EP4291636A1 - Method of producing an organoid - Google Patents

Method of producing an organoid

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Publication number
EP4291636A1
EP4291636A1 EP22705172.9A EP22705172A EP4291636A1 EP 4291636 A1 EP4291636 A1 EP 4291636A1 EP 22705172 A EP22705172 A EP 22705172A EP 4291636 A1 EP4291636 A1 EP 4291636A1
Authority
EP
European Patent Office
Prior art keywords
cells
breast
organoid
organoids
certain embodiments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22705172.9A
Other languages
German (de)
English (en)
French (fr)
Inventor
Anne Caroline Rios
Johanna Florentia Dekkers
Maj-Britt BUCHHOLZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Prinses Maxima Centrum Voor Kinderoncologie BV
Original Assignee
Prinses Maxima Centrum Voor Kinderoncologie BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Prinses Maxima Centrum Voor Kinderoncologie BV filed Critical Prinses Maxima Centrum Voor Kinderoncologie BV
Publication of EP4291636A1 publication Critical patent/EP4291636A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0631—Mammary cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/195—Heregulin, neu differentiation factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • This invention relates to breast organoids, method for production thereof, and uses of the organoid as various research tools.
  • BC Breast cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth receptor 2
  • breast tissue models available in the prior art which are used to study breast tissue health and disease.
  • 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.
  • 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.
  • Single cell RNA sequencing of human milk-derived cells reveals subpopulations of mammary epithelial cells with molecular signatures of progenitor and mature states: a novel, non-invasive framework for investigating human lactation physiology.
  • Journal of Mammary Gland Biology and Neoplasia 25.4 (2020): 367-387 reported a negligible basal/stem cell population, that they did not detect expression of myoepithelial cells or other basal MECs nor did they detect markers of pluripotency. Thus concluding that the milk samples analyzed lacked appreciable quantities of basal cells.
  • breast organoids may be generated from cells found in breast milk.
  • the inventors have found that it is possible to generate organoids that contain all epithelial cell compartments of breast. 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.
  • 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.
  • 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.
  • a method for producing a breast organoid comprising:
  • breast milk is obtained from a mammalian subject. In certain embodiments, breast milk is obtained from a human subject.
  • 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.
  • 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.
  • the isolated breast cells are stem cells. In certain embodiments, the isolated breast cells are epithelial stem cells. In certain embodiments, the epithelial cells are basal epithelial cells.
  • the culture medium comprises a Wnt agonist.
  • Wnt agonists increase the efficiency of organoid establishment.
  • the Wnt agonist is a surrogate wnt, chir or wnt3a.
  • the Wnt agonist is provided by Wnt conditioned media at a concentration of 5 to 50%v/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.
  • 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.
  • the Wnt agonist is provided by Wnt conditioned media at a concentration of 20%v/v of the final culture medium volume.
  • the Wnt agonist is wnt3a.
  • 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.
  • 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.
  • 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.
  • the culture medium further comprises at least one Lgr5 agonist.
  • the culture medium further comprises at least one BMP inhibitor.
  • the culture medium further comprises at least one ROCK inhibitor.
  • the culture medium further comprises at least one ErbB3/4 ligand.
  • the culture medium further comprises at least one FGFR2b ligand.
  • the culture medium further comprises at least one TGF-beta inhibitor.
  • the culture medium further comprises at least one receptor tyrosine kinase ligand.
  • the culture medium further comprises B27 supplement plus Vitamin A.
  • 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.
  • the culture medium further comprises nicotinamide.
  • the culture medium further comprises N-Acetylcysteine. [0035] In certain embodiments, the culture medium further comprises at least one antimicrobial agent.
  • the culture medium comprises the Lgr5 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 pg/ml hydrocortisone. [0044] 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.
  • the culture medium comprise 1 to 50 mM forskolin. In certain embodiments, the culture medium comprise 5 to 15 pM forskolin. In certain embodiments, the culture medium comprise 10 pM forskolin.
  • the culture medium comprise 1 to 50 pM ROCK inhibitor. In certain embodiments, the culture medium comprise 1 to 10 pM ROCK inhibitor. In certain embodiments, the culture medium comprise 5 pM ROCK inhibitor.
  • 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.
  • the culture medium comprise 0.1 to 5 pM TGF-beta inhibitor. In certain embodiments, the culture medium comprise 0.1 to 1 pM TGF-beta inhibitor. In certain embodiments, the culture medium comprise 0.5 pM TGF-beta inhibitor.
  • 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.
  • 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 pg/ml. In certain embodiments, comprise at least one antimicrobial agent at a concentration of 20 pg/ml.
  • 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
  • the at least one Lgr5 agonist comprises R-spondin1 ;
  • the at least one BMP inhibitor comprises Noggin
  • the at least one ROCK inhibitor comprises Y-27632
  • the at least one ErbB3/4 ligand comprises heregulin B1 ;
  • the at least one FGFR2b ligand comprises FGF-10;
  • the at least one TGF-beta inhibitor comprises A83-01 ;
  • the at least one p38 inhibitor comprises SB202190;
  • the at least one receptor tyrosine kinase ligand comprises EGF.
  • 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- spondinl conditioned media. In certain embodiments, the culture medium comprises 10% v/v R-spondin1 conditioned media.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 pg/ml hydrocortisone. [0061] 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.
  • the culture medium comprise 1 to 50 mM forskolin. In certain embodiments, the culture medium comprise 5 to 15 pM forskolin. In certain embodiments, the culture medium comprise 10 pM forskolin.
  • the culture medium comprise 1 to 50 pM Y-27632. In certain embodiments, the culture medium comprise 1 to 10 pM Y-27632. In certain embodiments, the culture medium comprise 5 pM Y-27632.
  • 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.
  • the culture medium comprise 0.1 to 5 pM A83-01. In certain embodiments, the culture medium comprise 0.1 to 1 pM A83-01.ln certain embodiments, the culture medium comprise 0.5 pM A83-01.
  • 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.
  • 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.
  • 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.
  • 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.
  • the culture medium comprises:
  • the culture medium consists of:
  • the culture medium consists of:
  • the ROCK inhibitor is removed from the culture medium 2 to 3 days after organoid establishment, passaging or thawing.
  • the Y- 27632 is removed from the culture medium 2 to 3 days after organoid establishment, passaging or thawing.
  • the breast cells prior to culturing the breast cells, are suspended in a matrix and are seeded onto a surface.
  • the matrix comprises one or more of laminin, collagen IV, entactin and heparan sulfate proteoglycan, preferably wherein the matrix is basement membrane extract (BME).
  • BME basement membrane extract
  • the matrix is a BME.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the breast organoid comprises: polarized progenitor luminal cells; matured luminal cells; and basal cells.
  • the breast organoid comprises: an inner compartment of polarized progenitor luminal cells and matured luminal cells and an outer of network of basal cells.
  • the breast organoid comprises a spherical shape.
  • a breast organoid according to the second aspect for use as a medicament.
  • the breast organoid is for use in drug discovery.
  • a breast organoid formed from healthy cells may be used as control for testing the specificity or toxicity of compounds.
  • the organoid may be used to test the efficacy of compounds such as anti-cancer agents and/or chemotherapeutics.
  • the breast organoid is for use in toxicity assays.
  • the organoid may be used as a control for testing the specificity or toxicity of compounds.
  • the breast organoid is for use in cancer research.
  • the breast organoid may be subjected to genetic manipulation in order to determine the effects of mutations and formation of cancerous cells.
  • 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.
  • the breast organoid is for use in research of breast milk production and composition.
  • the breast organoid is for use in recombinant breast milk production.
  • step (c) combining the cells from step (a), and step (b);
  • step (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).
  • the combined cells are cultured according to the method of the first aspect.
  • 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.
  • cell type refers to healthy and un-healthy cell types such as cancerous or tumorous cells.
  • both cell types are breast cells. In certain embodiments, both cell types are breast epithelial stem cells. In certain embodiments, both cell types are basal epithelial stem cells.
  • the assembloid may be formed from 2, 3, 4, 5 or more cell types or cell 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.
  • step (a) and step (b) are cultured in a culture medium as described herein.
  • 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.
  • the cells of step (b) comprise non-autologous cells to the cells of step (a).
  • the cells of the step (a) and the cells of the step (b) are obtained from the different subjects.
  • the cells of step (b) comprise cells derived from a tumour tissue.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • each organoid of the assembloid is formed from different cell lines or types.
  • cell line refers to morphologically or phenotypically distinct cell forms within a species.
  • the further organoid is derived from breast cells, immune cells, ovarian cells or any other cell line or type.
  • 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.
  • the further organoid is cultured in a culture medium as described herein.
  • the further organoid comprises autologous cells to the cells of the first organoid.
  • the cells of the first organoid and further organoid are obtained from the same subject.
  • the further organoid comprises non-autologous cells to the cells of the first organoid.
  • the cells of the first organoid and further organoid are obtained from the different subjects.
  • the further organoid comprises cells derived from a tumour tissue.
  • the further organoid is derived from tumour tissue.
  • the first organoid is derived from breast milk breast cells.
  • Assembloids may be for use according to the third and fourth aspects.
  • assembloids are for use as a medicament.
  • assembloids are for use in any one or more of:
  • FIG. 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.
  • FIG. 1 Organoid derivation, culturing, and passaging,
  • FIG. 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 pm.
  • 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.
  • FIG. 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 V medium, Type 2’ medium, or Type 2’ medium without Wnt3A.
  • C Bright-field images of morphologies observed in milk-derived organoid cultures. Scale bar 50 pm.
  • 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 pm.
  • 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 pm.
  • 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 pm.
  • FIG. 1 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.
  • FIG. 8 MDOs express markers of all main breast lineages.
  • A Heatmap of Milk Derived Organoids (MDO) and Tissue Derived Organoids (TDO) RNA sequencing results for the expression of key mammary subpopulation markers, as shown in Fig 11C.
  • B Main subset classification of MDO and TDO lines based on the expression of basal, luminal progenitor (LP) and mature luminal (ML) markers derived from RNA sequencing.
  • LP luminal progenitor
  • ML mature luminal
  • Figure 9 Quantification of milk cellular content, viability and gating strategy.
  • A Percentage viability of cells harvested from milk per donation per donor. The box represents the interquartile range and the whiskers the maximum and minimum range of the data.
  • B Total number of cells harvested per donation per donor in million cells.
  • C Gating strategy for analyzing subpopulations of milk-derived cells. Debris was excluded based on FCS/SSC, followed by exclusion of doublets by gating on FSC-A against FSC-H. Viable cells were gated by exclusion of fixable L/D dye and lineage- cells by excluding CD45 + , CD31 + and CD235a + cells.
  • Epithelial cells were selected by expression of EpCAM and/or CD49f and stromal cells by excluding EpCAM + and CD49P cells. Gates used for subsequent gating indicated in orange.
  • FIG. 10 Human breast milk can be used to establish mammary gland-resembling organoids.
  • A Schematic overview of milk cell isolation and organoid generation procedure.
  • C Cell sub-populations found in human breast milk through flow-cytometry analysis as percentage of live single cells. Bars represent mean + SD.
  • D Organoid forming efficiency in tumor-optimized Type 1 medium, Type 2 medium without Wnt3a-conditioned medium (no WCM) and Type 2 medium, depicted as organoids formed per million cells seeded normalized to the average amount of organoids formed among all conditions.
  • FIG. 11 MDO formation and culture characteristics and bulk RNA sequencing analysis.
  • A Organoids established per million cells seeded per donation per donor.
  • B Biweekly passaging ratios of MDOs per stage of culture represented as mean ⁇ SD. * p ⁇ 0.033, one-way ANOVA with multiple comparisons.
  • C Heatmap of sorted basal, luminal progenitor and mature luminal cells based on a previously published dataset (Pal et al,
  • MDOs express markers of all main breast lineages and can be amplified using a 2D culture step.
  • A Representative bright-field images of cultures derived from previously 2D amplified cells and non-amplified cultures. Scale bar 200 pm.
  • B Fold increase in organoid formation per million cells seeded of amplified cultures compared to non- amplified MDOs. Bars represent mean + SD. * p ⁇ 0.033, two-tailed paired T test.
  • C Heatmap of RNA sequencing results from non-amplified and amplified MDOs for the expression of key mammary subpopulation markers.
  • D Directional plots comparing breast- specific marker expression between non-amplified and amplified cultures.
  • SC stem cell
  • PC progenitor cell. * p ⁇ 0.033, ** p ⁇ 0.002, *** p ⁇ 0.001, two-tailed paired T test.
  • FIG. 13 MDOs are amenable to genetic engineering and can serve as healthy control tissue in breast cancer drug evaluation.
  • A Representative ml_SR-3D image of MDOs lentivirally transduced with nuclear histone 2B-mNeonGreen and membranal CAAX motive- mScarletl. Scale bar 10 pm.
  • B Representative bright-field images of control and TP53/PTEN genetically edited MDOs cultured in the absence or presence of Nutlin 3a. Scale bars 500 pm.
  • C Viability of MDOs and PDOs exposed to chemotherapy paclitaxel (left) and the PARP-inhibitor niraparib (right). Significant differences between the conditions depicted in the tables below. Results represented as mean ⁇ SEM. NS p > 0.12, * p ⁇ 0.033, ** p ⁇ 0.002, *** p ⁇ 0.001, two-way ANOVA with multiple comparisons (Dunnett’s correction).
  • FIG. 14 MDOs serve as a healthy control tissue for studying off-target killing of cellular immunotherapies.
  • C Quantification of PRAME-T cell-mediated killing as percentage cytotoxicity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • breast organoids of the present invention contain basal/stem cells, luminal progenitors (LPs) and mature luminal (ML) cells.
  • the methods and organoids of the invention may be used to study the development and function of the human mammary gland.
  • 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/BRCA2 mutations, can be used.
  • 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.
  • 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.
  • 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.
  • myoepithelial cells may be interchangeably used with the term basal cells.
  • 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.
  • the organoids formed by the methods of the invention may have an RNA expression profile that is more similar to wild type or naturally occurring breast tissue in comparison to a tissue derived breast organoid formed using the same or similar conditions.
  • the organoids formed by methods of the invention may have an expression level that is more similar to an expression level in wild type or naturally occurring breast tissue in comparison to an expression level in a tissue derived breast organoid formed using the same or similar conditions of any one or more of: KRT5; KRT14; ACTA2; MYLK; SNAI2; MME; ITGA6; OXTR; KIT; SOX10; ELF5; MKI67; EPCAM; GATA3; PRLR; CDH1; MUC1; KRT8; KRT18; FOXA1 ; RPGR; and/or ESR1.
  • the organoids formed by methods of the invention may comprise basal cells (myoepithelial cells) that have a level of expression more similar to an expression level in wild type or naturally occurring breast tissue in comparison to an expression level in a tissue derived breast organoid formed using the same or similar conditions of any one or more of: KRT5; KRT14; ACTA2; MYLK; SNAI2; MME; ITGA6; and/or OXTR.
  • basal cells myoepithelial cells
  • the organoids formed by methods of the invention may comprise luminal progenitor cells that have a level of expression more similar to an expression level in wild type or naturally occurring breast tissue in comparison to an expression level in a tissue derived breast organoid formed using the same or similar conditions of any one or more of: KIT; SOX10; ELF5; MKI67; and/or EPCAM.
  • the organoids formed by methods of the invention may comprise mature luminal cells that have a level of expression more similar to an expression level in wild type or naturally occurring breast tissue in comparison to an expression level in a tissue derived breast organoid formed using the same or similar conditions of any one or more of: GATA3; PRLR; CDH1; MUC1; KRT8; KRT18; FOXA1 ; RPGR; and/or ESR1.
  • the organoids formed by methods of the invention may comprise a lower ratio of basal cells to the other epithelial cell types in comparison to a tissue derived organoid formed using the same or similar conditions.
  • organoids formed by methods of the invention may comprise a ratio of basal cells to luminal progenitor cells and mature luminal cells of around at most 1 :3.
  • organoids formed by methods of the invention may comprise a ratio of basal cells to luminal progenitor cells and mature luminal cells of around at most 1 :4.
  • organoids formed by methods of the invention may comprise a ratio of basal cells to luminal progenitor cells and mature luminal cells from about 1:4 to about 1:8.
  • the organoids formed by methods of the invention may comprise a higher ratio of luminal progenitor cells to the other epithelial cell types in comparison to a tissue derived organoid formed using the same or similar conditions.
  • organoids formed by methods of the invention may comprise a ratio of luminal progenitor cells to basal cells and mature luminal cells of around at least 1 :1.8.
  • organoids formed by methods of the invention may comprise a ratio of luminal progenitor cells to basal cells and mature luminal cells from about 1 : 1.7 to about 1:1.
  • the methods and organoids of the present invention may be used for a variety of therapeutic and medical methods.
  • 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.
  • organoids of the present invention may be used for recombinant production of breast milk.
  • organoids may be stimulated with prolactin in order to induce production of breast milk.
  • 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.
  • 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.
  • 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.
  • 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.
  • breast organoids of the invention may be for use as a medicament.
  • breast organoids of the invention may be for use in methods of treating diseases such as cancer.
  • 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.
  • the methods and culture medium disclosed herein may be used for the production of breast organoids from breast cells derived from breast tissue.
  • 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.
  • 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.
  • Tissue used to isolate cells may be healthy tissue or tumour tissue.
  • Tuour tissue includes tissue of a solid tumour, a semi-solid tumour, a primary tumour, a metastatic tumour, and the like.
  • tumor 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.
  • TAMs tumour associated macrophages
  • Cancer cell may be used interchangeably herein with “tumour cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of tumour tissue.
  • “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.
  • a subject may refer to any animal that has breast cells, such as any mammal. In certain embodiments, the subject is a human.
  • 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.
  • 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.
  • Breast milk may be obtained from any subject that is capable of producing breast milk, for example any mammal.
  • the breast milk is obtained from a human.
  • 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.
  • breast milk typically refers to human whole milk.
  • Whole milk means breast milk that has not had fat removed therefrom.
  • 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.
  • 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.
  • the epithelial cells may be basal epithelial cells.
  • 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 (CK5), CD34, CD44, CD90, CD271 , CD146, SSEA4, TRA-1-60, TRA-1-81, OCT4, Sox2 TRA-60-1, Nanog, and/or KLF4.
  • markers For example one or more of, Alpha36 Integrin (CD49f), p63, Cytokeratin 5 (CK5), CD34, CD44, CD90, CD271 , CD146, SSEA4, TRA-1-60, TRA-1-81, OCT4, Sox2 TRA-60-1, Nanog, and/or KLF4.
  • 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.
  • breast epithelial stem cells may be derived from basal epithelial cells.
  • Isolated breast cells, derived from tissue samples or breast milk may be cultured under conditions in order to form an organoid.
  • the breast cells may be suspended in on 3-dimensional support such as a matrix.
  • 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.
  • EHS Engelbreth-Holm-Swarm
  • the extract may provide a natural extracellular matrix hydrogel that polymerizes at 37°C to form a reconstituted basement membrane.
  • 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.
  • MatrigelTM can be used as alternative for BME.
  • the cells and supported may be deposited onto a surface.
  • 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.
  • 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.
  • 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.
  • the culture medium may be refreshed.
  • the culture medium may be refreshed at least twice a week.
  • the culture medium may be refreshed every 2 to 3 days or more.
  • 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.
  • the cells or organoids formed therefrom may be passaged at a ratio of 1 :2 to 1 :10.
  • the cells or organoids formed therefrom may be passaged at a ratio of 1 :2 to 1 :6.
  • the cells or organoids formed therefrom may be passaged at a ratio of 1 :2.
  • 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.
  • cells or organoids formed therefrom any known method may be used.
  • cells or organoids may be detached from their support using any suitable method.
  • cells or organoids may be detached using shake-off, scarping and/or enzymatic dissociation.
  • enzymatic dissociation may involve the use of trypsin, collagenase, dispase and/or other enzymes or enzyme mixtures which are commercially available, for example GibcoTM TrypLETM and TrypLE ExpressTM (Thermo Fisher).
  • culture medium 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.
  • 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.
  • 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
  • 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.
  • Wnt agonists may include surrogate wnt, chir and/or wnt3a.
  • 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.
  • the wnt agonist may be provided by a recombinant wnt agonist.
  • 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.
  • 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.
  • Lgr5 agonist such as R-spondin1
  • 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
  • ROCK inhibitors refer to Rho kinase inhibitors such as compounds that target rho kinase.
  • the Rock inhibitor may be included in the culture medium at a concentration of 5mM.
  • the ROCK inhibitor may be removed from the culture medium 2 to 3 days after organoids have been formed.
  • the ROCK inhibitor may be removed from the culture medium after passaging.
  • the ROCK inhibitor may be removed from the culture medium after thawing.
  • the ROCK inhibitor may be provided by a recombinant ROCK inhibitor.
  • 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.
  • 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.
  • the ROCK inhibitor may be included in the culture medium at a concentration of 5nM.
  • FGFR2b ligands refers to fibroblast growth factor receptor 2.
  • 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.
  • culture mediums of the invention do not include FGF- 7.
  • FGF-10 may be included at a concentration of 20ng/ml.
  • 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.
  • TGF-beta Transforming growth factor beta
  • the culture mediums of the invention TGF-beta inhibitors may be included at a concentration of 0.5mM.
  • 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.
  • stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock
  • the culture mediums of the invention p38 inhibitors may be included at a concentration of 1mM.
  • the culture mediums of the invention do not include p38 inhibitors.
  • 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.
  • FGFR fibroblast growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • DDR discoidin domain receptor
  • 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.
  • EGF may be included at a concentration of 5ng/ml.
  • culture mediums of the invention include noggin.
  • Noggin also known as NOG
  • 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.
  • BMP4 bone morphogenic protein
  • 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).
  • TGF-beta transforming growth factor-beta
  • BMP4 bone morphogenetic protein-4
  • 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.
  • 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.
  • culture mediums of the invention include R-spondin1.
  • R- Spondin-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 ⁇ -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.
  • 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.
  • the R-spondin1 may be provided by a recombinant Rspo protein such as recombinant R-spondin1 , R-spondin2, and/or R- spondin3.
  • culture mediums of the invention comprise at least 2%v/v R- spondinl 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.
  • 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.
  • culture mediums of the invention include hydrocortisone.
  • Hydrocortisone, or cortisol is a glucocorticoid secreted by the adrenal cortex.
  • 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.
  • 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.
  • 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.
  • the culture media comprises 1x concentrated B27 supplement plus vitamin A.
  • B27 supplement plus Vitamin A include, B-27TM Plus Supplement (50X) (available from ThermoFisher under catalogue number A3582801).
  • 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.
  • the culture medium includes /V-acetylcysteine.
  • the culture medium includes /V-acetyl-L-cysteine.
  • /V-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.
  • 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.
  • the culture medium includes at least one antimicrobial agent.
  • 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.
  • antimicrobial antibodies e.g., antibodies that bind to and directly kill organisms or enhance their clearance during infection
  • antimicrobial peptides e.g., antibodies that bind to and directly kill organisms or enhance their clearance during infection
  • phages e.g., phage lysins
  • 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
  • other alternative class or nonstandard agents developed as therapeutic agents for treating infections caused by one or more microbial organisms e.g., antibodies that bind to and directly kill organisms or enhance their clearance during infection
  • antimicrobial peptides e.g., antibodies that bind to and directly kill organisms or enhance their clearance during infection
  • phages e.g., phage lysins (e.g., bacteriophage endolysins, which are phage- encoded peptidogly
  • the culture medium includes 20 pg/ml of an antimicrobial agent.
  • the antimicrobial agent is primocin.
  • Type 2 medium (as defined in Table 2) is used to refer to a certain embodiment of culture mediums of the invention.
  • 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.
  • culture mediums of the invention consist of Type 2 medium (as defined in Table 2).
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell types or lines or the two organoids formed therefrom are isolated or formed from two different tissue types.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • organoids derived from tissue For example, organoids formed from cells derived from a breast tissue sample and not isolated from breast milk.
  • 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.
  • Fig 1 details step-by-step protocol outlines the derivation, manipulation, and xenotransplantation of human breast organoids.
  • 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 (2016).
  • the inventors used home-made R-spondin-1, Noggin and Wnt3a conditioned media, for which detailed production protocols are available, for example, Cattaneo, C. M. etal. Tumour organoid-T-cell coculture systems. Nat. Protoc. 15, 15-39 (2020), Drost, J. etal. 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. etal. 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).
  • Fig. 2g-l 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.
  • FIG. 3a-c 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).
  • 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. etal. 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.
  • a large quantity of organoids is necessary for engraftment (steps 102-113), usually varying between 0.25 c 10 6 -1 c 10 6 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.
  • Organoid injection was carried out by injecting 30 pi transplantation medium with 5%Trypan Blue, followed by dissection to confirm localization in the mammary fad pad.
  • Bovine serum albumin (BSA), modified fraction V (Sigma-Aldrich, cat. no. A9418)
  • Collagenase Type II (Thermo Fisher Scientific, cat. no. 17101-015)
  • 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.
  • DPBS Dulbecco’s phosphate-buffered saline, no calcium, no magnesium, 1 c ; Thermo Fisher Scientific, cat. no. 14190-144)
  • Nicotinamide (Sigma-Aldrich, cat. no. N0636)
  • 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 ofCattaneo, C. M. et ai. Tumour organoid-T- cell coculture systems. Nat. Protoc. 15, 15-39 (2020).
  • Noggin e.g. U- Protein Express BV, cat. no. N002, at a final concentration of 1-2% (vol/vol)
  • Noggin e.g. U- Protein Express BV, cat. no. N002, at a final concentration of 1-2% (vol/vol)
  • recombinant Noggin Peprotech, cat. no. 120-10C, at a final concentration of 100 ng/ml
  • Penicillin-Streptomycin (Thermo Fisher Scientific, cat. no. 15140-122)
  • FGF-7 Recombinant human fibroblast growth factor (FGF)-7 (Peprotech, cat. no. AF-100- 19)
  • Red blood cell lysis buffer (Sigma Aldrich, cat. no. 11814389001)
  • 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 ai. 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.
  • R-spondin-3 e.g. R&D Systems, cat. no. 3500-RS/CF, at a final concentration of 250 ng/ml
  • R&D Systems cat. no. 3500-RS/CF, at a final concentration of 250 ng/ml
  • SB 202190 Sigma-Aldrich, cat. no. S7067
  • 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).
  • Wnt surrogate U-Protein Express BV, cat. no. N001 , at a final concentration of 0.2 nM
  • Isoflurane (100% (wt/wt); inhalation anesthetic (e.g. Isoflutek; Laboratories Karizoo) Reagent Setup Collagenase Type II
  • DMEM with 0.1% (wt/vol) BSA D-BSA
  • Supplement 500 ml of DMEM GlutaMAXwith 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.
  • the medium components with their end concentrations are listed in Table 2.
  • 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 50 c B27 supplement, 2 ml of nicotinamide (1 M in DPBS), 500 pi N-acetyl-L- cysteine (500 mM in H20), 400 mI Hydrocortisone (250 mg/ml), 200 mI b-estradiol (100 mM), 200 mI Forskolin (10 mM in DMSO), 400 mI Primocin (1 mg/ml), 10 mI Y-27632 (100 mM in DMSO), 100 mI Heregulin b1 (10 mM in DPBS-B), 100 mI human FGF
  • Wnt3a-conditioned medium production has been described in box 1 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. etal. 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.
  • Growth medium DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin and 300pg/ml Zeocin. Store at 4 °C for up to one week.
  • 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 1.5x106 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, passage cells as in step 2 but reseed 500cm plates with 4.5x106 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 production has been described in box 1 of Drost, J. et at. 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.
  • Growth medium DMEM supplemented with 10% FBS and 150pg/ml Zeocin. Store at 4 °C for up to one week.
  • 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 production has been described in box 2 of Cattaneo, C. M. et at. Tumour organoid-T-cell coculture systems. Nat. Protoc. 15, 15-39 (2020).
  • Noggin-conditioned medium can be stored at 4 °C for 6 months.
  • 4 °C-stored expansion medium can be used at any moment for organoid propagation with good performance within a week from preparation.
  • expansion medium 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.
  • CoolCellTM LX Freezing Container (Corning®, cat. no. CLS432001)
  • Counting chamber e.g. Burker-Turk, Merck, cat. no. BR719520
  • Electroporation Chambers (2mm), Cuvettes Plus (Fisher Scientific, cat. no. BTX620) [00328] Falcon tubes, 15 ml, conical bottom (Greiner Bio-One, cat. no. 188271)
  • Microcentrifuge e.g. Eppendorf, cat. no. 5424R
  • Multi-well suspension plates 12, 24, 48-well (Greiner Bio-One, cat. nos. 665102, 662102, 677102)
  • Orbital shaker e.g. Panasonic, model no. MIR-S100-PE
  • Pipetman L, P20L, P200L, P1000L (Gilson, cat. nos. FA10003M, FA10005M, FA10006M)
  • Pipette tips with filter e.g. Gilson Pipetman, Diamond tips
  • NEPA21 electroporator was set to the following settings:
  • Pulse length 5 ms 50 ms
  • mice Female NSG ( N O D . C g - Prkdc scid H2rg tm1WJI / SzJ) mice, between 6-8 weeks of age.
  • This section describes the isolation of epithelial cells from breast tissue resections for the establishment of organoid cultures (Fig. 2a).
  • Fig. 2a When pipetting disrupted tissue, always prewet 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).
  • 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.
  • This tissue sample can be used as the reference DNA sample for SNP analysis (Human Material section).
  • 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.
  • 1 ml of red blood cell lysis buffer is sufficient.
  • 2 ml of red blood cell lysis buffer is added 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. 3. Centrifuge the cells and aspirate the medium as described in step 30.
  • 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). 4. Resuspend the pellet in an appropriate amount of BME. As a guideline, 200 pi is sufficient for a pellet of approximately 50 mI. 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 mI 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. 5. Plate 100 mI BME containing cells in multiple small drops ( ⁇ 20 mI 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). 6. Turn the plate upside down and leave in the biosafety cabinet for 5 min. 7. 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. 8.
  • Type 1 or Type 2 medium Add 750 mI 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% CO2.
  • Table 3 refers to appropriate media volumes per type of culture plate.
  • Test both Type 1 and Type 2 expansion medium (Fig. 2e; Table 2).
  • Fig. 2e 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).
  • 9. (Optional) Take microscopic pictures of representative drops for documentation of organoid density and morphology (2.5x and 10x magnifications). 0.
  • 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.
  • 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 pm (further referred to as a ‘confluent well’). For organoid passaging via single cells follow option A, and via fragments follow option B.
  • step (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.
  • step (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.
  • step (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).
  • step (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.
  • step (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.
  • step (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 pm in diameter). 4. Transfer single cells or organoid fragments into a 15 ml Falcon tube with 10 ml ice- cold adDMEM/F12+++, using a p1000 pipette.
  • the cells can be counted by resuspending the cell pellet in 250- 500 pi expansion medium per harvested well, taking 10 pi of cells and mixing it with 10 pi 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.
  • spin down the non- counted cells at 300g for 5 min and remove the supernatant.
  • step 48 To directly plate organoids for passaging, continue to step 49.
  • 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.
  • Table 3 provides appropriate number of BME drops and total BME volume for different culture plates.
  • organoid density is a critical factor for a well-growing, viable culture (Fig. 2g-i).
  • ROCK inhibitor is added to enhance recovery from freeze-thawing.
  • Fig. 3a 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).
  • step 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.
  • transfected DNA contains a gene encoding a fluorescent protein (Fig. 3d,e)
  • expression can be detected within 1 day after transfection.
  • a selection agent follow steps 90-92.
  • the transfection efficiency is usually between 5 and 50%.
  • step 77 Dissociate and pellet the organoids as described in steps 43[00353]A/43B-46. After aspirating the supernatant from the organoid pellet as described in step 46, continue with step 78.
  • 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.
  • transfected DNA contains a gene encoding a fluorescent protein (Fig. 3d,e)
  • expression can be detected within 1 day after transfection.
  • a selection agent follow steps 90-92.
  • the transfection efficiency is usually between 20 and 80%.
  • step 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.
  • lentivirus concentrated to a titer of 50 x 10 L 6 infectious viral particles per ml (pfu/ml).
  • transduced DNA contains a gene encoding a fluorescent protein (Fig. 3d, e)
  • expression can be detected within 2-3 days after transduction.
  • selection agent follow steps 90-92.
  • the transduction efficiency is usually between 20 and 90%.
  • 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 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.
  • step 66-76 or 77-84 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.
  • step 91 Gently add 750 mI expansion medium with the appropriate concentration of selection agent, in a drop-wise manner to each well.
  • step 96 Use an inverted brightfield microscope (10x or20x objective) to check the dissociation.
  • 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
  • number of times to resuspend usually 5-30 times total
  • 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.
  • 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.
  • step 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.
  • step 102-108 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.
  • 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.
  • Tumouroids were seeded in basement membrane extract (BME; Cultrex) in uncoated 24-well plates (Thermo Fisher; 4 drops of 10 mI 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 (2016): 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).
  • tumouroids of a 5-12-day old culture 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 pm strainer (Greiner) to remove large tumouroids and pelleted before co-culturing.
  • 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).
  • 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.
  • 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).
  • 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 pi; 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.
  • BME basement membrane extract
  • TEGs which are peripheral blood ab T cells engineered to express a Vy9/V52 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).
  • E:T effector to tumour cell
  • CD4+ and CD8+ TEGs were mixed in a 1 :1 ratio just before plating.
  • TEGs Prior to co-culturing, TEGs were incubated with eBioscienceTM Cell Proliferation Dye eFIuorTM 450 (referred to as eFluor-450; 1 :4000; Thermo Fisher) to fluorescently label all TEGs.
  • eFluor-450 eBioscienceTM Cell Proliferation Dye eFIuorTM 450
  • 200 mI 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% C02) 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 pm 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).
  • 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.
  • TNBC triple negative breast cancer
  • Fig. 6a 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. 6c, d). CYTOF analysis confirmed the presence of basal, LP and ML cells in cultured normal organoids (Fig.
  • 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.
  • 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).
  • Fig. 7d 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).
  • Example 4 Human 3D breast tissue modelling using breast milk-derived organoids
  • MDOs Milk-derived organoids
  • MDOs could be used as healthy control tissue to screen for toxicity of molecular targeted breast cancer treatments and cellular immunotherapy.
  • human breast milk as an easily accessible and widely available cell source, a robust human 3D tissue model with population-wide biobanking potential is established, that can be applied for understanding mammary gland biology and pathology, as well as drug safety evaluation.
  • TDO_1 and TDO_2 refer to 16HD11 and 14MHD11, respectively(Dekkers et al, 2019b; Rosenbluth et al, 2020; Dekkers et al, 2021b).
  • milk-derived cells were resuspended in 750 mI_ organoid medium and seeded into tissue-culture treated 12-well plates (Corning) at a density of 5-20 x 10 6 cells per well. Once seeded cells reached confluency, cells were trypsinized and 1 x 10 5 cells were seeded in BME for organoid generation, as described above.
  • PDOs were cultured as described previously(Sachs et al, 2018; Dekkers et al, 2021b). Briefly, PDOs were resuspended in BME (Cultrex) and seeded in 12-well plates for suspension culture (Greiner Bio-One) and 750 mI_ of the following medium was added per well: adDMEM/F12+++ supplemented with 10% RCM, 10% NCM, 1x B27 + Vitamin A (Thermo Fisher), 10 mM Nicotinamide (Sigma), 1.25 mM N- acetylcysteine (Sigma), 100 pg/mL Primocin (Invivogen), 5 mM Y-27632 (Abmole), 5 nM Heregulin B1 (PeproTech), 5 ng/mL FGF-7 (PeproTech), 20 ng/ml FGF-10 (PeproTech), 0.5 mM A83
  • PDO_1, PDO_2, PDO_3, PDO_4 and PDO_5 refer to 10T, 27T, 13T, 62T and 169M, respectively, as published previously (Sachs et al, 2018).
  • Antibodies used to subset main cell populations were: CD31-BUV395 (1 :200; BD Biosciences, clone L133.1), CD49f-BV421 (1 :100; BioLegend, clone GoH3), CD45-V500 (1 :200; BD Biosciences, clone HI30), CD235a-BV750 (1 :100; BD Biosciences, clone GA-R2 (HIR2)) and EpCAM-PerCP-eFluor 710 (1 :50; Thermo Fisher, clone 1 B7). Data acquisition was performed on a CytoFlex LX system (Beckman Coulter). Data was analyzed using FlowJo version 10.8 (BD Life Sciences).
  • Organoids were fixed for 60 min at 4°C in 4% PFA, incubated in PBT for 10 min at 4°C and washed in organoid washing buffer (OWB; 0.1 % (v/v) Triton X-100, 0.2 % (v/v) of 10 % (w/v) SDS, 0.2 % (w/v) bovine serum albumin (BSA) in PBS) for at least 15 min at 4°C.
  • organoid washing buffer OLB
  • Triton X-100 0.1 % (v/v) Triton X-100
  • 0.2 % (v/v) of 10 % (w/v) SDS 0.2 % (w/v) bovine serum albumin (BSA) in PBS
  • BSA bovine serum albumin
  • Organoids were stained with primary antibodies overnight at 4 °C and washed in organoid washing buffer for 6h followed by overnight incubation with secondary antibodies, DAPI (1:1000; Thermo Fisher) and Phalloidin-Alexa Fluor 488 (1:200; Thermo Fisher) at 4°C in OWB. The following day, organoids were washed and cleared in FunGI(Rios et al, 2019) for 1 hour at 4°C before 3D imaging using an LSM880 confocal microscope (Zeiss).
  • Histon2B-NeonGreen/Scarletl-CAAX constructs were kindly provided by the Snipped lab, and inserted by In-Fusion HD Cloning Plus (Takarabio) into a lentiviral vector, including an EF1a promoter and IRES-puromycin-resistance cassette (pLV-EF1a-IRES- Puro(Hayer et al, 2016), a gift from Dr. Tobias Meyer).
  • the FUCas9Cherry plasmid used for expression of Cas9 and FgH1t-UCW plasmid used for the expression of sgRNAs targeting TP53 and PTEN were generated as described previously (Dekkers etal, 2019b).
  • HEK293T DSMZ ACC-635 cells were seeded 24 hours prior to transfection into 10 cm petri dishes at a density of 4 million cells per plate in HEK culture medium containing advanced DMEM/F12 supplemented with 100 U/mL P/S and 10 % FCS.
  • Lentiviral plasmids were transfected in HEK293T cells and medium was refreshed after 4 hours with HEK culture medium and again after 24 hours with harvest medium (adDMEM/F12+++).
  • Viral supernatant was collected 24 hours later and concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane tubes (Merck).
  • organoid transduction with pLV-EF1a-IRES-Puro-Histon2B-mNeonGreen-P2A-Scarletl- CAAX virus organoids from 7 day cultures were processed into single cells using TrypLE Express (Thermo Fisher Scientific). Cell pellets containing 0.1 x 10 6 - 0.3 x 10 6 cells were resuspended in concentrated virus diluted with Type 2 medium (total volume 100 pi; MOI of 5-10) and incubated for 30-45 minutes at 37°C.
  • CRISPR-cas9 gene editing Cell transductions and selection were performed as previously described (Dekkers et al, 2019b). Concentrated virus was produced as described above for lentiviral transductions with the additional step of coating the 10 cm petri dishes with Poly-L-Lysine (Sigma-Aldrich) prior to HEK293T seeding.
  • MDOs were processed into single cells using TrypLE Express (Thermo Fisher Scientific) and 0.2 x 10 6 cells were seeded in adherent 24-well cell culture plates (Greiner Bio-One).
  • Type 2 medium (total volume 100 pi; MOI of 5-10) was added on top of the cells and incubated for 3 hours at 37°C and 5% CO2. Cells were washed in adDMEM/F12+++ three times and Type 2 medium was added. After one week, cells were seeded in BME (Cultrex) in uncoated 24-well plates (Thermo Fisher) and cultured as described above. Three days after transduction, Nutlin-3a (5 mM; Cayman Chemicals) was added to the culture medium to select for cells with genetically modified TP53. Additionally, presence of FUCas9Cherry and FgH1t-UCWwas validated using fluorescence microscopy.
  • paclitaxel Sigma-Aldrich
  • niraparib Selleck Chemicals
  • concentration ranges of paclitaxel (Sigma-Aldrich) and niraparib (Selleck Chemicals) were prepared at double the final concentrations in Type 1 medium.
  • 0.1% (v/v) DMSO in Type 1 medium was included.
  • 100 mI of drug or control solution was added, and plates were incubated at 37 °C and 5% CO2 for 3 (paclitaxel) or 7 (niraparib) days.
  • cell viability was determined using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) as per the manufacturer’s instructions.
  • CD8 + T-cells were lentivirally transduced with a PRAMESLLQHLIGL/A2-specific T cell receptor (TCR)(Amir et al, 2011 ; Spel et al, 2015).
  • TCR PRAMESLLQHLIGL/A2-specific T cell receptor
  • PBMCs peripheral blood mononuclear cell
  • Daudi and LCL-TM cells CD19 + EBV transformed lymphoblastoid cell line
  • IL-2 50 U/ml
  • PHA-L 1 mV/hiI
  • T cell- organoid co-cultures were performed as described previously (Dekkers et al, 2021a). Briefly, MDOs with HLA-2A1 haplotype (MDO_1, MDO_6) and PDOs with either HLA-2A1 (PDO_4, PDO_5) or a different HLA haplotype (PDO_1) were recovered from BME by resuspension in TrypLE Express (Thermo Fisher) and collected in adDMEM/F12+++. Organoids were filtered through a 70pm strainer (Greiner) for removal of large organoid structures and pelleted before co-culture.
  • MDOs or PDOs were co-cultured with PRAME T-cells in an effector to target cell ratio of 2:3 in 96-well glass bottom SensoPlates (Greiner).
  • PRAME-T cells were fluorescently labelled with eBioscienceTM Cell Proliferation Dye eFIuorTM 450 (1 :3500 in PBS; Thermo Fisher) prior to co-culture.
  • Cells were imaged in 200 pl_ imaging medium per well, consisting of 50% Type 1 medium, 50% T cell medium (RPMI1640 supplemented with 2.5% human serum and 1% P/S), 2.5% BME (Cultrex), NucRedTM Dead 647 ReadyProbesTM (1 drop/mL; Thermo Fisher) and TO-PRO-3 (1 :3000; Thermo Fisher) for labelling of live and dead cells. Imaging was performed on a LSM880 (Zeiss) microscope equipped with an incubation chamber (37°C, 5% CO2) for up to 15 hours using a Plan-Apochromat 20x/0.8 NA dry objective.
  • LSM880 Zeiss microscope equipped with an incubation chamber (37°C, 5% CO2) for up to 15 hours using a Plan-Apochromat 20x/0.8 NA dry objective.
  • RNA-sequencing of milk and tissue-derived organoid lines For bulk RNA- seq analysis, MDO and TDO samples at passages below p10 were collected in duplicates at day 7 of culture, one well of a 6-well plate per duplicate. Total RNA was isolated from the collected organoids using QIAsymphony RNA Kit (QIAGEN) according to the manufacturer's protocol, including DNasel treatment. Quality and quantity of isolated RNA were checked and measured with Bioanalyzer2100 RNA Nano 6000 chips (Agilent). Library preparation was started with 100 ng of total RNA using the TruSeq Stranded mRNA kit (lllumina).
  • Human breast milk is a suitable cell source for organoid derivation.
  • breast milk was collected from healthy donors and separated cells by density centrifugation (Fig 10A).
  • the cellular composition of human breast milk was fist characterised to explore its potential as an epithelial cell source for organoids.
  • Good viability was obtained following our isolation procedure, with 78% of samples displaying a cell viability above 70% and live cell proportions for some donations close to 90% (Fig 9A).
  • the amount of cells retrieved varied between individual donors and different times of donation from the same donor (Fig 9B), yet was not related to the duration of lactation (Fig 10B).
  • the cell subset composition of these milk-derived cells was analyzed through flow cytometry (Fig 9C). It was found that human breast milk contained endothelial (CD31 + ; 1.9 ⁇ 5.0%), immune (CD45 + ; 1.4 ⁇ 1.1%), erythroid (CD235a + ; 7.6 ⁇ 7.1%), epithelial (lineage-negative EpCAIVT; 11.5 ⁇ 6.7%) and stromal cells (lineage-negative EpCAM CD49f ; 71.8 ⁇ 15.6%) (Fig 1C).
  • EpCAM and CD49f appear down-regulated in cells desquamated into milk (Fig 9C), compared to breast tissue-derived epithelial cells (Dekkers et al, 2019b), as has been described before (Twigger et al, 2020). Together, these data confirm the presence of epithelial cells in human breast milk, illustrating its potential as a cell source for organoid formation.
  • Organoids can be established with high efficiency from human breast milk- derived cells.
  • the organoids were cultured in a 3D matrix (Fig 10A), supplemented with medium optimized to propagate TDOs (Type 2 medium) (Dekkers et al, 2021b).
  • Outgrowth of MDOs was critically dependent on Wnt3a-conditioned medium (WCM), and minimal outgrowth was observed in ‘tumor optimized’ Type 1 medium, used previously to propagate TDOs (Sachs et al, 2018; Rosenbluth et al, 2020; Dekkers et al, 2021b, 2019b).
  • FIG 11 A Inter- and intra-donor dependent variation in organoid establishment efficiency was observed (Fig 11 A), which again did not correlate to weeks of lactation (Fig 10E). This indicates that breast milk can be used at any time for up to at least 16 months during lactation to obtain MDOs. Importantly, MDOs could be established with a 100% success rate and maintained in culture for up to 18 passages, showing robustness of the culture method. Growth speed declined at high passage number (> p10) (Fig 11 B), a well- known feature of healthy tissue organoids, including breast TDOs (Dekkers et al, 2021b). A biobank of 27 lines derived from 27 breast milk donations from 12 donors has been established.
  • MDOs recapitulate key cellular and morphological features of the mammary gland. MDO cultures displayed heterogenous architectures, predominantly manifesting with alveolar or round-shaped morphology and frequent lumen formation, as shown by representative bright-field images of two MDO lines (Fig 10F).
  • MDOs comprised both CK5 + basal and CK8/18 + luminal cells (Fig 10G), or appeared with a fully basal (Fig 10H, left panel) or luminal (Fig 10H, right panel) phenotype, similar to the phenotype of TDOs formerly described (Rosenbluth et al, 2020).
  • RNA-seq analysis was performed.
  • a gene signature for the three main breast lineages; basal, luminal progenitor and mature luminal cells was retrieved from a published human healthy breast dataset (Pal et al, 2021) (Fig 11C) and subsequently the relative expression of these signature genes in MDOs and TDOs was analyzed (Fig 8A).
  • the obtained gene expression values were consolidated into a relative identity contribution, showing that both MDOs and TDOs expressed markers characteristic for all three lineages, indicating that these cultures capture the key cell types present in the in vivo mammary gland (Fig 8B).
  • MDOs can be applied to asses tumorigenesis and breast cancer drug safety.
  • MDOs were transduced to express fluorescently labelled histone 2B and CAAX motive, which resulted in successful labelling of nuclei and cell membranes, respectively (Fig 13A).
  • CRISPR-Cas9 gene editing was implemented to knock-out tumour suppressor genes that have been used recently to transform TDOs to a malignant state (Dekkers et al, 2019b).
  • Successful knockout of TP53 through survival of transduced MDOs in cultures supplemented with Nutlin-3a is shown (Fig 13B, right panel).
  • MDOs can serve as a tool to study cancer initiation.
  • MDOs were exposed to clinically applied breast cancer therapies.
  • the effect of the chemotherapeutic paclitaxel was firstly evaluated and it was found that it reduced the viability of various breast cancer PDOs and, to a lesser extent, MDOs (Fig 13C, left panel). This result matches with a higher proliferation rate of PDOs (Dekkers et al, 2021b), making them more susceptible to chemotherapy.
  • treatment response to a targeted therapy was investigated; niraparib, a poly(ADP-ribose) polymerase (PARP) inhibitor used for the treatment of BRCA1/2 mutant breast cancer (Lee et al, 2014).
  • PARP poly(ADP-ribose) polymerase
  • MDO co-culture models can be used to screen for off-target activity of T cell therapy.
  • TCR antigen-specific T cell receptor
  • engineered T cells can offer a highly specific treatment option.
  • this requires the expression of tumor-restricted antigens that are not present on healthy tissue (Baruch et al, 2017; Restifo etal, 2012).
  • human healthy tissue models are highly valuable for confirming tumor-specific targeting of novel engineered T cell therapies.
  • mammoplasty samples generally represent healthy tissue, surgeries are performed infrequently (i.e. in 0.014% of the total female population in 2018) (Nations, 2019; ISAPS, 2019), making acquisition of these samples logistically complicated and time-consuming, also due to extensive ethical procedures.
  • human breast milk offers a widely available cell source, less subjected to complicated logistics and ethical approvals, that is exploited here to generate a human healthy breast organoid model.
  • An additional advantage of using human breast milk as starting material is the option to isolate immune cells from the same sample for more complex co-cultures to model immune interactions.
  • bioengineering approaches such as extracellular matrix engineering, microfluidics, and bioprinting could be implemented in a more controlled and directed manner to generate an even more intricate breast tissue model (Brassard & Lutolf, 2019). While MDOs spontaneously developed into lumenized organoids, using bioprinting MDO-derived cells could be further guided towards a physiologically more relevant spatial organization, including extensive architectures, such as epithelial ducts and terminal end buds, relevant for mammary gland development and function. Therefore, a synergy between the scalability of the organoid methods herein and upcoming developments in the field of bioengineering is anticipated.
  • T cell therapies Fuentes-Antras et al, 2020.
  • Such therapies have shown great potential for treating bloodborne malignancies (Baruch etal, 2017; Dudley et al, 2002; Dudley & Rosenberg, 2003), sparking efforts to translate this success to solid tumors (Paucek et al, 2019; Waldman et al, 2020; Yamamoto et al, 2019).
  • MDOs Due to their non-invasive and easily accessible cell source, MDOs furthermore have the important potential of offering a population-wide biobank of healthy tissue for these purposes. Within a reasonable (1-year) timeframe, we were able to generate 27 MDO lines from 12 donors. This potential to create large donor biobanks, puts MDOs at an advantage for drug screening purposes, as they might be able to identify rare toxic events that only occur in a small fraction of the population. In addition, such a populationwide biobank can be exploited to study the impact of genetic variation on tissue development and function, a key aim of ongoing human cell atlas activities (Bock et al, 2021). Thus MDOs can offer a preferred tool to study population heterogeneity.
  • MDOs advance human tissue modeling by providing an easily accessible 3D breast tissue model that recapitulates the human mammary gland and offers high-throughput and population-wide biobanking potential.
  • utility can be further expanded through combination with contemporary advances in concomitant state-of-the-art technologies, such as genetic editing and bioengineering.
  • Table 1 Detailed information of 45 biobanked breast organoid cultures.
  • a Expression status of the indicated receptors for tissue and organoid indicated as percentage of positive cells (estrogen receptor, ER/ progesterone receptor, PR) and IHC score (0 - 3; human epidermal growth factor receptor 2, HER2).
  • N/A indicates that the sample has not been tested or data are not available.
  • Ref. 7 refers to in Sachs, Norman, et al. "A living biobank of breast cancer organoids captures disease heterogeneity.” Cell 172.1-2 (2018): 373-386 and Ref. 8 refers to Rosenbluth, J. M. et al. Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages. Nat. Commun. 11, (2020).
  • 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.
  • 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.
  • a method for producing a breast organoid comprising:
  • a method of producing a breast organoid comprising:
  • 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.
  • 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.
  • 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.
  • 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.
  • the at least one BMP inhibitor comprises Noggin
  • the at least one ROCK inhibitor comprises Y-27632
  • the at least one FGFR2b ligand comprises FGF-10;
  • the at least one TGF-beta inhibitor comprises A83-01 ;
  • the at least one receptor tyrosine kinase ligand comprises EGF.
  • 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.
  • the at least one Lgr5 agonist is provided by R-spondin1 at a concentration of 50 to 1000 ng/ml;
  • the at least one BMP inhibitor is provided by Noggin at a concentration of 10 to 500 ng/ml;
  • the at least one ROCK inhibitor is provided at a concentration of 1 to 50mM
  • the at least one FGFR2b ligand is provided at a concentration of 5 to 100 ng/mL;
  • the at least one p38 inhibitor is provided at a concentration of 0.1 to 10 mM;
  • the at least one receptor tyrosine kinase ligand is provided at concentration of
  • the Wnt agonist is provided by Wnt3A at a concentration of 0.01 to 2 nM;
  • the nicotinamide is provided at a concentration of 1 to 50 mM
  • N-Acetylcysteine is provided at a concentration of 0.1 to 15 mM
  • the hydrocortisone is provided at a concentration of 0.1 to 5 pg/ml;
  • the at least one antimicrobial agent is provided at a concentration of 1 to 100 pg/ml.

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EP22705172.9A 2021-02-11 2022-02-11 Method of producing an organoid Pending EP4291636A1 (en)

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NL2027546A NL2027546B1 (en) 2021-02-11 2021-02-11 Method of producing an organoid
PCT/NL2022/050070 WO2022173300A1 (en) 2021-02-11 2022-02-11 Method of producing an organoid

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