes up to 72 hours to reach the lab for isolation.

[00337] The procedures are described for working with 12-well plates. Table 3 describes the protocol in the case of a culture plate with a different number of wells is desired.

Tissue preparation

[00338]

1. Transfer each tissue sample (< 3-4 cm?) in a separate 50 ml Falcon tube filled with D- BSA. Keep tissues on ice until starting the isolation.

2. Register the new sample in the corresponding lab management system.

3. (Optional) Generate labels for collecting pieces of tissue for different purposes (DNA, RNA, Protein and/or histology).

4, Collect the 50 ml Falcon tube containing the normal or tumour tissue and transfer the tissue to a 10 cm glass Petri dish by decanting the 50 ml Falcon tube (Fig. 2b).

5. Take a microscopic picture of the tissue, including a ruler in the picture (Fig. 2b). Make a note of how the tissue looks (i.e. size, fatty, vascularized, necrotic etc.) in the corresponding lab note.

6. Aspirate the excess D-BSA and cut the tissue into small pieces (2-4 mm?) using 2 scalpels by holding one scalpel in each hand and slicing the tissue while pressing the blades of both scalpels against each other.

7. (Optional) Use a micro-dissecting forceps cleaned with 70% EtOH to transfer 1-2 pieces of 2-4 mm3tissue into a 2 ml Eppendorf tube labelled for DNA analysis, snap freeze on dry ice, and transfer it to -20 °C for storage.

This tissue sample can be used as the reference DNA sample for SNP analysis (Human Material section).

8. (Optional) Fix 2 pieces of 2-4 mm? tissue for histology by transferring them into a 15 ml Falcon tube labelled with Histology label and containing 5 ml of 4% formalin. Perform this step in a fume hood, as formalin is toxic.

9. (Optional) When future transcriptomic analyses are required, transfer 1-2 pieces of 2-4 mm? tissue into a 2 ml microtube labelled with RNA label, snap freeze on dry ice and transfer to a -80 °C freezer.

10. (Optional) When future proteomic analyses are required, transfer 1-2 representative pieces of tissue into a 2 ml microtube labelled with Protein label, snap freeze on dry ice and transfer to a -80 °C freezer. Be aware that optional steps 7-10 will reduce tissue volume for organoid generation, which may result in a longer organoid propagation phase before frozen stocks can be generated. On average, 8-10 mm? would be enough for one well of a 12-well plate, but this is highly variable depending on the cellularity of the tissue sample. For steps 7-10, make sure to select tissue pieces that represent different colors or regions of the resected tissue to better capture different cell populations present. Organoid isolation from tissue

[00339]

11. Mince the remaining tissue into smaller pieces (0.5-1 mm?) with 2 scalpels as stated in step 6, until the tissue mass looks uniform and appears somewhat viscous.

12. Add 5 ml of D-BSA into the glass Petri dish and transfer the minced tissue to a 50 ml Falcon tube using a 5 ml sterile serological pipette pre-wetted with D-BSA. Wash the same glass Petri dish with 5 ml of D-BSA and collect residual minced tissue into the same 50 ml Falcon tube.

13. Add 35 ml of D-BSA to the 50 ml Falcon tube and use a 10 ml sterile serological pipette pre-wetted with D-BSA to resuspend the minced tissue by pipetting up and down 5 times.

14. Let the tissue sediment by gravity for 2-3 min, until all tissue pieces are at the bottom of the tube, before aspirating all but 10 ml of D-BSA.

15. Repeat steps 13-14 two more times to wash away potential microorganisms, cell types not contributing to the establishment of organoids such as blood cells, and dead cells and debris.

16. Aspirate all supernatant carefully, so that only the sedimented tissue pellet remains, and add 10 ml of Type 1 medium.

17. Add 500 pl collagenase (20 mg/ml stock, final concentration 1 mg/ml) and 10 ul ROCK inhibitor (10 mM stock, final concentration 10 uM) to the 50 ml Falcon tube containing the minced tissue.

18. Wrap the 50 ml Falcon tube with parafilm and place the tube at a slight angle (~15°) on top of an orbital shaker located inside of a heated incubator oven at 37 °C. Set the orbital shaker at 140 rpm and digest the tissue for 1-2 hours. At this moment, the processing of the next tissue can be initiated. Placing the tube at a slight angle facilitates the distribution of minced tissue along the tube for proper digestion, as it increases the surface area of tissue exposed to the digestion reagent. The angle can be achieved by placing the top of the tube on a 0.5-1 cm platform and resting the end of the tube on the shaker.

19. Monitor the digestion of the tissue at least every 30 min, using an inverted brightfield microscope (5x, 10x, or 20x objective). When clusters of 5-10 cells are observed (Fig. 2f), digestion is complete. Pipette up and down 5-10 times every 30 min using a 5 ml sterile serological pipette pre-wetted with D-BSA medium to aid digestion. Normal tissue digestion typically takes 2 hours, tumour tissue digestion typically takes 0.5-2 hours. The time of digestion depends on several variables, such as the time from tissue isolation to digestion, tissue size and consistency, and the quality and activity of the digestion reagent. Prolonged tissue storage prior to processing or freshly made digestion reagent reduces the digestion time, while solid, firm tissue or an older digestion reagent increases the digestion time.

20. After incubation, take the tube from the heated incubator oven and clean with 70% EtOH. Add 1 ml of FBS directly to the 50 ml Falcon tube to stop the collagenase digestion.

21. Homogenize the digested tissue by pipetting it up and down vigorously with a 10 ml sterile serological pipette pre-wetted with D-BSA.

22. Pre-wet a 100 um strainer by pipetting 10 ml D-BSA on the strainer into a new 50 ml Falcon tube. Swirl the 50 ml tube to wet the sides of the tube and discard the D-BSA. Then strain the digested tissue over the 100 um strainer into the 50 ml Falcon tube (both pre-wetted with D-BSA).

23. Transfer the unfiltered tissue pieces back into the initial 50 ml Falcon tube by reversing the filter and flushing with 10 ml of D-BSA.

24. Pipet the digested tissue up and down vigorously, using a 10 ml sterile serological pipette pre-wetted with D-BSA.

25. Reverse the 100 um filter and strain the tissue again, collecting the cell suspension in the same 50 ml Falcon tube from step 22.

26. Repeat steps 23-25 two more times, using the same filter to avoid wasting precious tissue.

27. Centrifuge the flow-through from steps 22-26 at 450g for 5 min at 8 °C.

28. Aspirate the supernatant, leaving the last 4 ml to not disturb the pellet. Resuspend the tissue pellet in the remaining 4 ml with a sterile serological pipette and transfer itto ato a 15 ml Falcon tube pre-wetted with D-BSA.

29. Add 10 ml of D-BSA with a sterile serological pipette and homogenize by pipetting up and down several times.

30. Centrifuge the 15 ml Falcon tube at 450g for 5 min at 8 °C. Aspirate the supernatant very carefully with a vacuum system and clean tip until 1 ml is left. Let the tube rest for 1 min to allow the D-BSA from the side of the tube to settle at the bottom and remove the residual D- BSA with a p1000 pipette.

31. (Optional) If the pellet is partially red, add 1-2 ml of red blood cell lysis buffer with a p1000 pipette, resuspend the pellet by pipetting up and down several times and incubate for 2 min at RT before continuing with step 32. For pellets < 100 pl in size, 1 ml of red blood cell lysis buffer is sufficient. For pellets > 100 pl, use 2 ml of red blood cell lysis buffer.

32. Add 10 ml D-BSA to the 15 ml Falcon tube with a sterile serological pipette and pipet up and down several times using the same pipette.

33. Centrifuge the cells and aspirate the medium as described in step 30. At this point, cells can be frozen with Recovery Cell Freezing Medium by adding 1 ml, resuspending the cell pellet and transferring to a cryovial. Preserving part of the isolation yield will ensure a backup of the sample in case the culture is lost at an early passage before cryopreservation (e.g. due to bacterial or fungal contamination or improper handling by an inexperienced user).

34. Resuspend the pellet in an appropriate amount of BME. As a guideline, 200 pl is sufficient for a pellet of approximately 50 pl. The amount of BME to add scales linearly with the pellet volume. After filtering the digested tissue, the cell pellet should be easy to resuspend in BME. However, if the pellet is very sticky, cut the tip of the 200 ul low retention filter tip. BME must be kept on ice to prevent solidification. Work quickly to prevent the BME from solidifying, but carefully to prevent bubble formation.

35. Plate 100 pl BME containing cells in multiple small drops (< 20 pl each) per well in a 12- well plate (Fig. 2d). When tissue yield is limited, plating can be scaled down to 24-well format by adapting all volumes accordingly (Table 3).

36. Turn the plate upside down and leave in the biosafety cabinet for 5 min.

37. Transfer the plate, upside down, into a 37 °C incubator and leave to solidify for 30 min. Solidifying the BME upside down prevents the tissue fragments from sinking and attaching to the bottom of the plate.

38. Add 750 pl of pre-warmed (RT to 37 °C) Type 1 or Type 2 medium to each well and transfer to an incubator at 37°C and 5% CO:. Table 3 refers to appropriate media volumes per type of culture plate. For each culture, test both Type 1 and Type 2 expansion medium (Fig. 2e; Table 2). Occasionally monitor organoid growth with an inverted brightfield microscope (5x, 10x, or 20x objective) and continue with the type of medium that results in the highest organoid confluency, thus yielding the highest outgrowth. Type 1 is typically better for BC organoids, Type 2 is typically better for normal organoids (Table 1).

39. (Optional) Take microscopic pictures of representative drops for documentation of organoid density and morphology (2.5x and 10x magnifications).

40. Refresh culture medium every 2-4 days by following steps 41-42. After isolation, organoids can be split for the first time after 7-21 days by following steps 43-48.

After 3-4 days, it should be possible to identify organoids with an inverted brightfield microscope (5x, 10x, or 20x objective). Monitor organoid growth closely and ensure the majority has reached a size of at least 100 pm before passaging. At early passages, it is possible to observe highly elongated fibroblast growing on the bottom of the culture plate. Avoid scratching the bottom of the plate while passaging to prevent transferring the fibroblast to the next passage. Refreshing medium

[00340]

41. Tilt the plate at a 45° angle and aspirate all expansion medium from the well containing cultured organoids by placing the aspiration tip to the side of the well. Avoid getting close to the BME drops. Use a clean tip for each different organoid culture and remove the lid from one culture plate at a time to avoid cross-contamination.

42. Tilt the plate at a 45° angle and gently add dropwise 750 ul expansion medium to each well using a p1000 pipette when refreshing a few wells, or 5 ml or 10 ml sterile serological pipette when refreshing many wells. Aim at the bottom corner of the well to avoid disturbing the BME drops. Do not touch the well while adding expansion medium to avoid cross- contamination.

Organoid maintenance and cryopreservation Organoid dissociation

[00341]

43. This section describes the culture of normal breast and BC organoids in a 12-well plate. Organoids can be passaged 7-21 days after organoid establishment or after the previous split at a 1:2-1:8 ratio. Table 1 provides further guidelines. The time of passage and split ratio should be optimized for each newly established organoid culture based on confluency. The best moment to passage is just before organoids in the center of the BME drop start dying, characterized by shedding of debris or a darker appearance, or when organoids reach a diameter larger than 300 um (further referred to as a ‘confluent well’). For organoid passaging via single cells follow option A, and via fragments follow option B.

[00342] The preferred way of passaging for most organoid cultures is via fragments rather than single cells (see Table 1), because single cells are slower in growing into new organoids and have a higher chance of dying compared to fragments. Both methods can be tried for newly established cultures, especially for fast-growing cultures. In some occasions, for instance when cell counts are critical, single cell passaging can be used. To prevent cross-contamination of different organoid cultures, it is advised to work cautiously when handling different cultures simultaneously.

[00343] (A) Dissociating organoids into single cells by TrypLE digestion (i) Aspirate all expansion medium from a well containing organoids by holding the plate at a 45° angle and placing the aspiration tip to the side of the well, not getting close to the BME drops. When passaging multiple wells of organoids of the same culture, all medium of those wells can be aspirated at once with the same tip. (i) Add 1 ml of TrypLE to the well and dissociate the BME by forcefully pipetting up and down 6-10 times per well with a p1000 pipette, aiming at the BME drops.

(iii) (Optional) Repeat step (ii) with other wells of the same organoid culture by transferring the 1 ml of organoids in TrypLE to the next well with organoids without expansion medium and repeating the pipetting process. Up to 3 wells of organoids can be combined in 1 ml of TrypLE.

(iv) Directly after step (iii), hold the plate at a 45° angle and forcefully pipet the BME- TrypLE mixture with organoids up and down 10 times. This will dissociate most of the BME, but the organoids will remain intact. Bubble formation is not harmful, but can accelerate the dissociation.

(v) Incubate the plate at RT for 5-10 min.

(vi) Hold the plate at a 45° angle and forcefully pipet the BME-TrypLE mixture with organoids up and down 10 times to mechanically dissociate the organoids.

(vii) Determine if a single cell solution has been achieved by checking the well with an inverted brightfield microscope (5x, 10x, or 20x objective) (Fig. 2f). If partially- dissociated organoids are still visible, repeat steps (v)-(vi) until a single cell solution has been achieved. Incubation time varies between donors, but the total incubation time should ideally not take more than 20-25 min. For cultures that are hard to dissociate, the plates can be incubated at 37 °C instead of RT in step (v).

(B) Dissociating organoids to fragments by shearing with TrypLE

(i) Remove 500 pl expansion medium from the well containing organoids, leaving ~250 ul in the well, by holding the plate at a 45° angle and placing the aspiration tip to the side of the well, not getting close to the BME drops.

(i) Add TrypLE to the well, to a final volume of 1 ml, and dissociate the BME by forcefully pipetting up and down 6-10 times per well with a p1000 pipette, aiming at the BME drops.

(iii) (Optional) Repeat step (ii) with other wells of the same organoid culture by transferring the 1 ml of organoids in TrypLE to the next well with organoids without expansion medium and repeating the pipetting process. Up to 3 wells of organoids can be combined in 1 ml of TrypLE.

(iv) Directly after step (iii), hold the plate at a 45° angle and forcefully pipet the organoids in TrypLE up and down 10 times. This will dissociate most of the BME, but the organoids will still be intact.

(v) To shear the organoids, hold the plate at a 45° angle and forcefully pipet up and down 10-20 times, firmly pressing the 1 ml tip to the bottom corner of the well to create a small opening for organoid disruption.

(vi) Check the organoids with an inverted brightfield microscope (5x, 10x, or 20x objective). Repeat step (v) until the desired fragment size has been achieved (Fig. 2f). Continue with step 44. Organoid shearing will result in a suspension of single cells and small-large organoid fragments. Shearing is optimal when the majority of the cells are present in the form of small organoid fragments (usually containing 5- 20 cells, or 20-50 um in diameter).

44. Transfer single cells or organoid fragments into a 15 ml Falcon tube with 10 ml ice-cold adDMEM/F12+++, using a p1000 pipette. Rinse the well with 1 ml adDMEM/F12+++ to ensure no dissociated organoids remain in the well. A maximum of 6 wells with organoids from the same culture can be pooled in one 15 ml Falcon tube. The 15 ml Falcon tube with processed organoids can be stored on ice for 1 hour in order to first harvest multiple cultures before continuing with step 45, with minimal effect on organoid recovery after passaging.

45. Centrifuge the 15 ml Falcon tube with organoids at 300g for 5 min, at 4 °C and confirm presence of a BME-free pellet by eye (Fig. 2¢). Presence of BME in the pellet should be avoided.

46. Remove the supernatant by decanting the fluid in a waste bottle and put the pellet on ice for 1 min. Remove the remaining supernatant carefully with a p1000 pipette.

47. (Optional) At this stage the cells can be counted by resuspending the cell pellet in 250- 500 pl expansion medium per harvested well, taking 10 ul of cells and mixing it with 10 HI Trypan Blue solution, and counting using a counting chamber and an inverted brightfield microscope (10x or 20x objective). One confluent well of a 12-wells plate commonly contains

200,000-500,000 cells. Before continuing with step 48, spin down the non-counted cells at 300g for 5 min and remove the supernatant.

48. To directly plate organoids for passaging, continue to step 49. To genetically manipulate organoids, continue to step 66 for Lipofectamine 2000-based transfection, to step 78 for electroporation-based transfection and to step 85 for lentiviral transduction. Plating organoids for passaging (TIMING 15-20 min)

[00344]

49. Table 3 provides appropriate number of BME drops and total BME volume for different culture plates. Use a p200 or p1000 pipette to resuspend the pellet of dissociated organoids in the 15 ml tube in ice-cold BME, by carefully pipetting up and down ~ 10 times. Plate in multiple small drops (< 20 pl each) per well (Fig. 2d). In addition to using a new tip, we recommend to clean the p200 or p1000 pipette with 70% EtOH before using it to resuspend the pellet of the next culture, to prevent cross-contamination.

50. Flip the plate upside down before placement in the incubator to prevent organoids from sinking to the bottom and attaching to the plate.

51. Place the plate in an incubator (5% CO, 37 °C) for 10-20 min to allow the BME to solidify.

52. Supplement the well with freshly-passaged organoids with 750 pl of pre-warmed (RT-37 °C) Type 1 or Type 2 medium, in a dropwise manner. Refer to Table 3 for the appropriate media volumes when using a 6, 24, 48, or 96-well culture plate.

53. Refresh expansion medium every 2-4 days, by following steps 41-42. Occasionally check organoid density and morphology with an inverted brightfield microscope (5x, 10x, or 20x objective). Organoids secrete factors that stimulate cell proliferation. Therefore, organoid density is a critical factor for a well-growing, viable culture (Fig. 2g-i).

Freezing intact organoids

[00345] Make sure to freeze organoids 2-7 days before they reach optimal confluency for splitting, usually when they reach a diameter of 100-150 um. This ensures a recovery efficiency after thawing of close to 90%.

54. Follow steps 43A, (i)—(iv) to remove intact organoids from the BME.

55. Follow steps 44-46 to wash and pellet the intact organoids.

56. Resuspend the pelleted organoids in Recovery Cell Freezing Medium. Combine up to 4 wells of a 12-well plate of the same donor in 1 ml of freezing medium. Transfer the organoids in freezing medium to a 2 ml cryovial, storing 1 ml per vial.

57. Transfer the cryovials to a -80 °C cell freezing container. Store at -80 °C at least overnight before transferring to a longer-term storage methods. Organoids can be stored at -80 °C for up to one month, or in liquid nitrogen indefinitely. Thawing organoids

[00346]

58. Transport the cryovial containing organoids from the -80 °C or liquid nitrogen storage to the cell culture laboratory on dry ice.

59. Quickly thaw the cryovial containing organoids in a 37 °C water bath, clean the vial using 70% EtOH and dropwise add 1 ml pre-warmed (37 °C) adDMEM/F12 +++ supplemented with 10 HM ROCK inhibitor with a p1000 pipette while tapping the bottom of the vial. ROCK inhibitor is added to enhance recovery from freeze-thawing.

60. Gently resuspend 2 times, transfer the content of the vial to a 15 ml Falcon tube and dropwise add 10 ml of pre-warmed (37 °C) adDMEM/F12 +++ supplemented with 10 uM ROCK inhibitor.

61. Centrifuge the 15 ml Falcon tube with organoids at 300g for 5 min at 4 °C

62. Remove the supernatant and put the pellet on ice for a few minutes to allow the residual supernatant to reach the bottom of the tube. Remove the remaining supernatant carefully with a p200 pipette.

63. Follow steps 49-53 to plate the organoids.

64. Refresh expansion medium every 2-4 days by following steps 41-42. Genetic manipulation

[00347]

65. This section describes the genetic manipulation of organoids (Fig. 3a) using Lipofectamine 2000 (steps 66-76}, electroporation (steps 77-84), or lentiviral transduction (steps 85-89; Fig. 3b). Subsequently, we describe how to select organoids with selection agents (steps 90-92; Fig. 3c) and how to generate clonal organoid cultures (steps 93-101; Fig. 3d,e).

[00348] Use 20-50 % of one confluent well of organoids (12-well plate) or 100,000-200,000 single organoid cells for each transfection or transduction condition.

[00349] Plate the manipulated organoids at least twice as dense as compared to standard passaging.

[00350] For optimal efficiency and organoid recovery, make sure to use an organoid culture with minimal presence of cell debris and dying organoids as starting material (Fig. 2h,i), and process organoids into small fragments containing several cells (Fig. 2f).

[00351] When it is desired to generate clonal organoid cultures after transfection, it is advised to use an organoid culture that allows passaging via single cells. Using single cells, as opposed to fragments, will increase the likelihood that the organoids growing out after selection (e.g. by puromycin) indeed originate from single cells, and are thus clonal.

[00352] Following genetic manipulation of organoids, expression of transgenes can disappear due to silencing or genetic rearrangements, especially in tumour organoids. It is therefore important to supplement the expansion medium with the appropriate selection agent from the manipulation onward to maintain transgene expression in close to 100% of cells.

Transfection of organoids using Lipofectamine 2000

[00353]

66. Dissociate and pellet the organoids as described in steps 43A/43B-46. After aspirating the supernatant from the organoid pellet as described in step 46, continue with step 67.

67. Resuspend the pellet of dissociated organoids in 450 ul Type 1 or Type 2 expansion medium per condition using a p1000 pipette, by pipetting 5 times up and down and transfer 450 ul to each well of a 24-well suspension plate. Place the plate at 37 °C, 5% CO: while the transfection mix is being prepared.

68. Prepare two 1.5 ml Eppendorf tubes. To the first, add Lipofectamine 2000 (4 ul + 10% per transfection) and Opti-MEM (21 ul + 10% per transfection). To the second, add plasmid DNA (4 pl of a 1 pg/ul stock + 10% per transfection) and Opti-MEM (21 ul + 10% per transfection).

69. Vortex both Eppendorf tubes briefly at maximum speed and incubate at RT for 5 min.

70. Transfer the content of the first to the second Eppendorf tube, using a p200 pipette, vortex the Eppendorf tube briefly and incubate at RT for 20 min.

71. Add 50 pl transfection mix to 450 pl expansion medium with organoids, using a p200 pipette.

72. Mix the transfection mix with the organoids by gently pipetting up and down 3 times, using a p1000 pipette, preventing bubble formation.

73. Incubate at 37 °C, 5% CO: for 4 hours.

74. Transfer the transfected organoids to a 15 ml Falcon tube containing 3 ml adDMEM/F12+++, using a p1000 pipette.

75. Centrifuge the Falcon tube with transfected organoids for 5 min at 300g and plate as described in steps 49-53.

76. When the transfected DNA contains a gene encoding a fluorescent protein (Fig. 3d,e), expression can be detected within 1 day after transfection. For selection of organoids using a selection agent follow steps 90-92. The transfection efficiency is usually between 5 and 50%. Transfection of organoids by electroporation (TIMING 50-60 min)

[00354]

77. Dissociate and pellet the organoids as described in steps 43[00341]A/43B-46. After aspirating the supernatant from the organoid pellet as described in step 46, continue with step 78. Place the Falcon tube with dissociated organoids on ice while preparing the transfection mix.

78. Prepare the transfection mix. Per condition, add 10 pg plasmid (in max 10 ul) to an 1.5 ml Eppendorf tube and top up with Opti-MEM to a final volume of 100 pl. Vortex the Eppendorf tube briefly and place on ice for 1 min.

79. Resuspend the pellet of dissociated organoids in the transfection mix by gently pipetting up and down 5 times and transfer the sample (~100 pl) into an 2 mm electroporation cuvette, using a p200 pipette. Make sure the sample reaches the bottom and prevent bubble formation.

80. Place the cuvette in the NEPA21 electroporator and check the resistance (ohm). The transfection is most optimal between 0.030 and 0.055 ohm. The sample should be diluted in transfection mix (prepared in step 78) if the resistance is too high.

81. Electroporate using the settings as described in the Equipment Setup. Vary in V (100- 300) and pulse length (2-8 ms) to optimize settings for each organoid culture.

82. Add 400 pl ice-cold expansion medium to the cuvettes and transfer the sample into a clean

1.5 ml Eppendorf tube using a p200 pipette.

83. Centrifuge the Eppendorf tube with electroporated organoids for 5 min at 300g and plate transfected cells as described in steps 49-53.

84. When the transfected DNA contains a gene encoding a fluorescent protein (Fig. 3d,e), expression can be detected within 1 day after transfection. For selection of organoids using a selection agent follow steps 90-92. The transfection efficiency is usually between 20 and 80%.

Lentiviral transduction of organoids

[00355] Follow institutional biosafety guidelines for working with infectious material, while working with lentivirus.

85. Dissociate and pellet the organoids as described in steps 43A/43B-46. After aspirating the supernatant from the organoid pellet as described in step 46, continue with step 86.

When performing a cell count is desired, perform step 47, before continuing with step 86. We describe transduction using lentivirus concentrated to a titer of 50 x 106 infectious viral particles per ml (pfu/ml).

86. Resuspend the pellet of dissociated organoids in 10 pl virus (500,000 infective virus particles) + 90 ul Type 1 or Type 2 expansion medium by gently pipetting up and down 5 times with a p200 pipette. When 100,000 organoid cells are used (often comparable to ~ 1/3 of a confluent well of a 12-well plate), the MOI is 5.

87. Incubate the 15 ml Falcon tube at 37 °C, 5% CO: for 30-60 min with a loose lid to allow diffusion of gas to the organoids.

88. Add 10 ml cold adDMEM/F12+++ and centrifuge the Eppendorf tube with transduced organoids for 5 min at 300g and plate as described in steps 49-53.

89. When the transduced DNA contains a gene encoding a fluorescent protein (Fig. 3d, e), expression can be detected within 2-3 days after transduction. For selection of organoids using a selection agent follow steps 90-92. The transduction efficiency is usually between and 90%. Organoid selection

[00356] Organoid selection (Fig. 3c) can be started 3 days after transfection or transduction, by adding the selection agent to the expansion medium. It is advised that the concentration of the 20 selection agent is first optimized by culturing organoids using a titration of the selection agent. We advise to use the lowest concentration that kills 100% of untreated organoids after 3-10 days of culture.

90. Three days after transfection (steps 66-76 or 77-84) or transduction (steps 85-89), aspirate all medium from a well containing organoids by holding the plate at a 45° angle and placing the aspiration tip to the side of the well, avoiding getting close to the BME drops.

91. Gently add 750 pl expansion medium with the appropriate concentration of selection agent, in a drop-wise manner to each well.

92. Refresh the expansion medium every 2-4 days following steps 41-42 until organoids have reached a size sufficient for further passaging (diameter of < 300 um; steps 43-53) or clone picking (diameter of 200-500 um; steps 93-101). Clone picking

[00357] To ensure that individual organoids used for clone picking originated from a single cell, it is best to generate a culture with sparsely growing organoids after selection (Fig. 3e). In case no selection is applied, this can also be achieved by passaging at low density.

93. Prepare 1.5 ml Eppendorf tubes, one per organoid, with 100 pl TrypLE.

94. Disrupt the BME drops with cultured organoids by pipetting the 750 pl expansion medium up and down 8-10 times with a p1000 pipette, aiming at the top of the BME drops, and transfer the organoids to a 10 cm culturing dish with 10 ml adDMEM/F12+++ at RT.

95. Use an inverted brightfield microscope (20x objective) and pick individual organoids with a p20 pipette, thereby aspirating as little volume as possible (< 10 pl}. Transfer each individual organoid to a separate 1.5 ml Eppendorf tube with TrypLE. When working outside of a biosafety cabinet with primary human cells, it is essential to work fast to avoid contamination, and wear appropriate PPE. Prewet the 20 pl tip with Type 1 or Type 2 expansion medium to prevent organoids sticking to the tip. Alternatively, D-BSA or adDMEM/F12+++ can be used.

96. Incubate the Eppendorf tubes with a single organoid each for 5 min at RT. Using a p200 pipette, add 100 pl FBS at RT to the Eppendorf tube and directly resuspend the clone by pipetting 3-10 times up and down.

97. Use an inverted brightfield microscope (10x or 20x objective) to check the dissociation.

Repeat step 96 until the organoid is dissociated into small fragments and/or single cells (see Fig. 2f as reference). The incubation time (usually 5-20 min total) and number of times to resuspend (usually 5-30 times total) need to be optimized per organoid culture. The digestion time of a clonal organoid is often comparable to the digestion time of the parental culture when digesting for passaging. Make sure not to over-digest the organoid, as this will lead to a decreased viability. Do not digest the single organoid for longer than 25 minutes.

98. Centrifuge the Eppendorf tubes at 300g for 5 min. Carefully aspirate the supernatant with a p200 pipette and resuspend the pellet in 30 pl ice-cold BME, by gently pipetting up and down 5 times.

99. Plate the dissociated organoid in a 48-well plate in 1 drop of 30 yl BME, in the center of the well, and incubate for 10-20 min at 37 °C, 5% CO:.

100.Tilt the plate at a 45° angle and gently add 250 pl expansion medium to each well in a dropwise manner, using a p1000 pipette. Aim at the bottom corner of the well, avoiding the BME drops. Continue to supplement the expansion medium with the selection agent to prevent potential outgrowth of organoids in which the transgene is silenced.

101.Refresh the expansion medium every 3—4 days. After approximately 7-21 days, when organoids reach a diameter of 200-500 pm, they can be passaged as described in steps 43A/43B and steps 44-53 to an appropriate culture plate (Table 3). The expected outgrowth efficiency is 20-80 % and depends on the organoid culture.

Xenotransplantation in mouse mammary fat pad

[00358] Before scaling-up organoid cultures for transplantation, we recommend to measure the number of cells harvested on average per well, using steps 102-108 below and use this to estimate the required number of wells to expand for transplantation.

[00359] Use organoids for transplantation when they are still in their proliferative state, 2-5 days before they would normally be split, as this will improve engraftment efficiency. As a guideline, we recommend to harvest organoids passaged at a weekly interval at day 5 and organoids passaged bi-weekly at day 10, counted from the day they were last split. Avoid growing organoids bigger than 70 | m, because they will be filtered out during organoid harvesting to avoid clogging the injection needle. Defining the number of wells required for transplantation

102.Place a 70 um cell strainer on a 50 ml Falcon tube and pre-wash the strainer with 5 ml of ice-cold adDMEM/F12+++.

103.Harvest intact organoids from 2 representative wells of a 12-well plate as described in steps 43A (i)—(iv). Pipette this solution over the 70 um strainer with a p1000 pipette and rinse the strainer with another 5 ml of adDMEM/F12+++. Transfer the filtrated solution to a 15 ml Falcon tube.

104.Centrifuge the 15 ml Falcon tube for 5 min at 300g at 4 °C. Remove the supernatant by decanting into a waste bottle, place the Falcon tube at RT for 1 min, remove the remaining supernatant with a p1000 pipette and resuspend the organoid pellet in 1 ml of TrypLE.

105.Incubate for 5 min at 37 °C and thoroughly pipette up and down 10 times with a p1000 pipette.

106.Use an inverted brightfield microscope (5x, 10x, or 20x objective) to assess digestion. If required, repeat step 105 until a single cell suspension has been achieved.

107.Add 9 ml of adDMEM/F12+++ and centrifuge the 15 ml Falcon tube for 5 min at 300g at 4 °C. Leave 1 ml of supernatant in the Falcon tube and resuspend the cells.

108.Mix 10 pul of the single cell suspension with 10 pl Trypan Blue solution and count using a counting chamber and an inverted brightfield microscope (10x or 20x objective). To calculate the average number of cells per well, take the mean of the two wells counted. From this, extrapolate the number of wells necessary for the desired number of injections. Account for at least 10-20% excess. For example, when injecting 10 mammary fat pads with 1*108 cells, prepare 10*10° plus 20% = 12*10° cells. Organoid harvesting for transplantation

109.To prepare the cells for transplantation, two different methods can be used to take the organoids out of the BME. For using Cell Recovery Solution, follow option A. For using TrypLE, follow option B. Compared to organoid harvesting by TrypLE, harvesting by Cell Recovery Solution requires less hands-on time and dissolves all BME more efficiently, usually resulting in a tight, BME-free organoid pellet. However, in rare occasions option A may result in organoid dissociation, which is more likely to happen for organoid lines that dissociate fast into single cells upon TrypLE digestion (step 43A). In case this happens, dissociated cells can again be propagated in BME following steps 44-53. However, we advise to test one well of organoid culture following option A prior to the day of transplantation, to test if they remain intact throughout the process. If not, follow option B.

(A) Removing organoids from BME with Cell Recovery Solution ( Remove the expansion medium from the number of wells needed , as calculated in step 102-108, by holding the plate at a 45° angle and aspirating with a vacuum system and clean tip.

(if) Add 1 ml of Cell Recovery Solution per well. Incubate for 30-60 min on a horizontal orbital shaker at 20 rpm at 4 °C. The incubation is ready when the BME drops are breaking up and detach from the plate.

(iii) Place a 70 um cell strainer on a 50 ml Falcon tube and pre-wash the strainer with 2 ml of ice-cold adDMEM/F12+++, (iv) Resuspend the organoid suspension by pipetting up and down 5-10 times with a p1000 pipette, holding the plate at a 45° angle. Pipette the suspension with intact organoids over the 70 um strainer into the 50 ml Falcon tube, combining up to 6 wells of the same culture. Rinse the strainer with 6 ml of adDMEM/F12+++ and transfer the filtrated solution to a 15 ml Falcon tube.

(v) Continue to step 110.

(B) Removing organoids from BME with TrypLE (i) Place a 70 um cell strainer on a 50 ml Falcon tube and pre-wash the strainer with 5 ml of ice-cold adDMEM/F 12+++. (ii) Remove the expansion medium from the number of wells needed, calculated in step 102-108 by holding the plate at a 45° angle and aspirating with a vacuum system and clean tip.

(iii) Resuspend up to 3 wells of the same culture in 1 ml TrypLE, as described in steps 43A (ii)-{iv), to break down the BME, but leave the organoids intact. (iv) Pipette the intact organoid solution over the 70 um strainer into the 50 ml Falcon tube, combining up to 6 wells of the same culture. Rinse the strainer with 5 ml of adDMEM/F12+++ and transfer the filtrated solution to a 15 ml Falcon tube.

110.Centrifuge the 15 ml Falcon tube at 300g for 5 min at 4 °C. Confirm by eye that a tight BME-free pellet is present (Fig. 2¢).

111.Remove the supernatant by decanting the fluid in a waste bottle. Let the tube rest for 1 min to allow the adDMEM/F12+++ from the side of the tube to settle at the bottom and remove the residual adDMEM/F12+++ with a p1000 pipette.

112.Pool the organoids from the same culture together in 1 ml of Type 1 or Type 2 expansion medium and transfer to a 1.5 ml Eppendorf tube. Centrifuge the tube at 300g for 5 min at 4 °C.

113.Remove all supernatant carefully with a p1000 pipette, estimate the volume of the pellet, and add ice-cold transplantation buffer up to a total volume of 40 ul x the number of injection sites (e.g. for 10 injections, the total volume of organoid pellet + transplantation buffer = 400 ul) Mix well by pipetting up and down 10 times with a p1000 pipette. Prevent bubble formation. Implantation of the estrogen pellet

[00360] All animal experiments should be carried out conform the local and national animal welfare regulations and guidelines.

114.Implant the estrogen pellet while the organoids are stored on ice until injection, or implant up to one day prior to organoid injection to minimize the time the organoids are stored on ice.

115.Prepare the implantation by placing the estrogen pellet in the trocar. Make sure the trocar has been cleaned and sterilized with 70% alcohol and is completely dry before inserting the estrogen pellet.

116.Anesthetize the mouse with 3—4 % (vol/vol) isoflurane in an induction chamber. 117 .Carefully take the mouse out of the induction chamber and place in front of you with its back facing up and towards you from the anterior-lateral side. The mouse will no longer receive anaesthesia at this point and it is thus important to proceed swiftly and carefully.

118.Fix the dorsal skin of the neck between thumb and index finger. Position the trocar with estrogen pellet flat on the head of the mouse (Fig. 4a) and inject the needle subcutaneously, with the tip facing down, into the skin of the neck.

119.Insert the estrogen pellet by pushing the inner cylinder forward, releasing the pellet into the skin pocket.

120.Carefully take out the trocar, while keeping the pellet steady in the skin between the thumb and index finger. When organoid injection directly follows estrogen pellet injection, continue with step 126. When organoids are injected the next day, continue with step 121.

121.Gently place the mouse back into the cage and monitor until recovery from anesthesia.

Xenotransplantation of breast cancer organoids

[00361]

122.Pre-cool insulin syringes with 29G needle on ice. Use one syringe for up to 5 injections.

123.Perform the procedures described in steps 124-130 for one mouse at a time.

124.Anesthetize the mouse with 3-4 % (vol/vol) isoflurane in an induction chamber.

125.Take the mouse out of the induction chamber and carefully place it on a foam board wrapped with aluminum foil.

126.Place the mouse with its back down and nose inserted into the nose piece that supplies isoflurane. Lower the anesthesia to 1.5-2 % (vol/vol) isoflurane. Tape down all 4 legs of the mouse for fixation. Continuously monitor breathing and movement reflexes to ensure the anesthesia is not too high or too low.

127.Shave the area around the 4!” nipple using a trimmer and disinfect with 70% EtOH (Fig. 4b). Shaving the injection area is required to better visualise the nipple.

128.Shortly resuspend the Eppendorf tube with organoids, prepared in steps 109-113, by flicking the sides a few times. Fill the pre-cooled insulin syringes with 40 pl of organoid suspension per injection for up to 5 injections per needle. Ensure to work swiftly to prevent premature BME polymerization, which may clog the syringe. Place the syringe on ice in between 2 injections.

129.Position the syringe flat on the mouse and insert the needle approximately 3 mm below the 4™ nipple and move the tip of the needle right under the nipple, ~ 1 mm deep into the fat pad (Fig. 4b). Steadily inject 40 pl of organoid suspension into the fat pad.

130.Take the mouse off anesthesia and gently place it into a clean cage placed on a heating pad to recover from anesthesia.

131.Monitor the mouse until awake and mobile.

132.Monitor tumour growth by palpation of the injection site at least 2x per week. The tumour onset depends on the organoid culture and typically ranges from 2 weeks to 4 months (Fig. 4c). The proportion of organoid cultures that can engraft in vivo is ~ 50% for cultures that can be passaged at least 1:2, every 2 weeks. Example 2 - HUMAN BREAST MILK-DERIVED ORGANOIDS Cell isolation from milk

[00362] Fresh milk was collected from donors and kept on ice until processed (processing within 2 hours). Milk was mixed with ice cold DPBS (Gibco) at a ratio of 1:2 and centrifuged at 2000 g for 10 min at 4 °C. The supernatant was discarded and the pellet was washed with DPBS and centrifuged at 2000 g for 10 min at 4 °C.

Milk organoid culture

[00363] Cell pellets were resuspended in a volume of DPBS and counted manually. Cells were centrifuged at 2000 g for 10 min at 4 °C. The supernatant was discarded and cell pellets were resuspended in a volume of basement membrane extract (BME; Cultrex) and seeded in uncoated 12-well plates (Thermo Fisher; each well containing either 500,000 or 1 million cells). The BME was allowed to solidify for 10 min at 37 °C and 750 ul. medium were added per well (for medium composition see Table 1). Culture medium was refreshed twice a week and organoids were passaged at a ratio of 1:2 every 7-14 days using TrypLE Express (Thermo Fisher).

Tumouroid culture

[00364] Tumouroids were seeded in basement membrane extract (BME; Cultrex) in uncoated 24-well plates (Thermo Fisher; 4 drops of 10 pl per well containing 10,000 cells per drop) and cultured as described previously (Sachs, Norman, et al. "A living biobank of breast cancer organoids captures disease heterogeneity.” Cell 172.1-2 (2018): 373-386.). Culture medium was refreshed every 2-3 days and tumouroids were passaged 1:2 — 1:6 every 7-21 days depending on the tumouroid using TrypLE Express (Thermo Fisher). For co-culturing, single tumouroids of a 5-12-day old culture (depending on the growth speed of the tumouroid) were recovered from the BME by resuspension in TrypLE Express and collected in Advanced DMEM/F12 supplemented with pen/strep, 10 mM HEPES and Glutamax (all from Thermo Fisher). The tumouroid suspension was filtered through a 70 um strainer (Greiner) to remove large tumouroids and pelleted before co-culturing. Imaging

[00365] BME was dissolved in ice-cold Cell Recovery Solution (Corning) and organoids were then fixed for 30 min in 4% Paraformaldehyde, washed with PBT (PBS, 0.1% Tween) and incubated overnight at 4 °C with primary antibodies, followed by washing steps and overnight incubation with secondary antibodies (Supplementary Table ST2)( Rios, A., Fu, N., Lindeman, G. et al. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322-327 (2014). https://doi.org/10.1038/nature12948). The following day, organoids were washed and incubated in 80% glycerol for 1 hour before 3D imaging using a SP8 confocal microscope. 3D rendering was performed using the Imaris software as described previously (Rios, A., Fu, N., Lindeman, G. et al. In situ identification of bipotent stem cells in the mammary gland. Nature 506,

322-327 (2014). https://doi.org/10.1038/nature12948). Confetti-fluorescent living cultures were imaged as described above, and scored using the 3D visualization module of Imaris. Cell transduction

[00366] Lentiviral plasmids were transfected in HEK293T cells. One day after transfection, media was refreshed with Advanced DMEM/F12 (Gibco) supplemented with 10 mM HEPES and Glutamax (Thermo Fischer Scientific). Virus was collected 24 hours later and then concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane tubes (Merck).

[00367] For organoid transduction, organoids from 7-day old cultures were processed into single cells using TrypLE Express (Thermo Fisher Scientific). The cell pellet containing 100,000 — 300,000 cells was resuspended in concentrated virus that was diluted in complete organoid culture medium (total volume 100 pl; MOI of 5-10) and incubated for 30-45 minutes at 37°C. Cells were then washed in Advanced DMEM/F12 supplemented with penicillin/streptomycin, HEPES and Glutamax (Thermo Fischer Scientific) and seeded in basement membrane extract (BME; Cultrex) in uncoated 24-well plates (Thermo Fisher) and cultured as described above.

Live cell imaging of TEG and breast tumouroid co-cultures

[00368] TEGs, which are peripheral blood af T cells engineered to express a Vy9/Vé2 T cell receptor (TCR) and have the ability to recognize cancer cells, (20,000) were co-cultured with breast tumouroids or milk-derived organoids in a effector to tumour cell (E:T) ratio of 1:3 (Fig. 1,2,4) or 1:25 (Fig. 3). CD4+ and CD8+ TEGs were mixed in a 1:1 ratio just before plating. Cells were incubated in 96-well glass-bottom SensoPlates (Greiner) in 200 ul imaging medium, containing a mix of 50% organoid culture medium (see above) and 50% TEG assay medium (RPMI-Hepes supplemented with 10% FCS and 1% pen/strep), supplemented with 2.5% BME, pamidronate for the accumulation of the phosphoantigen IPP to stimulate tumour cell recognition?, 46 (1:2000) and both NucRed™ Dead 647 (2 drops per ml; Thermo Fisher) and TO- PRO-3 (1:3000; Thermo Fisher) for fluorescent labelling of living and dead cells. Prior to co- culturing, TEGs were incubated with eBioscience™ Cell Proliferation Dye eFluor™ 450 (referred to as eFluor-450; 1:4000; Thermo Fisher) to fluorescently label all TEGs. To prevent evaporation while imaging, 200 pl PBS was added to the wells surrounding the co-culture wells. The plate was placed in a LSM880 (Zeiss) microscope containing an incubation chamber (37 °C; 5% CO2) and incubated for 30 min to ensure settling of TEGs and tumouroids at the bottom of the well. The plate was imaged for up to 24 hours with a Plan-Apochromat 20 x/0.8 NA dry objective with the following settings: online fingerprinting mode, bidirectional scanning, optimal Z-stack step size, maximum Z-stack of 60 um in total and time series with a 30 min (42 conditions simultaneously; resolution 512 x 512) or 2 min interval (4 conditions simultaneously; resolution 200 x 200 — 512 x 512). Example 3 - ASSEMBLOID FORMATION

[00369] We further advanced our methods to generate hybrid 3D cultures containing normal as well as primary breast cancer cells named ‘assembloids’.

[00370] Different cultures that will form the hybrid structure are first cultured separately to similar size. A healthy organoid and tumour organoid were cultured separately as described for other organoids above.

[00371] The two cultures were then mixed in a one to one ratio at a high density and incubated at 37°C for several hours so that different structures will fuse into ‘assembloids’ These are then directly used for functional assays.

DISCUSSION

[00372] Organoids derived from breast cancer tissue can display solid (~60 % of cultures), cystic (< 5%), grape-like (~20 %), or mixed morphologies (~20 %) (Fig. 5a, b). Breast cancer organoids highly vary in their growth rate, but can reach high expansion speed for some donors (1:6 every week; Table 1). Once established (e.g. > passage 5 in vitro), it is rarely observed that breast cancer organoids stop growing or reduce growth speed. Organoid establishment efficiency ranged between 55-70% for most BC subtypes, and was ~40 % for triple negative breast cancer (TNBC; Fig. 5c,d), suggesting that it is challenging for the often aggressive and genetically unstable TNBC cells to adapt to in vitro culture conditions. Immunohistochemical comparison to the original tumour specimen indicated that organoids sometimes lost expression of ER (~ 25% of samples), PR (~ 25% of samples) or HER2 (~ 20% of samples) during culturing and few samples gained ER (~ 10%) or HER2 (< 5%) expression (Fig. 5e, f; Table 1). The time in culture at which receptor expression reduces or increases can vary and should be assessed for each culture. Reasons for these changes are currently unclear, but could be influenced by propagating organoids in Type 1 or Type 2 expansion medium, culture-induced gene silencing or outgrowth of certain sub-clones. Organoids generated from tumour resections can be contaminated with normal cells, which is expected to be the case for < 10% of tumour samples. However, as normal organoids in general grow slow (Table 1) and ultimately stop growing, most likely normal cells will be lost during culture over time. It is not possible to distinguish normal cells from tumour cells by eye using light microscopy, but pathologists are often able to confirm normal or tumourous origin of a culture by immunohistochemistry. When the tumour harbors p53 mutations, Nutlin-3a can be added to the expansion medium to select for tumour cells, ensuring death of all normal cells that are potentially present (Fig. 3c). Confirmation of the cancerous nature of the sample can be achieved by assessing genomic alterations, either by whole-genome or whole-exome sequencing or by comparative genomic hybridization.

[00373] Regarding normal breast organoid cultures, the rate of establishment is near 100%. However, their growth rate is generally low (split ratio of 1:2 every 2 weeks at best; Table 1) and declines over time, and cultures have limited growth potential (up to in vitro passage ~ 20). Normal breast organoids display different morphologies (Fig. 6a), including mature luminal{ML )- type cystic structures, luminal progenitor(LP)-type solid, budding structures and basal/stem cell- type dense, solid structures (Fig. 6b). Normal organoids can display as branching or cystic structures containing a central lumen (Fig. 6¢,d). CYTOF analysis confirmed the presence of basal, LP and ML cells in cultured normal organoids (Fig. 6e). the present invention provides different options to alter the ratio of basal, LP and ML cells (Fig. 6f). These manipulations can be useful for short-term study of specific cell types, but can affect the morphology and long-term growth of the culture. Furthermore, we have genetically-edited normal breast organoids using CRISPR/Cas9 (Fig. 3b,c), showing that mutating P53, PTEN and RB1 in normal organoids is sufficient to transform them into organoids that form ER+ tumours upon xenotransplantation in vivo (Fig. 4c). This illustrates the potential utility of this model to increase our understanding of the early molecular events that culminate in specific subtypes of breast cancer.

[00374] Surprisingly, it was found that the use of Type-2 medium, which is used for culturing of ovarian organoids, provided better outgrowth and maintenance of normal tissue-derived organoids in comparison to Type 1 medium, which is the established culture medium for tissue- derived breast organoids (Fig. 7a).

[00375] In addition, we explored the use of human breast milk as a source for healthy human cells to study mammary tissue development and pathology. We defined culture conditions that enable generation of 3D breast organoids from milk with high efficiency. These milk-derived breast organoids display long-term culture capacity (>15 passages), suggesting presence of breast stem cells. It is also demonstrated outgrowth of the milk-derived mammary organoids is significantly more efficient in ‘Type 2’ medium compared to the regular Type 1 medium and that Whnt3a was important for proper outgrowth in type 2 medium (Fig. 7a). Furthermore, milk-derived organoids grown in ‘Type 2’ medium grow faster compared to tissue-derived organoids grown in ‘Type 2° medium (Fig. 7b). Organoids can have a ‘budding’ or ‘cystic’ morphology (Fig. 7¢).

[00376] It is also shown in Fig. 7d that high-resolution 3D imaging demonstrated that breast milk- derived organoids recapitulated important phenotypic features of non-lactating in vivo mammary epithelium; they comprised an inner compartment of polarized progenitor- and matured luminal cells and an outer of network of myoepithelial (basal) cells, Cells were also successfully engineered by viral transduction to express fluorescently labelled proteins (Fig. 7e).

[00377] Milk-derived organoids were also used to show that solely cancer cells were killed by engineered cancer-targeting T cells called ‘TEGs’, while healthy cells stayed alive (Fig. 7f).

[00378] Furthermore, we further advanced our methods to generate hybrid 3D cultures containing normal as well as primary breast cancer cells named ‘assembloids’. These were used to show that cancer cells were killed by chemotherapy, while healthy cells stayed alive (Fig. 79), thus illustrating the use of milk-derived organoids as healthy control tissue for drug toxicology studies. In summary, we describe breast milk as an easy-access source for multiple human cells, and optimized conditions for generation of breast organoid-based 3D live cell imaging models suitable for studying tissue dynamics and safety profiles of breast cancer therapies.

[00379] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00380] Features, integers, characteristics or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

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Table 2. Overview of the components to add to adDMEM/F12+++ to generate Type 1 and Type 2 media, with the respective final concentrations per type of medium.

Component ~~ Type Type2 WntBa ~~ - 20%conditioned medium

R-spondinl 10% conditioned medium 10% conditioned medium

Noggin 10% conditioned medium 10% conditioned medium

B27 + VitA 1x 1x

Nicotinamide 10 mM 10 mM

N-Acetylcysteine 1.25 mM 1.25 mM

Hydrocortisone - 0.5 pg/mL

Primocin 20 ug/mL 20 pg/mL

B-estradiol - 100 nM

Forskolin - 10 uM

Y-27632% 5 uM 5 uM

Heregulin B1 5nM 5nM

FGF-7 5 ng/mL -

FGF-10 20 ng/mL 20 ng/mL

A83-01 0.5 uM 0.5 uM

EGF 5 ng/mL 5 ng/mL

SB202190 1uM - * Optional: Y-27632 can be removed from the medium 2-3 days after organoid establishment, passaging, or thawing, as used in ref.

Rosenbluth, J.

M. et al.

Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages.

Nat.

Commun. 11, (2020). VitA = Vitamin A.

Table 3. Standard number of BME drops and volumes of BME and culture medium per type of culture plate.

Culture plate BME (pL) BME drops Medium (ul) 96-well 10 1 100 48-well 25 1 200 24-well 50 4 500 12-well 100 5-8 750 - 1000 6-well 200 10-16 1500 - 2000 Table 4. Troubleshooting table.

Kil ie [oe [or 19 The tissue is not Digestion was not sufficient.

Centrifuge the 15 ml Falcon tube at noticeably digested 4509 for 5 min at 8 °C and repeat after two hours. steps 16-19. Expansion medium | Plating density is too high.

Passage organoids at lower density. appears very yellow (and dead cells are present in the Not sufficient expansion Refresh expansion medium more culture). medium. often and/or use 1 ml expansion medium per well.

BME drops are The drops were not properly Skip step 51 and/or incubate for 30 floating. attached to the plate. min in step 50.

45, | The organoid pellet | The TrypLE treatment was (1) Resuspend the pellet in 1 ml after centrifugation | insufficient to dissolve the TrypLE, incubate for 5 min at RT and 110 is fuzzy and BME, or the amount of wash with adDMEM/F12+++, or (2) contains adDMEM/F12+++ was Wash another time in >10 ml undissolved BME. insufficient to wash away the adDMEM/F12+++ following steps 44- BME, or the adDMEM/F12+++ | 46, or (3) place the tube on ice for 10 was not cold enough to aid min, resuspend the well using a 10 ml dissolving the BME. pipet, and centrifuge at 300g for 5 min at 4 °C. The organoid pellet | Single organoid cells or Add 2% (vol/vol) FBS to the tube, after centrifugation | fragments stuck to the side of | resuspend well using a 10 ml pipet, is smaller than the tube. and centrifuge at 300g for 5 min 4 °C. expected.

53 The culture is Organoids are growing at too Increase plating density by passaging growing slowly or low density. with low split ratio (sometimes lower reduces growth than 1:1, thus plating in a smaller rate. total volume of BME compared to the previous passage). The organoid Passaging conditions are too Passage with milder conditions by culture contains a harsh. reducing TrypLE concentration, lot of dead cells/cell incubation time, or times of debris. resuspension. Culture is changing | The culture is cross- Avoid cross-contamination by in growth speed or | contaminated with a different | following the steps at 41-43 and 49. morphology. culture. Perform a SNP analysis (see section Human Material) to check for culture purity. Discard the culture and continue with an early passage.

76 Lipofectamine 2000 | The specific organoid culture is | Try electroporation or select a transfection difficult to transfect. different organoid culture. efficiency is low.

The plasmid is difficult to transfect, e.g. too large. / Try electroporation. 81, | Organoid survival The settings are too harsh for Use lower voltage and pulse length 84 after transfection by | the cells. settings. electroporation is low.

The specific organoid culture is | Use a different organoid culture or try sensitive to electroporation.

Lipofectamine 2000-based transfection.

Transfection The settings are too mild.

Use higher voltage and pulse length efficiency after settings. transfection by electroporation is low.

Organoid survival MOI is too high Lower the MOI by adding less virus or after lentiviral by increasing the number of organoid transduction is low cells (up to 300,000). Use a different organoid culture.

The specific organoid culture is sensitive to lentiviral transduction.

Transduction MOI or incubation time is too Increase the MOI (up to 10) by adding efficiency is low. low. more virus or by decreasing the number of organoid cells (> 50,000), or increase incubation time. 101 | Low efficiency of Passaging conditions were too | Reduce TrypLE incubation time or clone outgrowth. harsh. times of resuspending.

Organoids were small while Grow larger organoids {e.g. > 300 um picking. diameter) before picking.

Clauses Also disclosed are examples according to the following clauses: The inventors have also surprisingly found a culture medium that is particularly efficient at growing breast organoids from breast milk and breast tissue. The breast organoids developed using the culture medium show improved outgrowth and long-term maintenance of the organoid through more passages than observed with breast organoids in the prior art. Clauses:

1. A method for producing a breast organoid comprising: (a) isolating breast cells from breast milk; and (b) culturing the breast cells in a culture medium and under conditions suitable to form a breast organoid.

2. The method of clause 1, wherein breast milk is obtained from a mammalian subject.

3. The method of clause 1, wherein breast milk is obtained from a human subject.

4. A method of producing a breast organoid comprising: (a) isolating breast cells from breast tissue; and (b) culturing the breast cells in a culture medium and under conditions suitable to form a breast organoid, wherein the culture medium is as recited in accordance with the first aspect of the invention.

5. The method of clause 4, wherein the breast tissue is healthy breast tissue or tumour breast tissue.

6. The method of clause 4, wherein the breast tissue is healthy breast tissue.

7. The method of clause 4, wherein the breast tissue is tumour breast tissue.

8. The method any of clauses 1 to 3 or 4 to 7, wherein the breast cells are epithelial stem cells.

9. The method of any previous clause, wherein the culture medium comprises a Wnt agonist.

10. The method of clause 9, wherein the Wnt agonist is a surrogate wnt, chir or wnt3a.

11. The method any of clauses 9 or 10, wherein the Wnt agonist is provided by Wnt conditioned media at a concentration of 5 to 50%v/v of the final culture medium volume.

12. The method any of clauses 9 or 10, wherein, the Wnt agonist is provided by Wnt conditioned media at a concentration of 10 to 30% v/v of the final culture medium volume.

13. The method any of clauses 9 to 11, wherein the culture medium comprises the wnt agonist at concentration of 0.01 to 2 nM.

14. The method any of clauses 9 to 11, wherein culture medium comprises the wnt agonist at concentration of 0.05 to 1 nM.

15. The method any of clauses 9 to 11, wherein culture medium comprises the wnt agonist at concentration of 0.2 nM.

16. The method any of clauses 9 to 11, wherein the Wnt agonist is provided by Wnt conditioned media at a concentration of 20%v/v of the final culture medium volume.

17. The method any of clauses 9 to 16, wherein the Wnt agonist is wnt3a.

18. The method of any previous clause, wherein the culture medium further comprises one or more of at least one Lgr5 agonist, at least one BMP inhibitor, at least one ROCK inhibitor, at least one ErbB3/4 ligand, at least one FGFR2b ligand, at least one TGF-beta inhibitor, at least one p38 inhibitor, at least one receptor tyrosine kinase ligand, B27 supplement plus Vitamin A, nicotinamide, at least one antimicrobial agent, and/or N-Acetylcysteine.

19. The method of any of clauses 9 to 16, wherein the culture medium further comprises one or more of at least one Lgr5 agonist, at least one BMP inhibitor, at least one ROCK inhibitor, at least one ErbB3/4 ligand, at least one FGFR2b ligand, at least one TGF-beta inhibitor, at least one receptor tyrosine kinase ligand, B27 supplement plus Vitamin A, nicotinamide, at least one antimicrobial agent, and/or N-Acetylcysteine.

20. The method any of clauses 1 to 17, wherein the culture medium further comprises at least one Lgr5 agonist.

21. The method any of clauses 1 to 17 and 20, wherein culture medium further comprises at least one BMP inhibitor.

22. The method any of clauses 1 to 17 and 20 to 21, wherein the culture medium further comprises at least one ROCK inhibitor.

23. The method any of clauses 1 to 17 and 20 to 22, wherein the culture medium further comprises at least one ErbB3/4 ligand.

24. The method any of clauses 1 to 17 and 20 to 23, wherein the culture medium further comprises at least one FGFR2b ligand.

25. The method any of clauses 1 to 17 and 20 to 24, wherein the culture medium further comprises at least one TGF-beta inhibitor.

26. The method any of clauses 1 to 17 and 20 to 25, wherein the culture medium further comprises at least one receptor tyrosine kinase ligand.

27. The method any of clauses 1 to 17 and 20 to 26, wherein the culture medium further comprises B27 supplement plus Vitamin A.

28. The method any of clauses 1 to 17 and 20 to 27, wherein the culture medium further comprises hydrocortisone and/or forskolin.

29. The method any of clauses 1 to 17 and 20 to 27, wherein the culture medium further comprises hydrocortisone.

30. The method any of clauses 1 to 17 and 20 to 27, wherein the culture medium further comprises forskolin.

31. The method any of clauses 1 to 17 and 20 to 27, wherein the culture medium further comprises hydrocortisone and forskolin.

32. The method any of clauses 1 to 17 and 20 to 31, wherein the culture medium further comprises nicotinamide.

33. The method any of clauses 1 to 17 and 20 to 32, wherein the culture medium further comprises N-Acetylcysteine.

34. The method any of clauses 1 to 17 and 20 to 33, wherein the culture medium further comprises at least one antimicrobial agent.

35. The method any of the previous clauses wherein: (i) the at least one Lgr5 agonist comprises R-spondin1; (ii) the at least one BMP inhibitor comprises Noggin; (iii) the at least one ROCK inhibitor comprises Y-27632; (iv) the at least one ErbB3/4 ligand comprises heregulin B1; (v) the at least one FGFR2b ligand comprises FGF-10; (vi) the at least one TGF-beta inhibitor comprises A83-01; (vii) the at least one p38 inhibitor comprises SB202190; and (viii) the at least one receptor tyrosine kinase ligand comprises EGF.

36. The method any of the previous clauses, wherein the culture medium comprises Wnt3a, R-spondin1, Noggin, B27 plus Vitamin A, nicotinamide, N-Acetylcysteine, hydrocortisone, B- estradiol, forskolin, Y-27632, heregulin B1, FGF-10, A83-01, primocin, and EGF.

37. The method any of the previous clauses, wherein the culture medium consists of Wnt3a, R-spondin1, Noggin, B27 plus Vitamin A, nicotinamide, N-Acetylcysteine, hydrocortisone, B- estradiol, forskolin, Y-27632, heregulin B1, FGF-10, A83-01, primocin and EGF.

38. The method any of the previous clauses, wherein: (i) the at least one Lgr5 agonist is provided by R-spondin1 at a concentration of 50 to 1000 ng/ml; (ii) the at least one BMP inhibitor is provided by Noggin at a concentration of 10 to 500 ng/ml; (iii) the at least one ROCK inhibitor is provided at a concentration of 1 to 50uM; (iv) the at least one ErbB3/4 ligand is provided at a concentration of 1 to 50nM; (v) the at least one FGFR2b ligand is provided at a concentration of 5 to 100 ng/mL; (vi) the at least one TGF-beta inhibitor is provided at a concentration of 0.1 to 5uM; (vii) the at least one p38 inhibitor is provided at a concentration of 0.1 to 10 uM; (viii) the at least one receptor tyrosine kinase ligand is provided at concentration of 1 to 50 ng/mL; (ix) the Wnt agonist is provided by Wnt3A at a concentration of 0.01 to 2 nM; (x) the B27 supplement plus Vitamin A is provided at a concentration of 1 to 20% v/v of the final volume; (xi) the nicotinamide is provided at a concentration of 1 to 50 mM; (xii) the N-Acetylcysteine is provided at a concentration of 0.1 to 15 mM; (xiii) the hydrocortisone is provided at a concentration of 0.1 to 5 ug/ml; (xiv) the B-estradiol is provided at a concentration of 50 to 500 nM; (xv) the forskolin is provided at a concentration of 1 to 50 uM; (xvi) the at least one antimicrobial agent is provided at a concentration of 1 to 100 ug/ml.

39. The method any of the previous clauses, wherein the culture medium comprises: (i) 20%v/v Wnt3a conditioned media; (ii) 10% v/v R-spondin1 conditioned media; (iii) 10% v/v Noggin conditioned media of the final volume; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A;

(v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 ug/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 uM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

40. The method any of the previous clauses, wherein the culture medium consists of: (i) 20%v/v Wnt3a conditioned media; (ii) 10% v/v R-spondin1 conditioned media; (iii) 10% v/v Noggin conditioned media; (iv) 2% 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 ug/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 HM forskolin; (x) 5 HM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 uM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

41. The method any of clauses 1 to 39, wherein the culture medium comprises:

(i) 0.2nM Wnt agonist; (ii) 250 ng/ml Rspondin1; (iii) 100 ng/ml Noggin; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 uM Y-27632; (xi) 5 nM Heregulin B1; (xii) 20 ng/ml FGF-10; (xiii) 0.5 uM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

42. The method any of clauses 1 to 39, wherein the culture medium consists of: (i) 0.2nM Wnt agonist; (ii) 250 ng/ml Rspondin1; (iii) 100 ng/ml Noggin; (iv) 2% v/v 50 times concentrated B27 supplement plus Vitamin A; (v) 10 mM nicotinamide; (vi) 1.25 mM N-Acetylcysteine; (vii) 0.5 pg/ml hydrocortisone; (viii) 100 nM B-estradiol; (ix) 10 uM forskolin; (x) 5 HM Y-27632; (xi) 5 nM Heregulin B1;

(xii) 20 ng/ml FGF-10; (xiii) 0.5 uM A83-01; (xiv) 20 pg/ml primocin; and (xv) 5 ng/ml EGF.

43. The method any of the previous clauses, wherein the ROCK inhibitor is removed from the culture medium 2 to 3 days after organoid establishment, passaging or thawing.

44. The method any of the previous clauses, wherein prior to culturing the breast cells, the breast cells are suspended in a matrix and are seeded onto a surface.

45. The method of the clause 45, wherein the matrix comprises one or more of laminin, collagen IV, entactin and heparan sulfate proteoglycan, preferably wherein the matrix is basement membrane extract (BME).

46. The method of the clause 45, wherein the matrix is a BME.

47. The method any of the previous clauses, wherein culturing the breast stem cells comprises refreshing the culture medium at least twice a week.

48. The method any of the previous clauses, wherein culturing the breast stem cells comprises passaging the cells.

49. The method any of the previous clauses, wherein the breast stem cells and/or breast organoids are passaged at a ratio of 1:2 to 1:10.

50. The method any of the previous clauses, wherein the breast stem cells and/or breast organoids are passaged every 7 to 21 days.

51. The method any of the previous clauses, wherein the breast stem cells and/or breast organoids are passaged at least 4 times, at least 6 times, at least 10 times, at least 15 times.

52. A breast organoid obtainable by the method according any of the previous clauses, wherein the organoid withstands more than 4 passages, more than 10 passages and/or is maintained for at least 4 to 10 weeks.

53. The breast organoid of clauses 52 or 53, wherein the organoid is maintained for at least 4 weeks to at least 15 weeks.

54. The breast organoid of any one of clauses 52 to 54, wherein the breast organoid comprises: polarized progenitor luminal cells; matured luminal cells; and basal cells.

55. The breast organoid of any one of clauses 52 to 55, wherein the breast organoid comprises: an inner compartment of polarized progenitor luminal cells and matured luminal cells and an outer of network of basal cells.

56. The breast organoid of any one of clauses 52 to 56, wherein the breast organoid comprises a spherical shape.

57. A breast organoid according to any of the previous clauses, for use as a medicament.

58. A breast organoid according to any of the previous clauses, for use in any one or more of: (i) drug discovery; (ii) toxicity assay; (iii) research of nutrient and drug metabolism; (iv) cancer research; (v) research of tissue embryology, cell lineages, or differentiation pathways; (vi) research of breast milk production and composition; and/or (vii) recombinant breast milk production.

59. A method for producing an assembloid, comprising (a) isolating breast cells from breast milk; (b) isolating at least one second cell type or cell line from a tissue or breast milk; and (c) combining the cells from step (a), and step (b); (d) culturing the combined cells of step {c} in a culture medium and under conditions suitable to form an organoid comprising the cells of step (a) and step (b).

60. A method for producing an assembloid, comprising: producing a first organoid according the method of any one of clauses 1 to 52; producing at least one further organoid by (a) isolating cells from a tissue or breast milk; and (b) culturing the cells in a culture medium and under conditions suitable to form the at least one further organoid;

combining the first organoid and at least one further organoid; incubating the first organoid and the least one further organoid under conditions suitable to fuse the first organoid and the least one further organoid to form a hybrid organoid.

61. The method of clause 60, wherein the further organoid is cultured in a culture medium as described herein.

62. The method of clauses 60 or 61, wherein the second organoid comprises autologous cells to the breast cells of the first breast organoid optionally wherein the cells of the first organoid and second organoid are obtained from the same subject.

63. The method of clauses 60 or 61, wherein the second organoid comprises non-autologous cells to the breast cells of the first breast organoid, optionally wherein the cells of the first organoid and second organoid are obtained from the different subjects.

64. The method of any of clauses 60 to 63, wherein the second organoid comprises cells derived from a tumour tissue.

65. The method of any of clauses 60 to 64, wherein the second organoid is an organoid according to any of clauses 4 to 59.

66. The method of any of clauses 60 to 64, wherein the second organoid is derived from tumour tissue.

67. An assembloids for use according to clauses 58 or 59.

Claims (22)

-80 - CONCLUSIONS A method of producing a breast organoid comprising: (a) isolating breast cells from breast milk; and {b) culturing the breast cells in a culture medium and under conditions suitable to form a breast organoid. The method of claim 1, wherein the breast cells are epithelial stem cells. The method according to any of the preceding claims, wherein the culture medium comprises a Wnt agonist. The method of claim 3, wherein the Wnt agonist is a surrogate wnt, chir or wnt3a. The method of claim 3 or claim 4, wherein the culture medium further contains one or more of at least one Lgr5 agonist, at least one BMP inhibitor, at least one ROCK inhibitor, at least one ErbB3/4 ligand, at least one FGFR2b ligand , at least one TGF-beta inhibitor, at least one p38 inhibitor, at least one receptor tyrosine kinase ligand, B27 supplement plus vitamin A, nicotinamide, at least one antimicrobial and/or N-acetyl cysteine. The method of claim 5, wherein: (i) the at least one Lgr5 agonist comprises R-spondin1; (ii) the at least one BMP inhibitor comprises Noggin; (iid) comprises at least one ROCK inhibitor Y-27632; (iv) it comprises at least one ErbB3/4 ligand heregulin B1; (v) it comprises at least one FGFR2b ligand FGF-10; (vi) the at least one TGF-beta inhibitor comprises A83-01; (vii) the at least one p38 inhibitor comprises SB202190; and (viii) it comprises at least one receptor tyrosine kinase ligand EGF; (ix) the at least one antimicrobial agent comprises primocin. -81- The method of any one of claims 3 to 6, wherein the culture medium further comprises hydrocortisone and/or forskolin. The method according to any of the preceding claims, wherein the culture medium consists of Wnt3a, R-spondin1, Noggin, B27 supplement plus vitamin A, nicotinamide, N-acetylcysteine, hydrocortisone, β-estradiol, forskolin, Y-27632, heregulin B1 , FGF-10, A83-01, Primocin and EGF. The method of any one of claims 5 to 8, wherein: (i) the at least one Lgr5 agonist is provided by R-spondin1 at a concentration of 50 to 1000 ng/ml; (ii) the at least one BMP inhibitor is provided by Noggin at a concentration of 10 to 500 ng/ml; (iii) the at least one ROCK inhibitor is provided at a concentration of 1 to 50 µM; (iv) the at least one ErbB3/4 ligand is provided at a concentration of 1 to 50 nM; (v) the at least one FGFR2b ligand is provided at a concentration of 5 to 100 ng/ml; (vi) the at least one TGF-beta inhibitor is provided at a concentration of 0.1 to 5 µM; (vii) the at least one p38 inhibitor is provided at a concentration of 0.1 to 10 µM; (viii) the at least one receptor tyrosine kinase ligand is provided at a concentration of 1 to 50 ng/ml; (ix) the Wnt agonist is provided by Wnt3A at a concentration of 0.01 to 2 nM; (x) the B27 supplement plus vitamin A is provided at a concentration of 1 to 20% v/v of the final volume; (xi) the nicotinamide is provided at a concentration of 1 to 50 mM; (xii) the N-acetylcysteine is provided in a concentration of 0.1 to 15 mM: -82- (xiii) the hydrocortisone is provided at a concentration of 0.1 to 5 pg/ml; (xiv) the β-estradiol is provided at a concentration of 50 to 500 nM; (xv) the forskolin is provided in a concentration of 1 to 50 HM; (xvi) the at least one antimicrobial agent is provided at a concentration of 1 to 100 pg/ml. The method of any one of claims 1 to 9, wherein prior to culturing the breast cells, the breast cells are suspended in a matrix and seeded onto a surface. The method according to claim 10, wherein the matrix comprises one or more of laminin, collagen IV, entactin and heparan sulfate proteoglycan, preferably wherein the matrix is a basement membrane extract (BME). The method of any one of claims 1 to 11, wherein culturing the breast stem cells comprises changing the culture medium at least twice a week. A breast organoid obtainable by the method of any one of claims 1 to 12, wherein the organoid is resistant to more than 4 passages, more than 10 passages and/or is maintained for at least 4 to at least 10 weeks . The breast organoid of claim 13, comprising: polarized luminal progenitor cells; mature luminal cells; and basal cells. The breast organoid of claim 13 comprising: an inner compartment of polarized luminal progenitor cells and mature luminal cells and an outer network of basal cells. -83- The breast organoid of any one of claims 13 to 15, for use as a medicament. Use of the breast organoid of any one of claims 13 to 15 in: (i) drug discovery; (i) toxicity testing; (iii) research on the metabolism of nutrients and drugs; (iv) cancer research; (Vv) research into tissue embryology, cell lineages or differentiation pathways; (vi) research on the production and composition of human milk; and (vi) recombinant production of human milk. A method of producing an assembloid comprising: (a) isolating breast cells from breast milk; (b) isolating at least one second cell type or cell line from a tissue or breast milk; and (c) combining the cells of step (a) and step (b); (d) culturing the combined cells of step (c) according to the method of any one of claims 1 to 13. The method of claim 18, wherein at least one second cell type or cell line comprises autologous cells to the breast cells of the first breast organoid. The method of claim 18 or 19, wherein the at least one second cell type or cell line is cells derived from a tumor tissue. The method of any of claims 18 to 20, wherein the cells of step (a) are cultured to form an organoid of any of claims 13 to 15 and the cells of step (b) are cultured to form at least one further organoid; and -84 - wherein step (d) comprises incubating the organoid formed from the cells of step (a) and the at least one further organoid formed from the cells of step (b) under conditions suitable to incubate the first organoid and the fuse at least one further organoid to form an assembloid. Use of a culture medium for producing a breast organoid derived from breast cells isolated from breast milk, wherein the culture medium is as recited in any one of claims 3 to 9.