WO2025262230A1 - In vitro method of producing milk - Google Patents

Abstract

The invention relates to a lactiferous unit adapted to be cultured in suspension in a bioreactor, to a method for producing said lactiferous unit and also to a method for producing a milk-like product therefrom.

Description

IN VITRO METHOD OF PRODUCING MILK

FIELD OF THE INVENTION

[0001] The invention relates to an in vitro method of producing a milk-like product. More particularly the invention makes use of mammary epithelial cells which are adapted to be cultured in suspension and therefore producing milk-like product at a cost effective and industrial scale. The invention relates also to a lactiferous unit, a cell aggregate made of those mammary epithelial cells able to produce said milk-like product while being cultured in suspension. The present invention also relates to the method of producing said lactiferous unit.

BACKGROUND OF THE INVENTION

[0002] Dairy farming and global dairy industry have a significant environmental impact.

[0003] In 2007, the dairy sector emitted 1 969 million tonnes CO2 equivalent (emissions of carbon dioxide, methane and nitrous oxide) and of which 1 328 million tonnes are attributed to milk, 151 million tonnes to meat from culled animals, and 490 million tonnes to meat from fattened calves. It represents 4.0 percent to the total global anthropogenic greenhouse gas emissions. Restricted to milk production, processing and transportation it represents 2.7 percent of total anthropogenic emissions (FAO, in Greenhouse Gas Emissions from the Dairy Sector-A Life Cycle Assessment, 2010).

[0004] The dairy industry is also the cause of air and water pollution, deforestation, and question animal health and welfare.

[0005] Besides consumers goods (milks, cheeses, creams, yogurt...) dairy industry produces industrial inputs and additives derived from milk like caseins, whey, lactose etc.. There is a constant increase in need of dairy products (milk production increase by 30 percent between 2005 and 2015, FAO 2019) while there is a need to provide a mix of solutions for a more sustainable dairy production.

[0006] Also, a particular and specific product milk product is infant milk, an ultra- processed food designed and marketed for feeding to babies and infants under 12 months of age.

[0007] The most commonly used infant formulas contain purified cow's milk whey and casein as a protein source, a blend of vegetable oils as a fat source, lactose as a carbohydrate source, a vitamin-mineral mix, and other ingredients depending on the manufacturer (Institute of Medicine (US) Committee on the Evaluation of the Addition of Ingredients New to Infant Formula. Infant Formula: Evaluating the Safety of New Ingredients. Washington (DC): National Academies Press (US); 2004. PM ID: 25009867). Other infant formulas using soybean, rice or goat milk as an alternative for cow's milk or protein hydrolysates for allergic infants exist.

[0008] Obviously, such formulations only partially reflect human milk composition and lack key compounds specific to human milk (e.g. human milk oligosaccharides, lactoferrin, fatty acids, specific casein or other proteins) beneficial to the health of the infant. Furthermore, some constituents, such as cow proteins, are structurally different which make the formulation allergenic or hard to digest for infants.

[0009] Plant based milks exist. They are produced by emulsifying a vegetable flour suspended in water, they are similar in appearance to animal milks, but their composition is totally different from that of any animal milk.

[0010] Recent years have seen the advent of cell culture to manufacture therapeutical or nutraceutical components such as the production of monoclonal antibodies by CHO cells. These processes rely on cellular biology and biochemistry technologies: cells isolated from animals or plants, including cells from meat, seafood, fat and offal, or eggs, are grown in a controlled environment, and stimulated to produce key bioactive components.

[0011] Some cell therapy techniques are made from stem cells that can be induced to differentiate into cells of a particular lineage or type, in order to produce cell types of interest for particular diseases.

[0012] Producing a milk-like product containing bioactive components by cell culture, implies an enhanced degree of complexity as animal milk is the product of the mammary gland, an exocrine gland in humans and other mammals. Hence, producing an in vitro milk implies the recreation of an in vitro cellular functional unit mimicking the function a mammary acinus able to secrete milk.

[0013] A human mammary like organoid derived from human iPSC (hiPSC) has been developed by Qu et al (2017), which use 3D floating mixed gel culture which expresses common breast tissue, luminal, and basal markers. Also, Rauner et al. (2023) developed an organotypic 3D culture method, based on a biomimetic ECM scaffold made of a mix of fibronectin, laminin, and hyaluronic acid.

[0014] WO2021141762 discloses a three-dimensional scaffold onto which mammary cells grow, the three-dimensional scaffold defining two separated compartments one containing the culture medium and the other, which would correspond to the luminal part of this reconstituted acini, where the milk is to be secreted.

[0015] WQ2022054053 discloses also a system for in vitro production of milk ; it uses an array of mammary organoids seeded on tertiary-branched duct scaffolding, the system delineating two compartments, such that the milk collected does not comprise nutrients supplied. Despite of their interest as scientific tools for studying mammary secretory gland, these methods which are de facto limited by the cellular composition and structure of the system, and therefore suffer from being difficult to implement at an industrial level for in vitro producing milk or components thereof at a satisfactory level.

[0016] There is thus a need for a method for in vitro producing milk, a component thereof or a mix of components thereof at a satisfactory level and yields.

SUMMARY

[0017] The method developed by the inventor actually solves this problem. Inventors have been able to develop a method allowing the production of lactiferous units able to self-form and to secrete a milk product while being grown and maintained in suspension, such as in a suspension bioreactor. The invention therefore provides a lactiferous unit, a method for producing said lactiferous unit and a method for producing a milk-like product from said lactiferous unit.

[0018] Up to now the only systems that have been developed for in vitro producing milk make use of scaffolds and/or molecular matrices in order to obtain cellular structures mimicking the organization of acini found in mammary glands.

[0019] Inventors have been able to develop lactiferous units able to be formed in suspension without the help of any scaffold or support, and which can produce milk components directly in the culture medium when cultured in suspension and therefore provide an effective tool in terms of cost and yields.

[0020] Accordingly, one object of the invention is a lactiferous unit characterized in that it is adapted to be cultured in suspension, such as in a suspension bioreactor, and in that it comprises epithelial mammary cells. Said epithelial mammary cells preferably comprise myoepithelial cells and secretory cells.

[0021] The lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, may comprise the following optional features, alone or combined :

• It is individually encapsulated within a hydrogel capsule,

• when present, the hydrogel is preferably selected from alginate, chitosan, silk, poly(vinyl alcohol), dextran, polyacrylamide, polyethylene glycol, pectin, guar gum, gelatin, zein, agar-methylcellulose, a cellulose derivative hydrogel, casein, or a mix thereof, preferably alginate, therefore protecting said lactiferous unit from shear stress,

• In an alternative embodiment, the lactiferous unit is not encapsulated within a hydrogel capsule, • It comprises a mix of myoepithelial mammary cells and of secretory mammary cells. Preferably, myoepithelial mammary cells are mainly localized inside of the lactiferous unit and secretory mammary cells are mainly localized at the outside of said lactiferous unit, said secretory mammary cells being oriented such that their apical face is on the outside of said lactiferous unit, and/ or

• the epithelial mammary cells are selected from epithelial mammary cells of human origin, bovine origin, canine origin, feline origin, ovine origin, caprine origin, cetacean origin, seal origin, elephant origin, equidae origin, or a mix thereof, preferably selected from epithelial mammary cells of human origin.

[0022] It will be understood that the lactiferous unit of the present invention is obtained or obtainable according to the method described below.

[0023] A further object of the invention is a method for producing at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, as described above, comprising, or consisting essentially of, the steps of: i. Seeding in suspension at least one mammary cell selected from a progenitor of mammary cells, a mammary luminal epithelial cell, or a mix of said cells, preferably in a suspension bioreactor, ii. Amplifying said at least one mammary cell in suspension, iii. Optionally promoting cell aggregation, and iv. Inducing differentiation of the cells into epithelial mammary cells, if the mammary cell seeded at step i is the progenitor of mammary cells or the mix of said cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[0024] In an embodiment, the method for producing at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, as described above, comprises, or consists essentially of, the steps of: i. Seeding in suspension at least one progenitor of mammary cells, such as in a suspension bioreactor, ii. Amplifying said progenitor cells in suspension, iii. Optionally promoting cell aggregation, iv. Inducing differentiation of the progenitor cells into epithelial mammary cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor. [0025] In another embodiment, the method for producing at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, as described above, comprises, or consists essentially of, the steps of: i. Seeding in suspension at least one mammary luminal epithelial cell, such as in a suspension bioreactor ii. Amplifying said cell in suspension, iii. Optionally promoting cell aggregation, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[0026] Yet, in another embodiment, the method for producing at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, as described above, comprises, or consists essentially of, the steps of: i. Seeding in suspension at least one progenitor of mammary cells and at least one mammary luminal epithelial cell, ii. Amplifying said cells in suspension, iii. Optionally promoting cell aggregation, iv. Inducing differentiation of the progenitor cells into epithelial mammary cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[0027] This method of producing at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor, may comprise the following optional features alone or combined:

• it is a method free of exogenously added component(s) of the extracellular matrix (ECM),

• the at least one mammary cell seeded at step i is a progenitor of mammary cells,

• the at least one progenitor of mammary cells is a bipotent or unipotent mammary progenitor cell, or a mix of said cells,

• the at least one progenitor of mammary cells is a luminal progenitor cell, a basal progenitor cell, or a mix of said cells, preferably is a luminal progenitor cell,

• if the progenitor of mammary cells is a basal progenitor cell, it is a bipotent progenitor cell,

• the at least one mammary cell of step i is isolated from a mammary tissue sample or a milk sample. The mammary tissue sample is preferably a mammary resection, • if at least one progenitor of mammary cells is seeded in step i, said progenitor is preferentially differentiated into mammary luminal epithelial cells, more preferably into mammary secretory cells,

• it preferentially comprises step iii of promoting cell aggregation, especially in a stirred-tank or orbital shaking or wave bioreactor,

• it is implemented in a culture medium comprising a basal medium, a proliferation supplement and preferably a differentiation-promoting agent,

• the differentiation-promoting agent comprises an agent promoting differentiation into mammary luminal epithelial cells, more preferably into mammary secretory cells. Said agent is preferably a TGF-beta inhibitor,

• it may comprise a step of encapsulating the cells in a hydrogel capsule,

• it may comprise a preliminary step of adapting progenitor of mammary cells to culture in suspension, and/or

• the culture medium is constituted of human-food grade components.

[0028] Another object of the invention is a method of in vitro producing a milk-like product, comprising, or consisting essentially of: a) Providing at least one lactiferous unit according to the invention in suspension in a culture medium, preferably in a suspension bioreactor unit, such as by implementing a method of the invention for producing at least one lactiferous unit adapted to be cultured in suspension as described above, b) Inducing lactation by preferably adding to said medium prolactin at a preferred final concentration from 0.1 to 10 mg/L, c) Maintaining lactation induction till at least one milk component is produced in the culture medium, said component being preferably selected from alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, a milk oligosaccharide, or any combination thereof, more preferably till the component reaches a target concentration in said culture medium, d) Optionally, collecting the culture medium which itself constitutes the milk-like product.

[0029] This method of in vitro producing a milk-like product may comprise the following optional features alone or combined :

• the epithelial mammary cells of said at least one lactiferous unit comprise epithelial mammary cells of human origin and the at least one milk component of step (c) is selected from alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, at least one Human Milk Oligosaccharide (HMO), or any combination thereof,

• it further comprises supplementing the culture medium or the milk-like product with Vitamin D, Iron, selenium, at least one antibody, at least one HMO, at least one long chain polyunsaturated fatty acid, or a combination thereof,

• It further comprises a step of processing said milk-like product comprising : extracting, centrifuging, filtrating, separating, concentrating, lyophilising, hydrolysing, and/or precipitating said milk-like product or a fraction thereof,

• it further comprises a step of purifying at least one compound selected from a HMO, a casein, a lactoferrin, a lactalbumin, whey protein, long chain polyunsaturated fatty acids from said milk-like product,

• it further comprises a further step of dissociating the at least one lactiferous unit,

• the culture medium is constituted of human-food grade components.

FIGURE LEGENDS

[0030] Figures 1 A-B: representation of embodiments A and B of the method of producing a lactiferous unit according to the invention.

[0031] Figures 2 A-B: representation of embodiments A and B of the method of producing a milklike product according to the invention.

[0032] Figures 3 A-E: Lactiferous units obtained from mammary cells isolated from human milk, according to the method of the invention A: Flow-cytometry profile of mammary epithelial cells isolated from milk. B: Phase-contrast microscopy images of the lactiferous units obtained from milk cells in A. Scale bar = 350 pM. C: Analysis of the expression of mammary epithelial genes and lactation-related genes of interest by RT-qPCR before and after 16 days post-differentiation. Heat-map representation of ACt for each gene D: Relative expression of cell identity characterisation markers at day 16 post-differentiation in comparison to day 0. E: Relative expression of lactation markers at day 16 post-differentiation in comparison to day 0.

[0033] Figure 4 A-B: Lactiferous units obtained from mammary cells isolated from a mammary resection, according to the method of the invention A: Phase-contrast microscopy images of lactiferous units of the invention taken at different time points from induction of differentiation in suspension ; scale bar = 200 pm. B: size distribution of the lactiferous units at DO and D10 from induction of differentiation. [0034] Figure 5 A-B: Lactiferous units obtained from mammary cells isolated from a mammary resection, according to the method of the invention, with and without TGF-B inhibition. A: Phasecontrast microscopy images of lactiferous units of the invention obtained with or without TGF-B inhibition. B: Relative expression of lactation markers at day 14 post-differentiation in comparison to day 0, for lactiferous units of the invention obtained with TGF-B inhibition, in relation to lactiferous units obtained without TGF-B inhibition.

[0035] Figure 6 A-B: Lactiferous units obtained from mammary cells isolated from a mammary resection, according to the method of the invention. A: Phase-contrast microscopy images of lactiferous units of the invention generated from basal (CD10+) cells or from luminal (CD24+) cells. B: Relative expression of lactation markers post-differentiation, for lactiferous units of the invention generated from basal (CD10+) cells or from luminal (CD24+) cells.

[0036] Figure 7 A-C: Lactiferous units obtained from mammary cells isolated from a mammary resection, according to the method of the invention. A: Phase-contrast microscopy images of lactiferous units of the invention generated from basal (EpCam- or low ; CD49+) cells or from luminal (EpCam+; CD49f- or med) cells. B: Analysis of the expression of mammary epithelial genes and lactation-related genes of interest by RT-qPCR at post-differentiation, for lactiferous units of the invention generated from luminal (EpCam+; CD49f- or med) cells or from a mix of luminal (EpCam+; CD49f- or med) cells and basal (EpCam- or low ; CD49f+) cells. C: Analysis of lactoferrin secretion by Western-Blot in supernatant from lactiferous units of the invention generated from luminal (EpCam+; CD49f- or med) cells or from a mix of luminal (EpCam+; CD49f- or med) cells and basal (EpCam- or low ; CD49f+) cells.

DETAILED DESCRIPTION AND ADDITIONAL EMBODIMENTS

[0037] The invention relates, inter alia, to an in vitro method of producing a milk-like product.

Definitions

[0038] The terms "comprise", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". This term is thus inclusive or open-ended and does not exclude additional, unrecited element or method step.

[0039] By contrast, the term “consisting of” excludes the presence of any additional, unrecited element or method step.

[0040] The term “consisting essentially of” or “comprising substantially” means that additional, unrecited element or method step can be present, under the proviso that such element or step does not materially affect the essential characteristics of the described or claimed subject-matter. [0041] In some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of” or, alternatively, by “consisting essentially of’.

[0042] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0043] In some embodiments not otherwise explicitly recited, any instance of “a”, “an”, or “the” may be replaced by “one or more”, or “plurality of”.

[0044] When, referring to the value of a datum, an interval is herein specified, this interval is thought to include its limits, except otherwise specifically stated. Also, it should be understood said interval should be considered as covering all the possible individual numerical values within that interval, regardless of the breadth of the interval.

[0045] By “about” or “around”, it is meant that the measured value may vary within a certain range depending on the margin of error of the method or device used to evaluate the parameter of interest. In the context of the present invention, it preferably means that the measured value may be 15%, 10% away, advantageously 5%, 4%, 3%, 2%, or 1% away, from the given numerical value and that the recited range includes both endpoints.

[0046] As used herein “lactiferous unit” refers to an aggregate comprising epithelial mammary cells and, more particularly, comprising a mix of myoepithelial mammary cells and secretory mammary cells, and which is found able to produce a milk-like product, a component of milk or a mix of components thereof under induced lactation culture conditions. Said aggregate can be three-dimensionally organized in an acinus-like structure. In an embodiment, said acinus-like structure presents a layer of myoepithelial mammary cells that supports a layer of secretory mammary cells and defines a lumen that is separated from the culture medium by the layers of myoepithelial and secretory mammary cells, the myoepithelial cells making the interface between the culture medium and the lactiferous unit. In another embodiment, said “lactiferous unit” presents an inverted acinus-like structure wherein the myoepithelial mammary cells which provide support to the layer of secretory mammary cells is localized inside the unit whereas a secretory mammary cells layer makes the interface with the lactiferous unit and the culture medium. In another embodiment, in said lactiferous unit, myoepithelial mammary cells and secretory mammary cells are basically aggregated together without any specific three-dimensional organization.

[0047] The term “amplifying”, as applied to the cells used in the present invention, means herein proliferating, multiplying, growing or renewing said cells. In other words, the step of amplifying in the method of the invention leads to the production of a plurality of cells. To do so, the cells used in the present invention may be capable of self-renewal, and/or be immortalized cells. [0048] As used herein an “aggregate” refers to a cluster of cells which has formed thanks to the tendency of dissociated cells to in vitro group themselves under appropriate conditions. “Promoting aggregation” therefore means inducing the formation of such aggregate. The formation of so-called organoids usually needs the provision of a matrix-like membrane matrix (as, e.g. Matrigel™) as a basement for supporting the formation or anchorage of a cell aggregate as a first nucleation step for organoid formation. The lactiferous unit as developed by the inventors is “adapted to be cultured in suspension”. In other words, there is no need of a membrane matrix, or a component thereof (ECM), or of any other basement to support the formation, the maintenance and/or milk (or component(s) thereof) production in a culture medium of said lactiferous unit. In other words, said epithelial cells of the lactiferous according to the invention do not need to be attached to a membrane matrix for forming said lactiferous unit, for the maintaining said lactiferous unit culture in suspension and/or for producing milk-like product or milk component(s) upon the induction of lactation.

[0049] By “inducing differentiation” or “differentiating”, as applied to the cells used in the present invention, it is meant herein inducing or promoting the transformation of said cells into a more specialized cell type, preferably into a cell that is morphologically and functionally mature. Such step typically applies herein to progenitor cells.

[0050] The term “TGF-beta inhibitor” or “TGF-pi” refers herein to an agent capable of inhibiting the activity of transforming growth factor beta (TGF-beta or TGF-P), such as the signalling pathway of this growth factor in epithelial cells. Such agent may be provided herein in the form of a small molecule (e.g. small organic molecule), an antibody, a nucleic acid (RNAi, antisense, aptamer), the like, or a combination thereof. Particularly preferred TGF-beta inhibitors according to the invention are inhibitors of the TGF-p type I receptor, more specifically of TGF-p type I receptor/ALK5. Examples of TGF-beta inhibitors that are suitable according to the invention include, without limitation, RepSox (2-(3-(6-Methylpyridine-2-yl)-1 H-pyrazol-4-yl)-1 ,5- naphthyridine), SB 431542 (4-[4-(1 ,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H-imidazol-2- yl]benzamide), A83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1 H-pyrazole-1- carbothioamide), GW788388 (4-[4-[3-(2-Pyridinyl)-1 H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro- 2H-pyran-4-yl)-benzamide), and any combination thereof.

[0051] As used herein, "bioreactor" means a device or system that supports the growth of cells or of tissues; more particularly it enables the amplification differentiation and maintenance of mammary epithelial cells, the formation of lactiferous units, their maintenance and/or the production by said lactiferous unit of a milk product, a component thereof or a mix of components thereof. [0052] Stem cells are, by definition, able to self-renew and to generate differentiated progeny cells. In adult organs, stem cells show the property to extensively self-renew, allowing basal tissue maintenance thanks to their ability to generate all cell types found in the tissue. Mammary progenitor cells or mammary stem cells found in the mammary gland are involved in the regeneration of the mammary gland. These cells have also been identified in and can be isolated from maternal milk samples (Indumathi et al, 2013; Coni et al, 2023; Inman et al, 2015], Mammary progenitor cells and mammary stem cells can differentiate into cell components of mammary gland, namely luminal epithelial mammary cells and basal epithelial mammary cells. The term “luminal epithelial mammary cells” designates herein mature cells (i.e. differentiated cells) with a luminal epithelial phenotype, which can be divided into secretory mammary cells and hormoneresponsive mammary cells. By “basal epithelial mammary cells”, it is meant herein mature cells (i.e. differentiated cells) with a basal epithelial phenotype, also known as myoepithelial mammary cells. These two lineages of epithelial cells are the main cell populations found in normal mammary gland. Other cell types present in the mammary gland are stromal cells (e.g. adipocytes, fibroblasts, perivascular cells, vascular endothelial cells and lymphatic endothelial cells) and immune cells (e.g. T/NK cell, B cells and myeloid cells). In an adult mammary gland, luminal cells are typically found in the inner layer of the gland, while basal cells are typically found in an outer layer.

[0053] By contrast to pluripotent stem cells which are undifferentiated cells that can propagate indefinitely and give rise to every other cell type in the body, “progenitor cells” are undifferentiated cells capable of self-renewal - though not of indefinite self-renewal - which are lineage-committed and therefore give rise to a specific cell type. Unipotent progenitor cells can differentiate into one specific cell type, while bipotent progenitor cells can differentiate into two specific cell types.

[0054] A “luminal progenitor cell” refers to a progenitor cell as defined herein, which is essentially committed to a luminal lineage. A luminal progenitor cell is more precisely capable of differentiating into a luminal epithelial mammary cell as defined herein. A luminal progenitor cell is herein preferably a unipotent progenitor cell, meaning that it is restricted to a luminal lineage.

[0055] A “basal progenitor cell” refers to a progenitor cell as defined herein, which is essentially committed to a basal lineage. A basal progenitor cell is more precisely capable of differentiating into a basal epithelial mammary cell as defined herein. A basal progenitor cell is herein preferably a bipotent progenitor cell, meaning that it can also give rise to a luminal lineage.

[0056] All the above-mentioned cell types can be distinguished based on marker expression, as further detailed below. [0057] The term “hydrogel” designates herein a porous three-dimensional network of polymers, in which polymers are held together by covalent or noncovalent crosslinks, and in which water has been absorbed thereby forming a gel. Such polymers are preferably synthetic polymers.

[0058] The terms “capsule” or “hydrogel capsule” refers, in the context of the invention, to an hollow structure whose wall is made of said hydrogel. The space within said capsule is adapted to receive either mammary cells which will subsequently to encapsulation, be amplified, aggregate and form lactiferous units according to the invention, or to receive an already formed lactiferous unit. That is to say, the space within said capsule is adapted to encapsulate anchorageindependent mammary cells which will be subsequently amplified.

[0059] The term “extracellular matrix component” (ECM) refers herein to a component capable of providing support, adherence, movement, and/or regulation of cells within a cellular environment. Such components are typically extracellular macromolecules that are naturally present in connecting tissue and other tissues of multicellular organisms such as animals or plants and include e.g. proteins, peptides, proteoglycans, and the like. In vivo, an extracellular matrix is made of a set of extracellular matrix components, which in animals are mainly made of glycoproteins and pure proteins, as well as polysaccharides - though its composition may vary between tissue types. Major components of the extracellular matrix of the breast tissue, such as of the human breast tissue, include, without limitation, collagen (e.g. type IV), laminin (e.g type 111 , 332, 511 , 521), fibronectin and tenascin. Plant extracellular matrix, on the other hand, is composed primarily of polysaccharides of which cellulose is the major component. In vitro, an extracellular matrix (i.e. matrix) may be provided as an homogenous extracellular matrix (essentially made of a single ECM), or as an heterogenous extracellular matrix (essentially made of a plurality of ECM). A well-known example of homogenous extracellular matrix is a collagen matrix. A well-known example of heterogenous extracellular matrix is Matrigel™, which is a reconstituted basement membrane prepared from Engelbreth- Holm-Swarm (EHS) mouse sarcoma cells.

[0060] As used herein, the term “milk-like product" refers to an edible product which is the result of the induction of lactation in the at least one lactiferous unit adapted to be cultured in suspension, preferably in a suspension bioreactor, of the invention in the culture medium according to the method of producing a milk-like product of the invention. In other words, the milk-like product of the invention is constituted both of i) culture medium in which said the lactiferous unit of the invention is cultured in suspension at the end of the induction lactation step and ii) of the component produced upon the induction of lactation by the lactiferous unit adapted to be cultured in suspension in a bioreactor of the invention. [0061] The cells, lactiferous units, and methods of the invention can be cultured in batch, fed- batch, or continuous mode. Briefly, fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients are fed to the bioreactor during cultivation and in which the product remains in the bioreactor until the end of the run.

[0062] The fed-batch strategy is typically used in bio-industrial processes to reach a high cell density in the bioreactor. Mostly the feed solution is highly concentrated to avoid dilution of the bioreactor, increase of pH and osmolality. The controlled addition of the nutrient directly affects the growth rate of the culture and helps to avoid nutrient depletion, overflow metabolism and oxygen limitation.

[0063] The constantly-fed-batch culture is the one in which the feed rate of a growth-limiting substrate is constant, i.e. the feed rate is invariant during the culture. If the feed rate of the growthlimiting substrate is increased in proportion to the exponential growth rate of the cells, it is possible to maintain exponential cell growth rate for a long time, called exponentially-fed-batch culture.

[0064] Perfusion culture means to maintain a cell culture in bioreactor in which equivalent volumes of media are simultaneously added and removed while the cells are retained in the reactor. This provides a steady source of fresh nutrients and constant removal metabolites and/or components produced by the cells.

[0065] The cultivation vessel of the present invention may be selected from, but is not limited to, agitated flask, Erlenmeyer flask, spinner flask, and stirred paddled or wave bioreactors. Particularly, the cultivation vessel may be selected among, but not limited to, continuous stirred tank bioreactor, Wave ™ Bioreactor, Bello ™ bioreactor, Mobius bioreactor, agitated bioreactor (e.g, Orbshake), bioreactor with perfusion systems. For scaled up production, the preferred cultivation vessel is a bioreactor. The volume of bioreactor may be equal or larger than 20 liters, larger than 100 liters, larger than 1 ,000 liters, preferably up to 10,000 liters. According to the preferred embodiment, the cultivation vessel is a continuous stirred tank bioreactor that allows control of temperature, aeration, pH and other controlled conditions and which is equipped with appropriate inlets for introducing the cells, sterile oxygen, various media for cultivation and outlets for installing probes, removing cells and media and means for agitating the culture medium in the bioreactor.

Lactiferous unit

[0066] As stated above the inventors have been able to produce lactiferous units able to be cultured in suspension, for example in a bioreactor. Indeed, lactiferous unit according to the invention is able to resist to shear stress, and to be maintained in suspension culture without the need of any cellular matrix or a component thereof (ECM) and/or scaffold. Lactiferous unit of the invention does not necessarily present the structural organization as described e.g. in Lee et al. (2023). Representative images of lactiferous units of the invention are presented in the Figures.

[0067] Such property of lactiferous units of the invention is particularly advantageous as it allows an industrial scale production of milk-like product of the invention, and more particularly the production at an industrial scale of milk component(s) secreted by the secretory mammary cells comprised in said lactiferous unit according to the invention.

[0068] Even if a lactiferous unit of the invention is able to be maintained and to produce milk components in suspension culture while resisting to shear stress, in some embodiments, the lactiferous unit of the invention is encapsulated, in order, notably but not only, to make it even more resistant to shear stress produced upon the stirring of the culture medium.

[0069] Encapsulation methods are well known in the art, and an example is provided below.

[0070] Any material suitable for said encapsulation and suitable to encapsulating living cells in culture can be used, such as hydrogel. It shall nevertheless be understood that such encapsulation is not required for the mammary epithelial units of the invention to be lactiferous.

[0071] Said encapsulation material can be an hydrogel selected from: alginate, chitosan, silk, poly(vinyl alcohol), dextran, polyacrylamide, polyethylene glycol, pectin, guar gum, gelatin, zein, agar-methylcellulose, a cellulose derivative hydrogel, casein or a mix thereof.

[0072] When producing a human milk-like product is considered, a human food grade encapsulation system is particularly preferred, for example it can be selected from sodium alginate, gum arabic, chitosan (or modified chitosan).

[0073] Alginate, more particularly sodium alginate or calcium alginate, is particularly preferred. Alginate capsules can form and dissociate by a mere variation of concentration of calcium ions (Bennacef et al., 2023), without the need of mechanical mean or of other chemicals, thereby avoiding unwanted contamination of milk-like product with other components.

[0074] Further alginate being a food grade ingredient even for infant food, its use in a method for making milk-like product according to the invention is particularly preferred, especially when it is considered for an infant nutrition. More generally, alginate is of particular interest, notably, but not only, when considering producing milk-like product for human consumption.

[0075] In some other embodiments, as an alternative to alginate capsules which are hollow structures, lactiferous unit of the invention, once formed in suspension, may have been integrated within alginate beads, in order to provide a further protection.

[0076] Alginate capsules or beads also present the advantage of being able to be functionalized by coupling with specific proteins or small molecules participating in the stimulation of mammary epithelial cells (e.g. prolactin, FGF or any other compound of interest) for the secretion of the milklike product thus isolating said small molecules from the milk-like product itself.

[0077] As mentioned previously, mammary epithelial cells and lactiferous unit of the invention are adapted to grow in suspension. In other words, they do not need to interact with a matrix, as e.g. membrane matrix or, when encapsulated, a hydrogel component as listed above to aggregate and/or differentiate.

[0078] A lactiferous unit according to the invention can comprise a mix of myoepithelial mammary cells (also called basal epithelial cells) and secretory mammary cells (also called luminal cells). Said cells can be easily identified by assessing the presence or the absence of specific markers, for example, as specified in Table 1 below. The level of expression of some markers, such as CD49f or EpCam, may also distinguish cell types.

Table 1

[0079] It is to be understood that among the cells in a lactiferous unit according to the invention, a mammary myoepithelial cell is typically found positive for at least one of the following markers CK14, EpCam, p63, Keratin 17, CD10, ACTA 2, TAGLN, MYLK, TPM2 or a combination thereof. In an embodiment, a myoepithelial cell is found positive for at least 2, 3, 4, 5, 6, 7, 8 or 9 of the following markers : CK14, EpCam, p63, Keratin 17, CD10, ACTA 2, TAGLN, MYLK, TPM2. In a particular embodiment, a myoepithelial cell is found positive for the following markers : CK14, EpCam, p63, Keratin 17, CD10, ACTA 2, TAGLN, MYLK, TPM2.

[0080] It is to be understood that among the cells in a lactiferous unit according to the invention, a mammary secretory cell should be typically found positive for at least one of the following markers EpCam, CK18, CK8, CD24, optionally CD49f, or a combination thereof. In an embodiment, a mammary epithelial secretory cell is found positive for at least 2, 3, 4 or 5 of the following markers : EpCam, CK18, CK8, CD24, optionally CD49f. For example, a mammary secretory cell should be typically found positive for at least one of the following markers CD49f, EpCam, CK18 or a combination thereof. In an embodiment, a mammary epithelial secretory cell is found positive for at least 2 or 3, preferably at least 3 of the following markers : CD49f, EpCam, CK18.

[0081] In a particular embodiment, a basal epithelial cell is found positive for the following markers : CD49f, CK14, EpCam, p63, Keratin 17, CD10, ACTA 2, TAGLN, MYLK, TPM2.

[0082] In some embodiments, a further marker such as those listed in Table 7 or 8, can be used also, possibly in a combination with any of the above markers.

[0083] In an embodiment, mammary myoepithelial cells and secretory mammary cells constitute the majority of the cells of the lactiferous unit of the invention. In other words, the majority of the cells in the lactiferous unit according to the invention have undergone lactogenic differentiation. That is to say, the lactiferous unit according to the invention consists essentially of mammary secretory mammary cells and myoepithelial cells.

[0084] In a particular embodiment, said myoepithelial cells and secretory mammary cells represents more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, even more than 95% of the cells present in the lactiferous according to the invention. In another particular embodiment, progenitors (myoepithelial (basal), secretory (luminal) and/or bipotent progenitors) of mammary cells represents less than 60%, less than 50%, less than 40% less than 30%, less than 20%, less than 10 % even less than 5% of the total cell population of a lactiferous according to the invention. In a preferred embodiment, the population of secretory mammary cells exceeds that of myoepithelial cells in the lactiferous unit of the invention; in other words, there is a greater proportion of secretory mammary cells compared to myoepithelial cells in said lactiferous unit. The proportion of myoepithelial cells and secretory mammary cells can be measured by quantification, within representative members of a lactiferous unit population according to the invention, of the cells positive for the corresponding markers as listed above. Detection of said markers can be made by any means known in the art, for example by flow cytometry, immunohistochemistry, or RT-qPCR as described in the below Examples. [0085] The ability of the lactiferous unit according to the invention to produce a milk product can be detected by the measurement of the expression of at least one the genes or proteins that are known to have their expression modulated as a function of lactogenic differentiation, as, e.g. for those listed identified by Sornapudi et al (2018) or other as prolactin receptor (Twigger et al, 2022). Examples of such genes are listed in Table 2. Their expression can vary as a function of the species mammary epithelial cells comprised in the lactiferous unit are derived from. The skilled in the art will know which are the most appropriate to assess lactiferous differentiation depending of the species the cells originate from.

Table 2

*upregulated upon induction with prolactin t not expressed in human and/or ruminants

[0086] Accordingly, in an embodiment, an overexpression at least one of the genes, or corresponding proteins thereof, selected from : Csn2, Cns3, Wap, Lalba, Spp1 , Lyz, Ptgds, Ltf, Klk1 b9, Arl4d, Pigr, Pip, Prlr, Stat5, Scd, Olah, Gk5, Fabp7, Slc25a1 , Jak2, Akt1 , Fut2, Fut3, St6galnac2, Slc2a1 , B4galt1 , Slc35a2, Galt, Aldoc, Ugp2, Fabp3, Gpam, Lipg, Acly, Acaca, Lpinl , Thrsp, Xdh in the lactiferous unit according to the invention under lactation induction conditions (e.g. upon exposure to prolactin), when compared to the expression level of the corresponding genes under conditions without lactation induction, shows the lactogenic differentiation. Preferably, an overexpression at least one of the genes selected from : Csn2, Wap, Lalba, Spp1 , Lyz, Ptgds, Ltf, Klk1b9, Arl4d, Pigr, Pip, Prlr, Stat5, Scd, Olah, Gk5, Fabp7, Slc25a1 , Jak2, Akt1 , Fut2, Fut3, St6galnac2, Slc2a1 , B4galt1 , Slc35a2, Galt, Aldoc, Ugp2, Fabp3, Gpam, Lipg, Acly, Acaca, Lpinl , Thrsp, in the lactiferous unit according to the invention under lactation induction conditions (e.g. upon exposure to prolactin), when compared to the expression level of the corresponding genes under conditions without lactation induction, shows the lactogenic differentiation.

[0087] Said overexpression can be detected by RT-qPCR on a sample of a suspension culture of lactiferous units according to the invention. Said overexpression can also be detected by qualifying the presence of the corresponding protein, either within the cells or in the culture medium of the lactiferous unit of the invention, by methods well-known in the art such as by Western-Blot. Preferably, said genes and/or proteins are overexpressed by a factor of at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, or even more when related to a gene that is not expressed at all in non-lactogenic cells.

[0088] In a particular embodiment, the lactiferous unit according to the invention shows an overexpression of at least one gene or protein selected from Csn2, Wap, Lalba, Ltf, Spp1 , Lyz, and Pip, upon induction of lactation. The corresponding protein are milk protein and can consequently easily be detected in the medium, depending of course, of the species mammary epithelial cells are derived from.

[0089] In a particular embodiment, the lactiferous unit according to the invention shows an overexpression of at least one gene or protein selected from Fabp3, Gpam, Scd, Lipg, Olah, Acly, Gk5, Acaca, Fabp7, Lpinl , Slc25a1 , Thrsp upon induction of lactation. These genes are known to be implied in the lipid metabolism of mammary cells.

[0090] In a particular embodiment, the lactiferous unit according to the invention shows an overexpression of at least one gene or protein selected from Slc2a1 , B4galt1 , Slc35a2, Galt, Aldoc, Ugp2 upon induction of lactation. These genes are known to be implied in lactose metabolism in mammary cells.

[0091] In a particular embodiment, the lactiferous unit according to the invention shows an overexpression of at least one gene or proteins selected from Stat5, PrIR, Jak2, Akt1 upon induction of lactation. These genes are known to be implied the lactation signalling pathway.

[0092] In a particular embodiment, the lactiferous unit according to the invention shows an overexpression of at least one gene or proteins selected from Fut2, Fut3, St6galnac2, upon induction of lactation. These genes are known to be implied in HMOs metabolism in mammary cells.

[0093] In some embodiments, a further marker such as those listed in Table 7 or 8, can be used also, possibly in a combination with any of the above markers.

[0094] As exposed above, the lactiferous unit according to the invention can present several organizations depending on the way the cells aggregate, grow and/or differentiate. It has been found that, surprisingly, in any of the observed organizations, lactiferous units of the invention are able to produce a milk-like product.

[0095] In an embodiment, said lactiferous unit presents an acinus like structure, in other words, a layer of myoepithelial mammary cells supports a layer of secretory mammary cells which defines a lumen that is separated from the culture medium by the layers of myoepithelial and secretory mammary cells, the myoepithelial cells making the interface between the culture medium and the lactiferous unit. Milk-like product components secreted by the secretory mammary cells upon induction of lactation inside of said acinus-like structure. In this embodiment, myoepithelial mammary cells are mainly localized outside of the lactiferous unit and secretory mammary cells are mainly localized at the inside of said lactiferous unit, said secretory mammary cells being oriented such that their apical face is at the inside of said lactiferous unit. Otherwise said, in this embodiment, at least 50%, at least 60%, at least 70% at least 80%, at least 90% of the myoepithelial mammary cells can be localized at the outside of the lactiferous unit. Independently, in this embodiment, at least 50%, at least 60%, at least 70% at least 80%, at least 90% of the secretory mammary cells can be localized at the inside of the lactiferous unit.

[0096] In another embodiment, said lactiferous unit presents an inverted acinus-like structure wherein a layer of myoepithelial mammary cells which provides support to secretory mammary cells is localized inside the lactiferous unit whereas a secretory mammary cells layer makes the interface with the lactiferous unit and the culture medium. Milk-like product components secreted by the secretory mammary cells upon induction of lactation are then secreted directly in the culture medium. In this embodiment, myoepithelial mammary cells are mainly localized inside of the lactiferous unit and secretory mammary cells are mainly localized at the outside of said lactiferous unit, said secretory mammary cells being oriented such that their apical face is at the outside of said lactiferous unit. In other words, in this embodiment, at least 50%, at least 60%, at least 70% at least 80%, or at least 90% of the myoepithelial mammary cells can be localized at the inside of the lactiferous unit. Independently, in this embodiment, at least 50%, at least 60%, at least 70% at least 80%, or at least 90% of the secretory mammary cells can be localized at the insides of the lactiferous unit.

[0097] In a further embodiment, said lactiferous unit comprises a mix of cells without particular organization, notably in regard with the distribution, notably, of myoepithelial mammary cells and secretory mammary cells which are distributed within said lactiferous unit without delineating any specific cellular organization.

[0098] In some embodiment the lactiferous unit according to the invention can comprise further cell types known to compose the acini, as for example adipocytes and/or lymphocytes. Presence of these cells may be useful as it can result in the presence in the milk-like product of the invention of fatty acids and/or immunoglobulins which is of particular interest for example when considering producing infant milk formulas from milk-like product of the invention. It shall nevertheless be understood that the presence of such cells is not required for the mammary epithelial units of the invention to be lactiferous.

[0099] In some embodiments, a lactiferous according to the invention has at least one dimension of at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, but no more than 500 pm.

[00100] The lactiferous unit of the invention can be composed of mammary epithelial cells of any mammalian origin, in particular of human origin, bovine origin, canine origin, feline origin, ovine origin, caprine origin cetacean origin (more preferably of baleen whales’ origin), seal origin, elephant origin, equidae origin, or of a mix thereof. In some instance, a lactiferous unit comprising a mix of cells from different mammalian origin therefore allowing to produce one milk like product comprising a mix of compounds specific to different mammalian. Also, it can result also in obtaining a milk-like product with a lower content in an unwanted compound (for example because of immunogenicity or being an allergen) while content in other milk compounds is maintained. When human mammary epithelial cells are considered to be comprised in the lactiferous unit of the invention, which is a particular embodiment of the invention, they do not have an embryonic origin.

Method for producing (100) at least one lactiferous unit to be cultured in suspension, such as in a suspension bioreactor

[00101] A further object of the invention also lies in the specific conditions and means that inventors have been able to set up and which allow obtaining lactiferous unit adapted to be cultured in suspension. Obtaining lactiferous unit able to produce milk like product and/or component thereof in the culture medium is of an outmost advantage when considering scaling up of in vitro milk production. Inventors made the discovery that under specific conditions it was possible to develop a lactiferous unit that is able to form without the help of e.g. Matrigel™ matrix or the like or of any support, or of any scaffold that might serve as physical support in order to promote mammary cell aggregation and differentiation of the cells.

[00102] Accordingly, an object of the invention concerns a method (100) for producing at least one lactiferous unit as described above. Exemplary embodiments of said method (100) are illustrated in Figure 1. It shall be understood that all steps of this method can be implemented in suspension.

[00103] More precisely, the method (100) for producing at least one lactiferous unit according to the invention comprises, or consists essentially of, the steps of: i. Seeding (120) in suspension at least one mammary cell selected from a progenitor of mammary cells, a mammary luminal epithelial cell, or a mix of said cells, ii. Amplifying (140) said at least one mammary cell in suspension, iii. Optionally, promoting cell aggregation (150), and iv. Inducing differentiation of the cells (170) into epithelial mammary cells, if the mammary cell seeded at step i is a progenitor of mammary cells or a mix of said cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[00104] In an embodiment, which is herein particularly preferred, said method (100) comprises, or consists essentially of, the following steps : i. Seeding (120) at least one progenitor of mammary cells in suspension, such as in a suspension bioreactor, in a culture medium ii. Amplifying (140) said progenitor cells in suspension, iii. Optionally promoting cell aggregation (150), iv. Inducing differentiation of said progenitor cells (170) into epithelial mammary cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[00105] In another embodiment, said method (100) comprises, or consists essentially of, the steps of: i. Seeding (120) in suspension at least one mammary luminal epithelial cell in suspension, such as in a suspension bioreactor, in a culture medium ii. Amplifying (140) said cell in suspension, iii. Optionally promoting cell aggregation (150), thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

[00106] Yet, in another embodiment, said method (100) , comprises, or consists essentially of, the steps of: i. Seeding (120) in suspension at least one progenitor of mammary cells, and at least one mammary luminal epithelial cell, in a culture medium, ii. Amplifying (140) said cells in suspension, iii. Optionally promoting cell aggregation (150), iv. Inducing differentiation of the progenitor cells (170) into epithelial mammary cells, thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor. [00107] As explained above, it shall be understood that the present method according to the invention is a method preferably free of exogenously added component(s) of the extracellular matrix (ECM), especially of animal-derived component(s) of the extracellular matrix (ECM). For example, the present method is preferably free of exogenously added collagen or Matrigel™.

Seeding of mammary cells (120)

[00108] One first step of the method comprises the seeding of at least one mammary cell (120) in suspension.

[00109] The at least one mammary cell is herein selected from a progenitor of mammary cells, a mammary luminal epithelial cell, or a mix of said cells, more preferably is a progenitor of mammary cells.

[00110] In other words, in an embodiment, the at least one mammary cell is a progenitor of mammary cells. In another embodiment, the at least one mammary cell is a mammary luminal epithelial cell. Yet, in another embodiment, the at least one mammary cell is a mix of a progenitor of mammary cells and a mammary luminal epithelial cell.

[00111] In a preferred embodiment, the mammary cell as described herein is an immortalized mammary cell.

[00112] Mammary stem cells or so-called mammary progenitor cells are found in the mammary gland, and are involved in the regeneration of the mammary gland. Mammary stem cells or progenitors can also be found released in maternal milk upon lactation, though in a low amount. Likewise, primary epithelial cells such as mammary luminal epithelial cells can be found, besides mammary tissue, in milk. That is because, during lactation, mammary cells are gradually exfoliated from the mammary epithelium and become a minor component of the cell population present in milk. Accordingly, mammary cells suitable for the method of the invention can be isolated from maternal milk and/or dissection of sample from mammary resection, by methods known in the art, such as those described by Stingl et al. (2005), Chen et al. (2019), Plazaola et al. (2015), Qian et al. (2024), or as described in the Example section below.

[00113] Thus, in a preferred embodiment of the invention, the least one mammary cell is isolated from a milk sample or from a mammary tissue sample such as a mammary resection. Should the sample be a mammary tissue sample, it shall be understood that said sample is herein preferably a dissociated mammary tissue sample (single cells).

[00114] In the context of the present invention, it will be understood that the least one mammary cell used herein is preferably a healthy mammary cell. As such, the least one mammary cell can preferably be isolated from a healthy milk sample or from a healthy mammary tissue sample. [00115] In some embodiments, mammary cells can be selected and enriched using flow cytometry sorting methods well-known in the art, using lineage markers such as those specified in tables described herein such as Table 1 or 3, or as detailed in the Example section below.

[00116] Accordingly, in a preferred embodiment, the method according to the invention comprises, or consists essentially of, before step i:

• removing, from the sample, stromal cells and/or immune cells, and

• optionally recovering, from the sample, cells that are positive or negative for at least one marker selected from CD49f, EpCAM, CD10, CD24 or a combination thereof, thereby selecting a cellular population comprising, preferably enriched in, progenitors of mammary cells, mammary luminal epithelial cells, or a mix of said cells.

[00117] Accordingly, in a preferred embodiment, step i consists of, or essentially consists of, seeding a cellular population preferably enriched in progenitors of mammary cells, mammary luminal epithelial cells, or a mix of said cells, more preferably enriched into progenitors of mammary cells.

[00118] In a preferred embodiment, step i consists of, or essentially consists of, seeding a cellular population consisting of, or consisting essentially of, progenitors of mammary cells, mammary luminal epithelial cells, or a mix of said cells, more preferably progenitors of mammary cells.

[00119] In a preferred embodiment, step i consists of, or essentially consists of, seeding a cellular population consisting of, or consisting essentially of, progenitors of mammary cells.

[00120] In a preferred embodiment, step i consists of, or essentially consists of, seeding a cellular population consisting of, or consisting essentially of, mammary luminal epithelial cells.

[00121] In a preferred embodiment, step i consists of, or essentially consists of, seeding a cellular population consisting of, or consisting essentially of, a mix of progenitors of mammary cells and of mammary luminal epithelial cells.

[00122] In a preferred embodiment, the at least one progenitor of mammary cells is a bipotent or unipotent mammary progenitor cell.

[00123] In a preferred embodiment, mammary progenitor cells to be used in the method of the invention for producing at least one lactiferous unit are bipotent mammary progenitor cells. Indeed, bipotent mammary progenitor cells have the potency to differentiate either in secretory mammary cells or in myoepithelial mammary cells. Accordingly, a single population bipotent mammary progenitor cell can be used to produce lactiferous units according to the invention. [00124] In a preferred embodiment, the at least one progenitor of mammary cells is a mammary luminal progenitor cell or a basal progenitor cell or a mix thereof, preferably is a mammary luminal progenitor cell.

[00125] In a preferred embodiment, if the progenitor of mammary cells is a mammary basal progenitor cell, it is advantageously a bipotent mammary progenitor cell.

[00126] Seeding a mammary luminal progenitor cell may herein be preferred over a basal progenitor cell, especially over a bipotent mammary progenitor cell, in order to shorten the production timeline for obtaining a lactiferous unit of the invention.

[00127] The inventors have also discovered that seeding a mammary luminal progenitor cell is sufficient to obtain a lactiferous unit of the invention.

[00128] In another embodiment, the progenitors of mammary cells are a mix of two progenitor populations: a population of luminal progenitor mammary cells and a population of mammary basal progenitor cells.

[00129] As mentioned above, in this method (100) of the invention, the mammary cells can be from any mammalian organism. In some embodiments, they can be of human origin, bovine origin, canine origine, feline origine, ovine origin, caprine origin cetacean origin (more preferably of baleen whales’ origin), seal origin, elephant origin, equidae origin, or of a mix thereof. Indeed, in some other embodiments a mix of mammary progenitor cells from different origins can be used for producing lactiferous unit bearing secretory and myoepithelial cells from different origins, and therefore a milk-like product derived therefrom comprising a mix of compounds specific to these origins. In a particular embodiment, the mammary cells are human mammary cells.

[00130] In an embodiment, the at least one mammary cell is a human mammary progenitor cell, a human mammary luminal epithelial cell, or a mix thereof.

[00131] In another embodiment, herein preferred, the at least one mammary cell is a human mammary progenitor cell, preferably basal or luminal as described above, more preferably luminal.

[00132] In another embodiment, herein preferred, the at least one mammary cell is a mix of a human mammary progenitor cell, preferably basal or luminal as described above, more preferably luminal, and a human mammary luminal epithelial cell.

[00133] Suitable markers for identifying and selecting progenitors cells, and/or mammary luminal epithelial cells such as mammary secretory cells, are listed in Table 3 below, as well as in above Table 1. Table 3

[00134] According to some embodiments, at least 10⁴ cells/mL, 10⁵ cells/mL, 10⁶ cells/mL, at least 10⁷ cells/mL, at least 10⁸ cells/mL, or at least 10⁹ cells/mL or more are seeded in the culture medium.

[00135] For seeding step i, the at least one mammary cell can be placed in suspension in a culture medium.

[00136] Said culture medium is a medium suitable for the purpose of the present method.

[00137] The culture medium used herein typically comprises a basal medium, a proliferation supplement and preferably a differentiation-promoting agent.

[00138] Basal media typically provide essential nutrients, salts and buffering agents, and are well-known in the art.

[00139] According to some embodiments, any suitable basal medium can be used herein, like e.g. Minimum Essential Media (MEM), Essential 8 Media, Basal Medium Eagle (BME), Ham's F12, Ham's F-10, Fischer's Medium, CMRL-1066 Medium, Click's Medium, Medium 199, Dulbecco's Modified Eagle's Media (DMEM), RPMI-1640, L-15 medium, McCoy's 5A Modified Medium, William's Medium E, and Iscove's Modified Dulbecco's Medium (IMDM) or DMEM/F12 medium. A particularly preferred basal medium according to the invention is DMEM/F12.

[00140] Proliferation supplements typically promote cell growth and proliferation, or, in other words, amplification of the cells.

[00141] Examples of proliferation supplements suitable for the present method include without limitation growth factors such as EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), IGF (Insulin Growth Factor) and VEGF (Vascular Endothelial Growth Factor), or any combination thereof. Particularly preferred growth factors according to the invention are EGF and FGF, especially a combination of EGF and FGF2. [00142] The culture medium according to the invention may comprise any further additional supplement that can support the amplification of the mammary cells, and optionally also the differentiation of said cells.

[00143] Such additional supplement can include, for example: hydrocortisone, insulin, transferrin-selenium, amino acids such as glutamine, serum or derivatives or alternatives thereof, and any combination thereof.

[00144] Hydrocortisone is for example a supplement supporting both the amplification and the differentiation of cells. In other words, hydrocortisone is both a proliferation supplement and a differentiation-promoting agent.

[00145] If present, a serum derivative or alternative is herein preferred, such as Knock Out Serum Replacement medium (KOSr) or B27, in particular xeno-free, such as xeno-free KOSr or B27. That is to say, the culture medium used herein is preferably a xeno-free medium.

[00146] The culture medium may further comprise a Rho-associated protein kinase (ROCK) inhibitor, such as Y-27632.

[00147] In some embodiment, the basal medium is complemented with a proliferation supplement, heparin (e.g. around 4pg/mL) and hydrocortisone (e.g. around 0.48pg/mL).

[00148] In some embodiment the basal medium is DMEM/F12 complemented with a proliferation supplement, heparin (e.g. around 4pg/mL) and hydrocortisone (e.g. around 0.48 g/mL).

[00149] In a very particular embodiment, especially when producing human lactiferous units, the MammoCult medium or the EpiCult medium (StemCell Technologies), preferably the EpiCult medium, is used for amplifying mammary cells.

[00150] Yet, in an even more preferred embodiment, especially when producing lactiferous units of the invention, the culture medium is DM EM/F 12 supplemented with glutamine (e.g. around 2.5 mM), hydrocortisone (e.g. around 2 pg/ml), insulin (e.g. around 0.17 mM), transferrinselenium (e.g. around 6.87 pM), EGF (e.g. around 10 ng/mL), FGF2 (e.g. around 4.3 ng/mL), supplemented or not with FBS (e.g. around 5%) or 20% KOsr (e.g. around 20%) or B27. A representative culture medium according to the invention is described in the Example section below.

[00151] The culture medium may also comprise a differentiation-promoting agent. Such agent serves to induce differentiation of the seeded cells into mammary epithelial cells. To do so, said agent may be incorporated into the culture medium from the outset of the present method (i.e. from step i), or only at the subsequent differentiation step (i.e. step iv). [00152] In that regard, the inventors have herein observed that the lactiferous units of the invention exhibit enhanced lactogenic property when the cells are subjected to TGF-beta inhibition. Indeed, as demonstrated in the Example section, such inhibition significantly boosted the differentiation of the cells into a luminal cell type, especially into a secretory cell type.

[00153] Accordingly, in a preferred embodiment, the differentiation-promoting agent is an agent promoting differentiation into mammary luminal epithelial cells, more preferably into mammary secretory cells.

[00154] In a further preferred embodiment, the differentiation-promoting agent is a TGF- beta inhibitor, preferably selected from RepSox, SB 431542, or a combination thereof, more preferably is a combination thereof (e.g. around 25 pM RepSox, and/or around 10 pM SB 431542).

[00155] The invention accordingly also relates to the in vitro or ex vivo use of a TGF-beta inhibitor, as described herein, for differentiating a progenitor of mammary cells into a mammary luminal epithelial cell, more preferably into a mammary secretory cell.

[00156] The mammary progenitor cells to be used in the method (100) according to the invention have been adapted to grow in suspension. In other words, they do not need support to grow and colonize the culture medium. They can be obtained through the implementation of the preliminary step of adapting progenitor of mammary cells (110) to culture in suspension detailed below.

Amplifying ( 140) said mammary cells in suspension

[00157] In some embodiment, this step comprises culturing and passaging the mammary cells in a culture medium, for example a basal medium, like e.g. DMEM/F12 medium as described above.

[00158] The culture medium used herein typically comprises a basal medium, a proliferation supplement and preferably a differentiation-promoting agent, each being as described above.

[00159] In some embodiment, the basal medium is complemented with proliferation supplement, heparin (around 4pg/mL) and hydrocortisone (around 0.48 pg/mL).

[00160] In a very particular embodiment, especially when producing human lactiferous units, the MammoCult medium or the Epicult medium (StemCell Technologies), preferably the EpiCult medium, is used for amplifying mammary progenitor cells.

[00161] Yet, in an even more preferred embodiment, especially when producing lactiferous units of the invention, the culture medium is DMEM/F12 supplemented with glutamine (e.g around 2.5 mM), hydrocortisone (e.g. around 2 pg/ml), insulin (e.g. around 0.17 mM), transferrin-selenium (e.g. around 6.87 pM), EGF (e.g. around 10 ng/mL), FGF2 (e.g. around 4.3 ng/mL), supplemented or not with FBS (e.g. around 5%) or 20% KOsr (e.g. around 20%) or B27. A representative culture medium according to the invention is described in the Example section below.

[00162] In a preferred embodiment, the culture medium used in step ii comprises the same components as the culture medium used in step i. It shall nevertheless be understood that the culture medium may be renewed, if deemed necessary, between step ii and step i, and/or even between cell passages.

[00163] In some instance, the medium can be adapted as a function of e.g. the contemplated use or destination of the milk-like product to be produced. For example, it can be depleted of a particular compound or supplemented for another one. For example, in case where a milk-like product depleted or with a low content in phenylalanine is contemplated, then a medium with no or low content in phenylalanine can be used.

[00164] In some embodiments, the step of amplifying (140) comprises culturing and passaging the mammary cells for at least 24h, at least 48h, at least 60h, at least 72h or even more, notably to reach a cell concentration or a cell density suitable to trigger mammary cell aggregation, and/or to reach a sufficient number of lactiferous units.

[00165] In some embodiments, the step of amplifying (140) comprises merely gathering a sufficient number of mammary cells adapted to culture in suspension.

[00166] In some embodiments, other mammary cells (from either milk collection, or mammary resection tissue collection) in particular stromal cells, including adipocytes and/or lymphocytes, can be cocultured with the mammary cells. Accordingly, these cells are comprised in lactiferous units, which can be of a particular advantage, when considering producing a milklike product which comprises particular fatty acids or immunoglobulins such as those contained in milk produced by the mammal the cells come from. It shall nevertheless be understood that the presence of such cells is not required for the mammary epithelial units of the invention to be lactiferous.

[00167] In another embodiment, no stromal cells are co-cultured with the mammary cells in the method of the invention.

Optionally promoting cell aggregation (150)

[00168] Though mammary progenitor cells or mammary luminal epithelial cells may have a tendency to self-aggregate, in some embodiment an optional, yet particularly preferred, step of promoting cell aggregation (150) can be applied to the mammary cells amplified at step ii. [00169] The inventors have indeed observed that this step is beneficial for optimizing the production of lactiferous units of the invention, as it results in lactiferous units that are relatively uniform in size and/or shape, with minimal single cells remaining in suspension.

[00170] In some embodiment, this step (150) can comprise a step of lowering the speed of stirring of the culture medium, especially when the present method is implemented in a suspension bioreactor.

[00171] Alternatively, should the present method be implemented at lab-scale, one may plate the cells onto a culture vessel capable of promoting the formation of embryoid bodies or spheroids. Such plates are well-known in the art, and include, for example, the AggreWell™ microwell plates.

[00172] In some embodiments, this step comprises culturing and passaging the mammary cells in a culture medium, for example a basal medium, like e.g. DMEM/F12 medium as described above.

[00173] The culture medium used herein typically comprises a basal medium, a proliferation supplement and preferably a differentiation-promoting agent, each being as described above.

[00174] In a preferred embodiment, especially when producing lactiferous units of the invention, the culture medium is DMEM/F12 supplemented with glutamine (e.g around 2.5 mM), hydrocortisone (e.g. around 2 pg/ml), insulin (e.g. around 0.17 mM), transferrin-selenium (e.g. around 6.87 pM), EGF (e.g. around 10 ng/mL), FGF2 (e.g. around 4.3 ng/mL), supplemented or not with FBS (e.g. around 5%) or 20% KOsr (e.g. around 20%) or B27. A representative culture medium according to the invention is described in the Example section below.

[00175] In a preferred embodiment, the culture medium used in step iii comprises the same components as the culture medium used in prior step i and/or step ii. It shall also be understood that the culture medium may be renewed, if deemed necessary, between step ii and step iii, and/or even between cell passages.

[00176] In some embodiments, said step (150) can comprise adding to the culture medium a sufficient amount of calcium salt to force cells to aggregate. In some embodiment, calcium salt is calcium chloride. In some embodiment calcium chloride is used at a final concentration of at least 50 mg/L, at least 100mg/L, at least 150 mg/L at least 200 mg/L, at least 250 mg/L, at least 500mg/L.

[00177] In some embodiment, aggregation of mammary cells can be spontaneous; this implies that cell density is sufficient to promote cell aggregation. In some embodiments, the cell concentration is at least of 10⁵ cells/ml, 10⁶ cells/ml, at least 10⁷ cells/ml, at least 10⁸ cells/ml, or at least 10⁹ cells/ml or even more.

Inducing differentiation of mammary cells (170)

[00178] In some embodiment, this step comprises culturing and passaging the mammary cells in a culture medium, for example a basal medium, like e.g. DMEM/F12 medium as described above.

[00179] In some embodiment, to obtain lactiferous units, aggregated mammary cells are placed in a differentiation medium.

[00180] In some embodiment, the culture medium is made e.g. of a basal medium such as DMEM/F12 complemented with glutamine, insulin transferrin selenium, and FGF2 (e.g. 2nM).

[00181] In a very particular embodiment, when producing human lactiferous units, EpiCult™ medium (StemCell Technologies) can be used for inducing differentiation.

[00182] Yet, in an even more preferred embodiment, especially when producing lactiferous units of the invention, the culture medium is DMEM/F12 supplemented with glutamine (e.g around 2.5 mM), hydrocortisone (e.g. around 2 pg/ml), insulin (e.g. around 0.17 mM), transferrin-selenium (e.g. around 6.87 pM), EGF (e.g. around 10 ng/mL), FGF2 (e.g. around 4.3 ng/ml), supplemented or not with FBS (e.g. around 5%) or 20% KOsr (e.g. around 20%) or B27. A representative culture medium according to the invention is described in the Example section below.

[00183] In a preferred embodiment, the culture medium used in step iv comprises the same components as the culture medium used in any or all of prior steps i to iii. It shall be understood that the culture medium may be renewed, if deemed necessary, between step ii and step iv or between step iii and step iv, and/or even between cells passages.

[00184] Also, should the same culture medium be used throughout all steps of the present method, one will understand that the differentiation step iv can be achieved by merely culturing and passaging the cells in said medium over a sufficient period of time for the present purpose. It is within the skill of the person in the art to determine such time period, for example by assessing cell lineage markers as described above, so as to achieve a differentiation into mammary epithelial cells, notably into mammary luminal epithelial cells, preferably into mammary secretory cells.

[00185] In some instance, the medium can be adapted as a function of e.g. the contemplated use or destination of the milk-like product to be produced. For example, it can be depleted of a particular compound or supplemented for another one. For example, in case where a milk- like product depleted or with a low content in phenylalanine is contemplated, then a medium with no or low content in phenylalanine can be used.

[00186] In some embodiments, lactiferous units are harvested and characterized regularly in time, for example every 2, 3, 4, 5 or even 6 days. The differentiation into secretory mammary cells and myoepithelial mammary cells can be assessed and quantified using, for example, markers of tables described herein such as previous Table 1 and/or 3.

[00187] In one embodiment, step (170) lasts at least 10 days, at least 15 days, or even at least 20 days, during which differentiation is assessed on a regular basis, using e.g., either FACS, RT-qPCR or immunochemistry imaging. In some embodiment, induction of differentiation lasts until a significant part of cells in mammary cell aggregates presents the features of secretory or myoepithelial mammary cells.

[00188] In one embodiment, lactiferous units can be assessed for the presence of markers of lactogenic differentiation as described in tables provided herein such as in Table 2.

Optional steps of encapsulating (130, 160) mammary cells in an hydrogel capsule

[00189] As mentioned above, though lactiferous units of the invention are found surprisingly resistant, to a certain extent, to shear stress and suitable to be culture in a suspension in a bioreactor, in the absence of any support or matrix or component(s) thereof (ECM), encapsulation may provide further protection and even favour mammary progenitor differentiation. Also, it is possible to habituate mammary cells to growth in suspension and to form aggregates.

[00190] Several techniques exist for encapsulating cells or cell aggregates, for example, ones make use of coaxial nozzle (see e.g. Horiguchi and Skai (2015)), of microfluidic tools (Wang et al., 2024), some of them being commercially available (Fluigent).

[00191] Also, in some embodiments, the method (100) of the invention for producing at least one lactiferous unit adapted to be cultured in suspension bioreactor may comprise an optional step of encapsulating (130) mammary cells before the step of amplifying (140) these cells, or a step of encapsulating (160) mammary cells aggregates before differentiation induction.

[00192] An encapsulation step (130) before step (140) of amplifying progenitors cells is particularly preferred.

[00193] In some embodiments, encapsulation means and method are set such that one mammary cell is encapsulated within the hydrogel capsule. In that case said cell can be a mammary progenitor cell as described above, such as a bi-potent mammary progenitor cell, a luminal mammary epithelial cell, or a mix of said cells. [00194] In some other embodiments, encapsulation is set such that a plurality of mammary cells, e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, or even more, are encapsulated within the hydrogel capsule.

[00195] In one embodiment, said mammary cells can be a mix of bipotent mammary progenitor cell, secretory mammary progenitor cells and/or myoepithelial mammary progenitor cells, provided that if no bipotent mammary progenitor cell is present is said mix, then both myoepithelial mammary progenitor cells and secretory mammary progenitor cells are present, and encapsulation step is set such that capsules statistically contains both types of progenitors.

[00196] In another embodiment, herein preferred, said mammary cells consist essentially of, or consist of, luminal mammary progenitor cells.

[00197] In some embodiments, encapsulation means and methods are set such that allowing the individual encapsulation of or the development of lactiferous units having at least one dimension of at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, but no more than 500 pm.

[00198] As exposed above, numerous suitable hydrogels exist and are suitable for encapsulating the lactiferous units of the invention. Suitable examples are selected from alginate, chitosan, silk, poly(vinyl alcohol), dextran, polyacrylamide, polyethylene glycol, pectin, guar gum, gelatin, zein, agar-methylcellulose, a cellulose derivative hydrogel (e.g., Carboxymethyl Cellulose (CMC), Hydroxypropyl Methyl Cellulose, Hydroxypropyl Cellulose (HPC), casein, or a mix thereof. Alginate (e.g. sodium or calcium alginate) is particularly preferred as it is well known and commonly used as a food grade encapsulating agent.

[00199] In some instance, encapsulation has been found effective in favouring amplification and differentiation of cells.

[00200] In some alternative embodiments, the further protection of lactiferous unit can be provided through their embedment into alginate beads. Such embedment is implemented after the formation of the lactiferous unit using methods well known in the art (Alsobaie et al, 2023). It will be understood that in these embodiments, alginate does not form a capsule (i.e. a hollow sphere within which the lactiferous unit is received) but a plain sphere of alginate hydrogel which comprises a lactiferous unit according to the invention.

[00201] Notwithstanding the above, as explained above, the present method is preferably free of exogenously added extracellular matrix component(s) (ECM), especially of animal-derived component(s) of the extracellular matrix (ECM). This means for example that the cells are preferably not encapsulated in an hydrogel such as Matrigel™ nor in an hydrogel containing collagen. Preliminary step of adapting mammary cells (110) to culture in suspension

[00202] Accordingly in an embodiment the method of producing at least lactiferous unit adapted to be cultured in suspension bioreactor, comprises a preliminary step of adapting (110) the at least one mammary cell to culture in suspension.

[00203] Said preliminary step (110) can comprise a sub step of cultivating the at least one mammary cell under adherent conditions. Once the appropriate confluency is reached (e.g. 70- 90%), then a sub step of disaggregating the aggregates or adherent mammary cells with a dissociation reagent and/or to a dissociation force is applied. According to some embodiments, the dissociation reagent comprises at least one proteolytic enzyme and optionally at least one DNA degrading enzyme and/or chelating agent. According to some embodiments, the dissociation reagent comprises at least one proteolytic enzyme, at least one DNA degrading enzyme and a chelating agent. According to certain embodiments, the chelating agent is selected from EDTA and EGTA. According to some embodiments, the proteolytic enzyme is trypsin. According to some embodiments, the dissociation force is a shear force. According to certain embodiments, the shear force rate is set by an impeller embedded within the vessel. According to some embodiments, following the dissociation sub step, viability of the cells is at least of 60%, at least of 70% even more preferably at least of 80%. Viability of cells can be assessed using any techniques known in the art.

[00204] According to certain embodiments, the sub step of disaggregating further comprises washing the homogenous aggregates or adherent cells prior to exposing said aggregates or adherent cells to the dissociation reagent and/or dissociation force with an aqueous-based washing medium.

[00205] In a further sub step, the dissociated cells are then resuspended into serum free medium (a basal medium as described above) in a vessel with no inactivated feeder cells or organic matrix or of any other means (e.g. non-adherent plate or non-adherent Erlenmeyer flask) adapted to promote cell adherence or aggregation.

[00206] According to some embodiments, the step of adapting mammary cells (110) to culture in suspension as described above is a sequential step : it means that several sub steps of cultivating and dissociating adherent cells are successively applied using decreasing concentrations of serum and/or of feeder cells and/or of organic matrix or of any other means adapted to promote adherence or aggregation of the cells at each round until reaching culture condition wherein no serum and no means for promoting cells adherence or aggregation are used, the resulting mammary cells being able to be cultivated in suspension. Method of in vitro producing (200) a milk-like product

[00207] The lactiferous units of the invention can freely float within the culture medium, thereby allowing an easy scaling up of production milk like product and component thereof directly in the culture medium.

[00208] Accordingly, another object of the invention lies in a method of in vitro producing (200) a milk-like product, comprising, or consisting essentially of: i. Providing, in suspension in a culture medium, at least one lactiferous unit (210) adapted to be cultured in suspension in a bioreactor, as described above, such as by implementing a method for producing at least one lactiferous unit adapted to be cultured in suspension, as described above, ii. Inducing lactation (220) by preferably adding to said medium prolactin at a preferred final concentration from about 0.1 to about 10 mg/L, iii. Maintaining lactation induction (230) till at least one milk component is produced in the cultured medium, said component being preferably selected from alpha-casein, betacasein, kappa-casein, lactoferrin, alpha-lactalbumin, a milk oligosaccharide, or any combination thereof, more preferably till the milk component reaches a target concentration in said culture medium, iv. Optionally, collecting the culture medium (250) which itself constitutes the milk-like product.

[00209] In an embodiment, the method of in vitro producing (200) a milk-like product according to the invention, comprises, or consists essentially of: i. Providing, in suspension in a culture medium, at least one lactiferous unit (210) adapted to be cultured in suspension in a bioreactor, as described above, such as by implementing a method for producing at least one lactiferous unit adapted to be cultured in suspension, as described above, ii. Inducing lactation (220) by adding to said medium prolactin at a preferred final concentration from about 0.1 to about 10 mg/L, iii. Maintaining lactation induction (230) till at least one milk component is produced in the cultured medium, said component being preferably selected from alpha-casein, betacasein, kappa-casein, lactoferrin, alpha-lactalbumin, or any combination thereof, more preferably till the milk component reaches a target concentration in said culture medium, iv. Optionally, collecting the culture medium (250) which itself constitutes the milk-like product. [00210] In an embodiment, the method of in vitro producing (200) a milk-like product according to the invention, comprises, or consists essentially of: i. Providing, in suspension in a culture medium, at least one lactiferous unit (210) adapted to be cultured in suspension in a bioreactor, ii. Inducing lactation (220) by adding to said medium prolactin at 0.1 to 10 mg/L, iii. Maintaining lactation induction (230) till at least one milk component selected from alphacasein, beta-casein, kappa-casein, lactoferrin and/or alpha-lactalbumin, or any combination thereof, reaches a target concentration in said culture medium, iv. Collecting the culture medium (250) which itself constitutes the milk-like product.

[00211] Exemplary embodiments of said method of in vitro producing (200) is illustrated in Figure 2.

Providing at least one lactiferous unit (210)

[00212] Any of the lactiferous units according to the invention as described above can be used in the method (200) of in vitro producing a milk-like product.

[00213] In said method (200) lactiferous units are cultured for at least 10 days, at least 15 days, or even at least 20 days, in a culture medium as described above.

[00214] Accordingly, the culture medium used herein typically comprises a basal medium, a proliferation supplement, and optionally a differentiation-promoting agent, each being as described above.

[00215] In some instance, the culture medium is a basal medium supplemented with FGF2, for example DMEM/F12 complemented with glutamine, insulin transferrin selenium, and FGF2 (e.g. 2nM) as described above.

[00216] Yet, in an even more preferred embodiment, especially when producing lactiferous units of the invention, the culture medium is DMEM/F12 supplemented with glutamine (e.g around 2.5 mM), hydrocortisone (e.g. around 2 pg/ml), insulin (e.g. around 0.17 mM), transferrin-selenium (e.g. around 6.87 pM), EGF (e.g. around 10 ng/mL), FGF2 (e.g. around) 4.3 ng/ml, supplemented or not with FBS (e.g. around 5%) or 20% KOsr (e.g. around 20%) or B27. A representative culture medium according to the invention is described in the Example section below.

[00217] In a preferred embodiment, the culture medium used herein comprises the same components as the culture medium used in the above-described method for producing a lactiferous unit adapted to the cultured in suspension. It shall nevertheless be understood that the culture medium may be renewed, if deemed necessary, between the two methods, and/or even between cell passages.

[00218] In some instance, the medium can be adapted as a function of e.g. the contemplated use or destination of the milk-like product to be produced. For example, it can be depleted of a particular compound or supplemented for another one. For example, in case where a milk-like product depleted or with a low content in phenylalanine is contemplated, then a medium with no or low content in phenylalanine can be used.

[00219] In some embodiments, said cultivation step can be made in a medium that has been conditioned with the coculture of other cell like, for example, adipocytes or stromal cells that produce growth factors or precursors which are taken up by the secretory mammary cells to produce milk like product component(s) derived therefrom (like e.g. in Darcy et al, 2000).

[00220] As exposed above, lactogenic differentiation can be checked by measuring gene expression levels of gene as listed in tables described herein such as table 2. Also, presence of differentiated mammary cells can be detected in the cultured lactiferous units by assaying markers of tables described herein such as Tables 1 , 2 and/or 3.

[00221] Induction of lactation can be applied after 2, 5, 10, 15 or 20 days of culture in the above medium, preferably after 10, 15 or 20 days of culture.

Inducing lactation (220) and Maintaining lactation induction (230)

[00222] Induction of lactation (220) is made by preferably adding prolactin in the medium, preferably at a final concentration from about 0.1 to about 10 mg/L, from about 0.5 to about 5 mg/L preferably abound 1mg/L. Induction of lactation step can last till a reference milk component reaches a target concentration.

[00223] In some instance, the medium can be adapted as a function of e.g. the contemplated use or destination of the milk-like product to be produced. For example, it can be depleted of a particular compound or supplemented for another one. For example, in case where a milk-like product depleted or with a low content in phenylalanine is contemplated, then a medium with no or low content in phenylalanine can be used.

[00224] In some embodiments, such reference milk component can be for example alphacasein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, milk oligosaccharides, or any combination thereof, or can be alpha-casein, beta-casein, kappa-casein, lactoferrin and/or, alphalactalbumin, or any combination thereof. Of course, depending on the species the secretory cells of the lactiferous unit derived from, some of these reference milk components can be secreted at a various level. [00225] In some other embodiments, said reference milk component can be a component specific to the species the cells comprised in the lactiferous unit relate to. For example, when using a lactiferous unit comprising human mammary secretory cells, said reference compound could be selected from compounds known to be specific to human milk as for example one of the Human Milk Oligosaccharides (HMO) listed in Table 4 below :

Table 4

[00226] It shall be noted that the lactiferous units of the invention can produce HMO without the addition of prolactin to the culture medium.

[00227] Detection methods for HMOs have recently been developed and validated using HILIC-FLD (e.g. as described in Ellingson et al (2022) or another AOAC described method (Benet et al, 2024)).

[00228] Presence of key milk components can be assessed in the spent media (casein isoforms (alpha, beta, kappa), lactoferrin, alpha-lactalbumin) using ELISA kits.

[00229] Triglycerides can be detected using kits such as the Triglyceride assay kit from Abeam (Ab65336). Additionally, Gas-Liquid chromatography can be used as the reference method to analyse the fatty acid composition following the standard methods described in ISO 16958 | IDF 231 :2015 - Milk, milk products, infant formula, and adult nutritional - Determination of fatty acid composition - Capillary gas chromatographic method.

[00230] Caseins account for 20 to 45% of the total protein content and whey proteins the majority remaining in human milk. ELISA kits for each specific target proteins can be used in combination to liquid chromatography coupled to UV or mass spectrometry. The quantification of Lactoferrin can be done following the standard AOAC method described in Ellingson et al (2019). Analytical Method for Lactoferrin in Milk-Based Infant Formulas by Signature Peptide Quantification with Ultra-High Performance LC-Tandem Mass Spectrometry. Quantification methods for lactalbumin and caseins in breastmilk have also been described before (Chen et al, 2016).

[00231] Commercially available MilkoScan™ devices by FOSS Analytics™ can be used to analyse main components of milk (fat fraction, proteins, lactose etc depending on the model) based on Fourier Transform Infrared spectroscopy. The device is compliant with both AOAC and IDF standards.

Collecting the culture medium (250) which itself constitutes the milk-like product

[00232] In some embodiment, collecting milk like product implies the separation of the lactiferous unit from the culture medium which itself constitutes the milk like product. Several techniques exist that are well known from the skilled in the art.

[00233] In some embodiments, cells can be separated from the liquid fraction by tangential flow filtration (aka cross-flow filtration). This is advantageous as it allows to filter large structures like lactiferous unit or encapsulated lactiferous units, while smaller particles as e.g. milk fat globules (ranging in size from 0.1 to 15 pm), or protein aggregates or micelles can pass through the filter.

[00234] In some other embodiments, methods like centrifugation, or sedimentation can be used. Typically, centrifugation can be performed of between 1500g and 5000g preferably between 2000g and 4000g, for example 3000g for 10 min. In an embodiment the centrifugation speed is chosen such as not breaking cells and/ or not breaking lactiferous units or encapsulated lactiferous unit, on order to avoid contaminating milk like product with cell debris. In another embodiment sedimentation can be performed by adding calcium salt to culture medium. The calcium salt may be selected from the group consisting of, but not limited to, calcium chloride, calcium acetate, calcium carbonate, calcium citrate and calcium lactate. Preferably, calcium chloride is used. The final concentration of the calcium chloride is in the range from 300mg/l to 500 mg/l. As a result, the biomass will be sediment in the bottom of the container and the supernatant can be gathered. Nonetheless such concentration of salts might have an impact of some compounds of the milk like product.

[00235] In some embodiments, the hydrogel capsule and/or the lactiferous unit are broken or dissociated, in order to gather compounds that might be sequestered either in the capsule and/or the lactiferous unit. This step of dissociating lactiferous unit (240) can be performed either before collecting the milk-like product, or on the hydrogel capsules and/or the lactiferous unit once harvested apart from the milk-like product. In an embodiment this can be achieved by submitting the lactiferous units to a shear stress sufficient to break lactiferous unit but not mammary cells. Regarding the hydrogel capsule, it can be broken either by chemical or physical means. Regarding especially alginate capsules or beads, they typically can be broken by using calcium sequestrant like citrate (e.g.3.2% sodium citrate solution) or the like.

[00236] In some embodiments, the method of in vitro producing (200) a milk-like product can also comprise a step of processing the milk-like product (260).

[00237] As exposed above, it is particularly advantageous that medium components are chosen so that the milk like product is eventually constituted, at the end of lactation induction step by the milk components produced by the lactiferous units and the components of the spent culture medium. In that regard, in some embodiments, component of culture medium that are chosen are food grade compounds. In some embodiments, they do not interfere with the yield of components secreted by the mammary cells of the lactiferous unit. That way, the spent medium gathered at the end of lactation induction step, which comprises components secreted and produced by the lactiferous units, is directly used as a milk-like product.

[00238] In some embodiments, anyway, notably in order to purify some milk components like, e.g. a HMO, a casein, a lactoferrin, a lactalbumin, whey protein, long chain polyunsaturated fatty acids or the like, some a further step of processing (260) the spent culture medium (milk-like product) can be applied. Processing can comprise, hydrolysing, centrifuging, extracting, filtrating, separating, concentrating, lyophilising, and/or precipitating said milk-like product or a fraction thereof. Said step can serve for example to purify from a particular component from the milk-like product or to enrich said milk-like product in said component. For example, fat globules, casein micelles, and whey proteins in the cell culture medium can be separated using microfiltration, ultrafiltration, and nanofiltration (Carter et al, 2021). Conversely, said methods can also be used to remove unwanted components, if present in the milk-like product (e.g. hormones or antibiotics, in the event such compound are used in the culture medium of lactiferous unit of the invention). In another example, said step of processing can comprise a step of enzymatically hydrolysing the milk-like product or a fraction thereof, to lowering or depleting said milk-like product of unwanted compounds. In some instance an extracting step comprising super critical extraction can be applied (Singh et al (2018)).

[00239] In some embodiment, the method of in vitro producing (200) a milk-like can comprise a step of supplementing culture medium, the milk-like product, with vitamin D, iron, selenium, at least one immunoglobulin, at least one HMO, long chain polyunsaturated fatty acids, the like, or any combination thereof. Also, the milk-like compound can be completed or adapted to particular needs, or uses. For example, HMOs, vitamin D selenium and the like are currently used to complement human infant milk formulas, and can therefore be used to complement milk like product of the invention, even if, when using lactiferous units comprising human mammary epithelial cells, said milk like product is closer to human breast milk than current ultra- processed infant milk formulas.

Clauses

[00240] Various aspects of the invention are also described in the following clauses:

[00241] 1. A lactiferous unit characterized in that it is adapted to be cultured in suspension in a bioreactor and it comprises epithelial mammary cells.

[00242] 2. The lactiferous unit adapted to be cultured in suspension bioreactor according to clause 1 , characterized in that said lactiferous unit is individually encapsulated within a hydrogel capsule.

[00243] 3. The lactiferous unit adapted to be cultured in suspension bioreactor according to clause 2, wherein said hydrogel is selected from alginate, collagen, fibrin, chitosan, silk, poly(vinyl alcohol), dextran, polyacrylamide, polyethylene glycol, hyaluronic acid, pectin, guar gum, gelatin, zein, agar-methylcellulose, a cellulose derivative hydrogel, casein, or a mix thereof, preferably alginate, therefore protecting said lactiferous unit from shear stress.

[00244] 4. The lactiferous unit adapted to be cultured in suspension bioreactor according to any one of clauses 1-3 wherein the epithelial mammary cells are selected from epithelial mammary cells of human origin, bovine origin, canine origin, feline origin, ovine origin, caprine origin, cetacean origin, seal origin, elephant origin, equidae origin, or a mix thereof, preferably selected from epithelial mammary cells of human origin.

[00245] 5. A method (100) for producing at least one lactiferous unit adapted to be cultured in suspension bioreactor according to any of preceding clauses comprising the steps of: i. Seeding (120) at least one progenitor of mammary cells in a suspension bioreactor, ii. Amplifying (140) said progenitor cells in suspension, iii. Optionally promoting cell aggregation (150), iv. Inducing differentiation of said progenitor cells (170) into epithelial mammary cells thereby obtaining adapted to be cultured in suspension bioreactor.

[00246] 6. The method (100) for producing at least one lactiferous unit according to clause

5 further comprising the step of encapsulating (130, 160) progenitor mammary cells in a hydrogel capsule selected from alginate, fibrin, chitosan, silk, poly(vinyl alcohol), dextran, polyacrylamide, polyethylene glycol, hyaluronic acid, pectin, guar gum, gelatin, zein, agar-methylcellulose, a cellulose derivative hydrogel, casein, or a mix thereof, preferably alginate.

[00247] 7. The method (100) for producing at least one lactiferous unit according to any one of clauses 5 or 6, wherein said at least one progenitor of mammary cells is a bipotent mammary progenitor cell.

[00248] 8. The method (100) for producing at least one lactiferous unit according to any one of clauses 5-7, wherein said at least one progenitor of mammary cells is isolated from milk or from mammary resection.

[00249] 9. The method (100) of in vitro producing a milk-like product according to any one of clauses 5-8, comprising a preliminary step of adapting progenitor of mammary cells (110) to culture in suspension.

[00250] 10. A method of in vitro producing (200) a milk-like product, comprising: i. Providing at least one lactiferous unit (210) adapted to be cultured in suspension in a bioreactor as specified in any one of clauses 1-4 in a culture medium in a suspension bioreactor unit, ii. Inducing lactation (230) by adding to said medium prolactin at a final concentration from 0.1 to 10 mg/L, iii. Maintaining lactation induction (240) till at least one milk component selected from alpha-casein, beta-casein, kappa-casein, lactoferrin and/or alpha-lactalbumin, or any combination thereof, reaches a target concentration in said culture medium, iv. Collecting the culture medium (250) which itself constitutes the milk-like product.

[00251] 11. The method of in vitro producing (200) a milk-like product according to clause

10, wherein the epithelial mammary cells of said at least one lactiferous unit comprise epithelial mammary cells of human origin and wherein the at least one milk component of step iii. is selected from alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin and/or at least one Human Milk Oligosaccharide (HMO), or of any combination thereof.

[00252] 12. The method of in vitro producing (200) a milk-like product according to clause

10 or 11 , further comprising supplementing culture medium or the milk-like product, with Vitamin D, Iron, selenium, at least one antibody, at least one HMO, at least one long chain polyunsaturated fatty acid.

[00253] 13. The method of in vitro producing (200) milk according to any one of clauses

10-12, further comprising a step of purifying at least one compound selected from a HMO, a casein, a lactoferrin, a lactalbumin, whey protein, long chain polyunsaturated fatty acids from said milk-like product.

[00254] 14. The methods (100, 200) of any one of clauses 5-13 wherein the culture medium is constituted of human-food grade components.

[00255] The present invention is further explained with the following non-limiting examples.

EXAMPLES

[00256] The data provided below have been generated using cells of human origin. This can be adapted for cells from other mammals.

EXAMPLE 1 : Production of lactiferous units from mammary cells isolated from milk

I. Isolation of mammary epithelial cells comprising mammary progenitor/stem cells and amplification of said cells

A. From maternal milk samples

[00159] Fresh (human) maternal milk was diluted 1 :2 in PBS pH 7.4 and centrifuged at 500 g for 20 minutes. Cells of the pellet were gathered and rinsed up to 4 times with PBS. This step is in order to remove most of the fat fraction contained in milk.

[00160] The cell suspension in PBS was then filtrated using a 40 pm cell strainer to obtain single cell suspension. Cells were then resuspended into appropriate culture medium supplemented or not with FBS or a serum-free alternative thereof (KOsr or B27) such as medium A which is complete DMEM/F12 complemented with insulin, transferrin-selenium, hydrocortisone EGF and FGF2 (medium A: DMEM/F12 with 2.5 mM L-glutamine, 2 pg/ml hydrocortisone, 0.17 mM insulin, 6.87 pM transferrin-selenium, 10 ng/mL EGF, 4.3 ng/ml FGF2, supplemented or not with 5% FBS or 20% KOsr or B27). Cells were cultured for 2 passages to enable their amplification.

[00161] The cell suspension solution can optionally be treated for debris removal using the Debris Removal Solution from Miltenyi Biotec following instructions from the manufacturer. Once the final step of resuspending the cells in their appropriate cell culture medium has been performed, cells are counted and seeded in a proper culture vessel.

B. Cell sorting (optional), maintenance and characterization

[00162] Optionally, in order to allow enrichment of specific cell populations, single cells can be analysed and sorted by immunochemistry coupled to flow-cytometry before seeding the cells of interest into appropriate culture vessels. Minimum 50,000 cells are resuspended in 100 pl of staining solution (2 pl of antibody diluted in PBS 1X for a final dilution of 1/50). Cells are left to incubate for 10 minutes at 4°C. Cells are then resuspended into their medium before being sorted using flow- cytometry associated cell sorting (Table 5 describes an example of a panel of antibodies used to characterize mammary cell populations). Cells positives for CD45 (hematopoietic cells), CD31 (endothelial cells) or CD235a (erythropoietic cells) are removed from the cell population. Mammary cell population can then also be sorted in order to enrich cell populations of interest, preferably cells expressing CD49f and with low to absent expression of EpCAM representing the progenitor/stem cells enriched population.

[00163] Example of antibodies panel that can be used for cell sorting of mammary cells from a mammary tissue sample such as a mammary resection, or from a milk sample, are listed in Table 5 below:

Table 5

[00164] For example, the combination of the following markers can allow the identification of different mammary cell populations, which may be present in milk or not, as shown in Table 6a below.

Table 6a

[00165] Here, the cells isolated from milk were analysed by flow-cytometry using CD49f and EpCAM markers, but not sorted. Briefly, 50,000 cells were resuspended in 100 pl of staining solution (2 pl of antibody diluted in PBS 1X for a final dilution of 1/50) as well as 6 pl of EpCAM and CD49f markers (Miltenyi Biotech). Cells were incubated with antibodies for 10 minutes at 4°C. Cells were washed once with PBS 1X. Analysis by flow cytometry depicted a mixed population of 88% luminal progenitor cells and 7% basal cells: the vast majority of cells isolated from milk were identified as being luminal progenitors (Figure 3A).

II. Cell aggregation

[00166] Each well of the 24-well format Aggrewell™ 400 plates was pre-treated with 500 pL of Anti-Adherence Rinsing Solution, centrifuged at 1300 x g for 5 minutes (swinging-bucket rotor), and rinsed with 500 pL of warm basal medium.

[00167] Cells were seeded into Aggrewell plates at a density of 130,000 cells/well in a total volume of 2 mL of complete medium. Unless mentioned otherwise, the complete medium used herein was medium A. Plates were centrifuged at 100 x g for 3 minutes for proper homogenisation of cell distribution in microwells.

III. Differentiation of aggregates

[00168] After 76h of aggregation, cell aggregates were harvested by flushing the wells with the medium of the wells, pooled in a 15ml Falcon tube and centrifuged at 200 x g for 5 min. The spheroids were then resuspended in 6 ml of fresh complete medium A, and 3 mL were seeded into two wells of a ULA 6-well plate. All tips and pipette were coated with 1X PBS + 2.5% BSA to avoid any 3D-structure to adhere to the plastic. Plates were placed in an incubator at 37°C, with 5% CO2 with shaking (85 rpm). Media was changed every 2 to 3 days by replacing % of the media by fresh complete media.

[00169] Lactiferous units were harvested after 16 days of culture and transferred in ULA- plates for further analysis. To do so, all pipet tips were coated with 2.5% BSA in 1X PBS and lactiferous units and media were collected into 15ml falcon tubes. Each well was rinsed with PBS 1X to retrieve any remaining lactiferous units at the bottom of each well. The 15 ml falcon tubes were then centrifuged at 200g for 5 minutes. The dry pellet of lactiferous units was used for RNA extraction and subsequent RT-qPCR analysis.

IV. Characterization of lactiferous units

[00170] Lactiferous units derived from mammary cells present in milk are shown in Figure 3B.

[00171] Once the lactiferous units were harvested, total RNA was extracted using a kit such as RNeasy Mini kit from Qiagen™ following manufacturer’s instructions or the RNA purification method using Trizol and chloroform solutions for phase separation. RNA was quantified and 200 ng to 2 pg have been retro transcribed into cDNAs. Level of expressions of each gene was evaluated by RT-qPCR using SYBR Green as a specific double stranded DNA binding dye allowing quantification of DNA molecules in real time.

[00172] Table 7 below lists all the genes used to characterize different stages of differentiation as well as lactation capability of the cells.

Table 7

[00173] Results of this RT-qPCR are displayed on Figure 3C.

[00174] Briefly, as shown in Figure 3D, CD24 and CD133 mRNA levels were slightly upregulated by a fold change between 1.3 and 1.7 while in contrast, KRT14 (CK14) and KRT18 (CK18) expression was reduced by 3-fold and 1.2-fold. Most importantly, expression of ELF5, a transcription factor associated with alveolar lineage commitment and milk-secreting cell differentiation, exhibited a robust induction with a 6-fold increase. Concurrently, as shown in Figure 3E, expression of key milk genes was increased such as LALBA (2.5-fold), LYZ (14-fold), FABP3 (2-fold), SPP1 (12-fold), FLIT2 (2-fold) which further confirmed lactogenic activation at the transcriptional level. IV. Conclusion

[00175] These results demonstrate that the differentiated aggregates were functional since they formed lactiferous units.

[00176] Culturing mammary cells, predominantly enriched in luminal progenitor cells, as aggregates in suspension as described herein induced transcriptional reprogramming of milk- derived mammary epithelial cells into a cell state capable of initiating a lactation-associated gene expression program. Such a system provides a reproducible method for producing lactation- competent lactiferous units in vitro in suspension using non-invasively sourced primary material.

EXAMPLE 2: Production of lactiferous units from mammary cells isolated from a mammary resection

[00177] Because the cellular composition of human milk differs from that of mammary tissue (it is notably less heterogenous with a lower density of cells, notably viable cells), the Inventors proceeded to apply the method disclosed in above-described Example 1 to a mammary resection sample.

EXAMPLE 2A

I. Isolation of mammary epithelial cells comprising mammary progenitor/ stem cells and amplification of said cells

A. From mammary resection

[00178] Tissue from (human) mammary resection was first manually dissected in order to remove most of the fat pads using a scalpel to obtain 1 mm² square pieces before washing the pieces with the wash solution composed of DMEM/F12 complemented with 1% BSA. Cells were then enzymatically dissociated using hyaluronidase and collagenase (1 mg/ml final concentration) overnight at 37°C and 100 rpm. Cells were then treated with DNase. Optionally, red blood cells lysis can be performed. Once cells were dissociated, these were filtered using a cell strainer and placed in complete cell culture medium such as complete DM EM/F 12 complemented with insulin and transferrin, EGF and FGF2 supplemented or not with 5% FBS (DMEM/F12 with 2.5 mM L- glutamine, 2 pg/ml hydrocortisone, 0,17 mM insulin, 6,87 pM transferrin, 10 ng/mL EGF and 4.3 ng/ml FGF2), or in other words in medium A.

[00179] Optionally, in order to allow enrichment of specific cell populations, single cells can be analysed by immunochemistry coupled to flow-cytometry before seeding the cells into appropriate culture vessels. Minimum 50000 cells are resuspended in 100 pl of staining solution (2 pl of antibody diluted in PBS 1X for a final dilution of 1/50. Cells are left to incubate for 10 minutes at 4°C. Cells are then resuspended into their medium before being sorted using flow- cytometry associated cell sorting using a similar panel than described before (Table 5). Cells positives for CD45 (hematopoietic cells), CD31 (endothelial cells) orCD235a (erythropoietic cells) are removed from the cell population. Mammary cell population can then also be sorted in order to enrich cell populations of interest, preferably cells expressing CD49f and with low to absent expression of EpCAM representing the progenitor/stem cells enriched population.

B. Cell sorting (Optional), maintenance and characterization

[00180] Once cells were placed in the appropriate culture vessel, they were left to recover, meaning when confluency has reached 70 to 90% and viability above 80%. Cells were then passaged at a regular frequency such as every 2 to 3 days.

[00181] Cell populations were regularly characterized by RT-qPCR, western blot, immunofluorescence coupled to flow-cytometry or immunostaining coupled to fluorescent microscopy (Tables 1, 2, 5, 8).

Analysing mammary cell populations using immunofluorescence coupled to flow-cytometry

[00182] Adherent cells were dissociated using TryplE for 15 minutes at 37°C. A minimum of 50,000 cells were resuspended in 100 pl of staining solution (2 pl of antibody diluted in PBS 1X for a final dilution of 1/50 or 1 pl of Viobility™ (Miltenyi Biotec) live/dead marker in PBS 1X). Cells were left to incubate for 10 minutes at 4°C. Cells are washed once with PBS 1X.

[00183] Mammary cell populations were analysed by flow-cytometry according to combinations of markers indicated in Table 6b below.

Table 6b

Analysing mammary cell populations using RT-qPCRs

[00184] Approximately 2 million cells were harvested, total RNA was extracted using a kit such as RNeasy Mini kit from Qiagen™ following manufacturer’s instructions or the RNA purification method using Trizol and chloroform solutions for phase separation. RNA was quantified and 500 ng to 1 pg have been retro transcribed into cDNAs. Level of expressions of each gene was evaluated by RT-qPCR using SYBR Green as a specific double stranded DNA binding dye allowing quantification of DNA molecules in real time. [00185] In Table 8 are listed all the genes used to characterize different stages of differentiation as well as lactation capability of the cells.

Table 8

C. Results - Cell isolation from a mammary resection

[00186] A mammary resection tissue sample was obtained following a mammary resection and gifted from a consenting donor. The tissue piece was dissected and cut into 1 mm² smaller pieces. Overnight digestion was performed as described above. After digestion, cells were washed and red blood cell lysis was performed. After lysis, approximately 1 million cells have been collected and seeded in a 24 well plate in DM EM supplemented with penicillin/streptomycin, amphotericin, 2.5 mM L-glutamine, 0,17 mM insulin, 6,87 pM transferrin, 10 ng/mL EGF and 4.3 ng/ml FGF2 with 5% FBS. After 3 days, cells had attached and reached 70% confluency. Cells were passaged and seeded in a 12 well plate format. Cells were then passaged every 2 or 3 days and amplified from 12 well plate format to 6 well plate, to T-flask format of 25 cm² (= T25-flask).

[00187] After 7 passages, cells from the 25-flask format were characterized by flowcytometry. Most of the cell population expressed CD49f and expressed very low amounts EpCAM which is characteristic of epithelial progenitor and basal cells. This cell population has been renamed Population_A for the remaining of the experiments.

D. Cell banking

[00188] In order to produce bank of cells of interests, cells were amplified until reaching 70 to 90% of confluency. Cells were counted and 1 million cells are banked in 1 ml of freezing medium composed of 40% basal media, 50% FBS and 10% DMSO. Alternatively, freezing medium or solutions such as the Serum free cell freezing medium from ATCC could be used. Cells were left at -80°C for 24 hours in a CoolCell container (Corning) and then placed in a liquid nitrogen tank or left in a cryobox in a -80°C or -150°C freezer.

[00189] Cells from Population_A were frozen in 40% DMEM, 50% FBS and 10% DMSO.

II. Adaptation to suspension (Optional)

[00190] In order to adapt the mammary cells to suspension, cells are cultivated until reaching confluency ranging from 70 to 90 %. Adherent cells culture medium is removed from the culture vessel and appropriate volume of PBS is added on top of the cells. Dissociating agent such as TrypLE is used to detach the cells from the plasticware. Once dissociated, cells are counted and viability is evaluated. Viability should be above 80%. Cells are then pelleted using centrifugation at 200g for 5 minutes and resuspended into serum free culture medium. Cells are then seeded in an ultra-low attachment plate or alternatively directly in a vented Erlenmeyer flask of 125 ml to reach a concentration of 1 to 3 x 10⁵ cells/ml in 30 ml of medium.

III. Culturing Lactiferous Units

A. Generation of lactiferous units in suspension

[00191] To assess the potential of generating lactiferous units of cells of interest, cells were first differentiated in suspension followed by induction of lactation. Mammary epithelial progenitor or stem cells, adapted or not to suspension, were amplified until reaching the desired cell density. This step could be performed in a T-flask, ultra-low attachment plate, shaking or spinner flask or bioreactor of choice, in suspension using a medium promoting proliferation of mammary progenitor cells such as Mammocult from StemCell Technologies or DMEM/F12 supplemented with calcium, insulin, transferrin, EGF and FGF2 (medium A).

[00192] Once the desired density was reached, cells were placed in differentiation conditions in a medium such as EpiCult medium (Stem Cell Technologies) supplemented, with 1 pg/ml hydrocortisone, or in DMEM/F12 supplemented with insulin, transferrin, EGF, FGF2, oestrogen, progesterone, hydrocortisone and prolactin, in order to promote differentiation into luminal and basal epithelial mammary cells. Medium was changed every 2 to 3 days.

[00193] To monitor differentiation stages and/or lactation potential, lactiferous units were characterized by RT-qPCR using similar panel of markers as described in Table 8. Differentiated mammary cells were expected to lose expression of CD49f, and to gain expression of EPCAM, CD133, CK14, CK15, P63, CK18, CD49f; Secretory cells were expected to express secretory and lactation related genes such as prolactin receptor (PRLR), CSN2, the gene coding for casein beta or LTF and LALBA respectively encoding lactoferrin and lactalbumin.

[00194] Cells from Population_A were amplified until reaching 70% confluency, corresponding to day -2. On that day, cells were placed in suspension in an Aggrewell 800 (StemCell Technologies) plate. 125000 cells were seeded per 24-well. After 48 hours, corresponding to day 0 of the differentiation process, spheroids were observed. Approximately 300 spheroids, corresponding to the number of microwell per 24-well were obtained. These spheroids from Population_A were then transferred in differentiation condition in ultra-low attachment (ULA) plates in EpiCult medium. Differentiating lactiferous units were maintained in culture for 14 days while medium was changed every 2 to 3 days to maintain nutrient availability. After 14 days, lactation was induced by addition of 1 pg/ml of prolactin and 1 pg/ml of hydrocortisone into the media. After 4 days of induction of lactation, cells and medium supernatant representing the milk-like product were harvested for analysis.

B. Characterization of lactiferous units

[00195] In order to assess differentiation and maturation of cells composing the lactiferous units in suspension, cells were characterized morphologically by microscopy as exemplified in Figure 4A. It was observed that cells aggregated when placed in Aggrewell. Upon induction of differentiation, it was observed that, in this experiment, cells had self-organized as an external layer of myoepithelial cells surrounding cells of epithelial morphology while maintaining their diameter and capacity to resist to suspension conditions (Figure 4A).

[00196] Further characterization was done by assessing expression of specific cell markers of progenitor cells, secretory epithelial cells and myoepithelial cells as well as proteins involved in lactation and/or corresponding to some key milk components (Tables 2, 3 8 and 9).

[00197] Once lactiferous units were harvested, total RNA was extracted using a kit such as RNeasy Mini kit from Qiagen™ following manufacturer’s instructions or the RNA purification method using Trizol and chloroform solutions for phase separation. RNA was quantified and 200 ng to 2 pg have been retro transcribed into cDNAs. Level of expressions of each gene was evaluated by RT-qPCR using SYBR Green as a specific double stranded DNA binding dye allowing quantification of DNA molecules in real time.

[00198] Result of RT-qPCR analysis performed on lactiferous units at D18 is reported in Table 9 below.

Table 9

[00199] These results confirm that a lactiferous units gathered at D18 underwent differentiation and comprised secretory and myoepithelial cells in a relevant quantity. IV. Analysis of milk components

A. Harvest of milk-like product

[00200] From suspension culture vessels, cells were separated from the liquid fraction by tangential flow filtration. The milk like product composition was then analysed before further processing or formulation steps.

B. Detection of milk components

[00201] Presence of key milk components was assessed in the spent media (casein isoforms (alpha, beta, kappa), lactoferrin, alpha-lactalbumin) using commercially available ELISA kits.

[00202] Presence of key milk components was assessed in the spent media (casein isoforms (alpha, beta, kappa), lactoferrin, alpha-lactalbumin). The results that were observed for the tested milk components are reported in Table 10 below :

Table 10

+ : detected in the milk like product of the invention

[00203] Lactoferrin was quantified using Human beta Casein ELISA Kit (ref A77920, antibodies.com), alpha-lactalbumin using Human alpha Lactalbumin ELISA Kit (A77524, antibodies.com), and lactoferrin using Human Lactoferrin ELISA Kit (ref A2094, antibodies.com)

[00204] Other components such as lipids and HMO (notably 2’FL) were assessed and detected in the medium.

V. Conclusion

[00205] Analyses confirmed the lactogenic differentiation of lactiferous unit of the invention, and the detection within the spent medium of compounds characteristic of milk, thereby ascertaining that lactiferous unit of the invention were able to produce milk-like product and/or components thereof. The above methods, applied to human mammary cells can be easily adapted to mammary cells from other mammalian species.

[00206] It was further observed that epithelial cells isolated from mammary resection were quicker to adapt to the culture conditions than cells isolated from milk. EXAMPLE 2B

In the present experiment, medium A was supplemented with a TGF-B inhibitor (TGF-Bi) at the differentiation stage.

I. Isolation of mammary epithelial cells comprising mammary progenitor/ stem cells and amplification of said cells

[00207] A mammary tissue sample was obtained from a mammary resection gifted from another consenting human donor. This tissue sample was first manually dissected using a scalpel to obtain 1 mm² square pieces; cells were then enzymatically dissociated and treated with DNase. Once single-cell suspensions were obtained, the cells were filtered with a cell strainer and pooled before performing red blood cells lysis.

[00208] For amplification, cells were cultured in complete cell culture medium A until reaching the desired cell density, as well as a high viability (above 80%). Cells were then passaged at a regular frequency such as every 2 to 3 days.

[00209] Cell populations were regularly characterized by RT-qPCR, western blot, immunofluorescence coupled to flow-cytometry and/or immunostaining coupled to fluorescent microscopy (Tables 1 , 2, 5, 8), according to the same protocol as described in Example 2A.

[00210] Just like in Example 2A, most of the cell population isolated from the mammary resection expressed CD49f as well as very low amounts EpCAM which is characteristic of epithelial progenitors and basal cells.

II. Cell aggregation

[00211] Each well of the 24-well format Aggrewell™ plates was pre-treated with AntiAdherence Rinsing Solution, centrifuged at 1300 x g for 5 minutes (swinging-bucket rotor), and rinsed with warm basal medium.

[00212] Cells were plated into Aggrewells at a density of 200,000 cells/well in a total volume of 2 mL of complete medium. The complete medium used herein was medium A. Plates were centrifuged at 100 x g for 3 minutes for proper homogenisation in microwells.

III. Differentiation of aggregates and induction of lactation

[00213] After 72h of aggregation, cell aggregates were harvested and put in suspension differentiation conditions in ultra-low attachment (ULA) plates containing medium A supplemented or not with a TGF-B inhibitor. To do so, two TGF-Bi were tested in combination: RepSox (25 pM final concentration), and SB431542 (10 pM final concentration). [00214] Differentiating lactiferous units were maintained in culture for 14 days after initiating the differentiation in ULA plates, while the medium was renewed every 2 to 3 days to maintain nutrient availability. Spheroids were observed under the microscope throughout this process, and lactation was induced by adding prolactin in the media at day 7. Spheroids were harvested 7 days after inducing lactation for further analysis.

IV. Characterization of lactiferous units

[00215] Microscopic inspection showed that the lactiferous units had self-organized.

[00216] Those differentiated in absence of TGF-Bi displayed an acinus-like profile, with a lumen cavity clearly visible with DAPI-staining. Immunostaining with anti-CD14 and anti-CD8 antibodies showed the clear presence of basal and luminal cells (data not shown). The prolactin receptor (PRLR) was detected by immunostaining in cells identified as being luminal, which is indicative of hormonal responsiveness (data not shown).

[00217] Lactiferous units differentiated in presence of TGF-Bi were more dense than those not treated with TGF-Bi (Figure 5A).

[00218] The lactiferous units were further characterized by flow cytometry and RT-qPCR.

[00219] Flow cytometry analysis of the cells contained in the lactiferous units and stained for EpCam and CD49f showed a clear predominance of luminal cells over basal cells, whether treated or not with TGF-Bi (data not shown).

[00220] TGF-Bi treatment strikingly led to an enrichment of the lactiferous units in luminal cells, by about 51% (from 17 to 68% of luminal cells) (data not shown).

[00221] Result of RT-qPCR analysis performed on lactiferous units at D14 is reported in Table 11 below.

Table 11 [00222] Table 11 shows a significant enhancement in expression of lactation markers in presence of TGF-Bi, notably of several markers of luminal secretory cells such as LTF, LALBA, LYZ, SPPA and FLIT2, especially of LYZ which is one the most abundant bioactive components of natural human milk (Figure 5B).

[00223] The introduction of a TGF-B inhibitor in the media therefore increases the differentiation efficiency of mammary cells into the luminal lineage, more particularly towards a luminal secretory lineage.

[00224] These results were confirmed by Western-Blot for Casein B and LALBA (data not shown).

V. Conclusion

[00225] TGF-Bi is particularly useful to optimize the lactogenic capacity of the lactiferous units of the invention, and hence the production of milk-like product.

EXAMPLE 3: Production of lactiferous units from mammary basal or luminal cells isolated from a mammary resection

[00226] To assess the functional contribution of basal and luminal cell populations to lactogenic secretion, lactiferous units were directly produced from luminal and/or basal cells isolated from mammary surgical resection of human donors.

EXAMPLE 3A

I. Isolation of mammary basal and luminal cells

[00227] In a first set of experiments, mammary epithelial cells were isolated as described previously, and were placed in complete medium A.

[00228] Some cell pellets were frozen for subsequent RNA extraction and RT-qPCR while other cells were sorted using the TYTO fluorescence-activated cell sorter (Miltenyi Biotech).

II. Sorting of luminal and basal compartments and amplification of cells

[00229] Cells were stained using an antibody mix of 1/50 dilution of CD10/CD24/CD49f/CD31/CD45/CD235, so as to characterize and sort the type of cells from each sample, as indicated in Table 12 below.

Table 12

[00230] Cells were then resuspended in 10 ml TYTO Running Buffer (Miltenyi Biotech). Prior to sorting, 200 pl were used for flow-cytometry analysis. Cell population gating for cell sorting was then performed on CD49f, CD24 and CD10 to separate the luminal population from the basal population. Sorted cells were then subjected again to flow-cytometry analysis to validate proper separation of cell populations.

[00231] To do so, basal cells were first sorted based on CDIO and CD49f to retain the positive fraction for CD10^high/CD49f^high, which correspond to basal progenitors. The negative fraction was then marked with an anti-CD24 antibody to retain the fraction of cells displaying a CD24^high signal, i.e. luminal cells. CD24 is indeed predominantly expressed by luminal epithelial cells, whether mature or progenitors.

[00232] For each separated population, some sorted cells were frozen for subsequent RT- qPCR analysis while others were amplified in medium A as described above before performing the aggregation and differentiation step.

III. Cell aggregation and differentiation

[00233] Each population was plated into Aggrewells as described above to attempt producing lactiferous units either from 100% luminal cells or from 100% basal progenitors.

[00234] Plates were centrifuged at 100 x g for 3 minutes to facilitate homogenised cell seeding in microwells.

IV. Differentiation of aggregates

[00235] After 48h of aggregation, cell aggregates were differentiated as described in Example 1 or 2A (i.e. in absence of TGF-pi), and cultured in ULA-plates for several days (2 days) before being harvested.

V. Characterization of lactiferous units

[00236] Morphological differences of the structures were observed depending on the type of cells used. Entirely basal-derived organoids were indeed smaller and less expanded than their luminal counterparts (Figure 6A).

[00237] Interestingly, RT-qPCR analysis revealed a partial acquisition of luminal lineage in the 100% basal (CD10⁺)-derived organoids, which is suggestive of phenotypic plasticity of basal progenitors. Lactation markers were indeed expressed on those lactiferous units (Figure 6B).

[00238] 100% luminal (CD24+)-derived lactiferous units expressed approximately 10-fold higher levels of LTF and LYZ compared to those that were 100% basal-derived, while markers such as FABP3 and FLIT2 were expressed at similar levels in those two types of organoids (Figure 6B).

VI. Conclusion

[00239] The present experiments confirm that differentiation into a luminal phenotype is important to obtain lactiferous units.

[00240] It also demonstrates that seeding a luminal lineage is sufficient to produce such lactiferous units. Yet, basal progenitors may also serve as a latent reservoir of luminal cells under permissive conditions; lactiferous units could thus be produced solely from basal progenitors although the differentiation process may require a longer time period than with luminal cells.

EXAMPLE 3B

I. Isolation of mammary basal and luminal cells

[00241] In another set of experiments, mammary epithelial cells were isolated as described previously, and isolated cells were placed in complete medium A.

[00242] Some cell pellets were frozen for subsequent RNA extraction and RT-qPCR while other cells were sorted using the TYTO fluorescence-activated cell sorter (Miltenyi Biotech).

II. Sorting of luminal and basal compartments and amplification of cells

[00243] Cells were stained using an antibody mix of 1/50 dilution of Epcam/CD49f/CD31/CD45/CD235, so as to characterize and sort the type of cells from each sample, as indicated in Table 13 below.

Table 13

[00244] Cells were then resuspended in 10 ml TYTO Running Buffer (Miltenyi Biotech). Prior to sorting, 200 pl were used for flow-cytometry analysis. Cell population gating for cell sorting was then performed on CD49f and EpCam to separate the luminal population (the majority of which was made of luminal progenitors) from the basal population. Sorted cells were then subjected again to flow-cytometry analysis to validate proper separation of cell populations. [00245] For each separated population, some sorted cells were frozen for subsequent RT- qPCR analysis while others were amplified in medium A as described above before performing the aggregation and differentiation step.

III. Cell aggregation and differentiation

[00246] Each population was plated into Aggrewells as described above to attempt producing lactiferous units either from 100% luminal cells, 100% basal cells or a mix of both types of cells.

[00247] Plates were centrifuged at 100 x g for 3 minutes to facilitate homogenised cell seeding in microwells.

IV. Differentiation of aggregates

[00248] After 48h of aggregation, cell aggregates were differentiated as described in Example 1 or 2A (i.e. in absence of TGF-pi), and cultured in ULA-plates for several days (between 9 to 11 days) before being harvested.

V. Characterization of lactiferous units

[00249] Like in Example 3A, morphological differences of the structures were observed depending on the type of cells used. Basal-derived organoids were denser/more compact than their luminal counterparts (Figure 7A).

[00250] RT-qPCR analysis revealed that lactation markers such as LTF, CD133, FLIT2, CNS2, PRLR, XDH, OLAH and/or LALBA were expressed by 100% luminal-derived lactiferous units as well as by lactiferous units derived from a mix of luminal and basal cells (Figure 7B).

[00251] Analysis of 100% basal-derived lactiferous units was not conclusive due to insufficient yield of RNA extracted from these compact organoids.

[00252] In addition, the ability of lactiferous units to secrete lactoferrin in the medium was assessed by western blot. The results showed that both lactiferous units obtained from 100% luminal cells and a mix of basal and luminal cells could secrete lactoferrin (Figure 7C).

VI. Conclusion

[00253] The present experiments indicate that lactiferous units can be generated from a mix of cells comprising luminal cells, notably luminal progenitor cells, and confirm that cells of luminal lineage are sufficient to produce lactiferous units. REFERENCES

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Claims

1. A method (100) for producing at least one lactiferous unit adapted to be cultured in suspension, comprising the steps of: i. Seeding (120) in suspension at least one mammary cell selected from a progenitor of mammary cells, a mammary luminal epithelial cell, or a mix of said cells, ii. Amplifying (140) said at least one mammary cell in suspension, iii. Optionally, promoting cell aggregation (150), and iv. Inducing differentiation of the cells (170) into epithelial mammary cells, if the mammary cell seeded at step i is a progenitor of mammary cells or a mix of said cells. thereby obtaining at least one lactiferous unit adapted to be cultured in suspension, such as in a suspension bioreactor.

2. The method (100) according to claim 1 , which is a method free of exogenous component(s) of the extracellular matrix (ECM).

3. The method (100) according to claim 1 or 2, wherein the at least one mammary cell seeded at step i is a progenitor of mammary cells.

4. The method (100) according to any one of claims 1 to 3, wherein the at least one progenitor of mammary cells is a bipotent or unipotent mammary progenitor cell, or a mix of said cells.

5. The method (100) according to any one of the preceding claims, wherein the progenitor of mammary cells is a luminal progenitor cell, a basal progenitor cell or a mix of said cells, preferably with the proviso that the basal progenitor cell is a bipotent progenitor cell.

6. The method (100) according to any one of the preceding claims, wherein the at least one mammary cell of step i is isolated from a mammary tissue sample or a milk sample.

7. The method (100) according to any one of the preceding claims, comprising: iii. promoting cell aggregation (150), preferably in a stirred-tank or orbital shaking or wave bioreactor.

8. The method (100) according to any one of the preceding claims, comprising: iv. inducing differentiation of the progenitor cells (170) into epithelial mammary cells, preferentially into mammary luminal epithelial cells, more preferably into mammary secretory cells.

9. The method (100) according to any one of the preceding claims, which is implemented in a culture medium comprising a basal medium, a proliferation supplement and preferably a differentiation-promoting agent .

10. The method (100) according to claim 9, wherein the differentiation-promoting agent comprises an agent promoting differentiation into mammary luminal epithelial cells, more preferably into mammary secretory cells.

11. The method (100) according to claim 10, wherein said agent is a TGF-beta inhibitor.

12. The method (100) according to any one of the preceding claims, comprising a step of encapsulating (130, 160) the cells in a hydrogel capsule.

13. A lactiferous unit characterized in that it is adapted to be cultured in suspension, such as in a suspension bioreactor, and in that it comprises mammary epithelial cells, said lactiferous unit being preferably obtained or obtainable according to the method as defined in any one of claims 1 to 12.

14. The lactiferous unit according to claim 13, wherein said mammary epithelial cells comprise myoepithelial cells and secretory cells.

15. A method of in vitro producing (200) a milk-like product, comprising:

(a) Providing at least one lactiferous unit (210) as defined in claim 13 or 14 in suspension in a culture medium, preferably in a suspension bioreactor unit, such as by implementing a method for producing at least one lactiferous unit adapted to be cultured in suspension as defined in any one of claims 1 to 12;

(b) Inducing lactation (230) by preferably adding to said medium prolactin at a preferred final concentration from 0.1 to 10 mg/L,

(c) Maintaining lactation induction (240) till at least one milk component is produced in the culture medium, said component being preferably selected from alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, a milk oligosaccharide, or any combination thereof, more preferably till the component reaches a target concentration in said culture medium,

(d) Optionally, collecting the culture medium (250) which itself constitutes the milk-like product.

16. The method according to claim 15, wherein the epithelial mammary cells of said at least one lactiferous unit comprise epithelial mammary cells of human origin and wherein the at least one milk component of step (c) is selected from alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, at least one Human Milk Oligosaccharide (HMO), or any combination thereof.

17. The method according to claim 15 or 16, further comprising supplementing the culture medium or the milk-like product with Vitamin D, Iron, selenium, at least one antibody, at least one HMO, at least one long chain polyunsaturated fatty acid, or a combination thereof.

18. The method according to any one of claims 15 to 17, further comprising a step of purifying at least one compound selected from a HMO, a casein, a lactoferrin, a lactalbumin, whey protein, long chain polyunsaturated fatty acids from said milk-like product.

19. The method (100, 200) of any one of the preceding claims, wherein the culture medium is constituted of human-food grade components.