EA046437B1 - DAIRY PRODUCT COMPOSITIONS - Google Patents

Description

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This application claims the benefit of priority to U.S. Provisional Application No. 63/030149 filed May 26, 2020, U.S. Provisional Application No. 63/200480 filed March 9, 2021, and U.S. Application No. 17/301216 filed March 29, 2021. the contents of each of which are incorporated herein by reference in their entirety.

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This application contains a sequence listing that has been presented in ASCII format and is therefore incorporated by reference in its entirety. The specified ASCII copy, created on May 24, 2021, is called BMQ_002WO_SL.txt and is 29447 bytes in size.

Field of technology to which the present invention relates

This disclosure relates to dairy product compositions that contain protein, lipid and oligosaccharide components and concentrations of components that mimic human breast milk produced by a lactating woman, wherein the dairy compositions are produced in vitro and/or ex vivo by cultured mammary cells.

BACKGROUND OF THE INVENTION

Milk is a staple food for humans, both in infancy and throughout life. The American Academy of Pediatrics and the World Health Organization recommend that infants be exclusively breastfed for the first 6 months of life, and consumption of dairy products after infancy is the mainstay of human nutrition, representing a $700 billion global industry. However, lactation is a labor-intensive and metabolically intensive process that can present biological and practical challenges for nursing mothers, and milk production is associated with environmental, social and animal welfare impacts in agricultural contexts.

The possibility of using mammalian cell cultures for food production has received increasing interest in recent years, and several successful prototype meat and seafood food products have been developed from cultured muscle and fat cells (Stephens et al. 2018 Trends Food Sci Technol. 78:155-166 ). In addition, work is underway to commercialize the production of egg and milk proteins using microbial expression systems. However, this fermentation-based method, involving expression of the genetically engineered product and purification of individual components, is unable to reproduce the complete molecular profile of milk or dairy products.

The essence of the invention

Disclosed herein in certain embodiments are methods for producing an isolated cultured dairy product from mammary cells, comprising: (a) culturing the cell construct in a bioreactor under conditions under which the cultured dairy product is produced, said cell construct comprising: (i) a three-dimensional scaffold, having an outer surface, an inner surface defining an inner cavity, and a plurality of pores extending from the inner surface to the outer surface; (ii) matrix material located on the outer surface of the three-dimensional scaffold; (iii) culture media located in the internal cavity and in liquid contact with the internal surface; (iv) many plasma cells located on the matrix material; and (v) a confluent monolayer of polarized mammary cells located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, wherein the polarized mammary cells contain an apical surface and basal surface; and (b) isolating the cultured dairy product. In some embodiments, the polarized mammary cell monolayer has a confluency of at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100%. In some embodiments, at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100% of the mammary cells are polarized in the same orientation. In some embodiments, the cultured dairy product contains secretory IgA (sIgA). In some embodiments, the bioreactor includes an apical compartment that is substantially isolated from the interior cavity of the cell construct. In some embodiments, the basal surface of the mammary cells is in fluid contact with the culture media. In some embodiments, the apical compartment is in fluid contact with the apical surface of the mammary cells. In some embodiments, the cultured milk product is secreted from the apical surface of the mammary gland cells into the apical compartment. In some embodiments, the culture media is not substantially contacted with the cultured dairy product. In some embodiments, the total mammary cell density in the bioreactor is at least 10 ¹¹ ; and alternatively, wherein the total surface area of the mammary cells in the bioreactor is at least 1.5 m ² In some embodiments, the total cell raft

- 1 046437 The number of plasma cells in the bioreactor is approximately 200 to 500 plasma cells per mm ² . In some embodiments, the cultivation is carried out at a temperature of from about 27°C to about 39°C. In some embodiments, cultivation is carried out at an atmospheric CO ₂ concentration of from about 4% to about 6%.

Disclosed herein in certain embodiments are cellular constructs comprising: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity/basal chamber, and a plurality of pores extending from the inner surface to the outer surface; (b) matrix material located on the outer surface of the three-dimensional scaffold; (c) culture media located in the internal cavity/basal chamber and in fluid contact with the internal surface; (d) a plurality of plasma cells located on the matrix material; and (f) at least a 70% confluent monolayer of polarized mammary cells located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells. In some embodiments, the polarized mammary cells comprise an apical surface and a basal surface. In some embodiments, the basal surface of the mammary cells is in fluid contact with the culture media. In some embodiments, at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100% of the mammary cells are polarized in the same orientation. In some embodiments, the polarized mammary cell monolayer has a confluency of at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100%. In some embodiments, the breast cells contain a constitutively active prolactin receptor protein. In some embodiments, the culture medium contains prolactin.

Disclosed herein in certain embodiments are cultured dairy products containing beta-casein, alpha-lactalbumin, kappa-casein, alpha^1-casein, lactoferrin, lactadherin, lysozyme, C12:0 (lauric acid), C14:0 (myristic acid). acid), C16:0 (palmitic acid), lactose, glucose, 3'-SL and 6'-SL; wherein the composition does not contain or is substantially free of persistent organic pollutants (POPs), heavy metals, prescription drugs, recreational drugs, allergens, cells, hormones or viruses, provided that the composition may contain human breast epithelial cell (hECM) or plasma cell (PC). In some embodiments, the cultured dairy product described herein contains C18:2n-6 (linoleic acid, LA), C18:3n-3 (alpha-linolenic acid, ALA), butrophilin, osteopontin, mucin-4, mucin- 1, C8:0 (caprylic acid), C10:0 (capric acid), C13:0 (tridecanoic acid, C16:1 (palmitoleic acid), C20:3n-6 (dihomo-gama-linolenic acid, DGLA), C20 :4p-6 (arachidonic acid, AA), C20:5p-3 (eicosapentanoic acid, EPA), C22:6p-3 (docosahexaenoic acid, DHA), C22:1p9 (erucic acid), C24:1 (nervonic acid) , LNT or LNnT. In some embodiments, the cultured dairy product described herein further contains at least one oxylipin metabolite of a polyunsaturated fatty acid or at least one endocannabinoid. In some embodiments, the oxylipin metabolite is a 9- hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 5,15-dihydroxyeicosatetraenoic acid (5,15-DiHETE), 17,18-dihydroxyeicosatetraenoic acid (17,18-DiHETE), 8,9- dihydroxyicosatrienoic acid (8,9-DiHETrE), 11,12dihydroxyicosatrienoic acid (11,12-DiHETrE), 9,10-dihydroxyoctadecenoic acid (9,10DiHOME), 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 14( 15)-epoxyeicosatrienoic acid (14(15)-EpETre), 19(20)-epoxydocosapentanoic acid (19(20)-EpDPE), 17hydroxydocosahexaenoic acid (17-HDoHE), 5-hydroxyeicosatetraenoic acid (5-HETE), 8hydroxyeicosatetraenoic acid (8-HETE), 9-hydroxyeicosatetraenoic acid (9-HETE), 11-hydroxyeicosatetraenoic acid (11-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 9-hydroxyoctadecatrienoic acid (9-HOTrE), 9-oxo-octadecadienoic acid (9 -okco-ODE) or 13-oxooctadecadienoic acid (13-okco-ODE), 17,18-epoxyeicosatetraenoic acid (17(18)EPETE, 6-keto-prostaglandin F1-alpha (6-keto-PGF1a) or 15^) -hydroxyeicosatrienoic acid (15(S)-HETrE). In some embodiments, the endocannabinoid is anandamide or 2-arachidonoylglycerol (2-AG). In some embodiments, the cultured dairy product described herein further comprises secretory IgA (sIgA). In some embodiments, the cultured dairy product contains no or substantially no persistent organic pollutants (POPs). In some embodiments, the cultured dairy product is free or substantially free of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), and pesticides. In some embodiments, the cultured dairy product contains no or substantially no DDT. In some embodiments, the culture

- 2 046437 prepared milk product does not contain or is substantially free of heavy metals. In some embodiments, the cultured dairy product contains no or substantially no mercury, lead, arsenic, cadmium, nickel, chromium, cobalt, or zinc. In some embodiments, the cultured dairy product contains no or substantially no pharmaceutical or recreational drugs. In some embodiments, the cultured dairy product contains no or substantially no chemotherapy agents, antidepressants, or anti-anxiety drugs. In some embodiments, the cultured dairy product contains no or substantially no benzodiazepines. In some embodiments, the cultured dairy product contains no or substantially no lithium, carbamazepine, chlorpromazine, and clozapine. In some embodiments, the cultured dairy product contains no or substantially no alcohol, marijuana, opioids, phencyclidine, or cocaine. In some embodiments, the cultured dairy product is free or substantially free of allergens. In some embodiments, the cultured dairy product is free or substantially free of ovomucoid, ovalbumin, conalbumin, arachine 6, arachine 3, conarachine, Arah1, and arachine Arah2. In some embodiments, the cultured dairy product does not contain or is substantially free of human stem cells, human immune cells, or bacterial cells, provided that the cultured dairy product contains one or more human mammary epithelial cells (hECBC) or plasma cells (PCs). In some embodiments, the cultured dairy product contains no or substantially no myoepithelial cells, myeloid progenitor cells, neutrophils, granulocytes, or T cells. In some embodiments, the cultured dairy product is free or substantially free of Staphylococcus, Acinetobacter, Streptococcus, Pseudomonas, Lactococcus, Enterococcus, or Lactobacillus. In some embodiments, the cultured dairy product is free or substantially free of virus. In some embodiments, the cultured dairy product is free or substantially free of hepatitis B virus, hepatitis C virus, cytomegalovirus, West Nile virus, human T-cell lymphotropic virus, and human immunodeficiency virus. In some embodiments, the cultured dairy product contains no or substantially no hormones. In some embodiments, the cultured dairy product contains no or substantially no leptin, ghrelin, adiponectin, thyroxine (T4), triiodothyronine (T3), thyroid-stimulating hormone (TSH), epidermal growth factor, beta-endorphin, relaxin, cortisol, or erythropoietin.

Disclosed herein in certain embodiments are cultured dairy products containing beta-casein, alpha-lactalbumin, kappa-casein, alpha^1-casein, lactoferrin, butrophilin, osteopontin, lactaderin, lysozyme, mucin-4, mucin-1, C8 :0 (caprylic acid), C10:0 (capric acid), C12:0 (lauric acid), C13:0 (tridecanoic acid), C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:2n-6 (linoleic acid, LA), C18:3n-3 (alpha-linolenic acid, ALA), C20:3n-6 (dihomo-gamalinolenic acid, DGLA), C20:4n-6 (arachidonic acid, AA), C20:5p-3 (eicosapentanoic acid, EPA), C22:6p-3 (docosahexaenoic acid, DHA), C22:1p9 (erucic acid), C24:1 (nervonic acid), lactose, glucose , 3'-SL, 6'-SL, LNT, LNnT, 9-hydroxyoctadecadienoic acid (9HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 5,15-dihydroxyeicosatetraenoic acid (5,15-DiHETE), 17,18 -dihydroxyeicosatetraenoic acid (17,18-DiHETE), 8,9-dihydroxyicosatrienoic acid (8,9-DiHETrE), 11,12-dihydroxyicosatrienoic acid (11,12-DiHETrE), 9,10-dihydroxyoctadecenoic acid (9,10- DiHOME), 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 14(15)-epoxyeicosatrienoic acid (14(15)-EpETre), 19(20)-epoxydocosapentanoic acid (19(20)-EpDPE), 17 -hydroxydocosahexaenoic acid (17-HDoHE), 5-hydroxyeicosatetraenoic acid (5-HETE), 8-hydroxyeicosatetraenoic acid (8-HETE), 9-hydroxyeicosatetraenoic acid (9-HETE), 11-hydroxyeicosatetraenoic acid (11-HETE), 12-hydroxyeicosatetraenoic acid (12- HETE), 9-hydroxyoctadecatrienoic acid (9-HOTrE), 9-oxooctadecadienoic acid (9-okco-ODE), or 13-oxooctadecadienoic acid (13-okco-ODE), 17,18-epoxyeicosatetraenoic acid (17(18)-EPETE, 6-keto-prostaglandin F1-alpha (6-keto-PGF1a) or 15^)-hydroxyeicosatrienoic acid (15(S)-HETrE), and sIgA, where the composition does not contain or is substantially free of persistent organic pollutants (POPs), heavy metals, prescription drugs, recreational drugs, allergens, cells, hormones or virus, provided that the composition may contain human mammary epithelial cells or plasma cells. In some embodiments, the cultured dairy product described herein further contains a preservative, stabilizer, antioxidant, emulsifier, or thickener. In some embodiments, the cultured dairy product described herein further contains lecithin. In some embodiments, the cultured dairy product described herein further comprises carrageenan. In some embodiments, the cultured milk

- 3 046437 The product described herein additionally contains beta-carotene. In some embodiments, the cultured dairy product described herein further comprises vitamin E. In some embodiments, the cultured dairy product described herein further comprises ascorbyl palmitate or ascorbic acid. In some embodiments, the cultured dairy product described herein further contains lecithin, ascorbyl palmitate, and vitamin E. In some embodiments, the cultured dairy product described herein is dehydrated, lyophilized, or frozen. In some embodiments, the cultured dairy product described herein is a food product.

Disclosed herein in specific embodiments are methods of feeding an individual in need thereof comprising administering to the individual a cultured dairy product disclosed herein. In some embodiments, the individual is a human. In some embodiments, the person is an infant. In some embodiments, the person has an immunodeficiency. In some embodiments, the individual has a disease selected from severe combined immunodeficiency (SCID), HIV/AIDS, cancer, or an autoimmune disease. In some embodiments, the individual has lupus or diabetes mellitus (eg, type I diabetes mellitus or type II diabetes mellitus). In some embodiments, the person is an organ or bone marrow transplant recipient. In some embodiments, the person is suffering from malnutrition. In some embodiments, the person has malabsorption syndrome. In some embodiments, the person has wasting syndrome. In some embodiments, the person is elderly or senile.

Disclosed herein in specific embodiments are methods of treating or preventing a microbial infection in an individual in need thereof, comprising administering to the individual a cultured dairy product disclosed herein. In some embodiments, the infection is a bacterial infection, fungal infection, or parasitic infection. In some embodiments, the bacterial infection is an infection of E. con, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Salmonella, Shigella, Campylobacter, Staphylococcus aureus, Helicobacter pylori, C. difficile, or Vibrio holerae. In some embodiments, the parasitic infection is an infection of Giardia lamblia, Entameba histolytica, Cryptosporidium spp. or Cystoisospora belli. In some embodiments, the fungal infection is an Aspergillus, Cryptococcus, or Pneumocystis jirovecii infection. In some embodiments, the microbial infection is a gastrointestinal infection. In some embodiments, the gastrointestinal infection is gastroenteritis. In some embodiments, the microbial infection is candidiasis.

Disclosed herein in specific embodiments are methods for producing sIgA from mammary gland cells, a method comprising: (a) culturing the cell construct in a bioreactor under conditions under which a cultured dairy product is produced, said cell construct comprising: (i) a three-dimensional scaffold, having an outer surface, an inner surface defining an inner cavity, and a plurality of pores extending from the inner surface to the outer surface; (ii) matrix material located on the outer surface of the three-dimensional scaffold; (iii) culture media located in the internal cavity and in liquid contact with the internal surface; (iv) many plasma cells located on the matrix material; and (v) a confluent monolayer of polarized mammary cells located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, wherein the polarized mammary cells contain an apical surface and basal surface; and (b) isolating sIgA from the cultured dairy product. In some embodiments, the polarized mammary cell monolayer has a confluency of at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100%. In some embodiments, at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100% of the mammary cells are polarized in the same orientation. In some embodiments, the cultured dairy product contains secretory IgA (sIgA). In some embodiments, the bioreactor includes an apical compartment that is substantially isolated from the interior cavity of the cell construct. In some embodiments, the basal surface of the mammary cells is in fluid contact with the culture media. In some embodiments, the apical compartment is in fluid contact with the apical surface of the mammary cells. In some embodiments, the cultured milk product is secreted from the apical surface of mammary gland cells to the apical

- 4 046437 division. In some embodiments, the culture media is not substantially contacted with the cultured dairy product. In some embodiments, the total mammary cell density in the bioreactor is at least 10 ¹¹ ; and alternatively, wherein the total surface area of the mammary gland cells in the bioreactor is at least 1.5 m ² . In some embodiments, the total cellular density of plasma cells in the bioreactor is from about 200 to 500 plasma cells per mm ² . In some embodiments, the cultivation is carried out at a temperature of from about 27 to about 39°C. In some embodiments, cultivation is carried out at an atmospheric CO ₂ concentration of from about 4 to about 6%.

Disclosed herein in specific embodiments are pharmaceutical compositions comprising (a) sIgA and (b) a pharmaceutically acceptable excipient; wherein sIgA is produced by the method described herein. In some embodiments, the pharmaceutically acceptable excipient is a stabilizer, surfactant, buffer, or tonicity agent. In some embodiments, the pharmaceutically acceptable excipient is sucrose, trehalose, mannitol, sorbitol, histidine, arginine, glycine, polysorbate 20, polysorbate 80, poloxamer 188, ethylenediaminetetraacetic acid or salts thereof (e.g., EDTA), glutathione, metacresol, phenol, benzyl alcohol, benzalkonium chloride, methionine or cysteine.

Disclosed herein in specific embodiments are methods of treating or preventing a microbial infection in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising (a) sIgA and (b) a pharmaceutically acceptable excipient; wherein sIgA is produced by the method described herein. In some embodiments, the pharmaceutically acceptable excipient is a stabilizer, surfactant, buffer, or tonicity agent. In some embodiments, the pharmaceutically acceptable excipient is sucrose, trehalose, mannitol, sorbitol, histidine, arginine, glycine, polysorbate 20, polysorbate 80, poloxamer 188, ethylenediaminetetraacetic acid or salts thereof (e.g., EDTA), glutathione, metacresol, phenol, benzyl alcohol, benzalkonium chloride, methionine or cysteine. In some embodiments, the person is an infant. In some embodiments, the person has an immunodeficiency. In some embodiments, the individual has a disease selected from severe combined immunodeficiency (SCID), HIV/AIDS, cancer, or an autoimmune disease. In some embodiments, the individual has lupus or diabetes mellitus (eg, type I diabetes mellitus or type II diabetes mellitus). In some embodiments, the person is an organ or bone marrow transplant recipient. In some embodiments, the person is suffering from malnutrition. In some embodiments, the person has malabsorption syndrome. In some embodiments, the person has a wasting syndrome. In some embodiments, the person is elderly or senile. In some embodiments, the person has cystic fibrosis, COPD, or bronchiectasis without cystic fibrosis. In some embodiments, the infection is a bacterial infection, fungal infection, or parasitic infection. In some embodiments, the bacterial infection is an infection of E. coli, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Salmonella, Shigella, Campylobacter, Staphylococcus aureus, Helicobacter pylori, C. difficile, or Vibrio holerae. In some embodiments, the parasitic infection is an infection of Giardia lamblia, Entameba histolytica, Cryptosporidium spp. or Cystoisospora belli. In some embodiments, the fungal infection is an Aspergillus, Cryptococcus, or Pneumocystis jirovecii infection. In some embodiments, the microbial infection is a gastrointestinal infection. In some embodiments, the gastrointestinal infection is gastroenteritis. In some embodiments, the microbial infection is candidiasis. In some embodiments, the microbial infection is a respiratory infection. In some embodiments, the respiratory infection is pneumonia, bronchitis, aspergillosis, or cryptococcosis. In some embodiments, the respiratory infection is an infection caused by B. cepacia, P. aeruginosa, S. aureus, Aspergillus, Cryptococcus, or Pneumocystis. In some embodiments, the composition is formulated for inhalation. In some embodiments, the composition is a powder. In some embodiments, the composition is a formulation for administration via a nebulizer.

The present disclosure relates to milk compositions that contain protein, lipid and oligosaccharide components and concentrations of components that mimic and/or are substantially similar to human breast milk produced by a lactating woman, wherein the milk compositions are produced in vitro and/or ex vivo by cultured milk cells glands.

Thus, one aspect of the disclosure relates to dairy products containing certain concentrations or amounts by weight of protein components, lipid components, human milk oligosaccharide components and lactose, wherein at least one of the protein components

- 5,046,437 tom, lipid components, HMO and lactose are produced by cultured human mammary epithelial cells.

In one embodiment, disclosed herein are methods for producing an isolated cultured dairy product from mammary gland cells, a method comprising: (a) culturing the cell construct in a bioreactor, under conditions capable of producing a cultured dairy product, said cell construct comprising: (i ) a three-dimensional frame having an outer surface, an inner surface delimiting an internal cavity, and a plurality of pores extending from the inner surface to the outer surface; (ii) matrix material located on the outer surface of the three-dimensional scaffold; (iv) many plasma cells located on the matrix material; and (v) a confluent monolayer of polarized mammary cells located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells, wherein the polarized mammary cells contain an apical surface and basal surface; and (b) isolating the cultured dairy product. In some embodiments, the cultured dairy product contains secretory IgA (sIgA).

In some embodiments, the bioreactor includes an apical compartment that is substantially isolated from the interior cavity of the cell construct. In some embodiments, the basal surface of the mammary gland cell is in fluid contact with the culture media. In some embodiments, the apical compartment is in fluid contact with the apical surface of the mammary cells. In some embodiments, the cultured milk product is secreted from the apical surface of the mammary gland cells into the apical compartment. In some embodiments, the culture media is not substantially contacted with the cultured dairy product. In some embodiments, the total cellular density of mammary cells in the bioreactor is at least 10 ¹¹ . In some embodiments, the total surface area of the mammary gland cells in the bioreactor is at least 1.5 m ² . In some embodiments, the matrix material contains one or more extracellular matrix proteins. In some embodiments, the total cellular density of plasma cells in the bioreactor is from about 200 to 500 plasma cells per mm ² . In some embodiments, the cultivation is carried out at a temperature of from about 27 to about 39°C. In some embodiments, cultivation is carried out at an atmospheric CO ₂ concentration of from about 4 to about 6%.

Specific embodiments herein describe cellular constructs comprising: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity/basal chamber, and a plurality of pores extending from the inner surface to the outer surface; (b) matrix material located on the outer surface of the three-dimensional scaffold; (c) culture media located in the internal cavity/basal chamber and in fluid contact with the internal surface; and (d) a plurality of plasma cells located on the matrix material; and (e) an at least 70% confluent monolayer of polarized mammary cells arranged on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells. In some embodiments, the polarized mammary cells comprise an apical surface and a basal surface. In some embodiments, the basal surface of the mammary cells is in fluid contact with the culture media.

In some embodiments, at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100% of the mammary cells are polarized in the same orientation. In some embodiments, the polarized mammary cell monolayer has a confluency of at least 70%, at least 80%, at least 90%, at least 95%, at least 99, or 100%. In some embodiments, the breast cells contain a constitutively active prolactin receptor protein. In some embodiments, the culture medium contains a carbon source, a chemical buffer system, one or more essential amino acids, one or more vitamins and/or cofactors, and one or more inorganic salts. In some embodiments, the culture medium further contains prolactin. In some embodiments, the matrix material contains one or more extracellular matrix proteins. In some embodiments, the three-dimensional scaffold comprises a natural polymer, a biocompatible synthetic polymer, a synthetic peptide, a composite material derived from any of the foregoing, or any combination thereof. In some embodiments, the natural polymer is collagen, chitosan, cellulose, agarose, alginate, gelatin, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, and/or hyaluronic acid. In some embodiments, the biocompatible synthetic

- 6 046437 The polymer is polysulfone, polyvinylidene fluoride, vinyl acetate-polyethylene copolymer, polyvinyl alcohol, sodium polyacrylate, acrylate polymer and/or polyethylene glycol.

In certain aspects, a dairy product is described herein containing approximately 6-14 g per liter (g/L) of protein components, approximately 18-89 g/L of lipid components, approximately 7-14 g/L of human milk oligosaccharides (HMO), approximately 64-77 g/L lactose, wherein at least one of protein components, lipid components, HMO and lactose is produced by cultured human mammary epithelial cells.

In some embodiments, the dairy product further contains one or more immunoglobulins. In some embodiments, the immunoglobulin is secretory IgA.

In some embodiments, the protein component comprises approximately 55-65% dry weight of the dairy product. In some embodiments, the protein component comprises beta casein, kappa casein, and alpha casein. In some embodiments, beta casein has a concentration of approximately 0.5-1.5 g/L, kappa casein has a concentration of approximately 0.5-0.6 g/L, alpha casein has a concentration of approximately 0.5 g/L , 1-0.5 g/l in a dairy product. In some embodiments, beta casein, kappa casein, and alpha casein together comprise approximately 35-45% of the dry weight of the protein component of the dairy product. In some embodiments, beta casein makes up more than about 50% of the total casein content.

In some embodiments, the protein component further comprises one or more of alpha-lactalbumin, lysozyme, lactoferrin, haptocorrin, butyrophilin, osteopontin, MC5 mucin, BrE3 mucin, and lactadherin. In some embodiments, the protein component further comprises serum albumin. In some embodiments, alphalactalbumin has a concentration of approximately 2.7-3.3 g/L in the dairy product. In some embodiments, lysozyme has a concentration of approximately 0.2-0.5 g/L in the dairy product. In some embodiments, lactoferrin has a concentration of approximately 1.0-2.0 g/L in the dairy product. In some embodiments, haptocorrin has a concentration of approximately 0.07-0.7 g/L in the dairy product. In some embodiments, butyrophilin has a concentration of approximately 0.03-0.05 g/L in the dairy product. In some embodiments, osteopontin has a concentration of approximately 0.05-0.2 g/L in the dairy product. In some embodiments, the MC5 mucin has a concentration of approximately 0.5-0.6 g/L in the dairy product. In some embodiments, the BrE3 mucin has a concentration of approximately 0.5-0.7 g/L in the dairy product. In some embodiments, lactadherin has a concentration of approximately 0.06-0.07 g/L in the dairy product. In some embodiments, the serum albumin has a concentration of about 0.025-3.5 g/L, or about 0.01-2 g/L, or about 0.15-1 g/L, or about 0.2-0.7 g /l. In some embodiments, the protein components are of human origin.

In some embodiments, the lipid component comprises one or more of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, phospholipids, plasmalogens, and sphingolipids. In some embodiments, the saturated fatty acids have a concentration of approximately 5-34 g/L in the dairy product. In some embodiments, the saturated fatty acids comprise palmitic acid, stearic acid, and lauric acid. In some embodiments, palmitic acid contains at least about 50% sn-2 form. In some embodiments, palmitic acid has a concentration of approximately 3-18 g/L in the dairy product. In some embodiments, stearic acid has a concentration of approximately 0.7-5 g/L in the dairy product. In some embodiments, lauric acid has a concentration of approximately 0.5-5 g/L in the dairy product. In some embodiments, the monounsaturated fatty acids have a concentration of approximately 7-46 g/L in the dairy product. In some embodiments, the monounsaturated fatty acid comprises oleic acid. In some embodiments, oleic acid contains at least about 50% sn-1 form. In some embodiments, oleic acid has a concentration of approximately 7-45 g/L in the dairy product. In some embodiments, the polyunsaturated fats have a concentration of approximately 2-20 g/L in the dairy product. In some embodiments, the polyunsaturated fats contain one or more of linoleic acid, alpha-linolenic acid, eicosadienoic acid, arachidonic acid, dihomo-gamma-linolenic acid, and docosahexadienoic acid. In some embodiments, linoleic acid has a concentration of approximately 2-19 g/L in the dairy product. In some embodiments, linoleic acid contains at least about 50% sn-3 form. In some embodiments, alpha-linolenic acid has a concentration of approximately 0.5-0.7 g/L in the dairy product. In some embodiments, eicosadienoic acid has a concentration of approximately 0.50.7 g/L in the dairy product. In some embodiments, the arachidonic acid has a concentration of approximately 0.5-0.7 g/L in the dairy product. In some embodiments, di

- 7 046437 homo-gamma-linolenic acid has a concentration of approximately 0.3-0.5 g/l in the dairy product. In some embodiments, docosahexadienoic acid has a concentration of approximately 0.02-0.4 g/L in the dairy product. In some embodiments, linoleic acid has a concentration of about 2-19 g/L, alpha-linolenic acid has a concentration of about 0.5-0.7 g/L, eicosadienoic acid has a concentration of about 0.5-0.7 g/L, arachidonic acid has a concentration of approximately 0.5-0.7 g/L, dihomo-gamma-linolenic acid has a concentration of approximately 0.3-0.5 g/L and docosahexadienoic acid has a concentration of approximately 0.02-0.4 g/L l in a dairy product. In some embodiments, the dairy product contains oleic acid, palmitic acid, and linoleic acid, wherein oleic acid contains at least about 50% sn-1 form, palmitic acid contains at least about 50% sn-2 form, and linoleic acid contains at least about 50% sn-2 form. at least approximately 50% of the sn-3 form. In some embodiments, the dairy product contains cholesterol having a concentration of approximately 0.09-0.15 g/L in the dairy product. In some embodiments, the phospholipids, plasmalogens, and sphingolipids together have a concentration of approximately 0.1-0.4 g/L in the dairy product.

In some embodiments, the oligosaccharide component of human milk contains one or more neutral oligosaccharides. In some embodiments, the oligosaccharide component of human milk contains one or more acid oligosaccharides. In some embodiments, the oligosaccharide component of human milk contains neutral oligosaccharides and/or acidic oligosaccharides. In some embodiments, the one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexose), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-N-hexaose), LNFPI (lacto-N-fucopentaose I), LNDFHI (lacto-N-difucosylhexaose I), LNT (lacto-N-tetraose), LNnT (lacto-N-neotetraose), DF-L (difucosyllactose) and 3-FL (3-fucosyllactose).

74. A dairy product according to claim 65 or 66, in which one or more acid oligosaccharides contain 6'-SL (6'-sialyllactose), DS-LNT (disialyl-lacto-N-tetraose), FS-LNnHI (fucosyl-sialyl-lactoN -neohexaose I), LSTc (sialyl-lacto-N-tetraose c) and 3'-SL (3'-sialyllactose). In some embodiments, one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexose), the oligosaccharide having a concentration of approximately 1-4 g/L in the dairy product. In some embodiments, one or more neutral oligosaccharides comprise 2'-FL (2'-fucosyllactose), wherein the oligosaccharide has a concentration of approximately 1-4 g/L in the dairy product. In some embodiments, one or more neutral oligosaccharides comprise DF-LNHII (difucosyllacto-N-hexaose), wherein the oligosaccharide has a concentration of approximately 1-4 g/L in the dairy product. In some embodiments, one or more neutral oligosaccharides comprise LNFP I (lacto-N-fucopentaose I), the oligosaccharide having a concentration of approximately 0.5-2 g/L in the dairy product. In some embodiments, one or more neutral oligosaccharides comprise LNDFHI (lacto-N-difucosylhexaose I), the oligosaccharide having a concentration of approximately 0.2-2 g/L in the dairy product.

80. A dairy product according to any one of claims 64, 66, 67 or 70-74, in which one or more neutral oligosaccharides contain LNT (lacto-N-tetraose), and the oligosaccharide has a concentration of approximately 0.3-1.5 g/ l in a dairy product. In some embodiments, one or more neutral oligosaccharides comprise LNnT (lacto-N-neotetraose), the oligosaccharide having a concentration of approximately 0.5-1.5 g/L in the dairy product. In some embodiments, one or more neutral oligosaccharides comprise DF-L (difucosyllactose), the oligosaccharide having a concentration of approximately 0.1-1 g/L in the dairy product. In some embodiments, the one or more neutral oligosaccharides comprise 3-FL (3-fucosyllactose), the oligosaccharide having a concentration of approximately 0.2-1.5 g/L in the dairy product. In some embodiments, the one or more acid oligosaccharides comprise 6'-SL (6'sialyllactose), wherein the oligosaccharide has a concentration of approximately 0.2-1.2 g/L in the dairy product. In some embodiments, one or more acid oligosaccharides comprise DSLNT (disialyl lacto-N-tetraose), the oligosaccharide having a concentration of approximately 0.05-1 g/L in the dairy product. In some embodiments, one or more acid oligosaccharides comprise FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), wherein the oligosaccharide has a concentration of approximately 0.05-0.7 g/L in the dairy product. In some embodiments, one or more acid oligosaccharides comprise LST c (sialyl-lacto-N-tetraose c), wherein the oligosaccharide has a concentration of approximately 0.05-0.7 g/L in the dairy product. In some embodiments, one or more acid oligosaccharides comprise 3'-SL (3'-sialyllactose), wherein the oligosaccharide has a concentration of approximately 0.1-0.3 g/L in the dairy product.

In some embodiments, the one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexaose), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-N-hexaose), LNFPI (lacto-N-fucopentaose I) , LNDFHI (lacto-N-difucosylhexaose I), LNT (lacto-N-tetraose), LNnT (lacto-N-neotetraose), DF-L (difucosyllactose) and 3-FL (3-fucosyllactose) and where one

- 8 046437 or several acidic oligosaccharides contain 6'-SL (6'-sialyllactose), DS-LNT (disialyl-lacto-Ntetraose), FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), LST c (sialyl-lacto -H-tetraose c) and 3'-SL (3'-sialyllactose). In some embodiments, the dairy product contains about 1-4 g/L TF-LNH (trifucosyllacto-H-hexaose), about 1-4 g/L 2'-FL (2'-fucosyllactose), about 1-4 g/L DF-LNHII (difucosyllacto-L-hexaose), approximately 0.5-2 g/l LNFPI (lacto-N-fucopentaose I), approximately 0.2-2 g/l LNDFHI (lacto-N-difucosylhexaose I), approximately 0. 31.5 g/l LNT (lacto-N-tetraose), approximately 0.5-1.5 g/l LNnT (lacto-N-neotetraose), approximately 0.1-1 g/l DF-L (difucosyllactose ), approximately 0.2-1.5 g/l 3-FL (3-fucosyllactose), approximately 0.2-1.2 g/l 6'-SL (6'-sialyllactose), approximately 0.05-1 g/l DS-LNT (disialyl lacto-Ntetraose), approximately 0.05-0.7 g/l FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), approximately 0.05-0.7 g/l LST c (sialyl-lacto-N-tetraose c), approximately 0.1-0.3 g/l 3'-SL (3'sialyllactose). In some embodiments, the neutral oligosaccharide content is at least about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8 times, or about 9 times, or about 10 times, or about 11 times, or about 12 times, or about 13 times, or about 14 times, or about 15 times by weight the content acid oligosaccharides.

In certain aspects, disclosed herein are dairy products containing approximately 6-14 grams per liter (g/L) of protein components, approximately 18-89 g/L of lipid components, approximately 7-14 g/L of human milk oligosaccharides (HMO), approximately 64-77 g/l lactose, where the protein components contain beta-casein, kappa-casein, alpha-casein, alphalactalbumin, lysozyme, lactoferrin, haptocorrin, butyrophylline, osteopontin, MC5 mucin, BrE3 mucin and lactadherin, where the lipid components contain palmitic acid, stearic acid and lauric acid, oleic acid, linoleic acid, alpha-linolenic acid, eicosadienoic acid, arachidonic acid, dihomo-gamma-linolenic acid, docosahexadienoic acid, cholesterol, phospholipids, plasmalogens and sphingolipids, where human milk oligosaccharides contain TF- LNH (trifucosyllacto-N-hexaose), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-N-hexaose), LNFPI (lacto-N-fucopentaose I), LNDFHI (lacto-N-fucopentaose I). -N-difucosylhexaose I), LNT (lacto-N-tetraose), LNnT (lacto-N-neotetraose), DF-L (difucosyllactose), and 3-FL (3fucosyllactose), 6'-SL (6'-sialyllactose) , DS-LNT (disialyl-lacto-N-tetraose), FS-LNnHI (fucosylsialyl-lacto-I-neohexaose I), LSTc (sialyl-lacto-N-tetraose c) and 3'-SL (3'-sialyllactose), and wherein at least one of protein components, lipid components, FIMO and lactose is produced by cultured human mammary epithelial cells.

In some embodiments, the dairy product contains about 0.5-1.5 g/L beta casein, about 0.5-0.6 g/L kappa casein, about 0.1-0.5 g/L alpha casein, approximately 2.7-3.3 g/l alpha-lactalbumin, approximately 0.2-0.5 g/l lysozyme, approximately 1.0-2.0 g/l lactoferrin, approximately 0.07-0, 7 g/l haptocorrin, approximately 0.03-0.05 g/l butyrophilin, approximately 0.05-0.2 g/l osteopontin, approximately 0.5-0.6 g/l MC5 mucin, approximately 0.50 .7 g/l BrE3 mucin, approximately 0.06-0.07 g/l lactadherin, approximately 2-19 g/l linoleic acid, approximately 0.5-0.7 g/l alpha-linolenic acid, approximately 0. 5-0.7 g/l eicosadienoic acid, approximately 0.5-0.7 g/l arachidonic acid, approximately 0.3-0.5 g/l dihomogamma-linolenic acid, approximately 0.02-0.4 g /L docosahexadienoic acid, approximately 0.09-0.15 g/L cholesterol, approximately 0.1-0.4 g/L phospholipids, plasmalogens and sphingolipids combined, approximately 1-4 g/L TF-LNH (trifucosyllacto- N-hexaose), approximately 1-4 g/l 2'-FL (2'-fucosyllactose), approximately 1-4 g/l DF-LNHII (difucosyllacto-P-hexaose), approximately 0.5-2 g/l LNFPI (lacto-N-fuconentaose I), approximately 0.2-2 g/l LNDFHI (lacto-N-fucosylhexaose I), approximately 0.3-1.5 g/l LNT (lacto-N-tetraose), approximately 0. 5-1.5 g/l LNnT (lacto-N-neotetraose), approximately 0.1-1 g/l DF-L (difucosyllactose), approximately 0.21.5 g/l 3-FL (3-fucosyllactose) , approximately 0.2-1.2 g/l 6'-SL (6'-sialyllactose), approximately 0.05-1 g/l DS-LNT (disialyl-lacto-N-tetraose), approximately 0.05-0, 7 g/l FS-LNnHI (fucosyl-sialyllacto-N-neohexaose I), approximately 0.05-0.7 g/l LSTc (sialyl-lacto-N-tetraose c) and approximately 0.1-0.3 g /l 3'-SL (3'-sialyllactose).

In certain aspects, disclosed herein are dairy products containing about 3-15 percent protein by weight, about 9-92 percent lipids by weight, about 4-15 percent human milk oligosaccharides (HMO) by weight, about 33-80 percent lactose by weight. mass, where at least one of protein, lipid, HMO and lactose are produced by cultured human mammary epithelial cells. In some embodiments, the dairy product further comprises serum albumin. In some embodiments, the dairy product contains about 0.025-3.5 g/L, or about 0.012 g/L, or about 0.15-1 g/L, or about 0.3-0.7 g/L serum albumin.

In some embodiments, the dairy product further contains one or more immunoglobulins. In some embodiments, the immunoglobulins contain one

- 9 046437 or several of IgA, IgG and IgM. In some embodiments, IgA comprises one or more of IgA2 (secretory) and IgA1 (non-secretory). In some embodiments, the dairy product contains approximately 0.2-1.0 g/L secretory IgA. In some embodiments, the dairy product contains approximately 0.15-1.6 g/L total IgA. In some embodiments, the dairy product contains approximately 0.03-0.3 g/L IgG. In some embodiments, the dairy product contains approximately 0.01-0.1 g/L IgM. In some embodiments, the dairy product contains approximately 0.2-2 percent immunoglobulins by weight.

In some embodiments, protein components, lipid components, HMO, and lactose are isolated from cultured mammary epithelial cells. In some embodiments, the cultured mammary epithelial cells contain one or more immortalized mammary cell lines. In some embodiments, cultured mammary epithelial cells are obtained from one or more primary mammary tissue samples. In some embodiments, one or more primary breast tissue samples are obtained from a surgical breast tissue explant. In some embodiments, one or more primary samples of primary breast tissue comprise alveolar and/or cavitary tissue or cells collected from the alveolar structure of the mammary gland. In some embodiments, one or more primary breast tissue samples are obtained by needle aspiration of the breast tissue. In some embodiments, the primary breast tissue further comprises one or more myoepithelial cells. In some embodiments, the primary breast tissue further comprises one or more stem cells. In some embodiments, the primary breast tissue further comprises one or more immune cells. In some embodiments, the immune cells comprise B cells. In some embodiments, the immune cells comprise plasma cells.

In some embodiments, the dairy product is isolated from cultured mammary epithelial cells from one or more specific individuals. In some embodiments, a dairy product isolated from cultured mammary epithelial cells from a particular donor is provided only to that donor or persons authorized by that donor. In some embodiments, cultured mammary epithelial cells are cocultured with one or more plasma cell lines. In some embodiments, cultured mammary epithelial cells are cocultured with one or more myoepithelial cell lines. In some embodiments, cultured mammary epithelial cells are co-cultured with one or more stem cell lines.

In some embodiments, the dairy product contains at least about 80%, at least about 85%, at least about 90%, or at least about 95%, or at least about 97%, or at least about 98% , or at least approximately 99% of the total molecular composition of human breast milk. In some embodiments, the non-protein nitrogen content is at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30% of the total nitrogen content .

In certain aspects, described herein are frozen dairy products containing a dairy product described herein that has been frozen. In some embodiments, the dairy product has been lyophilized. In certain aspects, a containerized dairy product is described herein, comprising a dairy product described herein that is packaged in the container. In certain aspects, a containerized frozen dairy product is described herein, comprising a frozen dairy product that is packaged in the container. In certain aspects, a containerized lyophilized dairy product is described herein, comprising a lyophilized dairy product that is packaged in the container.

In certain aspects, described herein are extracted dairy products consisting of one or more components extracted from a dairy product disclosed herein. In some embodiments, one or more components extracted from the collected dairy product is lyophilized or concentrated to produce a lyophilized or concentrated extracted dairy product component. In some embodiments, one or more components extracted from the collected dairy product are concentrated by membrane filtration or reverse osmosis. In some embodiments, one or more components extracted from the collected dairy product contain milk protein, lipid, carbohydrate, vitamins and minerals.

In certain aspects, described herein are containerized extragyro

- 10 046437 bathed dairy products, including the extracted dairy product described herein, which is packaged in a container. In some embodiments, the container is sterile. In some embodiments, the container is sealed. In some embodiments, the container is a food container. In some embodiments, the container is a canister, can, bottle, bag, box, or pouch.

In some embodiments, the dairy product contains approximately 500-1150 kcal/L of available energy. In some embodiments, 40 to 55% of the available energy of the dairy product comes from lipids. In some embodiments, the dairy product contains from about 95.8 to 195.2 g/L of macromolecular compounds.

In certain aspects, described herein are methods of treating a patient suffering from an infectious disease comprising administering to the patient an effective amount of a dairy product containing approximately 6-14 grams per liter (g/L) of protein components, approximately 18-89 g/L of lipid components, approximately 7-14 g/l human milk oligosaccharides (HMO), approximately 64-77 g/l lactose, where at least one of protein components, lipid components, HMO and lactose. In some embodiments, the dairy product further contains one or more immunoglobulins. In some embodiments, the immunoglobulins comprise one or more of IgA, IgG, and IgM. In some embodiments, IgA comprises one or more of IgA2 (secretory) and IgA1 (non-secretory).

In some embodiments, the dairy product contains approximately 0.2-1.0 g/L secretory IgA. In some embodiments, the dairy product contains approximately 0.15-1.6 g/L total IgA. In some embodiments, the dairy product contains approximately 0.03-0.3 g/L IgG. In some embodiments, the dairy product contains approximately 0.01-0.1 g/L IgM. In some embodiments, the dairy product contains approximately 0.2-2 percent immunoglobulins by weight. In some embodiments, the infectious disease is a gastrointestinal tract infection. In some embodiments, the patient is immunocompromised.

In certain aspects, described herein are methods of treating an infant suffering from a gastrointestinal infection comprising administering an effective amount of a dairy product containing approximately 6-14 grams per liter (g/L) of protein components, approximately 18-89 g/L of lipid components, approximately 7-14 g/l human milk oligosaccharides (HMO), approximately 64-77 g/l lactose, where at least one of protein components, lipid components, HMO and lactose. In some embodiments, the dairy product further contains one or more immunoglobulins. In some embodiments, the immunoglobulins comprise one or more of IgA, IgG, and IgM. In some embodiments, IgA comprises one or more of IgA2 (secretory) and IgA1 (non-secretory). In some embodiments, the dairy product contains approximately 0.2-1.0 g/L secretory IgA. In some embodiments, the dairy product contains approximately 0.15-1.6 g/L total IgA. In some embodiments, the dairy product contains approximately 0.03-0.3 g/L IgG. In some embodiments, the dairy product contains approximately 0.01-0.1 g/L IgM. In some embodiments, the dairy product contains approximately 0.2-2 percent immunoglobulins by weight.

These and other aspects of the disclosure are set forth in more detail in the description of the disclosure below.

Brief description of drawings

In fig. 1 shows an example of the collection of milk for food use from mammary epithelial cells grown as a confluent monolayer in a compartment culture apparatus in which either fresh or processed medium is supplied to the basal compartment and milk is collected from the apical compartment. TEER, transepithelial electrical resistance.

In fig. Figure 2 shows an example of polarized nutrient uptake and milk secretion through a confluent monolayer of mammary epithelial cells attached to a scaffold on the basal surface.

In fig. Figure 3 provides an example of a microstructured scaffold that provides increased surface area for separate nutrient absorption and milk secretion by a confluent monolayer of mammary epithelial cells.

In fig. Figure 4 shows three examples of a hollow fiber bioreactor, depicted as a bundle of capillary tubes (top), which can support a lining of mammary epithelial cells on either the outer (top and bottom left) or inner (bottom right) surface of the capillaries, allowing targeted and separate suction nutrients and milk secretion.

In fig. Figure 5 shows an example of a cross section of a three-dimensional cell structure. The structure consists of a frame having an internal surface delimiting the internal cavity/basal chamber, and an external surface. The internal cavity/basal chamber contains media

- 11 046437 dy for cell cultivation. The matrix material is located on the outer surface of the frame. The pores traverse the scaffold from the inner surface to the outer surface, allowing cellular media to contact the basal surface of the cell monolayer located on the scaffold material.

In fig. Figure 6 shows an example of a bioreactor for producing a cultured dairy product. The bioreactor consists of a cell structure and an apical chamber. The cellular structure consists of a frame having an internal surface delimiting the internal cavity/basal chamber, and an external surface. The cavity contains media for cell culture. The matrix material is located on the outer surface of the frame. The pores traverse the scaffold from the inner surface to the outer surface, allowing cellular media to contact the basal surface of the cell monolayer located on the scaffold material. The apical surface of the cell monolayer secretes milk/cultured milk product into the apical chamber. The apical chamber and the internal cavity/basal chamber are separated by a cell monolayer.

In fig. Figure 7 shows an example of a cell structure. The structure consists of a frame having an internal surface delimiting the internal cavity/basal chamber, and an external surface. The internal cavity/basal chamber contains the cell.

In fig. 8 shows an example of a cellular construct containing mammary epithelial cells (BCE) and plasma cells. Plasma cells are adjacent to the scaffold. The ECMG form a confluent monolayer above (and in some cases between) plasma cells, with the apical side of the ECMG facing the apical compartment (or mammary compartment). Plasma cells secrete IgA, which then binds to receptors on the basolateral surface of the ECMG, triggering internalization of the antibody-receptor complex and further processing of the antibody into sIgA as it moves to the apical surface (not shown).

Fig. 9: Production of human cell cultured milk. (A) Cultivation of mammary epithelial cells for milk production in a hollow fiber bioreactor. ECMs are propagated in tissue culture flasks (1) and then inoculated into the extracapillary space of a hollow fiber bioreactor (2), where they attach to extracellular matrix components covering the surface of the fibers for further proliferation over a larger surface area. The media (3) circulate through the fibers using a peristaltic pump (4). Cellular metabolism is monitored by daily measurement of glucose utilization (5). When ECMs reach confluency and become contact-inhibited, culture media are switched to a lactogenic formulation containing prolactin and product is collected from the extracapillary space (6). (B) Monitoring mammary epithelial cell growth and milk biosynthesis in a hollow fiber bioreactor. Glucose utilization (solid line) and lactose synthesis (dotted line) were monitored daily after inoculation of human mammary epithelial cells into the hollow fiber bioreactor using a diabetic glucose monitor (AccuCheck) and a lactose fluorometric assay (Sigma-Aldrich), respectively. (C) Detection of milk protein synthesis. Capillary electrophoresis of samples collected from the extracapillary space and immunodetection of human beta-casein were performed. (D) Quantification of total protein in the apical (extracapillary space) and basal (culture media) compartments of a hollow fiber bioreactor with human ECMF culture.

Detailed Description of the Present Invention

Milk is a nutrient-rich liquid food produced in the mammary gland of mammals. It is the primary source of nutrition for mammalian infants (including breastfed humans) before they can digest other types of food. Milk is a complex biological matrix consisting of thousands of unique molecules with nutritional and functional properties that are ideal for supporting the growth and development of mammalian infants.

Natural milk contains many macronutrients, including proteins, lipids, polysaccharides and lactose. Each species produces milk with a unique composition that reflects its different physiological needs (Beck KL, et al., JProteome Res. 2015;14(5):2143-2157). Because milk composition is optimized for each animal species, breast milk is considered the gold standard for human infant nutrition and is recommended by the World Health Organization as the exclusive source of nutrition for the first 6 months (Mordor Intelligence, Global dairy market (20162024)).

Compared to cow's milk, human milk contains less protein but more fat and carbohydrates (Ballard O, et al., Pediatr Clin North Am. 2013;60(1):49-74). Many components overlap but undergo different processing steps in humans and bovines and thus differ in their final conformations and physiological properties. For example, the positions of specific fatty acids in triglycerides differ between cow's milk and human milk, and these differences have been shown to affect absorption (Andreas NJ, et al., Early Hum Dev.; 91(11):629-635). In addition, human milk contains hundreds of unique oligosaccharides that promote gut maturation and immunity in the developing infant that are not found in bovine milk-based formulas (Totten SM, et al., JProteome Res. 2012;1 1(12 ):6124). -6133).

Human milk isn't just nutritious. Rather, human milk contains many factors with bioactive properties that play important roles in infant survival and health. Mammalian early milk contains antibodies that provide protection to the newborn. Additional factors that help replenish the active potential of natural milk against diseases include leukocytes, hormones, antimicrobial peptides, cytokines, chemokines and other biologically active factors.

Secretory IgA (sIgA) is found in natural breast milk. IgA is produced plasmatically by cells located inside the breast tissue. IgA binds to a receptor (polymeric Ig receptor) on the basal surface of mammary epithelial cells. IgA and the receptor are transported into the mammary epithelial cells, where the mammary cells process the IgA by cleaving the extracellular domain of the polymeric Ig receptor bound to IgA, leaving the soluble Ig receptor ectodomain (secretory component) bound to IgA. sIgA is secreted from the apical surface of mammary epithelial cells. sIgA, passed on to babies through breast milk, is an integral component of infants' immune systems, allowing them to fight infections. In addition, sIgA is an important component of the immune systems of all people, and sIgA deficiency is associated with increased susceptibility to disease. sIgA-based therapy makes it possible to prevent and treat infectious diseases at their sites of entry.

Immune cells, including CD20+ B cells, which are the precursors of sIgA-secreting plasma cells, are present in normal non-lactating breast tissue. During lactation, immune cell homing to the mammary gland promotes local mucosal immunity, which helps prevent infection within the mammary gland. In addition, homing of immune cells to the mammary gland allows the transfer of sIgA antibodies to the infant through breastfeeding.

Unfortunately, efforts to mass produce sIgA are hampered by the fact that it is a multivalent and highly post-translationally modified protein that requires interaction with mucosal epithelium for processing into its bioactive form. Thus, it is difficult to produce sIgA for use in non-natural breast milk or as therapeutics in humans.

These are several factors that contribute to the inability of infant formula to replicate the properties of breast milk. Thus, there is a need for improved dairy products for developing infants and children that more closely mimic the composition of human breast milk produced by a lactating woman.

It has previously been demonstrated that mammary epithelial cells (ECBC) in culture exhibit organization and behavior similar to that observed in vivo (Arevalo et al. 2016 Am J Physiol Cell Physiol. 310(5):C348-3 56; Chen et al. et al 2019 Curr Protoc Cell Biol 82(1):e65). In Arevalo et al. Specific biomarkers of ECMG populations were discovered in immortalized bovine mammary epithelial cells (BME-UV1) and immortalized bovine mammary alveolar cells (MAC-T) cultured on adhesive 2-D plates, ultra-low attachment surfaces, 3D microplates with ultra-low attachment surface and 3D tablets coated with Matrigel. In addition, Chen et al. details protocols for isolating and culturing human mammary epithelial stem/progenitor cells from human breast tissue and subsequently generating mammospheres using 3D organoid culture on gelatin sponges and Matrigel matrices. However, neither Arevalo nor Chen attempted to stimulate milk production in these ECM crops.

Specifically, when grown on a suitable extracellular matrix and stimulated with prolactin, cultured bovine mammary epithelial cells polarize and organize into structures capable of secreting specific milk components (Blatchford et al. 1999 Animal Cell Technology: Basic & Applied Aspects 10:141-145 ). In Blatchford et al. Cattle ECSF polarized and formed mammospheres. Casein and butyrophilin were isolated from the cultures. However, the cells did not polarize in one direction. Blatchford et al. noted that milk proteins were distributed between cells and dispersed throughout the mammospheres. Due to the lack of a uniform direction of polarization, it was necessary to isolate the isolated proteins from the culture media.

Additionally, 2D in vitro models such as those used by Blatchford et al. provide a low surface area to volume ratio (low density format). The surface area available for cell attachment limits the number of cells that can be grown.

The only known attempt to culture mouse mammary epithelial cells in a high-density format, such as a hollow fiber bioreactor, failed to achieve the separation necessary for the production and extraction of a cultured milk product (Sharfstein et al. 1992 Biotechnology and Bioengineering 40:672-680). In a study by Sharfstein et al. have been studied

- 13 046437 demonstrated the growth, long-term expression, functional differentiation and metabolism of COMMA-1D (an immortalized mouse mammary epithelial cell line) in two different systems: extended batch culture and hollow fiber reactor culture. Using Somma-ID seeded on Costar Transwell® polycarbonate membrane culture inserts, Sharfstein et al. created a confluent monolayer capable of barrier formation and polarized metabolism between the apical and basal sides, which maintained glucose and lactate gradients. However, using the culture in a hollow fiber bioreactor, Sharfstein et al. it was not possible to achieve separation of the basal and apical compartments. Additionally, it has not been determined whether nutrient uptake was polarized in hollow fiber culture (Sharfstein et al., 1992). It is important to note that no previous work has been able to culture mammary epithelial cells from humans or other nutritionally similar species in a high-density, 3D compartmentalized format.

The present disclosure is explained in more detail below. This description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented or all the features that may be added to the present description. For example, features with respect to one embodiment may be included in other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Numerous variations and additions to the various embodiments provided herein will be apparent to those skilled in the art in light of the present disclosure without departing from the present disclosure. Accordingly, the following description is intended to be an example of some specific embodiments of the disclosure and not to be an exhaustive indication of all permutations, combinations and variations thereof.

Unless the context indicates otherwise, the various disclosure features described herein are specifically intended to be used in any combination. In addition, the present disclosure also provides that in some embodiments of the disclosure, any feature or combination of features set forth herein may be excluded or omitted. By way of example, if the specification states that a complex contains components A, B, and C, it is specifically intended that any one of components A, B, or C, or a combination thereof, may be omitted and omitted alone or in any combination.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. The terminology used in the description of the disclosure herein is intended to describe specific embodiments only and is not intended to limit the disclosure.

The terminology used herein is intended to describe specific embodiments only and is not intended to be limiting. As used herein, singular forms also include plural forms unless the context clearly indicates otherwise. In addition, it should be understood that the terms contains and/or containing, when used in this specification, indicate the presence of the specified features, elements and/or components, but do not exclude the presence or addition of one or more other features, elements, components and/ or their groups. As used herein, the term and/or includes any and all combinations of one or more related listed elements. As used herein, phrases such as between X and Y and approximately between X and Y are to be interpreted to include X and Y. As used herein, phrases such as approximately between X and Y mean between approximately X and approximately Y. As used herein, phrases such as about X to Y mean from about X to about Y.

In addition, the present disclosure also provides that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted.

In addition, the term approximately, as used herein when referring to a measurable quantity such as an amount of a compound or agent of the present disclosure, dose, time, temperature, etc., is intended to cover variations of ±10%, ±5% , ±1%, ±0.5% or even ±0.1% of the specified amount.

As used herein, the transition phrase consisting essentially of shall be interpreted to include those materials or steps listed and those that do not materially affect the main and novel feature(s) of the disclosure. Thus, the term consisting essentially of, as used herein, should not be construed as equivalent to the term containing.

As used herein, the compositions described in this disclosure are referred to as

- 14 046437 are read interchangeably as (singular or plural forms) food compositions substantially similar to human milk, dairy products, milk compositions, cultured dairy products, or the equivalent as clear from the context.

As used herein, by isolating (or as grammatically equivalent, eg, extracting) a product, it is meant that the product is at least partially separated from at least some of the other components in the starting material.

As used herein, the term polypeptide covers both peptides and proteins, and does not require any specific amino acid length or tertiary structure unless otherwise noted.

The terms cultured milk product and dairy product are used interchangeably to refer to the product secreted from the apical surface of a living cell construct (or cell culture) containing human mammary epithelial cells (hECM). In some embodiments, the living cell construct is cultured in a bioreactor.

The term polarized, as used herein in relation to cells and/or cell monolayers, refers to the spatial status of cells where there are two distinct cell surfaces, for example, an apical surface and a basal surface, which may be different. In some embodiments, the individual surfaces of the polarized cell contain different surfaces and/or transmembrane receptors and/or other structures. In some embodiments, individual polarized cells in a continuous monolayer have similarly oriented apical surfaces and basal surfaces. In some embodiments, individual polarized cells in a continuous monolayer have contact structures between individual cells (eg, tight junctions) to provide cross-talk between individual cells and create separation (eg, compartmentalization) of apical and basal compartments.

As used herein, apical surface means the surface of a cell that faces the external environment or a cavity or chamber, such as the cavity of an internal organ. In relation to mammary epithelial cells, the apical surface is the surface from which the cultured milk product is secreted.

As used herein, basal surface means the surface of a cell that is in contact with the surface of, for example, a bioreactor matrix.

As used herein, a bioreactor means a device or system that maintains a biologically active environment allowing the production of a cultured dairy product described herein by mammary cells described herein.

The term lactogenic, as used herein, refers to the ability to stimulate milk production and/or secretion. A gene or protein (eg prolactin) can be lactogenic, like any other natural and/or synthetic product. In some embodiments, the lactogenic culture medium contains prolactin, thereby stimulating the cells to produce milk upon contact with the culture medium.

As used herein, the term food grade refers to materials considered non-toxic and safe for consumption (e.g., human and/or other animal consumption), for example, in accordance with standards established by the US Food and Drug Administration .

Cellular structures

Included herein are cell constructs for the production of a cultured dairy product that is a biosynthetic product of cultured mammary epithelial cells (immortalized or from primary tissue samples) and IgA-producing cells, such as plasma cells. In certain aspects, disclosed herein are cell constructs for producing cultured dairy products containing sIgA, cell constructs comprising: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an internal cavity/basal chamber, and a plurality of pores extending from the inner surface to outer surface; (b) a matrix material located on the outer surface of the three-dimensional scaffold; (c) culture media located in the internal cavity/basal chamber and in fluid contact with the internal surface; (d) a population of plasma cells (PCs) located on the matrix matrix and (e) a confluent monolayer of mammary cells located on the population of plasma cells, the mammary cells selected from the group consisting of: (i) mammary epithelial cells, (ii ) mammary myoepithelial cells, and (iii) mammary progenitor cells.

Breast cells

In some embodiments, the mammary cells comprise milk-producing mammary epithelial cells (MLECs), contractile myoepithelial cells, and/or progenitor cells, which may give rise to both breast epithelial cells (MLECs) and contractile mammary myoepithelial cells. Breast epithelial cells

- 15 046437 (ECM) are the only cells that produce milk. In some embodiments, the mammary cells comprise mammary epithelial cells (EBC), primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells. In some embodiments, breast cells are obtained from a biopsy of breast tissue.

In some embodiments, the breast cells are derived from stem cells derived from breast milk or breast stem cells obtained from a biopsy of breast tissue. The epithelial component of breast milk includes not only mature epithelial cells, but also their precursors and stem cells in culture. A subpopulation of stem cells derived from breast milk has a very high multilineage potential, reminiscent of that characteristic of human embryonic stem cells (hESCs). Breast stem cells can also be derived from breast tissue biopsies and include terminally differentiated ECBM. Both stem cells derived from breast milk and breast stem cells derived from breast tissue biopsies are multipotent cells that can give rise to ECMG or myoepithelial cells.

In some embodiments, at least 50% of the mammary cells from the cell culture are polarized. In some embodiments, at least 55% of the mammary cells from the cell culture are polarized. In some embodiments, at least 60% of the mammary cells from the cell culture are polarized. In some embodiments, at least 65% of the mammary cells from the cell culture are polarized. In some embodiments, at least 70% of the mammary cells from the cell culture are polarized. In some embodiments, at least 75% of the mammary cells from the cell culture are polarized. In some embodiments, at least 80% of the mammary cells from the cell culture are polarized. In some embodiments, at least 85% of the mammary cells from the cell culture are polarized. In some embodiments, at least 90% of the mammary cells from the cell culture are polarized. In some embodiments, at least 95% of the mammary cells from the cell culture are polarized. In some embodiments, at least 100% of the mammary cells from the cell culture are polarized. In some embodiments, substantially all of the mammary gland cellular constructs are polarized (ie, have an apical surface and a basal surface). In some embodiments, substantially all of the mammary gland cellular constructs are polarized, and substantially all of the polarized cells are oriented in the same direction. For example, in some embodiments, substantially all mammary cells have an apical surface and a basal surface, wherein the apical surface of substantially all cells is oriented in the same direction and the basal surface of substantially all cells is oriented in the same direction.

In some embodiments, the continuous monolayer of mammary cells is at least 70% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary cells is at least 75% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary gland cells is at least 80% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary gland cells is at least 85% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary cells is at least 90% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary gland cells is at least 95% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary gland cells is at least 99% confluent over the scaffold. In some embodiments, the continuous monolayer of mammary cells is at least 100% confluent over the scaffold.

Genetic modifications of breast cells

In some embodiments, the breast cells contain a constitutively active prolactin receptor protein. In some embodiments, the breast cells contain constitutively active human prolactin receptor protein. In cases where the primary mammary epithelial cell or immortalized mammary epithelial cell contains a constitutively active prolactin receptor, the culture medium does not contain prolactin.

In some embodiments, the constitutively active human prolactin receptor protein comprises a deletion of amino acids 9 to 187, where the numbering is based on the human prolactin receptor reference amino acid sequence identified as SEQ ID NO: 1.

- 16 046437

SEQ ID NO: 1: Human prolactin receptor (GenBank accession number AAD32032.1)

MKENVASATVFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTD

GGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSF SDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPE KAAEWEIHFAGQQTEFKILSLHPGQKYLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDTT VWISVAVLSAVICLIIVW AVALKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGC QDFPPTSDYEDLLVEYLEVDDSEDQHLMSVHSKEHPSQGMKPTYLDPDTDSGRGSCDSPSL LSEKCEEPQANPSTFYDPEVIEKPENPETTHTWDPQCISMEGKIPYFHAGGSKCSTWWPLPQPS QHNPRSSYHNITDVCELAVGPAGAPATLLNEAGKDALK SSQTIKSREEGKATQQREVESFH SETDQDTPWLLPQEKTPFGSAKPLDYVEIHKVNKDGALSLLPKQRENSGKPKKPGTPENNK EYAKVSGVMDNNILVLVPDPHAKNVACFEESAKEAPPSLEQNQAEKALANFTATSSKCRL QLGGLDYLDPACFTHSSFH

In some embodiments, the constitutively active human prolactin receptor protein contains a deletion of the following amino acids:

VFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTDGGLPTNYSLTYHREGETL MHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYVDVTYIVQPDPPL ELAVEVKQPEDRKPYLWIKWSPPTLTDLKTGWFTLLYEIRLKPEKAA (e.g., amino acid positions 10 to 178 of SEQ ID NO : 1).

In some embodiments, the mammary cells contain a loss of function mutation introduced into the circadian related gene PER2. In some embodiments, a loss-of-function mutation introduced into the circadian-related gene PER2 increases the synthesis of cultured milk components. In some embodiments, the loss of function mutation in the PER2 gene involves a deletion of 87 amino acids from position 348 to 434 in PER2, where the numbering is based on the human PER2 reference amino acid sequence identified as SEQ ID NO: 2.

SEQ ID NO: 2: Human periodic circadian protein homolog 2 (GenBank accession number NM 022817)

MNGYAEFPPSPSPSNPTKEPVEPQPSQVPLQEDVDMSSGSSGHETNENCSTGRDSQGSDCDDS GKJELGMLVEPPDARQSPDTFSLMMAKSEHNPSTSGCSSDQSSKVDTHKEL1KTLKELKVH LPADKKAKGKASTLATLKYALRSVKQVKANEEYYQLLMSSEGHPCGADVPSYTVEEMES VTSEHIVKNADMFAVAV SLVSGKILYISDQVASIFHCKRDAFSDAKFVEFLAPHDVGVFHSF TSPYKLPLWSMCSGADSFTQECMEEKSFFCRVSVRKSHENEIRYHPFRMTPYLVKVRDQQG AESQLCCLLLAERVHSGYEAPRIPPEKRIFTTTTHTPNCLFQDVDERAVPLLGYLPQDLIET PVLVQLHPSDRPLMLAIHKKILQSGGQPF DYSPIRFRARNGEYITLDTSWSSFINPWSR KISFIIGRHKVRVGPLNEDVFAAHPCTEEKALHPSIQELTEQIHRLLLQPVPHSGSSGYGSL GSNGSHEHLMSQTSSSDSNGHEDSRRRRAEICKNGNKTKNRSHYSHESGEQKKKSVTEMQ TNPPAEKKAVPAMEKDSLGVSFPEELACKNQPTCSYQQISCLDSVIRYLESCNEAAT K NRKLKSKRVKPRDSSESTGSGGPVSARPPLVGLNATAWSPSDTSQSSCPAVPFPAPVPAAY SLPVFPAPGTVAAPPAPPHASFTVPAVPVDLQHQFAVQPPPFPAPLAPVMAFMLPSYSFPSG TPNLPQAFFPSQPQFPSPHPTLTSEMASASQPEFPEGGTGAMGTTGATETAAVGADCKPGTSR DQQPKAPLTRDEPSDTQNSDALSTSSGLL NLLLNEDLCSASGSAASESLGSGSLGCDASPSG AGSSDTSHTSKYFGSIDSSENNHKAKMNTGMEESEHFIKCVLQDPIWLLMADADSSVMMT YQLPSRNLEAVLI<EDREI<LI<LLQI<LQPRFTESQI<QELREVHQWMQTGGLPAAIDVAECVY CENKEKGNICIPYEEDIPSLGLSEVSDTKEDENGSPLNH RIEEQT

In some embodiments, the loss of function mutation introduced in PER2 comprises a deletion of the following amino acids:

CLFQDVDERAVPLLGYLPQDLIETPVLVQLHPSDRPLMLAIHKKILQSGGQPFDYSPIRFRAR NGEYITLDTSWSSFINPWSRKISFIIGRHKV (e.g., amino acid positions 341 to 434 of SEQ ID NO: 2).

In some embodiments, the breast cells contain a polynucleotide encoding a prolactin receptor containing a modified intracellular signaling domain. In some embodiments, a loss-of-function mutation introduced into the circadian-related gene PER2 increases the synthesis of selected components of cultured milk. In some embodiments, the prolactin receptor comprises a truncation where position 154 of exon

- 17 046437 is spliced to the 3' sequence of exon 11. In some embodiments, the prolactin receptor comprises the sequence of SEQ ID NO: 3.

SEQ ID NO: 3: Human prolactin receptor isoform 4 (GenBank accession number AF416619;

Trott et al. 2003 J Mol Endocrinol 3Q(I):31-47)

MKENVASATVFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTD GGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSF SDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPE KAAEWEIHFAGQQTEFKILSLHPGQK YLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDTT VWISVAVLSAVICLIIVWAVALKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGC QDFPPTSDYEDLLVEYLEVDDSEDQHLMSVHSKEHPSQGDPLMLGASHYKNLKSYRPRKIS SQGRLAVFTKATLTTVQ

In some embodiments, the breast cells contain a polynucleotide encoding a modified (eg, recombinant) prolactin protein effector. In some embodiments, the modified prolactin protein effector comprises a Janus kinase 2 (JAK2) tyrosine kinase domain. In some embodiments, the modified effector comprises a JAK2 tyrosine kinase domain fused to a signal transducer and activator of transcription-5 (STAT5) tyrosine kinase domain (e.g., a polynucleotide encoding a JAK2 tyrosine kinase domain linked to the 3' terminus of a polynucleotide encoding a STAT5 tyrosine kinase domain). In some embodiments, the modified prolactin protein effector promotes increased synthesis of selected components of cultured milk. In some embodiments, the modified effector has the sequence set forth in SEQ ID NO: 4. The amino acids corresponding to the JAK2 kinase domain at amino acid positions 757 to 1129 of the human JAK2 reference amino acid sequence are shown in bold.

SEQ ID NO: 4: Human STA5A signal transducer and transcriptional activator 5A fused at the 3' end to amino acids 757-1129 from human tyrosine protein kinase JAK2

MAGWIQAQQL QGDALRQMQV LYGQHFPIEV RHYLAQWIES QPWDAIDLDN PQDRAQATQL LEGLVQELQK KAEHQVGEDG FLLKIKLGHY ATQLQKTYDR CPLELVRCIR HILYNEQRLV REANNCSSPA GILVDAMSQK HLQINQTFEE LRLVTQDTEN ELKKLQQTQE Y FIIQYQESL RIQAQFAQLA QLSPQERLSR ETALQQKQVS LEAWLQREAQ TLQQYRVELA EKHQKTLQLL RKQQTIILDD ELIQWKRRQQ LAGNGGGPPEG SLDVLQSWCE KLAEIIWQNR QQIRRAEHLC QQLPIPGPVE EMLAEVNATI TDIISALVTS TFIIEKQPPQ VLKT QTKFAA TVRLLVGGKL NVHMNPPQVK ATIISEQQAK SLLKNENTRN ECSGEILNNC CVMEYHQATG TLSAHFRNMS LKRIKRADRR

GAESVTEEKF TVLFESQFSV GSNELVFQVK TLSLPWVIV HGSQDHNATA TVLWDNAFAE PGRVPFAVPD KVLWPQLCEA LNMKFKAEVQ SNRGLTKENL VFLAQKLFNN

SSSHLEDYSG LSVSWSQFNR ENLPGWNYTF WQWFDGVMEV LKKHHKPHWN

DGAILGFVNK QQAHDLLINK PDGTFLLRFS DSEIGGITIA WKFDSPERNL WNLKPFTTRD FSIRSLADRL GDLSYLIYVF PDRPKDEVFS KYYTPVLAKA VDGYVKPQIK QWPEFVNAS ADAGGSSATY MDQAPSPAVC PQAPYNMYPQ NPDHVLDQDG EFDLDETMDV ARHVEELLRR PMDSLDSRLS PPAGLFTSAR GSLSLDSQ RKLQFYEDRH QLPAPKWAEL ANLINNCMDY EPDFRPSFRA IIRDLNSLFT PDYELLTEND MLPNMRIGAL GFSGAFEDRD PTQFEERHLK FLQQLGKGNF GSVEMCRYDP LQDNTGEWA VKKLQHSTEE HLRDFEREIE ILKSLQHDNI VKYKGVCYSA GRRNLKLIME YLP YGSLRDY LQKHKERIDH IKLLQYTSQI CKGMEYLGTK RYIHRDLATR NILVENENRV KIGDFGLTKV LPQDKEYYKV KEPGESPIFW YAPESLTESK FSVASDVWSF GWLYELFTY IEKSKSPPAE FMRMIGNDKQ GQMIVFHLIE LLKNNGRLPR PDGCPDEIYM IMTECWNNNV NQRPSFRDLA LRVDQIRDN.

In some embodiments, the breast cells are immortalized. In some embodiments, the mammary gland cells contain one or more nucleic acids encoding human telomerase reverse transcriptase (hTERT) or simian virus 40 (SV40). In some embodiments, the mammary cells contain short hairpin RNA (shRNA) to p16 (inhibitor of cyclin-dependent kinase 4) (p16(INK4)) and master regulator of cell cycle entry and proliferative metabolism (c-MYC).

In some embodiments, the method comprises introducing into a mammary gland cell: (a) a polynucleotide encoding a prolactin receptor comprising a modified intracellular signaling domain, optionally wherein the prolactin receptor comprises a truncation wherein position 154 of exon 10 is spliced to the 3' sequence of exon 11; (b) a polynucleotide encoding a chimeric prolactin receptor that binds to a ligand capable of activating milk synthesis

- 18 046437 ka in the absence of prolactin; (c) a polynucleotide encoding a constitutively or conditionally active prolactin receptor protein, optionally, wherein the polynucleotide encodes a constitutively active human prolactin receptor protein comprising a deletion of amino acids 9 to 187 (e.g., a deletion of amino acids 9 to 187, where the numbering is based on a reference amino acid sequence human prolactin receptor identified as SEQ ID NO: 1); (d) a polynucleotide encoding a modified (e.g., recombinant) prolactin protein effector comprising (i) a tyrosine kinase domain of Janus kinase 2 (JAK2), optionally wherein the tyrosine kinase domain of JAK2 is fused to a tyrosine kinase domain of signal transducer and activator of transcription-5 (STAT5) (eg, a polynucleotide encoding a JAK2 tyrosine kinase domain linked to the 3' end of a polynucleotide encoding a STAT5 tyrosine kinase domain); and/or (ii) the intracellular domain of the prolactin receptor fused to the tyrosine kinase domain of JAK2; (f) loss-of-function mutations in the circadian-related gene PER2 (periodic circadian protein homolog 2); and/or (f) a polynucleotide encoding one or more genes for the glucose transporters GLUT1 and/or GLUT12, thereby increasing the rate of nutrient uptake by the basal surface of the monolayer.

Plasma cells

Plasma cells are obtained from a human donor. In some embodiments, the plasma cells are obtained from a bone marrow, spleen and/or lymph node, primary breast tissue sample. In certain embodiments, the plasma cells are derived from mucosal epithelial cells other than mammary gland cells (eg, oronasal, gastrointestinal, or respiratory tissue). In some embodiments, the plasma cells are derived from a plasma cell line. In certain embodiments, the plasma cells are derived from a plasmacytic cell line. In some embodiments, plasma cells are isolated and sorted from non-plasma cells by fluorescence-activated cell sorting, magnetic-activated cell sorting, and/or a microfluidic cell sorter. In some embodiments, plasma cells, plasmablasts, or preplasmoblasts are sorted and isolated by FACS analysis using markers known in the art (eg, CD38, CD138, and/or CD19). In certain embodiments, plasma cells are cultured together with immortalized mammary epithelial cells on a scaffold, thereby producing a cell construct to produce a cultured dairy product with plasma cell and mammary cell secretory products (eg, sIgA). In certain embodiments, plasma cells are grown on a scaffold below a monolayer of mammary gland cells. In certain embodiments, plasma cells are grown as low-density populations of plasma cells covered by a monolayer of mammary cells. In certain embodiments, plasma cells are stimulated to produce immunoglobins when co-cultured with mammary cells. In certain embodiments, the plasma cells produce one or more immunoglobin classes selected from IgG, IgM, and IgA. In certain embodiments, the plasma cells produce IgA. In certain embodiments, plasma cells produce IgA, and the IgA is processed by mammary epithelial cells to produce sIgA, which is associated with a secretory component, and sIgA is secreted by the apical surface of the mammary cells.

Frames

In some embodiments, the cell structure includes a frame having a top surface/outer surface and a bottom surface/inner surface. In some embodiments, the framework is a two-dimensional surface or a three-dimensional surface (eg, a three-dimensional surface with a micropattern and/or a cylindrical structure assembled into bundles). A non-limiting example of a two-dimensional surface frame is the Transwell® filter. In some embodiments, the framework is a three-dimensional surface. Non-limiting examples of micropatterned 3D surface include a microstructured bioreactor, acellular tissue (eg, acellular breast tissue or acellular plant tissue), micropatterned scaffolds manufactured by casting or 3D printing from biological or biocompatible materials, textured surface. In some embodiments, the scaffold is made by electrospinning cellulose nanofibers and/or a cylindrical structure that can be assembled into bundles (eg, a hollow fiber bioreactor). In some embodiments, the frame is porous. In some embodiments, the frame is a three-dimensional frame. In some embodiments, the three-dimensional framework is any structure having a closed internal cavity/central cavity. In some embodiments, the three-dimensional frame connects to one or more surfaces to form an enclosed inner chamber/basal compartment. For example, the frame may connect to one or more walls of the bioreactor to form an internal chamber/basal compartment. In some embodiments, the scaffold is a hollow fiber bioreactor. In some embodiments, the 3D framework is

- 19 046437 tube in which the central cavity is formed by the inner surface of the frame. In some embodiments, the 3D frame is a hollow sphere in which a central cavity is formed by the inner surface of the frame.

In vitro culture methods for studying intestinal absorption have long used 2D surface scaffolds such as Transwells® as a standard because they provide both apical and basolateral space to mimic the gut-blood barrier and enable both active and passive transport medicines and nutrients. However, the phenotypes of cells seeded on flat substrates differ markedly from those of cells in vivo, in part due to poor representation of the three-dimensional extracellular microenvironment.

The three-dimensional scaffold allows cells (eg, ECMG and plasma cells) to grow or interact with the environment in all three dimensions. Unlike a 2D environment, 3D cell culture allows in vitro cells to grow in all directions, approximating the in vivo mammary gland environment. In addition, the 3D scaffold allows for increased surface area for cell culture and for the exchange of gases and metabolites, and also provides the necessary compartmentalization, allowing the cultured milk product to be secreted into one compartment while the cell culture media is in contact with mammary cells and plasma cells in another department. To date, it has not been possible to obtain a confluent monolayer with polarized separation of basal and apical cell surfaces using mammary epithelial cells on a three-dimensional surface (Sharfstein et al. 1992).

In some embodiments, the frame is porous. In some embodiments, the scaffold is permeable to cell media, allowing cell media to contact cells from a cell monolayer. In some embodiments, the scaffold has at least one pore that allows cell media to contact the cell basal surface of the cell monolayer.

In some embodiments, the top surface/outer surface of the frame is coated with a matrix material. In some embodiments, the matrix consists of one or more extracellular matrix proteins. Non-limiting examples of extracellular matrix protein include collagen, laminin, entactin, tenascin and/or fibronectin. In some embodiments, the scaffold comprises a natural polymer, a biocompatible synthetic polymer, a synthetic peptide, and/or a composite made from any combination thereof. In some embodiments, the natural polymer used in this invention includes, but is not limited to, collagen, chitosan, cellulose, agarose, alginate, gelatin, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, and/or hyaluronic acid. In some embodiments, the biocompatible polymer used in this invention includes, but is not limited to, cellulose, polysulfone, polyvinylidene fluoride, polyethylene-co-vinyl acetate, polyvinyl alcohol, sodium polyacrylate, acrylate polymer, and/or polyethylene glycol. In some embodiments, the top of the scaffold is coated with laminin and collagen.

In some embodiments, the matrix material is porous. In some embodiments, the matrix material is permeable to cell media, allowing the cell media to contact cells from the cell monolayer. In some embodiments, the matrix material has at least one pore that allows cell media to contact the cell basal surface of the cell monolayer.

In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.1 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.2 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.3 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.4 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.5 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.6 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.7 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.8 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 0.9 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.0 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.1 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.2 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.3 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.4 μm. In some embodiments, the pore size of the scaffold and/or matrix material is

- 20 046437 at least approximately 1.5 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.6 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.7 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.8 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 1.9 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.0 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.1 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.2 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.2 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.3 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.4 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.5 μm. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.6 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.7 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.8 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 2.9 microns. In some embodiments, the pore size of the scaffold and/or matrix material is at least about 3.0 μm.

In some embodiments, the cell structure comprises: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity/basal chamber, and a plurality of pores extending from the inner surface to the outer surface; (b) matrix material located on the outer surface of the three-dimensional scaffold; (c) culture media located in the internal cavity/basal chamber and in fluid contact with the internal surface; (d) a population of plasma cells (PCs) located on the matrix material and (e) a confluent monolayer of mammary cells located on the population of plasma cells, the mammary cells selected from the group consisting of: (i) mammary epithelial cells, (ii) ) mammary myoepithelial cells and (iii) mammary progenitor cells; where a confluent monolayer of mammary epithelial cells has an apical surface and a basal surface (eg, the cells form a polarized and confluent monolayer of cells).

Bioreactor

In specific embodiments, disclosed herein are bioreactors comprising: (a) an apical compartment containing a cultured dairy product; and (b) at least one cellular construct comprising: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity/basal chamber, and a plurality of pores extending from the inner surface to the outer surface; (b) matrix material located on the outer surface of the three-dimensional scaffold; (c) culture media located in the internal cavity/basal chamber and in fluid contact with the internal surface; (d) a population of plasma cells (PCs) located on the matrix material, and (e) a confluent monolayer of mammary cells located on the population of plasma cells, the mammary cells selected from the group consisting of: i) mammary epithelial cells, (ii ) mammary myoepithelial cells and (iii) mammary progenitor cells. In certain embodiments, the cellular bioreactor construct comprises at least a 70% confluent monolayer of polarized mammary cells disposed on a matrix material, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells glands; where the apical surface of the mammary gland cells is in fluid contact with the apical compartment.

In some embodiments, the bioreactor is a closed bioreactor. In some embodiments, the apical chamber is substantially isolated from the internal cavity/basal compartment.

The hollow fiber bioreactor is an exemplary bioreactor for use with the methods disclosed herein. The hollow fiber bioreactor is a high-density continuous perfusion culture system that closely approximates the environment in which cells grow in vivo. It consists of thousands of semi-permeable three-dimensional scaffolds (i.e., hollow fibers) in a parallel array inside a cartridge shell equipped with input and output ports. These bundles of fibers are potted or sealed at each end so that

- 21 046437 any liquid that gets onto the ends of the cartridge will necessarily flow through the inside of the fibers. Cells are typically seeded outside the fibers within the cartridge in the extracapillary space (ECS).

Three fundamental characteristics distinguish hollow fiber cell culture from other methods: (1) the cells are bound to a porous matrix in much the same way as in vivo rather than to a plastic dish, microcarrier, or other impermeable support, (2) the molecular weight threshold of the support matrix can be controlled , and (3) an extremely high surface area to volume ratio (150 cm ² or more per ml), which provides a large area for the exchange of metabolites and gases for efficient growth of host cells.

The structure of the bioreactor provides a fibrous matrix that allows nutrients, gases and other components of the main media, as well as cell waste products, to penetrate, but not cells, while the cells can be expanded. Hollow fiber bioreactor technology is used to produce high-density cell growth by using hollow fibers to create a semi-permeable barrier between the cell growth chamber and the media flow. Since the surface area provided by this design is large, the use of this fiber as a culture substrate allows the production of large numbers of cells. Cells growing in a three-dimensional environment inside the bioreactor are bathed in fresh media that penetrates through the hollow fibers.

To reproduce intestinal topography, Costello et al. developed a 3D printed bioreactor that can contain porous villous scaffolds through micromolding (Costello et al. 2017 Scientific Reports 7(12515): 1-10). This geometrically complex molded scaffold provides separation of the apical and basolateral spaces such that fluid flow exposes the intestinal epithelial cell to appropriate shear stresses (Costello et al. 2017). Similarly, long-term in vitro culture in a simulated intestinal-like environment was established by Morada et al. using a hollow fiber bioreactor that allowed the use of two controlled separate environments (biphasic) to provide host cells with oxygen and nutrients from the basal layer, while allowing the creation of a low-oxygen, nutrient-rich environment at the apical surface ( Morada et al 2016 International Journal for Parasitology 26: 21-29).

When selecting a hollow fiber bioreactor configuration, there are design factors and parameters that may vary depending on the cell propagation goals. One such design factor is the size of the pores in the fiber wall. Typically, this is done to allow nutrients to enter the cell, remove waste, provide the cell with needed products (such as growth factors), remove needed products from the cell, and prevent certain factors present from entering the cell. Thus, the pore size in the fiber walls can be varied to modify which components will pass through the walls.

For example, the size of the pores can allow the passage of large protein molecules, including growth factors, including but not limited to epidermal growth factor and platelet-derived growth factor. One skilled in the art will appreciate how pore size can vary depending on the components that are desired to pass through the fiber walls to reach the cell or deliver material from the cells.

In some embodiments, the pore size is approximately 0.2 microns. In some embodiments, the pore size is approximately 0.1. In some embodiments, the pore size is approximately 0.2 microns. In some embodiments, the pore size is approximately 0.3 microns. In some embodiments, the pore size is approximately 0.4 microns. In some embodiments, the pore size is approximately 0.5 microns. In some embodiments, the pore size is approximately 0.6 microns. In some embodiments, the pore size is approximately 0.7 microns. In some embodiments, the pore size is approximately 0.8 microns. In some embodiments, the pore size is approximately 0.9 microns. In some embodiments, the pore size is approximately 1.0 microns. In some embodiments, the pore size is approximately 1.1 microns. In some embodiments, the pore size is approximately 1.2 microns. In some embodiments, the pore size is approximately 1.3 microns. In some embodiments, the pore size is approximately 1.4 microns. In some embodiments, the pore size is approximately 1.5 microns. In some embodiments, the pore size is approximately 1.6 microns. In some embodiments, the pore size is approximately 1.7 microns. In some embodiments, the pore size is approximately 1.8 microns. In some embodiments, the pore size is approximately 1.9 microns. In some embodiments, the pore size is approximately 2.0 microns. In some embodiments, the pore size is approximately 2.1 microns. In some embodiments, the pore size is approximately 2.2 microns. In some embodiments, the pore size is approximately 2.2 microns. In some embodiments, the pore size is approx.

- 22 046437 positive 2.3 microns. In some embodiments, the pore size is approximately 2.4 microns. In some embodiments, the pore size is approximately 2.5 microns. In some embodiments, the pore size is approximately 2.6 microns. In some embodiments, the pore size is approximately 2.7 microns. In some embodiments, the pore size is approximately 2.8 microns. In some embodiments, the pore size is approximately 2.9 microns. In some embodiments, the pore size is approximately 3.0 microns.

Methods for creating cell constructs

In specific embodiments, disclosed herein are methods for creating a cell construct to produce a cultured dairy product containing immunoglobulins. In some embodiments, the method comprises (a) placing (i) isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells and (ii) isolated plasma cells on a top surface of a scaffold having a top surface and a bottom surface to obtain a mixed population of plasma cells and mammary cells (ie, mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells); (b) culturing a mixed population of mammary cells and plasma cells (a) on the scaffold to obtain a monolayer of polarized mammary cells located adjacent to and above the plasma cells, where the plasma cells are located adjacent to and above the top surface of the scaffold, where the top surface is located adjacent to and above the lower surface of the scaffold, and where the polarized mammary cells contain an apical surface and a basal surface, thereby creating a cellular construct for producing a cultured milk product. In some embodiments, the mammary cells are primary mammary cells. In some embodiments, the breast cells are obtained from a cell culture. In some embodiments, mammary epithelial cells, mammary myoepithelial cells, and/or mammary progenitor cells are isolated from bone marrow, spleen tissue, lymph node tissue, mammary gland explants from mammary gland tissue (e.g., mammary gland, udder, teat tissue) , or raw breast milk. In some embodiments, the breast cells comprise mammary epithelial cells. In some embodiments, the mammary cells comprise mammary myoepithelial cells. In some embodiments, the breast cells comprise mammary progenitor cells. In some embodiments, plasma cells are isolated from any suitable human tissue or cell culture. In some embodiments, mammary cells and plasma cells are placed simultaneously. In some embodiments, the plasma cells are placed on the surface of the scaffold before the mammary cells are placed.

In some embodiments, the method comprises (a) placing (i) isolated immortalized mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells, and (ii) isolated plasma cells on a top surface of a scaffold having a top surface and a bottom surface. surface, producing a mixed population of plasma cells and immortalized mammary cells (i.e., immortalized mammary epithelial cells, immortalized mammary myoepithelial cells, and/or immortalized mammary progenitor cells); (b) culturing a mixed population of immortalized mammary cells and plasma cells (a) on the scaffold, obtaining a monolayer of immortalized polarized mammary cells located adjacent to and above the plasma cells, where the plasma cells are located adjacent to and above the upper surface of the scaffold, where the upper the surface is adjacent to and above the lower surface of the scaffold, and where the polarized immortalized mammary cells contain an apical surface and a basal surface, thereby creating a cellular construct for producing a cultured milk product. In some embodiments, the immortalized mammary cells comprise immortalized mammary epithelial cells. In some embodiments, the immortalized mammary cells comprise immortalized mammary myoepithelial cells. In some embodiments, the immortalized mammary cells comprise immortalized mammary progenitor cells. In some embodiments, plasma cells are isolated from any suitable human tissue or cell culture. In some embodiments, immortalized mammary cells and plasma cells are placed simultaneously. In some embodiments, plasma cells are placed on the surface of the scaffold prior to placement of the immortalized mammary cells. In some embodiments, plasma cells are added to a culture of immortalized mammary epithelial cells to produce a co-culture of mammary cells and plasma cells. In certain embodiments, plasma cells are cultured with immortalized epithelial cells

- 23 046437 mi mammary gland cells on a scaffold, thereby obtaining a cell construct for producing a cultured dairy product with secretory products of immune cells and mammary gland cells (for example, sIgA). In certain embodiments, the isolated mammary cells are immortalized before the cells are co-cultured.

In certain embodiments, immune cells are stimulated to produce immunoglobins during co-culture. In certain embodiments, the immune cells produce one or more immunoglobins from a class selected from IgG, IgM, and IgA. In certain embodiments, the immune cells produce secretory IgA. The classes of immunoglobins produced by immune cells include one or more of IgA, IgM, and IgG. In certain embodiments, immune cells are co-cultured with ECM in a bioreactor by methods described herein. In certain embodiments, the bioreactor is a hollow fiber bioreactor as described herein.

In certain embodiments, mammary cells are modified and/or stimulated with prolactin, in the manner described herein, to stimulate and optimize milk production. In certain embodiments, breast cells are modified to express a constitutively active prolactin receptor protein.

In certain embodiments, breast cells are identified and isolated from breast tissue samples. In some embodiments, breast cells are isolated and sorted by fluorescence-activated cell sorting, magnetically activated cell sorting, and/or a microfluidic cell sorter. In certain embodiments, mammary epithelial cell populations are sorted by FACS analysis using markers known in the art to identify cell populations. In certain embodiments, mammary myoepithelial cells and mammary luminal epithelial cells are isolated using FACS analysis. In certain embodiments, mammary myoepithelial progenitor cells and/or mammary luminal epithelial progenitor cells are isolated using FACS analysis. Any suitable method known in the art can be used to sort mammary epithelial cells (eg, luminal epithelial cells), myoepithelial cells, progenitor cells, and immune cells. For example, breast cells can be sorted using the cell surface markers CD24, EPCAM and/or CD49f.

In some embodiments, plasma cells are identified and isolated from primary mucosal tissue (eg, oronasal, gastrointestinal, respiratory, or breast). In some embodiments, plasma cells are identified and isolated from primary breast tissue samples. In some embodiments, plasma cells are isolated and sorted by fluorescence-activated cell sorting, magnetically activated cell sorting, and/or a microfluidic cell sorter. In certain embodiments, plasma cells are sorted and isolated using FACS analysis. In certain embodiments, plasma cells, plasmablasts, or preplasmoblasts are sorted and isolated by FACS analysis using markers known in the art (eg, CD20, CD38, CD138, and/or CD19).

In some embodiments, culturing and/or growing mammary gland cells and plasma cells for the cell construct is carried out at a temperature of about 35 to about 39°C (e.g., a temperature of about 35°C, 35.5°C, 36°C, 36, 5°C, 37°C, 37.5°C, 38°C, 38.5°C, or about 39°C, or any value or range within these limits, for example, from about 35 to about 38°C, about 36 to about 39°C, about 36.5 to about 39°C, about 36.5 to about 37.5°C, or about 36.5 to about 38°C). In some embodiments, cultivation and/or growth is carried out at a temperature of approximately 37°C.

In some embodiments, culturing and/or growing mammary gland cells and plasma cells for cell construct is carried out at an atmospheric CO ₂ content of from about 4% to about 6%, for example, at an atmospheric CO ₂ content of about 4%, 4.25% , 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, or 6%, at any value or range within these limits, for example, from about 4 to about 5, 5%, about 4.5 to about 6%, about 4.5 to about 5.5%, or about 5 to about 6%). In some embodiments, cultivation and/or growth is carried out at approximately CO2 levels in the atmosphere.

In some embodiments, culturing and/or growing mammary gland cells and plasma cells to produce a cell construct involves culturing and/or growing in a culture medium that is replaced approximately every 1 to 10 days (e.g., every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, or any value or range

- 24 046437 zones within these limits, for example, from approximately once a day to approximately once every 3 days, from approximately once every 3 days to approximately once every 10 days, from approximately once every 2 days to approximately once every 5 days). In some embodiments, the cultivation and/or growth further involves culturing in a culture medium that is changed from about once a day to about every few hours to about every 10 days, such as about every 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours up to approximately every 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 days, or any value or range within these limits. For example, in some embodiments, the cultivation and/or growth involves culturing and/or growing in a culture medium that is replaced about every 12 hours to about every 10 days, about every 10 hours to about every 5 days, or from approximately once every 5 hours to approximately once every 3 days.

In some embodiments, the cell construct is stored in a freezer or in liquid nitrogen. Storage temperature depends on the desired storage duration. For example, a freezer temperature may be used (e.g., storage at a temperature of about 0 to about -80°C or less, e.g., about 0°C, -10°C, -20°C, -30°C, -40° C, -50°C, -60°C, -70°C, -80°C, -90°C, -100°C at any value or range within these limits) if the cells are used within 6 months ( for example, within 1, 2, 3, 4, 5 or 6 months). For example, liquid nitrogen can be used (for example, stored at a temperature of -100°C or below (for example, approximately -100°C, -110°C, -120°C, -130°C, -140°C, -150° C, -160°C, -170°C, -180°C, -190°C, -200°C or less) for longer shelf life (e.g. storage 6 months or longer, e.g. 6, 7, 8 , 9, 10, 11 or 12 months, or 1, 2, 3, 4, 5, 6 or more years.

Cultured dairy products

The present invention relates to cultured dairy products that contain protein, lipid and oligosaccharide components, and the concentrations of the components mimic human breast milk produced by a lactating woman, wherein the compositions are produced, at least in part, by in vitro and/or ex vivo cultured mammary cells glands.

In some embodiments, two products with specific compositions can be produced depending on cellular inputs and culture conditions: (a) a functional food product that is a biosynthetic product of cultured mammary epithelial cells (immortalized or from primary tissue samples) and (b) a similar product , additionally containing immunoglobulins obtained by co-cultivating plasma cells (PCs) with mammary epithelial cells. Plasma cells can be obtained from a sample of primary breast tissue or, for example, from a plasma cell line.

The proposed product compositions of the present invention can be determined by the total level of proteins, lipids and carbohydrates (Tables 1A-1C) and/or by the characteristics of specific macronutrient components (Tables 2A-2C) present in concentrations and proportions corresponding to human milk.

Table 1A. Macromolecular composition of functional foods collected from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l % wt. Source Protein 3-21 ^a 1-44 ECMJ Total immunoglobulin ¹³ 0.4-1.8 0.14-3.75 PC Lipid 9-135 3.1-95+ ECMZH CME Lactose General content macromolecules 3.5-21 32-115 48-293 1.2-44 11-95+ ECMZH ECMZH ECM ± PC Energy(kcal/l) 375-1725

HMO, human milk oligosaccharide. ECMC, mammary epithelial cell; PC, plasma cell.

^a Non-immunoglobulin protein content.

^b Immunoglobulin-free formulations can be obtained from ECMG culture in the absence of plasma cells. Compositions containing immunoglobulin can be obtained by co-cultivating ECMG with plasma cells.

^c The long-chain fatty acids linoleic acid and alpha-linoleic acid are not synthesized by mammalian cells and are added to cell culture media.

- 25 046437

Table 1B. Macromolecular composition of functional foods collected from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l % wt. Source Protein 4.5-17.5 ^a 1.8-24 ECMZH Total immunoglobulin ¹³ 0.6-1.5 0.25-2.1 PC Lipid 13.5-111 5.5-95+ ECMZH CME 5.25-17.5 2.2-24 ECMZH Lactose 48-96 20-95+ ECMZH General content macromolecules 71.9-244 ECM ± PC Energy (kcal/l) 375-1438

HMO, human milk oligosaccharide. ECMC, mammary epithelial cell; PC, plasma cell.

^a Non-immunoglobulin protein content.

^b Immunoglobulin-free formulations can be obtained from cultured ECMG in the absence of plasma cells. Compositions containing immunoglobulin can be obtained by co-cultivating ECMG with plasma cells.

^c The long-chain fatty acids linoleic acid and alpha-linoleic acid are not synthesized by mammalian cells and are added to cell culture media.

Table 1C. Macromolecular composition of functional foods collected from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l % wt. % about. Istoyanik Protein 6-14 ^a 3-15 1 ECMZH Total immunoglobulin* ⁵ 0.8-1.2 0.4-1.3 0.1 PC Lipid 18-89 9-92 4 ECMZH CME 7-14 4-15 2.4 ECMJ Lactose 64-77 33-80 4.6 ECMZH General content macromolecules 95.8-195.2 10-15 ECM ± PC Energy (kcal/l) 500-1150

HMO, human milk oligosaccharide. ECMC, mammary epithelial cell;

PC, plasma cell.

^a Non-immunoglobulin protein content.

^b Immunoglobulin-free formulations can be obtained from ECMG culture in the absence of plasma cells. Compositions containing immunoglobulin can be obtained by co-cultivating ECMG with plasma cells.

^c The long-chain fatty acids linoleic acid and alpha-linoleic acid are not synthesized by mammalian cells and are added to cell culture media.

In some embodiments, the concentrations of the components listed in table. 1A-1C may vary for each individual, for example, have a concentration that is greater than the specified one by 0.1, or 0.2, or 0.3 times, or 0.4 times, or 0.5 times, or 0 .6 times, or 0.7 times, or 0.8 times, or 0.9 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

In some embodiments, the concentrations of the components listed in table. 1A-1C may vary for each individual, for example, have a concentration that is less than the specified one by 0.1, or 0.2, or 0.3 times, or 0.4 times, or 0.5 times, or 0. 6 times, or 0.7 times, or 0.8 times, or 0.9 times.

In some embodiments, provided herein are dairy products that contain a subset of the components (ie, macromolecular fractions) of Table 1. 1A1S. In other embodiments, the dairy products provided herein may have one or more components (ie, macromolecular fractions) excluded from the table. 1A-1C.

- 26 046437

Table 2A. Concentration ranges and contents of macromolecules from functional foods derived from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l Protein R-Casein 0.25-1.9 K-Casein 0.25-0.9 a-casein 0.05-0.75 a-lactalbumin 1.35-4.9 Lysozyme 0.2-0.75 Lactoferrin 0.5-3 G aptocorrin 0.03-1.1 Butyrophilin 0.02-0.075 Osteopontin 0.025-0.3 Mucin MS5 0.25-0.9 BgEZ mucin 0.25-1.1 Lactadherin 0.03-1.1 Immunoglobulins 0.1-3 Secretory IgA 0.1-1.5 Total IgA Total IgG Total IgM 0.07-2.4 0.02-0.45 0.005-0.2 Lipids Saturated fatty acids Palmitic acid (C16:0) Stearic acid (C18:0) Lauric acid (C 12:0) Monounsaturated fatty acids Oleic acid (C18:1 n-9 Z) Polyunsaturated fatty acids Linoleic acid, LA (08:2 p-6 Z) α-linolenic acid, ALA (C 18:3 p-3) Eicosadienoic acid (C20:2) 2.5-51 2-27 0.35-7.5 0.25-7.5 3.5-69 3.5-68 1-30 1-29 0.25-1.1 0.25-1.1

- 27 046437

Arachidonic acid, AA (C20:4 p-6) Dihomo-y-linolenic acid, DGLA (C20:3 p-6) Docosahexadienoic acid, DHA (C22:6 p-3) Cholesterol Phospholipids, plasmalogens, sphingolipids 0.25-1.1 0.15-0.8 0.01-0.6 0.045-0.22 0.05-0.6 Human milk olisaccharides Neutral TF-LNH (trifucosyllacto-CG-hexose) 2'-FL (2'-fucosyllactose) 0.8-4.8 DF-LNHII (difucosyllacto-X-hexaose) 0.8-4.8 LNFPI (lacto-A-fucopentaose I) 0.8-4.8 LNDFHI (lacto-X-difucosylhexaose I) 0.4-2.4 LNT (lacto-A-tetraose) 0.16-2.4 LNnT (lacto-A-neotetraose) 0.23-1.8 DF-L (Difucosyllactose) 0.4-1.8 3-FL (3-fucosyllactose) 0.08-1.2 Sour 0.16-1.8 b'-SL (b'-sialyllactose) 0.16-1.44 DS-LNT (disialyl lacto-A-tetraose) 0.04-1.2 FS-LNnHI (fucosyl-sialyl-lacto-X-peohexaose I) 0.04-0.84 LSTc (sialyl-lacto-A-tetraose c) 0.04-0.84 3'-SL (3'-sialyllactose) 0.08-0.36 Lactose 34-104

Table 2B. Concentration ranges and contents of macromolecules from functional foods derived from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l Protein B-Casein K-Casein a-Casein 0.38-1.9 0.38-0.75 0.08-0.63

- 28 046437

α-lactalbumin 2.0-4.13 Lysozyme 0.15-0.63 Lactoferrin 0.8-2.5 G aptocorrine 0.05-0.89 Butyrophilin 0.02-0.063 Osteopontin 0.04-0.25 Mucin MS5 0.38-0.75 BgEZ mucin 0.38-0.89 Lactadherin 0.045-0.09 Immunoglobulins 0.15-2.5 Secretory IgA 0.15-1.25 Total IgA 0.11-2 Total IgG 0.02-0.38 Total IgM 0.0075-0.13 Lipids Saturated fatty acids 3.75-43 Palmitic acid (C16:0) 2.25-23 Stearic acid (C18:0) 0.53-6.3 Lauric acid (C 12:0) 0.38-6.3 Monounsaturated fatty acids 5.25-58 Oleic acid (C18:1 n-9 Z) 5.3-56 Polyunsaturated fatty acids 1.5-25 Linoleic acid, LA (08:2 p-6 Z) 1.5-25 α-linolenic acid, ALA (C 18:3 p-3) 0.38-0.89 Eicosadienoic acid (C20:2) 0.38-0.89 Arachidonic acid, AA (C20:4 p-6) 0.38-0.89 Dihomo-y-linolenic acid, DGLA (C20:3 p-6) 0.23-0.63 Docosahexadienoic acid, DHA (C22:6 p-3) 0.015-0.5 Cholesterol 0.07-0.19 Phospholipids, plasmalogens, sphingolipids 0.075-0.5 Human milk olisaccharides Neutral TF-LNH (trifucosyllacto-M-hexose) 0.9-4.4 2'-FL (2'-fucosyllactose) 0.9-4.4 DF-LNHII (difucosyllacto-X-hexaose) 0.9-4.4 LNFPI (lacto-A-fucopentaose I) 0.45-2.2 LNDFHI (lacto-X-difucosylhexaose I) 0.18-2.2 LNT (lacto-A-tetraose) 0.27-1.7 LNnT (lacto-A-neotetraose) 0.4-1.7 DF-L (Difucosyllactose) 0.09-1.1 3-FL (3-fucosyllactose) 0.18-1.7 Sour b'-SL (b'-sialyllactose) 0.18-1.3 DS-LNT (disialyl lacto-A-tetraose) 0.04-1.1 FS-LNnHI (fucosyl-sialyl-lacto-X-neohexaose I) 0.04-0.8 LSTc (sialyl-lacto-A-tetraose c) 0.04-0.8 3'-SL (3'-sialyllactose) 0.09-0.33 Lactose 50-98

- 29 046437

Table 2C. Concentration ranges and contents of macromolecules from functional foods derived from human mammary epithelial cells cultured in the presence or absence of plasma cells

Macromolecular fraction Concentration, g/l Protein B-Casein 0.5-1.5 K-Casein 0.5-0.6 a-casein 0.1-0.5 a-lactalbumin 2.7-3.3 Lysozyme 0.2-0.5 Lactoferrin 1-2 G aptocorrine 0.07-0.7 Butyrophilin 0.03-0.05 Osteopontin 0.05-0.2 Mucin MS5 0.5-0.6 BgEZ mucin 0.5-0.7 Lactadherin 0.06-0.07 Immunoglobulins 0.2-2 Secretory IgA 0.2-1.0 Total IgA 0.15-1.6 Total IgG 0.03-0.3 Total IgM 0.01-0.1 Lipids Saturated fatty acids 5-34 Palmitic acid (C 16:0) 3-18 Stearic acid (C 18:0) 0.7-5 Lauric acid (C 12:0) 0.5-5 Monounsaturated fatty acids 7-46 Oleic acid (C18:1 n-9 Z) 7-45 Polyunsaturated fatty acids 2-20 Linoleic acid, LA (C18:2n-6 Z) 2-19 α-linolenic acid, ALA (C 18:3 p-3) 0.5-0.7 Eicosadienoic acid (C20:2) 0.5-0.7 Arachidonic acid, AA (C20:4 p-6) 0.5-0.7 Dihomo-y-linolenic acid, DGLA (C20:3 p-6) 0.3-0.5 Docosahexadienoic acid, DHA (C22:6 p-3) 0.02-0.4 Cholesterol 0.09-0.15 Phospholipids, plasmalogens, sphingolipids 0.1-0.4 Human milk olisaccharides Neutral TF-LNH (trifucosyllacto-M-hexose) 1-4 2'-FL (2'-fucosyllactose) 1-4 DF-LNHII (difucosyllacto-M-hexaose) 1-4 LNFPI (lacto-A-fucopentaose I) 0.5-2

- 30 046437

LNDFHI (lacto-I-difucosylhexaose I) 0.2-2 LNT (lacto-A-tetraose) 0.3-1.5 LNnT (lacto-A-neotetraose) 0.5-1.5 DF-L (Difucosyllactose) 0.1-1 3-FL (3-fucosyllactose) Sour 0.2-1.5 b'-SL (b'-sialyllactose) 0.2-1.2 DS-LNT (disialyl lacto-A-tetraose) 0.05-1 FS-LNnHI (fucosyl-sialyl-lacto-M-neohexaose I) 0.05-0.7 LSTc (sialyl-lacto-A-tetraose c) 0.05-0.7 3'-SL (3'-sialyllactose) 0.1-0.3 Lactose 67-78

In some embodiments, the concentrations of the components listed in table. 2A-C may vary for each individual, for example, have a concentration that is greater than the specified one by 0.1, or 0.2, or 0.3 times, or 0.4 times, or 0.5 times, or 0 .6 times, or 0.7 times, or 0.8 times, or 0.9 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

In some embodiments, the concentrations of the components listed in table. 2A-C may vary for each individual, for example, have a concentration that is less than the specified one by 0.1, or 0.2, or 0.3 times, or 0.4 times, or 0.5 times, or 0. 6 times, or 0.7 times, or 0.8 times, or 0.9 times.

In some embodiments, provided herein are dairy products that contain a subset of the components (ie, macromolecular fractions) of Table 1. 2A-C. In other embodiments, the dairy products provided herein may have one or more components (ie, macromolecular fractions) excluded from the table. 2A-C.

In some embodiments, provided herein are dairy products that further contain serum albumin. In some embodiments, serum albumin may have a concentration of approximately 0.025-3.5 g/L. In some embodiments, serum albumin may have a concentration of approximately 0.01-2 g/L. In some embodiments, serum albumin may have a concentration of approximately 0.15-1 g/L, and in some embodiments, serum albumin may have a concentration of approximately 0.2-0.7 g/L.

One aspect of the present disclosure relates to a dairy product containing approximately 6-14 g per liter (g/L) protein components, approximately 18-89 g/L lipid components, approximately 7-14 g/L human milk olisaccharides. (HMO), approximately 64-77 g/L lactose, wherein at least one of protein components, lipid components, HMO and lactose is produced by cultured human mammary epithelial cells.

In some embodiments, the protein component comprises approximately 55-65% dry weight of the dairy product. In some embodiments, the protein component comprises one or more of beta casein, kappa casein, and alpha casein, and in some embodiments, beta casein may have a concentration of approximately 0.5-1.5 g/L, kappa casein may have a concentration of approximately 0.5-0.6 g/L and alpha casein may have a concentration of approximately 0.1-0.5 g/L in the dairy product. In some embodiments of the dairy product, betcasein, kappa casein, and alpha casein together constitute approximately 35-45% of the dry weight of the protein component. In some embodiments, beta casein contains more than about 50% of the total casein content.

In some embodiments, the protein component further comprises, for example, one or more of alpha-lactalbumin, lysozyme, lactoferrin, haptocorrin, butyrophilin, osteopontin, MC5 mucin, BrE3 mucin, and lactadherin. Some embodiments additionally contain serum albumin. In some embodiments of the dairy product, alpha-lactalbumin may have a concentration of about 2.7-3.3 g/L, and in some embodiments, lysozyme may have a concentration of about 0.2-0.5 g/L, and in some embodiments, lactoferrin may have a concentration of approximately 1.0-2.0 g/l. In some embodiments, haptocorrine may have a concentration of about 0.07-0.7 g/L, and in some embodiments, butyrophilin may have a concentration of about 0.03-0.05 g/L. In some embodiments, osteopontin may have a concentration of approximately 0.050.2 g/L, and in some embodiments, MC5 mucin may have a concentration of approximately 0.5-0.6 g/L. In some embodiments, BrE3 mucin may have a concentration of about 0.5-0.7 g/L, and in some embodiments, lactadherin may have a concentration of about 0.06-0.07 g/L. In some embodiments, serum albumin may

- 31 046437 have a concentration of approximately 0.025-3.5 g/l, or approximately 0.01-2 g/l, or approximately 0.15-1 g/l, or approximately 0.2-0.7 g/l.

In some embodiments of the dairy product, the protein components are of human origin.

In some embodiments, the lipid component comprises one or more of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, phospholipids, plasmalogens, and sphingolipids. In some embodiments of the dairy product, the saturated fatty acids may have a concentration of approximately 5-34 g/L. The saturated fatty acids may contain, for example, one or more of palmitic acid, stearic acid and lauric acid and combinations thereof. In some embodiments, palmitic acid contains at least about 50% sn-2 form. In some embodiments of the dairy product, palmitic acid may have a concentration of approximately 3-18 g/L; in some embodiments, stearic acid may have a concentration of approximately 0.7-5 g/L; and in some embodiments, lauric acid may have a concentration of approximately 0. 5-5 g/l.

In some embodiments, the monounsaturated fatty acids may have a concentration of approximately 7-46 g/L in the dairy product. The saturated fatty acid component may comprise, for example, oleic acid, in some embodiments the oleic acid contains at least about 50% sn-1 form, and in some embodiments the oleic acid may have a concentration of about 7-45 g/L in the dairy product .

In some embodiments of the dairy product, the polyunsaturated fats may have a concentration of approximately 2-20 g/L. Polyunsaturated fats may contain, for example, one or more of linoleic acid, alpha-linolenic acid, eicosadienoic acid, arachidonic acid, dihomo-gamma-linolenic acid and docosahexadienoic acid. In some embodiments, linoleic acid contains at least about 50% sn-3 form, and in some embodiments, linoleic acid can have a concentration of about 2-19 g/L, and in some embodiments, alpha-linolenic acid can have a concentration of about 0 .5-0.7 g/l. In some embodiments, eicosadienoic acid may have a concentration of about 0.5-0.7 g/L, and in some embodiments, arachidonic acid may have a concentration of about 0.5-0.7 g/L. In some embodiments, dihomo-gamma-linolenic acid may have a concentration of about 0.3-0.5 g/L, and in some embodiments, docosahexadienoic acid may have a concentration of about 0.02-0.4 g/L. Some embodiments of the dairy product contain all of the above polyunsaturated fats, wherein linoleic acid may have a concentration of approximately 2-19 g/L, alpha-linolenic acid may have a concentration of approximately 0.5-0.7 g/L, eicosadienoic acid may have a concentration of approximately 0.5-0.7 g/L, arachidonic acid may have a concentration of approximately 0.5-0.7 g/L, dihomo-gamma-linolenic acid may have a concentration of approximately 0.3-0.5 g/L, and DHA may have a concentration of approximately 0.02-0.4 g/L in the dairy product.

In some embodiments, cholesterol may have a concentration of about 0.09-0.15 g/L in the dairy product, and in some embodiments, phospholipids, plasmalogens, and sphingolipids together may have a concentration of about 0.1-0.4 g/L in dairy product.

In some embodiments, the dairy product contains one or more neutral oligosaccharides, one or more acid oligosaccharides, or one or more neutral oligosaccharides and acid oligosaccharides individually.

In some embodiments, the one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexose), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-Nhexaose), LNFPI (lacto-N-fucopentaose I) , LNDFHI (lacto-N-difucosylhexaose I), LNT (lacto-Ntetraose), LNnT (lacto-N-neotetraose), DF-L (difucosyllactose) and 3-FL (3-fucosyllactose).

In some embodiments, the one or more acid oligosaccharides comprise 6'-SL (6'-sialyllactose), DS-LNT (disialyl-lacto-X-tetraose), FS-LNnHI (fucosyl-sialyl-lacto-Npeohexaose I), LSTc (sialyl- lacmo-N-tetraose c) and 3'-SL (3'-sialyllactose).

In some embodiments, one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexose), wherein the oligosaccharide may have a concentration of approximately 1-4 g/L in the dairy product. In some embodiments, the one or more neutral oligosaccharides comprise 2'-FL (2'-fucosyllactose), wherein the oligosaccharide may have a concentration of approximately 1-4 g/L in the dairy product, and in some embodiments, the one or more neutral oligosaccharides contain DF -LNHII (difucosyllacto-N-hexaose), wherein the oligosaccharide may have a concentration of approximately 1-4 g/l in the dairy product. In some embodiments, the one or more neutral oligosaccharides comprise LNFPI (lacto-Nfucopentaose I), wherein the oligosaccharide may have a concentration of about 0.5-2 g/L in the dairy product, and in some embodiments, the one or more neutral oligosaccharides

- 32 046437 reads contain LNDFHI (lacto-N-difucosylhexaose I), and the oligosaccharide can have a concentration of approximately 0.2-2 g/l in the dairy product. In some embodiments, the one or more neutral oligosaccharides comprise LNT (lacto-N-tetraose), wherein the oligosaccharide may have a concentration of approximately 0.3-1.5 g/L in the dairy product, and in some embodiments, the one or more neutral oligosaccharides contain LNnT (lacto-N-neotetraose), and the oligosaccharide may have a concentration of approximately 0.5-1.5 g/l in the dairy product. In some embodiments, the one or more neutral oligosaccharides contain DF-L (difucosyllactose), wherein the oligosaccharide may have a concentration of approximately 0.1-1 g/L in the dairy product, and in some embodiments, the one or more neutral oligosaccharides contain 3-FL (3-fucosyllactose), wherein the oligosaccharide may have a concentration of approximately 0.2-1.5 g/l in the dairy product.

In some embodiments, one or more acid oligosaccharides comprise 6'-SL (6'-sialyllactose), wherein the oligosaccharide may have a concentration of approximately 0.2-1.2 g/L in the dairy product, and in some embodiments, one or more acid oligosaccharides contain DS-LNT (disialyl lacto-N-tetraose), and the oligosaccharide may have a concentration of approximately 0.05-1 g/l in the dairy product. In some embodiments, one or more acid oligosaccharides comprise FS-LNnHI (fucosyl-sialyl-lacto-N-peohexaose I), wherein the oligosaccharide may have a concentration of approximately 0.05-0.7 g/L in the dairy product, and in some In embodiments, one or more acid oligosaccharides comprise LSTc (sialyl-lacto-Ntetraose c), wherein the oligosaccharide may have a concentration of approximately 0.05-0.7 g/L in the dairy product. In some embodiments, one or more acid oligosaccharides comprise 3'-SL (3'-sialyllactose), wherein the oligosaccharide may have a concentration of approximately 0.1-0.3 g/L in the dairy product.

In some embodiments of the dairy product, one or more neutral oligosaccharides comprise TF-LNH (trifucosyllacto-N-hexose), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-N-hexaose), LNFPI (lacto-N -fucopentaose I), LNDFHI (lacto-N-difucosylhexaose I), LNT (lacto-N-tetraose), LNnT (lacto-N-neotetraose), DF-L (difucosyllactose) and 3-FL (3-fucosyllactose) and one or more acid oligosaccharides contain 6'-SL (6'-sialyllactose), DSLNT (disialyl-lacto-N-tetraose), FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), LSTc (sialyllacto-N-tetraose c) and 3 '-SL (3'-sialyllactose).

In some embodiments, the dairy product contains about 1-4 g/L TFLNH (trifucosyllacto-N-hexose), contains about 1-4 g/L 2'-FL (2'-fucosyllactose), contains about 1-4 g/L DF-LNHII (difucosyllacto-N-hexaose), contains approximately 0.5-2 g/L LNFPI (lacto-N-fucopentaose I), contains approximately 0.2-2 g/L LNDFHI (lacto-N-fucosylhexaose I), contains approximately 0.3-1.5 g/l LNT (lacto-N-tetraose), contains approximately 0.5-1.5 g/l LNnT (lacto-N-neotetraose), contains approximately 0.1-1 g/ l DF-L (difucosyllactose), contains approximately 0.2-1.5 g/l 3-FL (3-fucosyllactose), contains approximately 0.21.2 g/l 6'-SL (6'-sialyllactose), contains approximately 0.05-1 g/l DS-LNT (disialyl-lacto-N-tetraose), approximately 0.05-0.7 g/l FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), contains approximately 0. 05-0.7 g/l LSTc (sialyl-lacto-N-tetraose C) and contains approximately 0.1-0.3 g/l 3'SL (3'-sialyllactose).

In some embodiments, the neutral oligosaccharide content is at least about 2 times, or about 3 times, or about 4 times, or about 5 times, or about 6 times, or about 7 times, or about 8 times times, or approximately 9 times, or approximately 10 times, or approximately 11 times, or approximately 12 times, or approximately 13 times, or approximately 14 times, or approximately 15 times more by weight than the content of acid oligosaccharides .

Another aspect of the disclosure provides a dairy product containing approximately 6-14 grams per liter (g/L) of protein components, approximately 18-89 g/L of lipid components, approximately 7-14 g/L of human milk olisaccharides (HMO), approximately 64 -77 g/l lactose, where the protein components contain one or more of beta casein, kappa casein, alpha casein, alphalactalbumin, lysozyme, lactoferrin, haptocorrin, butyrophilin, osteopontin, MC5 mucin, BrE3 mucin and lactadherin, where the lipid components contain one or more of palmitic acid, stearic acid and lauric acid, oleic acid, linoleic acid, alphalinolenic acid, eicosadienoic acid, arachidonic acid, dihomo-gamma-linolenic acid, docosahexadienoic acid, cholesterol, phospholipids, plasmalogens and sphingolipids, where milk olisaccharides human contain one or more of TF-LNH (trifucosyllacto-N-hexoses), 2'-FL (2'-fucosyllactose), DF-LNHII (difucosyllacto-N-hexaoses), LNFPI (lacto-N-fucopentaose I), LNDFHI (lacto-N-difucosylhexaoses I), LNT (lacto-N-tetraoses), LNnT (lacto-N-neotetraoses), DF-L (difucosyllactoses) and 3-FL (3-fucosyllactoses), 6'-SL (6' -sialyllactose), DS-LNT (disialyl-lacto-N-tetraose), FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), LSTc (sialyl-lacto-N-tetraose c) and 3'-SL (3'-sialyllactose ), and where at least one of protein components, lipid components,

- 33 046437

HMO and lactose are produced by cultured human mammary epithelial cells. In some embodiments, the dairy product further comprises serum albumin.

In some embodiments, the dairy product contains about 0.5-1.5 g/L beta casein, contains about 0.5-0.6 g/L kappa casein, contains about 0.1-0.5 g/L alpha casein, contains approximately 2.7-3.3 g/l alpha-lactalbumin, contains approximately 0.2-0.5 g/l lysozyme, contains approximately 1.0-2.0 g/l lactoferrin, contains approximately 0.070.7 g/l haptocorrin, contains approximately 0.03-0.05 g/l butyrophilin, contains approximately 0.05-0.2 g/l osteopontin, contains approximately 0.5-0.6 g/l mucin MC5 , contains approximately 0.5-0.7 g/l BrE3 mucin, contains approximately 0.06-0.07 g/l lactadherin, contains approximately 2-19 g/l linoleic acid, contains approximately 0.5-0.7 g/l alpha-linolenic acid, approximately 0.5-0.7 g/l eicosadienoic acid, approximately 0.5-0.7 g/l arachidonic acid, approximately 0.3-0.5 g/l dihomo-gamma -linolenic acid, contains approximately 0.02-0.4 g/l docosahexadienoic acid, contains approximately 0.09-0.15 g/l cholesterol, together contains approximately 0.1-0.4 g/l phospholipids, plasmalogens and sphingolipids, contains approximately 1-4 g/l TFLNH (trifucosyllacto-N-hexose), contains approximately 1-4 g/l 2'-FL (2'-fucosyllactose), contains approximately 1-4 g/l DF-LNHII ( Difucosyllacto-N-hexaose), contains approximately 0.5-2 g/l LNFP I (lacto-N-fucopentaose I), contains approximately 0.2-2 g/l LNDFHI (lacto-N-fucosylhexaose I), contains approximately 0. 3-1.5 g/l LNT (lacto-N-tetraose), contains approximately 0.5-1.5 g/l LNnT (lacto-N-neotetraose), contains approximately 0.1-1 g/l DF- L (difucosyllactose), contains approximately 0.2-1.5 g/l 3-FL (3-fucosyllactose), contains approximately 0.21.2 g/l 6'-SL (6'-sialyllactose), contains approximately 0 .05-1 g/l DS-LNT (disialyl-lacto-N-neohexaose), contains approximately 0.05-0.7 g/l FS-LNnHI (fucosyl-sialyl-lacto-N-neohexaose I), contains approximately 0.05- 0.7 g/l LSTc (sialyl-lacto-N-tetraose c), and contains approximately 0.1-0.3 g/l 3'-SL (3'-sialyllactose). In some embodiments, the dairy product further comprises serum albumin that has a concentration of about 0.025-3.5 g/L, or about 0.01-2 g/L, or about 0.15-1 g/L, or about 0. 3-0.7 g/l.

Another aspect of the disclosure provides a dairy product containing about 3-15% protein by weight, about 9-92% lipid by weight, about 4-15% by weight human milk olisaccharides (HMO), and about 33-80% by weight lactose, wherein at least one of protein, lipid, HMO and lactose is produced by cultured human mammary epithelial cells.

In some embodiments of certain aspects of the disclosure, the dairy product contains at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% of the total macromolecular composition of human breast milk.

In some embodiments of the dairy product disclosed, the non-protein nitrogen content is at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30 % of total nitrogen content.

In some embodiments, the dairy product contains approximately 500-1150 kcal/L of available energy, and in some embodiments, approximately 40-55% of the available energy comes from lipids. In some embodiments, the dairy product contains from about 95.8 to 195.2 g/L of macromolecules.

In some embodiments, the cultured dairy product contains betcasein, alpha-lactalbumin, kappa-casein, alpha-casein, lactoferrin, lactadherin, lysozyme, C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid). acid), lactose, glucose, 3'-SL and 6'-SL.

In some embodiments, the cultured dairy product contains betcasein, alpha-lactalbumin, kappa-casein, alpha-casein, lactoferrin, lactadherin, lysozyme, C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid). acid), C18:2n-6 (linoleic acid, LA), C18;3n-3 (alpha-linolenic acid, ALA), lactose, glucose, 3'-SL and 6'-SL.

In some embodiments, the cultured dairy product contains betcasein, alpha-lactalbumin, kappa-casein, alpha-casein, lactoferrin, butrophilin, osteopontin, lactadherin, lysozyme, mucin-4, mucin-1, C8:0 (caprylic acid), C10 :0 (capric acid), C12:0 (lauric acid), C13:0 (tridecanoic acid), C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:2p -6 (linoleic acid, LA), C18;3n-3 (alpha-linolenic acid, ALA), C20:3n-6 (dihomo-gama-linolenic acid, DGLA), C20:4n-6 (arachidonic acid, AA) , C20:5p-3 (eicosapentanoic acid, EPA), C22:6p-3 (docosahec

- 34 046437 saenoic acid, DHA), C22:1n9 (ervic acid), C24:1 (nervonic acid), lactose, glucose, 3'SL, 6'-SL, LNT and LNnT.

In some embodiments, the cultured dairy products described herein further contain at least one oxylipin polyunsaturated fatty acid metabolite or at least one endocannabinoid. In some embodiments, the oxylipin metabolite is 9-hydroxyoctadecadienoic acid (9HODE); 13-hydroxyoctadecadienoic acid (13-HODE); 5,15-dihydroxyeicosatetraenoic acid (5,15-DiHETE); 17,18-dihydroxyeicosatetraenoic acid (17,18-DiHETE); 8,9-dihydroxyicosatrienoic acid (8,9-DiHETrE); 11,12-dihydroxyicosatrienoic acid (11,12-DiHETrE); 9,10-dihydroxyoctadecenoic acid (9,10-DiHOME); 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME); 14(15)-epoxyeicosatrienoic acid (14(15)-EpETre); 19(20)-epoxydocosapentanoic acid (19(20)-EpDPE); 17-hydroxydocosahexaenoic acid (17-HDoHE); 5-hydroxyeicosatetraenoic acid (5-HETE); 8-hydroxyeicosatetraenoic acid (8-HETE); 9hydroxyeicosatetraenoic acid (9-HETE); 11-hydroxyeicosatetraenoic acid (11-HETE); 12hydroxyeicosatetraenoic acid (12-HETE); 9-hydroxyoctadecatrienoic acid (9-HOTrE); 9-oxo-octadecadienoic acid (9-okco-ODE); or 13-oxo-octadecadienoic acid (13-okco-ODE); 17,18-epoxyeicosatetraenoic acid (17(18)-EPETE; 6-keto-prostaglandin F1-alpha (6-keto-PGF1a); or 15(B)-hydroxyeicosatrienoic acid (15(S)-HETrE). In some embodiments The implementation of the endocannabinoid is anandamide or 2-arachidonoylglycerol (2-AG).

In some embodiments, the cultured dairy product further comprises a preservative, stabilizer, antioxidant, emulsifier, or thickener. In some embodiments, the cultured dairy product further comprises lecithin. In some embodiments, the cultured dairy product further comprises carrageenan. In some embodiments, the cultured dairy product additionally contains beta-carotene. In some embodiments, the cultured dairy product further comprises vitamin E. In some embodiments, the cultured dairy product further comprises ascorbyl palmitate or ascorbic acid. In some embodiments, the cultured dairy product further comprises lecithin, ascorbyl palmitate, and vitamin E. In some embodiments, the cultured dairy product contains lecithin, ascorbic acid, and vitamin E.

Another aspect of the present disclosure provides a containerized dairy product comprising a dairy product according to certain embodiments of the disclosure that is packaged in the container.

In another aspect, the disclosure provides a frozen dairy product comprising a dairy product according to certain embodiments of the disclosure that has been frozen. In some embodiments, the frozen dairy product is packaged in a container, resulting in a frozen dairy product in the container. In some embodiments, the cultured dairy product is stored at a temperature of from about -18°C to about -80°C. In some embodiments, the cultured dairy product is stored at approximately -18°C. In some embodiments, the cultured dairy product is stored at approximately -20°C. In some embodiments, the cultured dairy product is stored at approximately -80°C.

In another aspect of the disclosure, a lyophilized dairy product is provided, comprising a dairy product according to certain embodiments of the disclosure that has been lyophilized. In some embodiments, the lyophilized dairy product is packaged in a container, resulting in a lyophilized dairy product in a container.

Another aspect of the disclosure provides an extracted dairy product comprising one or more components extracted from the dairy product according to certain embodiments of the disclosure. Non-limiting examples of components that can be extracted from a dairy product include protein, lipid, carbohydrate, vitamin and/or minerals. In some embodiments, one or more components extracted from the collected dairy product is lyophilized or concentrated to produce a lyophilized or concentrated extracted dairy product component. In some embodiments, one or more components extracted from the collected dairy product is concentrated by membrane filtration or reverse osmosis, while in other embodiments, all of the unextracted dairy product is concentrated by membrane filtration or reverse osmosis. In some embodiments, one or more components extracted from the collected dairy product contain milk proteins, lipids, carbohydrates, vitamins and minerals. In some embodiments, one or more extracted components of the dairy product are packaged in a container, and in some embodiments, the concentrated whole unextracted dairy product is packaged in the container.

In some embodiments, the container is sterile, vacuum sealed

- 35 046437 or classified as food, or any combination thereof. Non-limiting examples of containers include a canister, can, bottle, bag, box or pouch. In some embodiments, the container is a canister, can, bottle, bag, box, or pouch.

Immunoglobulins

In some embodiments, the cultured dairy product contains one or more immunoglobulins or sIgA. In some embodiments, the cultured dairy product contains one or more of IgA, IgG, and IgM. In some embodiments, the cultured dairy product contains IgA2 (secretory) and IgA1 (non-secretory). In some embodiments, plasma cells of the cell construct produce IgA and mammary epithelial cells process IgA to produce sIgA (IgA2). sIgA contains a secretory component, the extracellular domain of the polymeric Ig receptor, attached to IgA. Breast epithelial cells process IgA by cleaving the extracellular domain of the polymeric Ig receptor to form sIgA. In some embodiments, sIgA is secreted from the apical surface of mammary epithelial cells.

In certain embodiments, immunoglobulins bind to an antigen of a microorganism (ie, bacteria or virus). In certain embodiments, immunoglobulins bind to viral or bacterial antigens capable of causing an infectious disease. In certain embodiments, immunoglobulins bind to viral or bacterial antigens that cause infections of the respiratory or gastrointestinal epithelium. In certain embodiments, immunoglobulins bind to antigens of microorganisms that cause enterocolitis or sepsis in infants.

In certain embodiments, the dairy product contains approximately 0.2-1.0 g/L secretory IgA. In certain embodiments, the dairy product contains approximately 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0. 7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.0 g/l secretory IgA. In certain embodiments, the dairy product contains approximately 0.15-1.6 g/L total IgA. In certain embodiments, the dairy product contains approximately 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0. 65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55 or 1.6 g/l total IgA.

In some IgG-containing embodiments, the milk product contains approximately 0.03-0.3 g/L IgG. In some IgG-containing embodiments, the milk product contains approximately 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0. 12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3 g/l. In some IgM-containing embodiments, the dairy product contains approximately 0.01-0.1 g/L IgM. In certain embodiments, the dairy product contains approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0. 1 g/l IgM. In some embodiments, the dairy product contains approximately 0.2-2.0 percent total immunoglobulin by weight. In some embodiments, the dairy product contains 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 percent by weight of total immunoglobulin.

Included herein are immunotherapeutic compositions containing (a) IgA and sIgA derived from the secreted products of mammary epithelial cells co-cultured with plasma cells, and (b) a pharmaceutically acceptable excipient. In some embodiments, IgA and sIgA are isolated from a product resulting from co-culture of mammary epithelial cells and plasma cells (eg, a cultured dairy product). In some embodiments, the pharmaceutically acceptable excipient is a stabilizer, surfactant, buffer, or tonicity agent. In some embodiments, the pharmaceutically acceptable excipient is sucrose, trehalose, mannitol, sorbitol, histidine, arginine, glycine, polysorbate 20, polysorbate 80, poloxamer 188, edetic acid/or salts thereof (e.g., EDTA), glutathione, metacresol, phenol, benzyl alcohol, benzalkonium chloride, methionine or cysteine.

In some embodiments, IgA is produced by plasma cells in a cellular construct. IgA binds to a receptor (polymeric Ig receptor) on the basal surface of mammary epithelial cells. IgA and receptors are transported into mammary epithelial cells. Breast cells are treated with IgA. The extracellular domain of the receptor bound to IgA (secretory component) is cleaved by a proteinase within mammary epithelial cells, and IgA bound to the secretory component is secreted from the apical surface of mammary epithelial cells to form sIgA. In some embodiments, sIgA is secreted within the cultured milk product from the apical surface of mammary cells into the apical compartment. In some embodiments, sIgA is isolated from a cultured dairy product. In some embodiments, sIgA is not isolated from the cultured dairy product.

Basic culture media and lactogenic media

In some embodiments, the culture medium contains a carbon source,

- 36 046437 chemical buffer system, one or more essential amino acids, one or more vitamins and/or cofactors and one or more inorganic salts. In some embodiments, the carbon source, chemical buffer system, one or more essential amino acids, one or more vitamins and/or cofactors, and/or one or more inorganic salts are food grade.

In some embodiments, the culture medium is a lactogenic culture medium. In some embodiments, the culture medium further comprises prolactin (eg, mammalian prolactin, eg, human prolactin), linoleic and alpha-linoleic acid, estrogen and/or progesterone. For example, in some embodiments, the culture medium contains prolactin (or prolactin is added) in an amount of from about 20 to about 200 ng/L of culture medium, for example, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng/ml or any value or range within these limits. In some embodiments, the culture medium contains prolactin (or prolactin is added) in an amount of about 20 to about 195 ng/ml, about 50 to about 150 ng/ml, about 25 to about 175 ng/ml, about 45 to about 200 ng/ml, or about 75 to about 190 ng/ml culture medium. In some embodiments, the culture medium further contains other factors to improve efficacy, including, but not limited to, insulin, epidermal growth factor, and/or hydrocortisone.

In some embodiments, the culture medium contains a carbon source in an amount of from about 1 to about 15 g/L of culture medium (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g/L or any value or range within these limits), or from about 1, 2, 3, 4, 5 or 6 g/L to about 7, 8, 9 or 10, 11, 12 , 13, 14 or 15 g/l culture medium. Non-limiting examples of the carbon source include glucose and/or pyruvate. For example, in some embodiments, the culture medium contains glucose in an amount of from about 1 g/L to about 12 g/L of culture medium, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 g/l, or any value or range within these limits. In some embodiments, the culture medium contains glucose in an amount of about 1 to about 6 g/L, about 4 to about 12 g/L, about 2.5 to about 10.5 g/L, about 1.5 to about 11.5 g/L, or about 2 to about 10 g/L culture medium. In some embodiments, the culture medium contains glucose in an amount of from about 1, 2, 3, or 4 g/L to about 5, 6, 7, 8, 9, 10, 11, or 12 g/L, or from about 1, 2. 3, 4, 5 or 6 g/l to approximately 7, 8, 9, 10, 11 or 12 g/l. In some embodiments, the culture medium contains pyruvate in an amount of from about 5 to about 15 g/L of culture medium, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g/L. l or any value or range within these limits. In some embodiments, the culture medium contains pyruvate in an amount of about 5 to about 14.5 g/L, about 10 to about 15 g/L, about 7.5 to about 10.5 g/L, about 5 .5 to about 14.5 g/L or about 8 to about 10 g/L culture medium. In some embodiments, the culture medium contains pyruvate in an amount of from about 5, 6, 7, or 8 g/L to about 9, 10, 11, 12, 13, 14, or 15 g/L, or from about 5, 6, 7. 8, 9 or 10 g/l to approximately 11, 12, 13, 14 or 15 g/l.

In some embodiments, the culture medium contains a chemical buffer system in an amount of about 1 to about 4 g/L (e.g., about 1, 1.5, 2, 2.5, 3, 3.5 or 4 g/L, or any value or range within these limits) of culture medium or from about 10 to about 25 mM (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mM or any value or range within these limits). In some embodiments, the chemical buffer system includes, but is not limited to, sodium bicarbonate and/or 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). For example, in some embodiments, the culture medium contains sodium bicarbonate in an amount of about 1 to about 4 g/L of culture medium, such as about 1, 1.5, 2, 2.5, 3, 3.5, or 4 g/L. l or any value or range within these limits. In some embodiments, the culture medium contains sodium bicarbonate in an amount of about 1 to about 3.75 g/L, about 1.25 to about 4 g/L, about 2.5 to about 3 g/L, about 1.5 to about 4 g/L, or about 2 to about 3.5 g/L culture medium. In some embodiments, the culture medium contains HEPES in an amount of about 10 to about 25 mM, such as about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mM or any value or range within these limits. In some embodiments, the culture medium contains HEPES in an amount of about 11 to about 25 mM, approx.

- 37 046437 specifically 10 to about 20 mM, about 12.5 to about 22.5 mM, about 15 to about 20.75 mM, or about 10 to about 20 mM.

In some embodiments, the culture medium contains one or more essential amino acids in an amount of about 0.5 to about 5 mM (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mM or any value or range within these limits) or from about 0.5, 1, 1.5, 2 mM to about 2.5, 3, 3.5, 4, 4.5 or 5 mM. In some embodiments, the one or more essential amino acids is histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, and/or arginine. For example, in some embodiments, the culture medium contains arginine in an amount of about 0.5 to about 5 mM, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mM or any value or range within these limits. In some embodiments, the culture medium contains essential amino acids in an amount of about 0.5 to about 4.75 mM, about 2 to about 3.5 mM, about 0.5 to about 3.5 mM, about 1 to about 5 mM, or about 3.5 to about 5 mM.

In some embodiments, the culture medium contains one or more vitamins and/or cofactors in an amount of from about 0.01 to about 50 μM (e.g., about 0.01, 0.02, 0.03, 0.04, 0.05 , 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0 ,9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2 , 2.3, 2.4, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45 , 46, 47, 48, 49, 49.025, 49.05, 49.075 or 50 µM or any value or range within these limits) or approximately 0.01, 0.02, 0.03, 0.04, 0.05 , 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0 .9 µM to approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2 ,2, 2.3, 2.4, 2.5, 3, 4, 5, 6 µM or approximately 0.02, 0.025, 0.05, 0.075, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM to approximately 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075 or 50 µM. In some embodiments, the one or more vitamins and/or cofactors include, but are not limited to, thiamine and/or riboflavin. For example, in some embodiments, the culture medium contains thiamine in an amount of from about 0.025 to about 50 μM, for example, about 0.025, 0.05, 0.075, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075 or 50 µM or any value or range within these limits. In some embodiments, the culture medium contains thiamine in an amount of about 0.025 μM to about 45.075 μM, about 1 μM to about 40 μM, about 5 μM to about 35.075 μM, about 10 μM to about 50 μM, or about 0.05 µM to approximately 45.5 µM. In some embodiments, the culture medium contains riboflavin in an amount of from about 0.01 μM to about 3 μM, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0 .07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1 ,1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2 .4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 µM or any value or range within these limits. In some embodiments, the culture medium contains riboflavin in an amount of about 0.01 to about 2.05 μM, about 1 to about 2.95 μM, about 0.05 to about 3 μM, about 0.08 to about 1.55 µM, or about 0.05 to about 2.9 µM.

In some embodiments, the culture medium contains one or more inorganic salts in an amount of from about 100 to about 150 mg/L of culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg/L or any value or range thereof) or about 100 mg/L to about 150 mg/L culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg/l or any value or range within these limits). In some embodiments, the one or more inorganic salts include, but are not limited to, calcium and/or magnesium. For example, in some embodiments, the culture medium contains calcium in an amount of from about 100 to about 150 mg/L of culture medium, such as about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg /l or any value or range within these limits. In some embodiments, the culture medium contains arginine in an amount of about 100 to about 125 mg/L, about 105 to about 150 mg/L, about 120 to about 130 mg/L, or about 100 to about 145 mg/L l culture medium. In some embodiments, the culture medium contains magnesium in an amount of about 0.01 mM to about 1 mM, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0 .07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1 mM or any value or range within these limits. In some embodiments, the culture medium contains magnesium in an amount of about 0.05 mM to about 1 mM, about 0.01 mM to about 0.78 mM, about 0.5 mM to about 1 mM, about 0. 03 mM to about 0.75 mM, or about 0.25 mM to about 0.95 mM.

- 38 046437

In some embodiments, the culture medium contains a carbon source in an amount of from about 1 to about 15 g/L of culture medium (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g/L or any value or range within these limits), or from about 1, 2, 3, 4, 5 or 6 g/L to about 7, 8, 9 or 10, 11, 12 , 13, 14 or 15 g/l culture medium. In some embodiments, the carbon source includes, but is not limited to, glucose and/or pyruvate. For example, in some embodiments, the culture medium contains glucose in an amount of from about 1 to about 12 g/L of culture medium, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 g/l or any value or range within these limits. In some embodiments, the culture medium contains glucose in an amount of about 1 to about 6 g/L, about 4 to about 12 g/L, about 2.5 to about 10.5 g/L, about 1.5 to about 11.5 g/L, or about 2 to about 10 g/L culture medium. In some embodiments, the culture medium contains pyruvate in an amount of from about 5 to about 15 g/L of culture medium, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g/L or any value or range within these limits. In some embodiments, the culture medium contains pyruvate in an amount of about 5 to about 14.5 g/L, about 10 to about 15 g/L, about 7.5 to about 10.5 g/L, about 5 .5 to about 14.5 g/L or about 8 to about 10 g/L culture medium.

In some embodiments, the culture medium contains a chemical buffer system in an amount of about 1 to about 4 g/L (e.g., about 1, 1.5, 2, 2.5, 3, 3.5 or 4 g/L, or any value or range within these limits) culture medium or from about 10 to about 25 mM (for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mM or any value or range within these limits). In some embodiments, the chemical buffer system includes, but is not limited to, sodium bicarbonate and/or HEPES. For example, in some embodiments, the culture medium contains sodium bicarbonate in an amount of about 1 to about 4 g/L of culture medium, such as about 1, 1.5, 2, 2.5, 3, 3.5, or 4 g/L. l or any value or range within these limits. In some embodiments, the culture medium contains sodium bicarbonate in an amount of about 1 to about 3.75 g/L, about 1.25 to about 4 g/L, about 2.5 to about 3 g/L, about 1.5 to about 4 g/L, or about 2 to about 3.5 g/L culture medium. In some embodiments, the culture medium contains HEPES in an amount of about 10 to about 25 mM, such as about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mM or any value or range within these limits. In some embodiments, the culture medium contains HEPES in an amount of about 1 mM to about 25 mM, about 10 mM to about 20 mM, about 12.5 mM to about 22.5 mM, about 15 mM to about 20, 75 mM, or about 10 mM to about 20 mM.

In some embodiments, the culture medium contains one or more essential amino acids in an amount of about 0.5 to about 5 mM (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mM or any value or range within these limits) or from about 0.5, 1, 1.5, 2 mM to about 2.5, 3, 3.5, 4, 4.5 or 5 mm. In some embodiments, one or more of the essential amino acids is arginine and/or cysteine. For example, in some embodiments, the culture medium contains arginine in an amount of about 0.5 to about 5 mM, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4 .5 or 5 mM or any value or range within these limits. In some embodiments, the culture medium contains arginine in an amount of about 0.5 mM to about 4.75 mM, about 2 mM to about 3.5 mM, about 0.5 mM to about 3.5 mM, about 1 mM to about 5 mM, or about 3.5 mM to about 5 mM. For example, in some embodiments, the culture medium contains cysteine in an amount of from about 0.5 mM to about 5 mM, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 , 4.5 or 5 mM or any value or range within these limits. In some embodiments, the culture medium contains cysteine in an amount of from about 0.5 mM to about 4.75 mM, from about 2 mM to about 3.5 mM, from about 0.5 mM to about 3.5 mM, from about 1 mM to about 5 mM, or about 3.5 mM to about 5 mM.

In some embodiments, the culture medium contains one or more vitamins and/or cofactors in an amount of from about 0.01 to about 50 μM (e.g., about 0.01, 0.02, 0.03, 0.04, 0.05 , 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0 ,9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2 , 2.3, 2.4, 2.5, 3, 4, 5, 6, 7, 8.9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45 , 46, 47, 48, 49, 49.025, 49.05, 49.075, or 50 µM, or any value or range therein

- 39 046437 cases) or approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2 , 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 µM to approximately 1, 1.1, 1.2, 1.3, 1.4, 1 .5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6 µM or approximately 0.02, 0.025, 0.05, 0.075, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM to approximately 12.5, 15, 17.5, 20 , 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075 or 50 µM. In some embodiments, the one or more vitamins and/or cofactors include, but are not limited to, thiamine and/or riboflavin. For example, in some embodiments, the culture medium contains thiamine in an amount of from about 0.025 to about 50 μM, such as 0.025, 0.05, 0.075, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075 or 50 µM or any value or range in these limits. In some embodiments, the culture medium contains thiamine in an amount of about 0.025 to about 45.075 μM, about 1 to about 40 μM, about 5 to about 35.075 μM, about 10 to about 50 μM, or about 0.05 to about 45.5 µM. In some embodiments, the culture medium contains riboflavin in an amount of about 0.01 to about 3 μM, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 µM or any value or range within these limits. In some embodiments, the culture medium contains riboflavin in an amount of about 0.01 to about 2.05 μM, about 1 to about 2.95 μM, about 0.05 to about 3 μM, about 0.08 to about 1.55 µM or about 0.05 to about 2.9 µM.

In some embodiments, the culture medium contains one or more inorganic salts in an amount of from about 100 to about 150 mg/L of culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg/L or any value or range thereof) or about 100 to about 150 mg/L culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg/l or any value or range within these limits). In some embodiments, one or more exemplary inorganic salts is calcium and/or magnesium. For example, in some embodiments, the culture medium contains calcium in an amount of from about 100 to about 150 mg/L of culture medium, such as about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg /l or any value or range within these limits. In some embodiments, the culture medium contains arginine in an amount of about 100 mg/L to about 125 mg/L, about 105 mg/L to about 150 mg/L, about 120 mg/L to about 130 mg/L, or about 100 mg/L to about 145 mg/L culture medium. In some embodiments, the culture medium contains magnesium in an amount of about 0.01 to about 1 mM, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 , 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0 .92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1 mM or any value or range within these limits. In some embodiments, the culture medium contains magnesium in an amount of about 0.05 mM to about 1 mM, about 0.01 mM to about 0.78 mM, about 0.5 mM to about 1 mM, about 0. 03 mM to about 0.75 mM or about 0.25 mM to about 0.95 mM.

In some embodiments, the carbon source, chemical buffer system, one or more essential amino acids, one or more vitamins and/or cofactors, and/or one or more inorganic salts are food grade.

In some embodiments, the culture medium is a lactogenic culture medium, for example, the culture medium further comprises prolactin (eg, mammalian prolactin, eg, human prolactin). For example, in some embodiments, the culture medium contains prolactin (or prolactin is added) in an amount of from about 20 to about 200 ng/L of culture medium, such as about 20, 30, 40, 50, 60, 70, 80, 90, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng/ml or any value or range within these limits. In some embodiments, the culture medium contains prolactin (or prolactin is added) in an amount of about 20 to about 195 ng/ml, about 50 to about 150 ng/ml, about 25 to about 175 ng/ml, about 45 to about 200 ng/m, or about 75 to about 190 ng/ml culture medium. In some embodiments, the methods further comprise adding prolactin to the culture medium, thereby providing a lactogenic culture medium. In some embodiments, prolactin is produced by a microbial and/or human cell expressing recombinant prolactin (e.g., prolactin containing a substitution of serine at position 179 of the prolactin gene for aspartate (S179D), e.g., S179D-prolactin). In some embodiments, adding prolactin to the culture medium provides conditioned culture medium by culturing cells expressing and secreting prolactin and applying the conditioned

- 40 046437 bath culture medium containing prolactin onto the basal surface of a monolayer of mammary cells (eg mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells).

In some embodiments, the culture medium further contains other factors to improve efficacy, including, but not limited to, insulin, epidermal growth factor, and/or hydrocortisone. In some embodiments, the methods of the present invention further provide for the addition of other factors (eg, insulin, epidermal growth factor and/or hydrocortisone) to the culture medium, for example, to improve efficiency.

Methods for producing cultured dairy products

Disclosed herein are methods for producing a cultured dairy product that is the result of the biosynthesis of cultured mammary epithelial cells (ECM) and plasma cells.

In some embodiments, the method involves culturing a cell construct containing plasma cells and mammary cells described herein in a bioreactor comprising a basal compartment and an apical compartment, wherein the basal compartment contains culture media and the plasma cells and mammary cells secrete the cultured dairy product containing immunoglobulins into the apical compartment.

In some embodiments, the method comprises: (a) isolating mammary epithelial cells, myoepithelial cells, and/or mammary progenitor cells from mammary explants from mammary tissue (e.g., mammary tissue, udder, teat), biopsy sample, or raw milk to obtain isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells; (b) isolating plasma cells from breast tissue, a biopsy sample, or raw milk to obtain isolated primary plasma cells; (c) culturing isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells; (d) culturing the isolated primary plasma cells; (f) placing cultured isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells and isolated primary plasma cells on a scaffold having a top surface and a bottom surface to obtain a mixed population of primary plasma cells on the scaffold and epithelial cells mammary gland, mammary myoepithelial cells and/or mammary progenitor cells, wherein the plasma cells are underneath the mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells; (f) culturing the mixed population of (e) on a scaffold to obtain a monolayer of polarized mammary gland cells to obtain a monolayer of polarized primary mammary epithelial cells located adjacent to and above the plasma cells, where the plasma cells are located adjacent to and above the top surface of the scaffold, where the top surface located adjacent to and above the lower surface of the scaffold, and where the polarized mammary cells contain an apical surface and a basal surface, and where the polarized monolayer contains an apical surface and a basal surface, thereby creating a cellular construct for producing a cultured dairy product containing immunoglobulins.

In some embodiments, the method comprises: a) isolating mammary epithelial cells, myoepithelial cells, and/or mammary progenitor cells from mammary gland explants from mammary tissue (e.g., mammary tissue, udder, teat), biopsy sample, or raw milk. to obtain isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells; (b) isolating plasma cells from breast tissue, a biopsy sample, or raw milk to obtain isolated primary plasma cells; (c) culturing isolated mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells to obtain a mixed population of primary mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells; (d) culturing the isolated primary plasma cells; (f) sorting the mixed population of mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells (eg, selecting primary mammary epithelial cells) to obtain a population of primary mammary epithelial cells; and (f) placing a sorted population of primary mammary epithelial cells, myoepithelial cells and/or mammary progenitor cells and isolated primary plasma cells on a scaffold having a top surface and a bottom surface, (g) culturing the sorted population of mammary epithelial cells and plasma cells cells on a scaffold to obtain a monolayer of polarized primary mammary epithelial cells located close to and above the plasma cells, where the plasma cells are located

- 41 046437 laid close to and above the upper surface of the framework, where the upper surface is located adjacent to and above the lower surface of the framework, and where the polarized mammary cells contain an apical surface and a basal surface, and where a polarized monolayer contains an apical surface and a basal surface, thereby creating a cellular design for producing a cultured dairy product containing immunoglobulins.

In some embodiments, the method comprises (a) culturing immortalized mammary epithelial cells to obtain an increased number of immortalized mammary epithelial cells; and (b) culturing plasma cells to obtain increased numbers of plasma cells; (c) culturing immortalized mammary epithelial cells and plasma cells (a) and (b) on a scaffold that has a top surface and a bottom surface to obtain a monolayer of polarized immortalized mammary epithelial cells above the plasma cells on the top surface of the scaffold, where the polarized monolayer has an apical surface and a basal surface, thereby forming a cellular construct for producing a cultured dairy product containing immunoglobulins.

In certain embodiments, plasma cells are added to a culture of immortalized mammary epithelial cells. In certain embodiments, plasma cells are cultured with immortalized mammary epithelial cells on a scaffold, resulting in a cultured milk product with secretory products of immune cells and mammary cells (eg, sIgA). In certain embodiments, the isolated mammary cells are immortalized before the cells are co-cultured with plasma cells.

Plasma cells can be obtained from a sample of primary breast tissue or, for example, from a plasma cell line. In certain embodiments, plasma cells are stimulated to produce immunoglobulins when co-cultured. In certain embodiments, the plasma cells produce IgA. In certain embodiments, the plasma cells produce secretory IgA. In possible embodiments, one or more classes of immunoglobulins include secretory IgA (sIgA). In certain embodiments, immunoglobulins bind to an antigen of a microorganism (ie, bacteria or virus). In certain embodiments, immunoglobulins bind to viral or bacterial antigens capable of causing infectious disease in humans. In certain embodiments, immunoglobulins bind to viral or bacterial antigens that cause infections of the respiratory or gastrointestinal epithelium. In certain embodiments, immunoglobulins bind to antigens of microorganisms that cause enterocolitis or sepsis in infants. In certain embodiments, plasma cells are co-cultured with ECM in a bioreactor by methods described herein. In certain embodiments, the bioreactor is a hollow fiber bioreactor described herein.

In certain embodiments, mammary cells are modified and/or stimulated with prolactin in the manner described herein to stimulate and optimize milk production. In certain embodiments, breast cells are modified to express a constitutively active prolactin receptor.

In certain embodiments, mammary epithelial cell populations are identified and isolated from primary mammary tissue. In some embodiments, breast cells are isolated and sorted by fluorescence-activated cell sorting, magnetically activated cell sorting, and/or a microfluidic cell sorter. In certain embodiments, mammary myoepithelial cells and mammary luminal epithelial cells are isolated using FACS analysis. In certain embodiments, mammary myoepithelial cell progenitor cells and/or mammary luminal epithelial cell progenitor cells are isolated using FACS analysis. Any suitable method known in the art can be used to sort mammary epithelial cells (eg, luminal epithelial cells), myoepithelial cells, progenitor cells, and immune cells. For example, breast cells can be sorted using the cell surface markers CD24, EPCAM and/or CD49f.

In some embodiments, plasma cells are isolated and sorted by fluorescence-activated cell sorting, magnetically activated cell sorting, and/or a microfluidic cell sorter. In certain embodiments, plasma cells are sorted by FACS analysis using markers known in the art to identify plasma cells. In a certain embodiment, plasma cells, plasmablasts or preplasmoblasts are sorted and isolated by FACS analysis using markers known in the art (eg, CD20, CD38, CD138 and/or CD19).

- 42 046437

In some embodiments, the cellular construct comprises a scaffold comprising a top surface and a bottom surface and a continuous monolayer of polarized mammary epithelial cells, a continuous monolayer of a polarized mixed population of mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and/or a continuous monolayer of polarized immortalized mammary epithelial cells, where a continuous monolayer is located on the upper surface of the scaffold.

In some embodiments, the lower surface of the frame is adjacent to the basal compartment. In some embodiments, the apical surface of the continuous monolayer is adjacent to the apical compartment. In some embodiments, the continuous monolayer releases milk and sIgA or IgA through its apical surface into an apical compartment, resulting in milk containing IgA and/or sIgA in culture.

In some embodiments, the monolayer of mammary cells forms a barrier that separates the apical compartment and the basal compartment, where the basal surface of the mammary cell is attached to the scaffold and the apical surface is oriented toward the apical compartment.

In some embodiments, the basal compartment is adjacent to the bottom surface of the frame. In some embodiments, the basal compartment comprises a culture medium in fluid contact with the basal surface of a monolayer of mammary epithelial cells (e.g., a polarized monolayer of mammary epithelial cells, a polarized monolayer of a mixed population of mammary cells, or a polarized monolayer of immortalized mammary epithelial cells).

In some embodiments, the culture medium contains a carbon source, a chemical buffer system, one or more essential amino acids, one or more vitamins and/or cofactors, and one or more inorganic salts.

In some embodiments, the bioreactor includes an apical compartment adjacent to the apical surface of the monolayer. In some embodiments, the apical compartment is adjacent to the upper surface of the frame.

In some embodiments, the total cellular density of mammary cells in the bioreactor is at least 10 ¹¹ mammary cells.

In some embodiments, the total cellular density of mammary cells in the bioreactor is at least 10 ¹² mammary cells. In some embodiments, the total cellular density of mammary cells in the bioreactor is at least 10 ¹³ mammary cells.

In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 20 to 55 cells per 100 microns ^. In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 20 cells per ¹⁰⁰ microns. In some embodiments, the total cellular density of mammary cells in the bioreactor is 25 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 30 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 35 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 40 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 45 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 50 cells per 100 μm ² . In some embodiments, the total cellular density of mammary cells in the bioreactor is approximately 55 cells per 100 μm ² .

In some embodiments, the total cellular density of plasma cells in the bioreactor is from about 200 to 500 plasma cells per mm ² . In some embodiments, the total cellular density of plasma cells in the bioreactor is approximately 200 plasma cells per mm ² . In some embodiments, the total cellular density of plasma cells in the bioreactor is approximately 300 plasma cells per mm ² . In some embodiments, the total cellular density of plasma cells in the bioreactor is approximately 400 plasma cells per mm ² . In some embodiments, the total cellular density of plasma cells in the bioreactor is approximately 500 plasma cells per mm ² .

In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 1.5 m ² In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 2 m ² . In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 2.5 m ² . In some embodiments, the total surface area of mammary gland cells in the bioreactor is

- 43 046437 at least approximately 3 m ² . In some embodiments, the total surface area of the mammary cells in the bioreactor is at least about 4 m ² . In some embodiments, the total surface area of the mammary gland cells in the bioreactor is at least about 5 m ² . In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 10 m ² In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 15 m ² . In some embodiments, the total surface area of the mammary gland cells in the bioreactor is at least about 20 m ² . In some embodiments, the total surface area of the mammary cells in the bioreactor is at least about 25 m ² . In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 50 m ² In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 100 m ² . In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 250 m ² In some embodiments, the total surface area of mammary cells in the bioreactor is at least about 500 m ² .

In some embodiments, the bioreactor is maintained at a temperature of about 27°C to about 39°C (e.g., a temperature of about 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34 °C, 35°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C or approximately 39° C, or any value or range within these limits, for example, from about 27 to about 38°C, from about 36 to about 39°C, from about 36.5 to about 39°C, from about 36.5 to about 37 .5°C, or about 36.5 to about 38°C). In some embodiments, the bioreactor is maintained at a temperature of approximately 37°C.

In some embodiments, the bioreactor has an atmospheric CO ₂ concentration of about 4 to about 6%, for example, an atmospheric CO ₂ concentration of about 4%, 4.25%, 4.5%, 4.75%, 5%, 5. 25%, 5.5%, 5.75% or 6%, or any value or range within these limits, for example, from about 4 to about 5.5%, from about 4.5 to about 6%, from about 4, 5 to about 5.5% or about 5 to about 6%). In some embodiments, the bioreactor has an atmospheric CO ₂ concentration of approximately 5%.

In some embodiments, the bioreactor has an atmospheric CO ₂ concentration of about 4 to about 6%, e.g., an atmospheric CO 2 concentration of about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25 %, 5.5%, 5.75% or 6% or any value or range within these limits, for example from about 4 to about 5.5%, from about 4.5 to about 6%, from about 4.5 to about 5.5% or about 5 to about 6%). In some embodiments, the bioreactor has an atmospheric CO2 concentration of approximately 5%.

In some embodiments, the method includes monitoring the concentration of dissolved O ₂ and CO ₂ . In some embodiments, the dissolved O ₂ concentration is maintained at about 10 to about 25%, or any value or range within these limits (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25%). For example, in some embodiments, the dissolved O ₂ concentration is maintained at about 12 to about 25%, about 15 to about 22%, about 10 to about 20%, about 15%, about 20%, or about 22%. In some embodiments, the CO2 concentration is maintained at about 4% to about 6%, e.g., the CO2 concentration is about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5% , 5.75% or 6%, or any value or range within these limits, such as about 4 to about 5.5%, about 4.5 to about 6%, about 4.5 to about 5.5%, or from about 5 to about 6%. In some embodiments, the CO ₂ concentration is maintained at approximately 5%.

In some embodiments, the culture medium is replaced from about once a day to about every 10 days (e.g., every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, or any value or range within these limits, for example, approximately once a day to once every 3 days, approximately once every 3 days to once every 10 days, approximately once every 2 days to once every 5 days). In some embodiments, the culture medium is replaced approximately every day, approximately every few hours, approximately every 10 days, for example, approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours to approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days or any value or range within these limits. For example, in some embodiments, the culture medium is replaced about every 12 hours to about every 10 days, about every 10 hours to about every 5 days, or about every 5 hours to about every 3 days.

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In some embodiments, the method involves monitoring the concentration of glucose and/or the rate of glucose consumption in the culture medium and/or in the lactogenic culture medium. In some embodiments, prolactin is added when the rate of glucose consumption in the culture medium is at steady state.

In some embodiments, the method further comprises the use of transepithelial electrical resistance (TEER) to measure the maintenance of a monolayer of epithelial cells. TEER measures the potential difference between fluids (eg, media) in two compartments (eg, between the apical and basal compartments), where if the barrier between the compartments loses its integrity, the liquids in the two compartments can mix. By stirring the liquid, the voltage difference will be reduced or eliminated; the difference in voltage indicates that the barrier is intact. In some embodiments, when voltage loss is detected by TEER, the scaffold (e.g., Transwell® filter, microstructured bioreactor, decellularized tissue, hollow fiber bioreactor, etc.) is reseeded with additional cells and allowed time to restore the barrier (e.g., monolayer) before resuming production of a cultured dairy product (for example, milk production).

In some embodiments, the method further includes collecting the cultured milk product from the apical compartment to obtain a collected cultured milk product. In some embodiments, collection is accomplished through a port, gravity, and/or vacuum. In some embodiments, a vacuum is attached to the port.

In some embodiments, the method comprises freezing the harvested cultured dairy product to obtain a frozen cultured dairy product and/or lyophilizing the harvested cultured dairy product to obtain a lyophilized cultured dairy product.

In some embodiments, the method comprises packaging a harvested cultured dairy product, a frozen cultured dairy product, and/or a lyophilized cultured dairy product into a container.

In some embodiments, the method further comprises extracting one or more components from the harvested cultured dairy product. Non-limiting examples of components from the harvested cultured dairy product include milk protein, lipid, carbohydrate, vitamin and/or minerals. In some embodiments, components from the collected cultured dairy product are lyophilized and/or concentrated to produce a lyophilized or concentrated cultured dairy product component. In some embodiments, components from the collected cultured dairy product are concentrated, for example, by membrane filtration and/or reverse osmosis. In some embodiments, the lyophilized or concentrated cultured dairy product component is packaged in a container, optionally where the container is a sterile and/or food grade container. In some embodiments, the container is sealed. In some embodiments, the container is a canister, can, bottle, bag, box, or pouch. In some embodiments, the cultured dairy product is a standardized, sterile cultured dairy product. In some embodiments, the cultured dairy product is intended for consumption.

In some embodiments, the cultured dairy product is produced by any method described herein.

Excluded Components

Due to the fact that cultured dairy products produced by the methods described herein are produced outside the human body, cultured dairy products contain little or no certain components found in natural breast milk. In some cases, the components are harmful (such as environmental pollutants and allergens), and in other cases, natural components are simply present due to the natural production of natural milk. In addition, the design of the living cell construct and bioreactor allows the production of cultured dairy products described herein that are free or substantially free of cell culture media and their components.

Cell culture media

Certain components of cell culture media may pass into the cultured milk product. For example, serum albumin, linoleic acid, and alpha-linolenic acid can be actively secreted by hECMF into the apical compartment with components of the cultured milk product produced by hECMF. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains from about 1% w/w to about 20% cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product,

- 45 046437 disclosed herein contains from approximately 5% w/w to approximately 10% cellular media components weighing more than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains approximately 5% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains from about 20% w/w to about 40% cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains from about 25% w/w to about 35% cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains from about 25% w/w to about 30% cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains approximately 25% w/w cell media components weighing less than 150 kDa.

In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 80% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 85% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 90% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 95% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 97% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 98% w/w cell media components weighing greater than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 99% w/w cell media components weighing greater than 150 kDa.

In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 60% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 65% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 70% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 75% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 77% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 78% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 79% w/w cell media components weighing less than 150 kDa. In some embodiments, the total dry weight of the cultured dairy product disclosed herein contains less than 80% w/w cell media components weighing less than 150 kDa.

Environmental pollutants and drugs

Breast milk contains small but measurable concentrations of environmental pollutants, harmful industrial chemicals and manufacturing products that are widely distributed in the environment. Environmental pollutants are partially excreted into breast milk. Contaminant levels in breast milk reflect contaminant levels in the mother's body and are ideal for monitoring exposure levels. Toxic environmental pollutants can be passed from mother to child through breastfeeding. Persistent organic pollutants (POPs) are a family of lipophilically stable chemicals that bioaccumulate in adipose tissue and create a long-term toxic burden on the body. Breastfeeding represents a significant source of exposure to POPs early in life, the consequences of which are unknown.

In some embodiments, the cultured dairy product is free or substantially free of one or more environmental contaminants. In some embodiments, the cultured dairy product contains no or substantially no persistent

- 46 046437 organic pollutants (POPs). In some embodiments, the cultured dairy product is free or substantially free of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and pesticides such as DDT.

Heavy metals such as mercury, lead, arsenic, cadmium, nickel, chromium, cobalt, zinc and other potentially toxic metals that are dispersed throughout the environment also have bioaccumulative properties and are known to accumulate in human milk and thus are a problem for an infant. The metal enters breast milk from exogenous sources, i.e., it enters through polluted air, food and drinking water, and endogenous excretion along with vital trace elements. For example, lead and mercury are equally distributed in the human food chain, and their effects on fetal development are largely determined by the mother's diet and nutritional status. Exposure to toxic metals is of significant public health importance; even at low concentrations and acute exposure, these metals remain toxic to humans. An infant may be exposed to toxic metals during the period of greatest susceptibility. Breastfed infants may be exposed to heavy metals through breast milk over time, and exposure may have consequences for the infant's health. In infants in particular, these exposures can have adverse effects on the developing central nervous system, leaving lifelong cognitive impairment.

Breast milk may contain residues of any pharmaceutical or recreational drugs used by the mother. Some prescription drugs, such as chemotherapy drugs, are contraindicated during breastfeeding due to risks to the infant. Commonly used recreational intoxicants such as cannabis and alcohol pass into breast milk. In addition, psychoactive substances have been found in the breast milk of women who use these drugs during lactation, and metabolites of illicit drugs have been found in the hair of breastfed infants of mothers who took these substances during breastfeeding.

In some embodiments, the cultured dairy product is free or substantially free of one or more heavy metals such as arsenic, lead, cadmium, nickel, mercury, chromium, cobalt, and zinc. In some embodiments, the cultured dairy product contains no or substantially no arsenic. In some embodiments, the cultured dairy product contains no or substantially no lead. In some embodiments, the cultured dairy product contains no or substantially no cadmium. In some embodiments, the cultured dairy product contains no or substantially no nickel. In some embodiments, the cultured dairy product contains no or substantially no mercury. In some embodiments, the cultured dairy product contains no or substantially no chromium. In some embodiments, the cultured dairy product contains no or substantially no cobalt. In some embodiments, the cultured dairy product contains no or substantially no zinc. In some embodiments, the cultured dairy product contains no or substantially no arsenic, lead, cadmium, nickel, mercury, chromium, cobalt, and zinc.

In some embodiments, the cultured dairy product contains no or substantially no drugs from the group consisting of chemotherapy drugs, antidepressants, anti-anxiety drugs, benzodiazepines, alcohol, marijuana, opioids, phencyclidine, cocaine, or metabolites thereof. In some embodiments, the cultured dairy product contains no or substantially no drugs from the group consisting of lithium, carbamazepine, chlorpromazine, and clozapine.

Allergens

Foreign allergenic proteins may be difficult to distinguish from endogenous human milk proteins. Food proteins with allergenic potential found in human milk include chicken egg and peanut proteins. There are eight major food allergens, known as the big eight, that are responsible for the majority of serious food allergic reactions in the United States. The Big Eight list consists of milk, egg, fish, shellfish, tree nut, peanut, wheat and soy allergens. Proteins that cause egg allergies include ovomucoid, ovalbumin, and conalbumin. Peanut proteins include araquine 6, araquine 3, conaraquine, major allergen Arah1, and araquine Arah2. As an example of the transfer of protein from the mother's diet into milk, consumption of one egg per day was shown to result in higher levels of the allergen hen's egg ovalbumin (OVA) in human milk compared to mothers avoiding eggs.

In some embodiments, the cultured dairy product is free or substantially free of one or more food allergens. In some embodiments, the cultured dairy product contains no or substantially no eggs, fish, shellfish, tree nuts, peanuts, wheat, and soy allergens. In some variants

- 47 046437 The cultured dairy product does not contain or is substantially free of egg allergens. In some embodiments, the cultured dairy product contains no or substantially no fish allergens. In some embodiments, the cultured dairy product contains no or substantially no crustacean allergens. In some embodiments, the cultured dairy product contains no or substantially no tree nut allergens. In some embodiments, the cultured dairy product contains no or substantially no peanut allergens. In some embodiments, the cultured dairy product contains no or substantially no wheat allergens. In some embodiments, the cultured dairy product contains no or substantially no soy allergens.

In some embodiments, the cultured dairy product contains no or substantially no arachine 6, arachine 3, conarachine, Arah1, and Arah2.

In some embodiments, the cultured dairy product contains no or substantially no ovalbumin (OVA).

Cells

Natural breast milk contains a significant population of living cells representing various cell types, including human stem cells, human immune cells and bacterial cells. Breast milk contains approximately 146,000 cells/ml, and this number decreases in transitional (8-12 days postpartum) and mature milk (26-30 days postpartum) to 27,500 and 23,650 cells/ml, respectively. Cells include myeloid cell precursors (9-20%), neutrophils (12-27%), immature granulocytes (8-17%), and noncytotoxic T cells (6-7%). The existence of the human milk microbiome was discovered just ten years ago. It is estimated that an infant fed 800 ml of breast milk per day can ingest 107-108 bacterial cells daily. The most commonly found bacteria in human milk are Staphylococcus, Acinetobacter, Streptococcus, Pseudomonas, Lactococcus, Enterococcus and Lactobacillus species.

In some embodiments, the cultured dairy product is cell-free or substantially cell-free. In some embodiments, the cultured dairy product comprises a human mammary epithelial cell and/or plasma cell. In some embodiments, the cultured dairy product contains no or substantially no cells other than one or more human mammary epithelial cells and/or one or more plasma cells. In some embodiments, the cultured dairy product contains no or substantially no cells selected from the group consisting of stem cells, myoepithelial cells, myeloid progenitor cells, neutrophils, granulocytes, T cells, Staphylococcus, Acinetobacter, Streptococcus, Pseudomonas, Lactococcus , Enterococcus and Lactobacillus. In some embodiments, the cultured dairy product comprises a human mammary epithelial cell and/or a human plasma cell. In some embodiments, the cultured dairy product contains no or substantially no cells other than one or more human mammary epithelial cells and/or one or more plasma cells.

Viruses

Natural breast milk can transmit certain infectious agents, such as hepatitis B virus, hepatitis C virus, cytomegalovirus, West Nile virus, human T-cell lymphotropic T-cell virus, and human immunodeficiency virus. In some embodiments, the cultured dairy product is free or substantially free of viruses. In some embodiments, the cultured dairy product is free or substantially free of viruses selected from the group consisting of hepatitis B virus, hepatitis C virus, cytomegalovirus, West Nile virus, human T-cell lymphotropic virus, and human immunodeficiency virus.

Hormones

Natural breast milk may contain hormones from the maternal circulation, such as leptin, ghrelin and adiponectin, which are synthesized outside the mammary glands and transported into the milk through the mammary epithelium. In some embodiments, the cultured dairy product does not contain the hormone. In some embodiments, the cultured dairy product does not contain hormones selected from the group consisting of leptin, ghrelin, adiponectin, thyroxine (T4), triiodothyronine (T3), thyroid stimulating hormone (TSH), epidermal growth factor, beta-endorphin, relaxin, cortisol and erythropoietin.

Methods of application

Provided herein in certain embodiments are methods of treating and/or preventing a microbial infection in an individual in need thereof, comprising administering a cultured dairy product containing IgA or sIgA described herein and/or an immunotherapeutic composition containing isolated IgA or sIgA, described in this document.

In some embodiments, the individual is a human. In some variants

- 48 046437 implementation of the person is an infant. In some embodiments, the person has an immunodeficiency. In some embodiments, the individual has a disease selected from severe combined immunodeficiency (SCID), HIV/AIDS, cancer, or an autoimmune disease. In some embodiments, the individual has lupus or diabetes mellitus (eg, type I diabetes mellitus or type II diabetes mellitus). In some embodiments, the person is an organ or bone marrow transplant recipient. In some embodiments, the person is suffering from malnutrition. In some embodiments, the person has malabsorption syndrome. In some embodiments, the person has a wasting syndrome. In some embodiments, the person is elderly or senile. In some embodiments, the individual has cystic fibrosis, COPD, or bronchiectasis without cystic fibrosis.

In certain embodiments, immunotherapeutic compositions and a cultured dairy product containing IgA and/or sIgA are administered to patients in an effective amount to treat or prevent a microbial infection. In some embodiments, the microbial infection is a bacterial infection, a fungal infection, or a parasitic infection.

In certain embodiments, the microbial infection is a bacterial infection. Non-limiting examples of bacterial infections that can be treated and/or prevented include infections caused by Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Streptococcus pyogenes, Salmonella, Shigella, Campylobacter, Staphylococcus aureus, Helicobacter pylori, E. coli, C. difficile or Vibrio cholerae

In certain embodiments, the microbial infection is a viral infection. Non-limiting examples of viral infections that can be treated and/or prevented include infections caused by influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus and human metapneumovirus.

In some embodiments, the microbial infection is a parasitic infection. In some embodiments, the parasitic infection is an infection of Giardia lamblia, Entameba histolytica, Cryptosporidium spp. or Cystoisospora belli.

In some embodiments, the microbial infection is a fungal infection. In some embodiments, the fungal infection is an Aspergillus, Cryptococcus, or Pneumocystis jirovecii infection.

In certain embodiments, a cultured immune milk product containing IgA or sIgA, or a therapeutic composition containing IgA or sIgA, is administered orally to an individual to treat or prevent a gastrointestinal infection. In certain embodiments, the gastrointestinal infection is bacterial or viral gastroenteritis. In certain embodiments, the individual has an ulcer caused by a Helicobacter pylori infection.

In some embodiments, the microbial infection is candidiasis.

In some embodiments, the microbial infection is a respiratory infection. In some embodiments, the respiratory infection is pneumonia, bronchitis, aspergillosis, or cryptococcosis. In some embodiments, the respiratory infection is an infection caused by B. cepacia, P. aeruginosa, S. aureus, Aspergillus, Cryptococcus, or Pneumocystis.

In certain embodiments, the immunotherapeutic compositions described herein are administered to an individual as a nasal inhalation agent for the treatment or prevention of a respiratory infection. In some embodiments, the compositions are formulated for inhalation. In some embodiments, the composition is a powder. In some embodiments, the composition is a formulation for administration via a nebulizer.

In certain embodiments, immunotherapeutic compositions and/or cultured dairy products are administered to the body with another antimicrobial agent (ie, antibiotic or antiviral agent). In certain embodiments, the cultured dairy product described herein is administered with an additional nutritional product to treat and/or prevent a gastrointestinal infection.

Having described the present disclosure, it will be explained in more detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the disclosure.

Examples

The examples that follow illustrate specific embodiments of the disclosure and their various applications. They are set forth for explanatory purposes only and should not be construed as limiting in any way the scope of the disclosure.

Example 1. Preparation of milk components from human mammary epithelium

In this example, human mammary epithelium is reproduced and milk is produced in vitro.

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The system and method described are exemplary and can be scaled up to produce multi-liter volumes of milk components. It is proposed that mammary epithelial cells form a polarized monolayer on bioreactor fibers precoated with one or more of laminin and collagen or other extracellular matrix proteins, as well as on uncoated fibers. When fused, the monolayer forms a barrier separating the intra- and extracapillary space (ECS), with the basal surface attached to the fibers and the apical surface oriented towards the ECS. The production of milk components is stimulated by adding prolactin to the medium. Secreted milk components are collected from the EPC and sent for subsequent analysis of protein, lipid and carbohydrate content in comparison with human breast milk produced in vivo.

Materials for use in this example are shown in Table. 3.

Table 3. Materials

Item Provider Catalog number Primary mammary epithelial cells ATSS PCS-600-010 Minimum environment for mammary epithelial cells ATSS PCS-600-30 Breast epithelial cell growth kit ATSS PCS-600-040 Phosphate buffered saline Dulbecco (D-PBS) ATSS ATCC 30-2200 Trypsin-EDTA ATSS PCS-999-003 Trypsin neutralizing solution ATSS PCS-999-004 EHS (Laminin-1/111) Sigma L2020-1MG Collagen-IV Sigma C5533-5MG Prolactin Shenandoah Biotechnology 100-45-500 mcg

Methods

Expansion of primary human breast epithelial cells (hECBC)

Human mammary epithelial cells (1 ampoule; 5x05 cells) are expanded in one T300 flask coated with collagen IV (or two T175 flasks) in mammary epithelial cell medium prepared with the additives presented in the kit indicated in Table. 3. After obtaining the appropriate number of cells, the cells are washed with D-PBS and collected from the plates using trypsin-EDTA. Once the cells are separated, trypsin activity is stopped using a trypsin neutralizing solution. Cells are resuspended in medium and seeded into a hollow fiber bioreactor (Fibercell Systems) prepared as described below.

Preparation of Hollow Fiber Bioreactor (C2025D, 20 kDa MWCO) Before seeding, prepare a bioreactor cartridge (Fibercell Systems) by pre-culture with PBS for at least 24 hours. The bioreactor cartridge is optionally pre-coated with the addition of approximately 50-100 μg of one or more of collagen I , collagen IV, laminin III (eg, laminin III isolated from Engelbreth-Holm-Swarm tumor), alpha-4, alpha-5, fibronectin, and/or entactin in 3.2 ml PBS and leave to ultrafiltrate along fibers at room temperature overnight. The uncoated or precoated cartridge is replaced with media and incubated overnight at room temperature. The medium is then replaced with cells collected from T300 (or T175) flasks. The tank volume is no more than 125 ml. The cartridge is rotated 180° after cell seeding.

Cell growth in a bioreactor and stimulation with prolactin

After seeding into the bioreactor, cells are grown in mammary epithelial cell culture medium supplemented with 10-25 ml DMEM/10% CDM-HD per 100 ml medium (Dulbecco's modified Eagle's medium; chemically defined medium for high-density cell culture) . The DMEM/CDM-HD ratio can be adjusted depending on the rate of glucose consumption.

Before stimulating milk secretion, the medium in the ECP is drained and replaced with PBS. To stimulate the secretion of the milk component, a medium supplemented with 100 ng/ml prolactin is added. The lactogenic environment can also be supplemented with an increased concentration of glucose and the necessary nutritional precursors of milk fatty acids, linoleic acid and α-linolenic acid. The bioreactor is maintained for 10 days with sampling as described below.

Collection and receipt of samples

Samples consisting of supernatant from the ECP and an equivalent volume of medium from the reservoir,

- 50 046437 dose once a day for 10 days after adding prolactin to the medium. Samples are centrifuged to collect debris and resuspended in an equivalent volume of PBS. Supernatants of ECP samples and media are divided into 0.5 ml aliquots in microcentrifuge tubes and frozen at -80°C. The debris pellet is resuspended in a volume of PBS equivalent to the original solution and frozen at -80°C. Samples are processed to determine the relative concentrations of milk components produced.

Scaling for multi-liter production

To scale up for multi-liter production, the previous method is carried out with appropriate adjustments to the volumes of reagents for a larger bioreactor (for example, Fibercell Systems, catalog number C2018).

Example 2.

A cell culture system designed for milk collection must maintain separated product secretion so that the milk is not exposed to media that provide nutrients to the cells. In the body, milk-producing epithelial cells line the inner surface of the mammary gland in a continuous monolayer. The monolayer is oriented such that the basal surface is attached to the underlying basement membrane and milk is secreted from the apical surface and stored in the luminal portion of the gland, or alveolus, until removed during milking or feeding. Tight junctions along the lateral surfaces of the cells provide a barrier between the underlying tissues and the milk present in the alveolar compartment. Thus, in vivo, mammary gland tissue is designed in such a way that milk secretion is compartmentalized, while the epithelial cells of the mammary gland themselves establish the phase separation and support the directed absorption of nutrients and milk secretion.

The present disclosure describes a cell culture apparatus that replicates the compartmentalization capacity of the mammary gland, which is used to collect milk from mammary epithelial cells grown outside the body. Such an apparatus may contain a scaffold to support the proliferation of mammary cells at the interface of the two compartments, such that the epithelial monolayer provides a physical boundary between the culture medium and the secreted milk. In addition to providing a surface for growth, the scaffold provides spatial cues that direct cell polarization and provide directionality for uptake and secretion. This invention describes the production, cultivation and stimulation of mammary epithelial cells in a compartmentalizing cell culture apparatus for the production and collection of milk for food purposes (see, for example, FIG. 1).

Obtaining mammary epithelial cells

Breast epithelial cells are obtained from surgical explants of dissected mammary tissue (eg, mammary gland, udder, teat), a biopsy specimen, or raw breast milk. Typically, after surgical dissection of the breast tissue, any fatty or stromal tissue is removed manually under aseptic conditions, and the remaining breast tissue is enzymatically digested with collagenase and/or hyaluronidase prepared in a chemically defined nutrient medium, which must be composed of components that are generally recognized safe (GRAS). The sample is kept at 37°C with gentle stirring. After digestion, a suspension of individual cells or organelles is collected either by centrifugation or by pouring the sample through a sterile nylon cell strainer. The cell suspension is then transferred to a tissue culture plate coated with appropriate extracellular matrix components (eg, collagen, laminin, fibronectin).

Alternatively, explant samples can be minced into small pieces, for example by mincing with a sterile scalpel. The tissue pieces are seeded onto a suitable surface, such as a gelatin sponge or plastic tissue culture plate coated with an appropriate extracellular matrix.

The seeded cells are kept at 37°C in an incubator with an atmosphere containing 5% CO ₂ . During incubation, the medium is replaced approximately every 1-3 days and the cells are subcultured until a sufficient number of viable cells is reached for subsequent processing, which includes obtaining the drug for storage in liquid nitrogen; development of immortalized cell lines by stable transfection of genes such as SV40, TERT or other genes associated with aging; isolating epithelial, myoepithelial and mammary stem cell types by, for example, fluorescence-activated cell sorting; and/or introduction into a compartmentalized tissue culture apparatus for the production and collection of milk for human consumption.

Cultivation of mammary epithelial cells for milk production. Milk for food purposes is produced by mammary epithelial cells isolated as described above and cultured in a format that supports compartmentalized secretion so that separation between culture medium and product is maintained. The system is based on the ability of mammary epithelial cells to form a continuous monolayer with appropriate apical-basal polarity when seeded on an appropriate scaffold located at the border between the apical

- 51 046437 nia, into which milk is secreted, and the basal compartment, through which the supply of nutrient media is ensured (see, for example, Fig. 2). Transwell® filters placed in tissue culture plates, as well as hollow fiber bioreactors or microstructured scaffolds, for example, are used to support these characteristics.

After isolation and expansion of mammary epithelial cells, the cells are suspended in a specific chemical nutrient medium consisting of food components and inoculated into a culture apparatus that has been previously coated with a mixture of extracellular matrix proteins such as collagen, laminin and/or fibronectin. A cell culture apparatus is any design that allows for separate absorption of nutrients and release of product from a polarized confluent epithelial monolayer. Examples include bioreactors with hollow fibers and microstructured scaffolds (see, for example, Figs. 3 and 4, respectively). Alternatives include other types of 3D tissue culture, such as obtaining an acellular mammary gland as a scaffold populated with stem cells to produce a functional organ in vitro, or harvesting milk from the lumen of mammary organoid epithelial cells or mammospheres grown either in a hydrogel matrix or in suspension .

The apparatus contains a sealed housing that maintains a temperature of approximately 37°C in a humid atmosphere containing approximately 5% CO ₂ . Glucose uptake is monitored to assess culture growth as cells proliferate in the bioreactor. Stabilization of glucose consumption indicates that cells have reached a confluent, contact-inhibited state. The integrity of the monolayer is ensured using transepithelial electrical resistance. Sensors monitor the concentration of dissolved O ₂ and CO ₂ in the environment in several places. A computerized pump circulates media through the bioreactor at a rate that balances the delivery of nutrients with the removal of metabolic wastes such as ammonia and lactate. The media can be recycled through the system after waste removal using lactate addition and adaptation technology (Freund et al. 2018 Int J Mol Sci. 19(2)) or by passing through a packed zeolite chamber.

Stimulation of milk production

In vivo and in cultured mammary epithelial cells, milk production and secretion are stimulated by prolactin. In culture, prolactin can be supplied exogenously to the culture medium at a concentration similar to that observed in the body during lactation, for example, from about 20 ng/ml to about 200 ng/ml. Purified prolactin can be obtained commercially; however, alternative methods are used to provide prolactin or stimulate lactation, including the expression and purification of recombinant proteins from microbial or mammalian cell cultures. Alternatively, conditioned medium prepared by culturing cells expressing and secreting prolactin can be applied to mammary epithelial cells to stimulate lactation. The bioreactors can be arranged in series such that the media passing through the culture of cells expressing prolactin or other key media additives is conditioned prior to exposure to mammary cells grown in the compartment culture apparatus as described.

Other approaches to enhance milk production and/or spare exogenous prolactin involve molecular manipulation of signal transduction pathways that are regulated by the binding of prolactin to its receptors on the surface of mammary epithelial cells, such as the following: (a) expression of constructs targeting post-translational modification of prolactin; (b) expression of alternative prolactin receptor isotypes; (c) expression of a chimeric prolactin receptor in which the extracellular domain is replaced by a binding site for another ligand; (d) introduction of a gene encoding a constitutively or conditionally active prolactin receptor or modified variants of its downstream effectors, such as STAT5 or Akt; (e) knockout or modification of the circadian PER2 gene; and/or (f) molecular approaches aimed at increasing the rate of nutrient uptake at the basal surface of the mammary epithelial monolayer.

Milk collection

The collection of secreted milk is carried out continuously or at certain intervals, for example, through a port installed in the apical compartment of the culture apparatus. A vacuum is attached to the port to facilitate collection and also to stimulate further production. Collected milk is packaged into containers and sealed for distribution, frozen or lyophilized for storage, or processed to extract specific components.

The present invention relates to cultures of mammary epithelial cells for the production of milk for food purposes. In addition to human breast milk, this method can be used to produce milk from other mammalian species, for example for human consumption or veterinary use. Since it was previously impossible to produce milk outside the body, this technology could lead to new commercial opportunities, in addition to providing an alternative way to produce existing products. The socio-economic implications of commercial development of this technology are vast and far

- 52 046437 walking. Producing human breast milk from cultured cells could provide a solution to infant malnutrition in food-scarce settings, provide essential nutrients to premature babies who cannot breastfeed, and offer mothers a new option for feeding their babies that provides optimal nutrition in the convenience of a nursery. mixtures. Producing cow's or goat's milk offers an opportunity to reduce the environmental, social and animal health impacts of livestock production. The method described here addresses important challenges in the emerging field of cellular agriculture and offers the opportunity to radically renew the human food supply without compromising our biological and cultural attachment to the most fundamental of our food sources.

Example 3. Production of dairy compositions containing immunoglobulins

Primary breast tissue is obtained from a healthy donor. CD20+ immune cell and mammary epithelial cell populations are sorted by FACS analysis using known markers for myoepithelial and luminal epithelial cell populations. Isolated mammary cells and immune cells are immortalized prior to cell co-culture. Immortalized immune cells are cocultured with immortalized isolated mammary epithelial cells in a hollow fiber bioreactor and grown to confluency. The culture is stimulated with prolactin to stimulate milk production. The secretory product of the cell culture, containing immunoglobulins, including secretory IgA, is then collected by a non-destructive withdrawal mechanism.

Example 4. Production of immunotherapeutic compositions from co-cultured mucosal epithelial cells and immune cells

Primary mammary epithelial cells and CD20+ immune cells are collected from tissue obtained from patients who have recovered from mucosal infections. Patients who tested positive for serum that exhibited virus neutralizing activity were selected. CD20+ immune cells are co-cultured with mammary epithelial cells in a hollow fiber bioreactor. Immune cells are grown in 2-D culture followed by inoculation into a hollow fiber bioreactor and grown to maximum density (i.e., superconfluence) within the bioreactor. Co-cultivation of mucosal epithelial cells and immune cells stimulates the production of immunoglobulins, including secretory IgA. The co-culture is stimulated with one or more stimulants selected from viral protein, viral protein fragment, prolactin, TGF-β and interleukin 10 (IL-10). Products of a co-culture of mucosal epithelial cells and stimulated immune cells are collected using a standard vacuum method. In certain embodiments, the secreted product is purified. Immunoglobulins are purified from the collected secreted product. The purified secreted product is formulated as an inhalation agent for pulmonary administration to humans and is effective in the treatment of respiratory infection.

Example 5. Treatment of respiratory infection with an immunotherapeutic composition of co-cultured mucosal epithelial cells and immune cells

The immunotherapeutic composition as described above in Example 3 is formulated as an inhalation agent and administered to a patient suffering from a respiratory infection in an amount effective to treat the infection. The immunotherapeutic composition is effective in reducing the severity of symptoms of infection in a patient.

Example 6 Cultured Dairy Product

Breast epithelial cells (EBCs) produce most of the components of milk and regulate the composition of milk in the body during lactation. In this document, the authors describe the composition of cell-cultured human dairy products that can be obtained by culturing human ECMF (hECMF) in a hollow fiber bioreactor (HFBR), which provides a permeable scaffold and allows the formation of an adhesive culture that maintains an epithelial barrier between the culture media cells that circulate through the capillaries of the bioreactor, and a secreted product that is collected from the extracapillary space (ECS).

Receipt and analysis

The product compositions described in this example are the biosynthetic product of hECM obtained from various sources, cultured in HFBR under lactogenic conditions (Fig. 9A). Cells administered include publicly available primary and immortalized hECM cells (Table 4).

- 53 046437

Table 4. Input cells and culture media compositions for the production of ____________________human dairy products_______________

Injected cells Basic Environments Environments for growing Lactogenic media Primary hECM Donor #1 Environments for e nutritional cells of the mammary gland ATCC Breast Epithelial Cell Growth Kit Environments for growing + 200400 ng/ml prolactin Primary hECM Donor #2 Environments for mammary epithelial cells (ATCC) Essential Media + Mammary Epithelial Cell Growth Kit (ATCC) Environments for cultivation + 200400 ng/ml prolactin (Sigma) Immortalized hECM PMC42-LA RPMI-1640 (Gibco) Basic media + 10% FBS (Sigma) Environments for growing + 200800 ng/ml prolactin (Sigma) Immortalized hECM MCF10A MEVM (Lonza) MEGM (Lonza) MEGM + 200-400 ng/ml prolactin (Sigma)

Briefly, after expansion in tissue culture flasks, hECMF were inoculated into HFBR ECPs and oxygenated culture medium was passed through capillaries using a peristaltic pump.

The growth media to support the proliferative phase of the method correspond to the designated basic media, supplemented with the necessary growth factors to support proliferation.

After inoculation, proliferation was monitored by assessing the daily glucose utilization ratio (GUR) using a glucometer (AccuCheck). An increase in GUR corresponds to increased metabolic activity resulting from an increase in the number of cells in the proliferative phase of the process. A stabilization or decrease in GUR indicates that the cell has reached a confluent state and growth has become contact-inhibited. Once confluent, the culture media circulating through the HFBR capillaries were replaced with lactogenic media supplemented with recombinant human prolactin (Sigma). It should be noted that it was observed that the transition to lactogenic media corresponded to the second phase of the increase in GUR, associated with increased metabolic demands of milk biosynthesis, including lactose production (Fig. 9B). During the lactogenic phase of the process, samples were taken from the ECP at regular intervals for analytical testing using a syringe to inject fresh PBS into one ECP access port while withdrawing an equal volume of product sample through the syringe through the other ECP access port.

Upon lactogenic stimulation, hECMs reached a state of terminal differentiation, forming a confluent monolayer with apical/basal polarity and tight junctions on the lateral surfaces, resulting in the formation of an impermeable barrier between the basal compartment and the apical compartment. The basal surface is attached to the extracellular matrix located above the capillaries, and the apical surface is oriented towards the EPC. With this orientation, hECM assembled their absorption and secretion machinery along the apical-basal axis, allowing for the uptake of nutrients at the basal surface from the medium perfused through the capillaries and the secretion of product at the apical surface into the EEC, where it accumulated until it was withdrawn. Product accumulation was demonstrated by the detection of lactose, beta-casein and an increase in total protein content in the ECP (Fig. 9B-D).

The rate of product accumulation in the ECP was determined by the number of cells that the HFBR could accommodate, which was limited by the available surface area for cell attachment and the perfusion of the medium. This process was carried out using available HFBRs that can accommodate approximately 10 ⁸ (Fibercell Systems, C2025D) to 10 ¹¹ (Fibercell Systems, C2018) cells. The authors estimated that the lactating human mammary gland, representing approximately 1 kg of tissue, contains approximately 10 ¹¹ cells, and that HFBR capable of supporting this number of cells would be able to produce a daily yield similar to that of the lactating mammary gland. The theoretical yield based on these estimates is confirmed by quantitative analysis of total protein content in our EPC samples obtained during lactogenic stimulation (Fig. 9D). In addition, the increased protein content of the EPC-derived product compared to culture media confirms that the cells provide a functional barrier between compartments and obstruction.

- 54 046437 is responsible for the diffusion transfer of medium components into the product.

Composition of macromolecular product

In addition to confirming that overall protein production by hECM grown in HFBR was consistent with that expected for milk, the authors performed a series of untargeted and targeted assays to evaluate the molecular composition of these products and confirm the presence of human milk components. Mass spectrometry was used to examine the protein, lipid, and carbohydrate content of these products over time during cycles spanning up to 90 days, and the 10 most common human milk components from each category are presented in Table 1. 5. Appropriate analysis of the compositions of the cell culture media used in each cycle confirms that the listed components were synthesized by hECM, as they were not present in the media.

Table 5. Major protein, lipid and carbohydrate components of human milk observed in samples collected from hECMF cultured under lactogenic stimulation in a hollow fiber bioreactor, listed in order of increasing abundance

Fraction Component Protein Beta-casein Alpha-lactalbumin Kappa-casein Alpha-S1-casein Lactoferrin Butrofilin Osteopontin Lactadherin Lysozyme Mucin-4 Mucin-1 Fatty acid C8:0, caprylic acid C 10:0, capric acid C 12:0, lauric acid C13:0, tridecanoic acid C 14:0, myristic acid C 16:0, palmitic acid C 16:1, palmitoleic acid C18:2p-6, linoleic acid, LA C18;3p-3, alpha-linolenic acid, ALA C20: Zn-6, dihomo-gama-linolenic acid, DGLA S20:4p-6, arachidonic acid, AA S20:5p-3, eicosapentanoic acid, EPA C22:6p-3, docosahexaenoic acid, DHA S22:1p9, erucic acid C24:1, nervonic acid Carbohydrate Lactose Glucose Z'-SL 6'-SL LNT LNnT

Besides

Claims (17)

immune system and infant immune defense, with gut development and brain development. Of particular interest, the authors also found numerous free oxylipin metabolites of polyunsaturated fatty acids, such as 9-hydroxyoctadecadienoic acid, which were reported to have anti-inflammatory and pro-inflammatory activities. Conclusion These results and our ongoing analyzes support the feasibility of producing human dairy products from cultured hECM, including key nutritional and bioactive components. The composition described herein represents combined product data, since a commercial process may involve post-production mixing of products from multiple units to obtain a specific composition. The above examples illustrate the present disclosure and should not be construed as limiting thereof. Although the disclosure has been described in detail with reference to preliminary embodiments, variations and modifications exist within the scope and spirit of the disclosure, which are described and defined in the following claims. CLAIM 1. A method for producing an isolated cultured dairy product containing secretory IgA (sIgA) from mammary gland cells and plasma cells, comprising: a) cultivating the cell construct in a bioreactor, under conditions allowing to obtain a cultured dairy product, wherein said cell construct contains: i) a three-dimensional frame having an outer surface, an inner surface defining an inner cavity, and a plurality of pores extending from the inner surface to the outer surface; ii) matrix material located on the outer surface of the three-dimensional scaffold; iii) a culture medium located in the internal cavity and in fluid contact with the internal surface; iv) many plasma cells located on the matrix material; And v) a confluent monolayer of polarized mammary cells located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells, wherein the polarized mammary cells comprise an apical surface and a basal surface surface; wherein said bioreactor comprises an apical compartment that is in fluid contact with the apical surface of living mammary cells, is substantially isolated from the interior cavity of the living cell construct, and is substantially free of cell culture media; And b) isolating a cultured milk product secreted into the apical compartment from the apical surface of living mammary cells and plasma cells. 2. The method of claim 1, wherein the monolayer of polarized mammary gland cells is at least 70% confluent, at least 80% confluent, at least 90% confluent, at least 95% confluent, at least at least 99% confluent or 100% confluent. 3. The method according to claim 1, wherein at least 70%, at least 80%, at least 90%, at least 95%, at least 99 or 100% of the mammary gland cells are polarized in the same orientation. 4. The method of claim 1, wherein the total cellular density of mammary cells in the bioreactor is at least 10 ¹¹ and, alternatively, wherein the total surface area of mammary cells in the bioreactor is at least 1.5 m ² . 5. The method of claim 1, wherein the total cellular density of plasma cells in the bioreactor is approximately 200 to 500 plasma cells per mm ² . 6. The method of claim 1, wherein the cultivation is carried out at a temperature of from about 27 to about 39°C. 7. The method of claim 1, wherein the cultivation is carried out at an atmospheric CO ₂ concentration of from about 4 to about 6%. 8. Living cell construct containing: a) a three-dimensional framework having an outer surface, an inner surface defining an inner cavity/basal chamber, and a plurality of pores extending from the inner surface to the outer surface; b) a matrix material located on the outer surface of the three-dimensional scaffold; c) many plasma cells located on the matrix material; d) at least 70% confluent monolayer of polarized mammary cells, - 56 046437 located on a plurality of plasma cells, wherein the mammary cells are selected from the group consisting of mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells, and wherein the living polarized mammary cells contain an apical surface from which milk is secreted product, and basal surface; And e) a culture medium located in the internal cavity/basal chamber and in fluid contact with the internal surface, the culture medium being substantially isolated from the apical surface of the living mammary cells. 9. The cell construct of claim 8, wherein the basal surface of the mammary gland cells is in fluid contact with the culture medium. 10. The cell construct according to claim 8, wherein at least 70%, at least 80%, at least 90%, at least 95%, at least 99 or 100% of the mammary gland cells are polarized in the same way same orientation. 11. The cell construct according to claim 8, wherein the monolayer of polarized mammary gland cells is at least 70% confluent, at least 80% confluent, at least 90% confluent, at least 95% confluent, according to at least 99% confluent or 100% confluent. 12. The cell construct according to claim 8, wherein the mammary gland cells contain a constitutively active prolactin receptor protein. 13. The cell construct according to claim 8, wherein the culture medium contains prolactin. 14. Cultured dairy product containing beta-casein, alpha-lactalbumin, kappacasein, alpha^1-casein, lactoferrin, lactadherin, lysozyme, C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid) acid), lactose, glucose, 3'-SL (3'-sialyllactose) and 6'-SL (6'-sialyllactose); wherein the cultured dairy product contains no or substantially no persistent organic pollutants (POPs), heavy metals, prescription drugs, recreational drugs, non-dairy allergens, cells, hormones or viruses. 15. Cultured dairy product containing beta-casein, alpha-lactalbumin, kappacasein, alpha^1-casein, lactoferrin, butrophilin, osteopontin, lactaderin, lysozyme, mucin-4, mucin1, C8:0 (caprylic acid), C10:0 (capric acid), C12:0 (lauric acid), C13:0 (tridecanoic acid), C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:2p-6 (linoleic acid, LA), C18,3n-3 (alpha-linolenic acid, ALA), C20:3n-6 (dihomo-gama-linolenic acid, DGLA), C20:4n-6 (arachidonic acid, AA), C20 :5p-3 (eicosapentanoic acid, EPA), C22:6p-3 (docosahexaenoic acid, DHA), C22:1p9 (erucic acid), C24:1 (nervonic acid), lactose, glucose, 3'-SL, 6' -SL, LNT, LNnT, 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 5,15-dihydroxyeicosatetraenoic acid (5,15-DiHETE), 17,18-dihydroxyeicosatetraenoic acid (17,18-DiHETE) , 8,9-dihydroxyicosatrienoic acid (8,9-DiHETrE), 11,12dihydroxyicosatrienoic acid (11,12-DiHETrE), 9,10-dihydroxyoctadecenoic acid (9,10DiHOME), 12,13-dihydroxyoctadecenoic acid (12,13- DiHOME), 14(15)-epoxyeicosatrienoic acid (14(15)-EpETre), 19(20)-epoxydocosapentanoic acid (19(20)-EpDPE), 17-hydroxydocosahexaenoic acid (17-HDoHE), 5-hydroxyeicosatetraenoic acid ( 5-HETE), 8-hydroxyeicosatetraenoic acid (8-HETE), 9-hydroxyeicosatetraenoic acid (9-HETE), 11-hydroxyeicosatetraenoic acid (11-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 9-hydroxyoctadecatrienoic acid (9-HOTrE), 9 -oxooctadecadienoic acid (9-okco-ODE) or 13oxooctadecadienoic acid (13-okco-ODE), 17,18-epoxyeicosatetraenoic acid (17(18)-EPETE, 6-keto-prostaglandin F1-alpha (6-keto-PGF1a) or 15^)-hydroxyeicosatrienoic acid (15(S)HETrE) and sIgA, wherein the cultured dairy product is free or substantially free of persistent organic pollutants (POPs), heavy metals, prescription drugs, recreational drugs, non-dairy allergens, cells , hormones or viruses. 16. The cultured dairy product according to claim 14, wherein the cells are human mammary epithelial cells (hECBC) or plasma cells (PC). 17. The cultured dairy product according to claim 15, wherein the cells are mammary epithelial cells or human plasma cells. -