Living cell constructs for producing cultured dairy products and methods of use thereof

Abstract

The present invention relates to living cell constructs for producing cultured milk products from mammary cells in vitro and / or ex vivo, methods for producing isolated cultured milk products from mammary cells, bioreactors for producing isolated cultured milk products, and cultured milk products.

Description

[0001] Cross-references

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 958,407, filed on January 8, 2020, and U.S. Provisional Application No. 63 / 199,164, filed on December 10, 2020, the contents of each of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present invention relates to living cell constructs and methods of using the same for producing cultured milk products from cultured mammary cells in vitro and / or ex vivo. Background of the Invention

[0005] Milk is a staple of the human diet, 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 six months of life, and dairy consumption beyond infancy is a mainstay of human nutrition, representing a $700 billion global industry. However, lactation is a physiologically demanding and metabolically intensive process that can present biological and practical challenges for breastfeeding mothers, and milk production is associated with environmental, social, and animal welfare impacts in agricultural settings.

[0006] In recent years, with the development of several successful prototypes of meat and marine foods from cultured muscle and fat cells, the possibility of producing food using mammalian cell cultures has become increasingly interesting (Stephens et al. 2018 Trends Food Sci Technol. 78: 155-166). In addition, efforts are underway to commercialize the production of egg and milk proteins using microbial expression systems. However, this fermentation-based method relies on genetically engineered expression and purification of individual components and cannot replicate the complete molecular profile of milk or dairy products.

[0007] The present invention overcomes the shortcomings of the art by providing living cell constructs and methods of using the same for producing cultured dairy products from cultured mammary cells in vitro and / or ex vivo. SUMMARY OF THE INVENTION

[0009] In certain embodiments, disclosed herein is a living cell construct comprising: (a) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity / base chamber, and more than one pore extending from the inner surface to the outer surface; (b) a matrix material disposed on the outer surface of the three-dimensional scaffold; (c) a culture medium disposed within the inner cavity / base chamber and in fluidic contact with the inner surface; and (d) a monolayer of at least 70% confluent polarized mammary cells disposed on the matrix material, wherein the mammary cells are selected from the group consisting of: living primary mammary epithelial cells, living mammary myoepithelial cells, living mammary progenitor cells, living immortalized mammary epithelial cells, living immortalized mammary myoepithelial cells, and living immortalized 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 fluidic contact with the culture medium. In some embodiments, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of the breast cells are polarized in the same direction. In some embodiments, the monolayer of polarized breast cells is at least 70% confluent, at least 80% confluent, at least 90% confluent, at least 95% confluent, at least 99% confluent or 100% confluent. In some embodiments, the breast cells include a constitutively active prolactin receptor protein. In some embodiments, the culture medium includes 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 also includes prolactin. In some embodiments, the matrix material includes one or more extracellular matrix proteins. In some embodiments, the three-dimensional scaffold includes a natural polymer, a biocompatible synthetic polymer, a synthetic peptide, a complex 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 polymer is polysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate, polyvinyl alcohol, sodium polyacrylate, acrylate polymers and / or polyethylene glycol.

[0010] In certain embodiments, disclosed herein is a method for producing an isolated cultured dairy product from mammary cells, the method comprising: (a) culturing a living cell construct in a bioreactor under conditions for producing a cultured dairy product, the living cell construct comprising: (i) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity / basal chamber, and more than one pore extending from the inner surface to the outer surface; (ii) a matrix material disposed on the outer surface of the three-dimensional scaffold; (iii) a culture medium disposed within the inner cavity / basal chamber and in fluidic contact with the inner surface; and (iv) a monolayer of at least 70% confluent polarized mammary cells disposed on the matrix material, wherein the mammary cells are selected from the group consisting of: living primary mammary epithelial cells, living mammary myoepithelial cells, living mammary progenitor cells, living immortalized mammary epithelial cells, living immortalized mammary myoepithelial cells, and living immortalized mammary progenitor cells; and (b) isolating and culturing the dairy product. 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 medium. In some embodiments, the bioreactor is a closed bioreactor. In some embodiments, the bioreactor includes an apical compartment that is substantially separated from the lumen / basal compartment of the living cell construct. In some embodiments, the apical compartment is in fluid contact with the apical surface of the mammary cells. In some embodiments, the cultured dairy product is secreted from the apical surface of the mammary cells into the apical compartment. In some embodiments, the culture medium does not substantially contact the cultured dairy product. In some embodiments, the total cell density of the mammary cells within the bioreactor is at least 10 11 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least 1.5 m 2. In some embodiments, the culture medium comprises 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 matrix material comprises one or more extracellular matrix proteins. In some embodiments, the scaffold comprises a natural polymer, a biocompatible synthetic polymer, a synthetic peptide, a composite 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 polymer is polysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate, polyvinyl alcohol, sodium polyacrylate, acrylate polymer, and / or polyethylene glycol. In some embodiments, the culture is carried out at a temperature of about 27° C. to about 39° C. In some embodiments, the culture is carried out at a temperature of about 30° C. to about 37° C. In some embodiments, the culture is carried out at an atmospheric concentration of CO2 of about 4% to about 6%. In some embodiments, the culturing is carried out at an atmospheric concentration of about 5% CO2.

[0011] In certain embodiments, disclosed herein is a bioreactor comprising: (a) an apical compartment comprising a cultured dairy product; and (b) at least one living cell construct comprising: (i) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity / basal compartment, and more than one pore extending from the inner surface to the outer surface; (ii) a matrix material disposed on the outer surface of the three-dimensional scaffold; (iii) a culture medium disposed within the inner cavity / basal compartment and in fluidic contact with the inner surface; and (iv) a monolayer of polarized mammary cells at least 70% confluent disposed on the matrix material, wherein the mammary cells are selected from the group consisting of: viable primary mammary epithelial cells, viable mammary myoepithelial cells, viable mammary progenitor cells, viable immortalized mammary epithelial cells, viable immortalized mammary myoepithelial cells, and viable immortalized mammary progenitor cells; wherein the apical surface of the mammary cells is in fluidic contact with the apical compartment. In some embodiments, the total cell density of the mammary cells within the bioreactor is at least 10 11 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least 1.5 m 2 .

[0012] Disclosed herein, in certain embodiments, are living cell constructs comprising mammary cells that compartmentalize the feeding of the cells and the secretion of cultured dairy products.

[0013] In certain embodiments, disclosed herein are living cell constructs comprising: a scaffold having an apical surface and a bottom surface; and a continuous monolayer of (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) viable immortalized mammary epithelial cells on the apical surface of the scaffold, wherein the continuous monolayer of (a), (b), and / or (c) has an apical surface and a basal surface (e.g., the cells form a polarized and confluent cell monolayer). layer): (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) immortalized mammary epithelial cells, wherein the construct comprises an apical compartment above and adjacent to the apical surface of a continuous monolayer of the following (a), (b), and / or (c), and a basal compartment below and adjacent to the bottom surface of the scaffold: (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) immortalized mammary epithelial cells.

[0014] In certain embodiments, disclosed herein are methods of producing milk in culture, the methods comprising culturing a living cell construct of the present invention, thereby producing milk in culture.

[0015] In certain embodiments, disclosed herein is a method for making a living cell construct for producing milk in culture, the method comprising (a) isolating primary mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells from a mammary explant, a biopsy sample, or raw breast milk from mammary tissue (e.g., mammary gland, breast, nipple tissue) to produce isolated mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells; (b) culturing the isolated primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells to produce a mixed population of primary mammary epithelial cells, mammary myoepithelial cells, and / or mammary progenitor cells; and (c) culturing the mixed population of (b) on a scaffold having an upper surface and a lower surface to produce a polarized continuous (i.e., confluent) monolayer of the mixed population of primary mammary epithelial cells, myoepithelial cells, and mammary progenitor cells on the upper surface of the scaffold, wherein the polarized continuous monolayer comprises an apical surface and a basal surface, thereby producing a living cell construct for producing milk in culture.

[0016] In certain embodiments, disclosed herein are methods of making a living cell construct for producing milk in culture, the methods comprising: a) isolating primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells from a mammary explant, a biopsy sample, or raw breast milk from mammary tissue (e.g., mammary gland, breast, nipple tissue) to produce isolated mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells; (b) culturing the isolated primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells to produce primary mammary epithelial cells; (c) sorting the mixed population of primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells to produce a population of primary mammary epithelial cells; and (d) culturing the population of primary mammary epithelial cells on a scaffold having an upper surface and a lower surface to produce a polarized continuous (i.e., confluent) monolayer of primary mammary epithelial cells on the upper surface of the scaffold, wherein the polarized continuous monolayer comprises an apical surface and a basal surface, thereby producing a living cell construct for producing milk in culture.

[0017] In certain embodiments, disclosed herein are methods of making a living cell construct for producing milk in culture, the methods comprising (a) culturing immortalized mammary epithelial cells to produce an increased number of immortalized mammary epithelial cells; (b) culturing the immortalized mammary epithelial cells of (a) on a scaffold having an upper surface and a lower surface to produce a polarized continuous (i.e., confluent) monolayer of the immortalized mammary epithelial cells on the upper surface of the scaffold, wherein the polarized continuous monolayer comprises an apical surface and a basal surface, thereby producing a living cell construct for producing milk in culture.

[0018] In certain embodiments, disclosed herein are methods for producing milk in culture, the methods comprising culturing a living cell construct comprising (a) a scaffold comprising an upper surface and a lower surface, and a continuous (i.e., confluent) polarized monolayer of viable primary mammary epithelial cells, a continuous polarized monolayer of a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or a continuous polarized monolayer of viable immortalized mammary epithelial cells having an apical surface and a basal surface; , and / or a continuous polarized monolayer of live immortalized mammary epithelial cells is located on the upper surface of the scaffold, (b) a basal compartment and an apical compartment, wherein the lower surface of the scaffold is adjacent to the basal compartment and the apical surface of the monolayer of live primary mammary epithelial cells, the monolayer of a mixed population of live primary mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells and / or the monolayer of live immortalized mammary epithelial cells is adjacent to the apical compartment, wherein the monolayer of live primary epithelial mammary cells, the live primary epithelial mammary cells in the monolayer of a mixed population of live primary mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells, or the monolayer of immortalized mammary epithelial cells secrete milk into the apical compartment through its apical surface, thereby producing milk in culture.

[0019] In certain embodiments, disclosed herein are methods for producing modified primary mammary epithelial cells or immortalized mammary epithelial cells, wherein the method comprises introducing into the cells: (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 has been spliced ​​to the 3' sequence of exon 11; (b) a polynucleotide encoding a chimeric prolactin receptor that binds to a ligand and that is capable of activating milk synthesis in the absence of prolactin; (c) a polynucleotide encoding a constitutively or conditionally active prolactin receptor. (d) a polynucleotide encoding a prolactin receptor protein, optionally wherein the polynucleotide encodes a constitutively active human prolactin receptor protein comprising a deletion of amino acids 9 to 187; (i) a polynucleotide encoding a modified (recombinant) effector of a prolactin protein comprising the following (i) and / or (ii): (i) a JAK2 tyrosine kinase domain fused to a STAT5 tyrosine kinase domain, and / or (ii) a prolactin receptor intracellular domain fused to a JAK2 tyrosine kinase domain; (e) a loss-of-function mutation introduced into the circadian rhythm-related gene PER2 (period circadian protein homolog 2); and / or (f) a polynucleotide encoding one or more glucose transporter genes GLUT1 and / or GLUT12, thereby increasing the rate of nutrient uptake at the basal surface of a cell monolayer of modified primary mammary epithelial cells or immortalized mammary epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is an example of milk harvesting for nutritional use from mammary epithelial cells grown as a confluent monolayer in a compartmentalized culture device, in which fresh or recycled culture medium is supplied to the basal compartment and milk is collected from the apical compartment. TEER, transepithelial electrical resistance.

[0022] Figure 2 Shown are examples of polarized nutrient uptake and milk secretion across a confluent monolayer of mammary epithelial cells anchored to a scaffold at a basal surface.

[0023] Figure 3 Shown are exemplary micropatterned scaffolds that provide increased surface area for compartmentalized nutrient uptake and milk secretion by a confluent monolayer of mammary epithelial cells.

[0024] Figure 4 Shown are three examples of a hollow fiber bioreactor, depicted as a bundle of capillaries (top), that can support mammary epithelial cells lining the outer (top and lower left) or inner (lower right) surfaces of the capillaries, providing targeted and compartmentalized nutrient absorption and milk secretion.

[0025] Figure 5 A cross-section of a three-dimensional living cell construct is illustrated. The construct comprises a scaffold having an inner surface defining a lumen / basal compartment and an outer surface. The lumen / basal compartment contains a cell culture medium. A matrix material is positioned above the outer surface of the scaffold. Pores traverse the scaffold from the inner surface to the outer surface, allowing the cell culture medium to contact the basal surface of cells in a cell monolayer disposed on the matrix material.

[0026] Figure 6 An example of a bioreactor for producing cultured dairy products is described. The bioreactor comprises a living cell construct and an apical chamber. The cell construct comprises a scaffold having an inner surface and an outer surface defining a lumen / basal chamber. The lumen contains the cell culture medium. A matrix material is positioned above the outer surface of the scaffold. Pores extend from the inner surface to the outer surface of the scaffold, allowing the cell culture medium to contact the basal surface of cells in a cell monolayer disposed on the matrix material. The apical surface of the cells in the cell monolayer secretes milk / cultured dairy products into the apical chamber. The apical chamber and the lumen / basal chamber are separated by the cell monolayer.

[0027] Figure 7 A living cell construct is illustrated. The construct comprises a scaffold having an inner surface defining a lumen / basal compartment and an outer surface. The lumen / basal compartment contains a cell culture medium. A matrix material is positioned above the outer surface of the scaffold. Pores traverse the scaffold from the inner surface to the outer surface, allowing the cell culture medium to contact the basal surface of cells of a cell monolayer disposed on the matrix material.

[0028] Detailed Description of the Invention

[0029] Milk is a nutrient-rich liquid food produced in the mammary glands of mammals. It is the primary source of nutrition for infant mammals (including breastfed humans) until they are able to digest other types of food. Human milk is more than just nutrition. Rather, it contains a variety of factors with biologically active properties that have profound effects on the survival and health of infants. Natural milk contains many other macronutrients, including proteins, lipids, polysaccharides, and lactose. Milk consumption occurs in two distinct general categories: the natural source of nutrition for all infant mammals, and food products.

[0030] In nearly all mammals, milk is delivered to infants through breastfeeding, either directly or by expression for storage and later consumption. Early milk from mammals contains antibodies that provide protection to the newborn infant, as well as nutrients and growth factors. Breast milk is not a homogeneous, unaltered, constant, factory-made product; rather, it is a biological product produced by women with markedly different genotypes, phenotypes, and diets. Further complicating matters, the composition of breast milk is influenced by a multitude of maternal, infant, and environmental factors. Human milk contains a rich and diverse array of proteins, carbohydrates, lipids, fatty acids, minerals, and vitamins, but much of its disease-fighting potential comes from its vast array of antibodies, leukocytes, hormones, antimicrobial peptides, cytokines, chemokines, and other bioactive factors.

[0031] It has been previously shown that mammary epithelial cells (MECs) in culture display similar organization and behavior to those observed in vivo (Arevalo et al. 2016 Am J Physiol Cell Physiol. 310(5):C348-356; Chen et al. 2019 Curr Protoc Cell Biol. 82(1):e65). In Arevalo et al., specific biomarkers for MEC populations were detected in immortalized bovine mammary epithelial cells (BME-UV1) and immortalized bovine mammary acinar cells (MAC-T) cultured on adherent 2-D plates, ultra-low attachment surface 3D microplates, and 3D plates coated with Matrigel. In addition, in Chen et al., a protocol for the isolation and culture of human primary mammary epithelial stem / progenitor cells from human mammary tissue and the subsequent generation of mammospheres using 3D organoid cultures on gelatin sponge and Matrigel matrices was described in detail. However, neither Arevalo nor Chen attempted to stimulate milk production from these MEC cultures.

[0032] In particular, when grown on an appropriate extracellular matrix and stimulated with prolactin, cultured bovine mammary epithelial cells polarize and organize into structures capable of secreting certain milk components (Blatchford et al. 1999 Animal Cell Technology: Basic & Applied Aspects 10: 141-145). In Blatchford et al., bovine MECs polarize and form mammospheres. Casein and lactophilin were isolated from the culture. However, the cells did not polarize in a uniform direction. Blatchford et al. noted that milk proteins were distributed between cells and dispersed throughout the mammospheres. Due to the lack of a uniform polarization orientation, Blatchford had to isolate the secreted proteins from the culture medium.

[0033] Furthermore, in vitro two-dimensional models, such as those used in 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.

[0034] 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 compartmentalization required for producing and extracting cultured dairy products (Sharfstein et al. 1992 Biotechnology and Bioengineering 40:672-680). In Sharfstein et al., the growth, long-term expression of functional differentiation and metabolism of COMMA-1D (an immortalized mouse mammary epithelial cell line) were examined in two different systems: extended batch culture and hollow fiber reactor culture. Using inoculation into Costar The COMMA-1D on polycarbonate membrane cell culture inserts, Sharfstein et al., produced a confluent monolayer capable of barrier formation and polarized metabolism between the apical and basal sides, which maintained a gradient of glucose and lactate. However, using hollow fiber bioreactors for cultivation, Sharfstein et al., were unable to achieve separation of the basal and apical compartments. In addition, it was not determined whether the nutrient uptake in hollow fiber culture was polarized (Sharfstein et al. 1992). Importantly, no work previously was able to cultivate mammary epithelial cells from humans or other nutritionally relevant species in a high-density, three-dimensional, compartmentalized form.

[0035] Disclosed herein, in certain embodiments, are living cell constructs, methods of making living cell constructs, and methods of using living cell constructs for producing cultured dairy products from cultured mammary cells in vitro and / or ex vivo.

[0036] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented or all the features that may be added to the present invention. For example, features described in connection with one embodiment may be incorporated into other embodiments, and features described in connection with a particular embodiment may be deleted from that embodiment. In addition, many variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure without departing from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0037] Unless the context indicates otherwise, it is specifically intended that the various features described herein can be used in any combination. Furthermore, in some embodiments, any feature or combination of features described herein may not be included or omitted. For illustration, if the specification states that a compound includes components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted or eliminated, individually or in any combination.

[0038] definition

[0039] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as alternatively ("or").

[0041] Furthermore, any feature or combination of features described herein may be excluded or omitted.

[0042] As used herein, when referring to a measurable value such as an amount of a compound or agent, dosage, time, temperature, and the like, the term "about" is meant to include variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] Nucleotide sequences are presented herein as a single strand only, in the 5' to 3' direction, from left to right, unless otherwise specifically indicated. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by the single-letter code or the three-letter code, both of which comply with 37 CFR § 1.822 and established usage.

[0045] Unless otherwise indicated, standard methods known to those skilled in the art can be used for the production of recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, viral vector constructs, and construction of transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor, NY, 1989); FM Ausubel et al., Current Protocols In Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0046] As used herein, the transitional phrase "consisting essentially of should be construed to include the recited materials or steps as well as those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Therefore, the term "consisting essentially of" as used herein should not be construed as being equivalent to "comprising."

[0047] As used herein, the term "polypeptide" includes both peptides and proteins, and does not require any particular amino acid length or tertiary structure unless otherwise indicated.

[0048] As used herein, the term "polarized" with respect to cells and / or cell monolayers refers to the spatial state of a cell in which there are two different surfaces of a cell, for example, a top surface and a base surface, which can be different. In some embodiments, the different surfaces of polarized cells include different surface receptors and / or transmembrane receptors and / or other structures. In some embodiments, a single polarized cell in a continuous monolayer has a top surface and a base surface of similar orientation. In some embodiments, a single polarized cell in a continuous monolayer has a communication structure (for example, a tight junction) between single cells to allow cross-communication between single cells and produce a separation (for example, compartmentalization) of a top compartment and a base compartment.

[0049] As used herein, "apical surface" means the surface of a cell that faces the external environment or toward a cavity or chamber, such as the lumen of an internal organ. With respect to mammary epithelial cells, the apical surface is the surface from which cultured milk products are secreted.

[0050] As used herein, "substrate surface" means the surface of cells that is in contact with a surface (eg, the matrix of a bioreactor).

[0051] As used herein, "bioreactor" means a device or system that supports a biologically active environment that enables the production of the cultured dairy products described herein from the mammary cells described herein.

[0052] As used herein, the term "galactagogue" refers to the ability to stimulate milk production and / or secretion. A gene or protein (e.g., prolactin) can be galactagogue, as can any other natural and / or synthetic product. In some embodiments, a lactogenic culture medium comprises prolactin, thereby stimulating cells in contact with the culture medium to produce milk.

[0053] As used herein, the term "food grade" refers to materials that are considered non-toxic and safe for consumption (eg, by humans and / or other animals), for example, as regulated by standards established by the U.S. Food and Drug Administration.

[0054] In some embodiments, milk produced by primary mammary epithelial cells (e.g., primary mammary epithelial cells from isolated, viable primary mammary epithelial cells, and / or primary mammary epithelial cells from a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and / or mammary progenitor cells) or immortalized mammary epithelial cells is secreted through the apical surface of the cells into the apical compartment. In some embodiments, the basal compartment comprises culture medium, and the culture medium is in contact with the basal surface of the viable primary mammary epithelial cells, the mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or the immortalized mammary epithelial cells.

[0055] Living cell constructs

[0056] Disclosed herein, in certain embodiments, are living cell constructs for producing milk in culture, comprising a continuous monolayer of living mammary cells selected from the group consisting of: (a) living primary mammary epithelial cells, (b) living mammary myoepithelial cells, (c) living mammary progenitor cells, and / or (d) living immortalized mammary epithelial cells.

[0057] In some embodiments, the mammary cells include milk-producing mammary epithelial cells, contractile myoepithelial cells and / or progenitor cells that can produce both mammary epithelial cells and mammary contractile myoepithelial cells. Mammary epithelial cells are the only cells that produce milk. In some embodiments, the mammary cells include mammary epithelial cells, primary mammary epithelial cells, mammary myoepithelial cells and mammary progenitor cells.

[0058] In some embodiments, the mammary cells are from mammary tissue, breast tissue, and / or nipple tissue of a mammal. In some embodiments, the mammary cells are from any mammal, such as primates (e.g., chimpanzees, orangutans, gorillas, monkeys (e.g., Old World monkeys, New World monkeys), lemurs, humans), dogs, cats, rabbits, mice, rats, horses, cows, goats, sheep, cattle (e.g., Bos spp.), pigs, deer, musk deer, bovines, whales, dolphins, hippos, elephants, rhinos, giraffes, zebras, lions, cheetahs, tigers, pandas, red pandas, and otters. In some embodiments, the mammary cells are from endangered species, such as endangered mammals. In some embodiments, the mammary cells are from humans. In some embodiments, the mammary cells are from bovines (e.g., cows).

[0059] In some embodiments, the continuous monolayer of mammary cells living derives from breast milk-derived stem cells or derives from the mammary stem cells of mammary tissue biopsy. The epithelial component of breast milk not only includes mature epithelial cells, but also includes its precursor and the stem cell in culture. The stem cell subpopulation derived from breast milk demonstrates very high multilineage potential, is similar to the typical characteristics of human embryonic stem cells (hESC). Mammary stem cells also may derive from mammary tissue biopsy and include terminally differentiated MEC. The mammary stem cells derived from breast milk and the mammary stem cells derived from mammary tissue biopsy are all multipotent cells that can produce MEC or myoepithelial cells.

[0060] In some embodiments, at least 50% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 55% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 60% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 65% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 70% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 75% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 80% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 85% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 90% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 95% of the mammary cells in the living cell culture are polarized. In some embodiments, at least 100% of the mammary cells in the living cell culture are polarized. In some embodiments, substantially all of the breast cells of the living cell construct are polarized (i.e., have an apical surface and a basal surface). In some embodiments, substantially all of the breast cells of the living cell construct are polarized, and substantially all of the polarized cells are oriented in the same direction. For example, in some embodiments, substantially all of the breast cells have an apical surface and a basal surface, wherein substantially all of the cells have apical surfaces oriented in the same direction, and substantially all of the cells have basal surfaces oriented in the same direction.

[0061] In some embodiments, the monolayer of epithelial breast cells is at least 70% confluent on the scaffold. In some embodiments, the monolayer of breast epithelial cells is at least about 75% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is at least about 80% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is at least about 85% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is at least about 90% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is at least about 95% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is at least about 99% confluent on the scaffold. In some embodiments, the monolayer of epithelial breast cells is 100% confluent on the scaffold.

[0062] Genetic modification of breast cells

[0063] In some embodiments, the mammary cells comprise a constitutively active prolactin receptor protein. In some embodiments, the mammary cells comprise a constitutively active human prolactin receptor protein. When the primary mammary epithelial cells or immortalized mammary epithelial cells comprise a constitutively active prolactin receptor, the culture medium does not contain prolactin.

[0064] In some embodiments, the constitutively active human prolactin receptor protein comprises a deletion of amino acids 9 to 187, wherein the numbering is based on the reference amino acid sequence of the human prolactin receptor identified as SEQ ID NO: 1.

[0065] SEQ ID NO: 1: Human prolactin receptor (GenBank accession number AAD32032.1)

[0066]

[0067] In some embodiments, the constitutively active human prolactin receptor protein comprises a deletion of the following amino acids:

[0068]

[0069] (eg amino acid positions 9 to 187 of SEQ ID No: 1).

[0070] In some embodiments, the mammary cells comprise a loss-of-function mutation introduced into the circadian rhythm-related gene PER2. In some embodiments, the loss-of-function mutation introduced into the circadian rhythm-related gene PER2 promotes the synthesis of cultured milk components. In some embodiments, the loss-of-function mutation in the PER2 gene comprises a deletion of 87 amino acids from positions 348 to 434 in PER2, wherein the numbering is based on the reference amino acid sequence of human PER2 identified as SEQ ID NO: 2.

[0071] SEQ ID NO: 2: Human circadian rhythm protein homolog 2 (GenBank accession number NM 022817)

[0072]

[0073] In some embodiments, the loss-of-function mutation introduced into PER2 comprises a deletion of:

[0074]

[0075] (eg, amino acid positions 348 to 434 of SEQ ID NO: 2).

[0076] In some embodiments, the mammary cells comprise a polynucleotide encoding a prolactin receptor comprising a modified intracellular signaling domain. In some embodiments, a loss-of-function mutation introduced into the circadian rhythm-related gene PER2 promotes the synthesis of individual cultured milk components. In some embodiments, the prolactin receptor comprises a truncation in which position 154 of exon 10 has been spliced ​​to the 3' sequence of exon 11. In some embodiments, the prolactin receptor comprises a sequence according to SEQ ID NO: 3.

[0077] SEQ ID NO: 3: Human prolactin receptor isoform 4 (GenBank Accession No. AF416619; Trott et al. 2003J.Mol.Endocrinol 3Q(l):31-47)

[0078]

[0079] In some embodiments, the mammary cells comprise a polynucleotide encoding a modified (e.g., recombinant) effector of a prolactin protein. In some embodiments, the modified effector of the prolactin protein 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 is connected to the 3' end of a polynucleotide encoding a STAT5 tyrosine kinase domain). In some embodiments, the modified effector of the prolactin protein promotes the increased synthesis of a separate cultured milk component. In some embodiments, the modified effector has a sequence according to SEQ ID NO: 4. The amino acids in bold correspond to the JAK2 kinase domain at amino acid positions 757 to 1129 of the reference human JAK2 amino acid sequence.

[0080] SEQ ID NO: 4. Fused at the 3' end to amino acids 757-1129 of the JAK2 human tyrosine protein kinase STA5A human signal transducer and activator of transcription 5A

[0081]

[0082]

[0083] In some embodiments, the mammary cells are immortalized. In some embodiments, the mammary cells comprise one or more nucleic acids encoding human telomerase reverse transcriptase (hTERT) or simian virus 40 (SV40). In some embodiments, the mammary cells comprise small hairpin RNA (shRNA) against p16 (inhibitor of cyclin-dependent kinase 4) (p16(INK4)) and a master regulator of cell cycle entry and proliferation metabolism (c-MYC).

[0084] In some embodiments, the method comprises introducing into a 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 has been spliced ​​to a sequence 3' to exon 11; (b) a polynucleotide encoding a chimeric prolactin receptor that binds to a ligand and that is capable of activating milk synthesis 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, wherein numbering is based on the sequence identified as SEQ ID NO: 1). NO:1 of the reference amino acid sequence of the human prolactin receptor); (d) a polynucleotide encoding a modified (e.g., recombinant) effector of a prolactin protein comprising the following (i) and / or (ii): (i) a janus kinase-2 (JAK2) tyrosine kinase domain, optionally wherein the JAK2 tyrosine kinase domain is fused to a signal transducer and activator of transcription 5 (STAT5) tyrosine kinase domain (e.g., a polynucleotide encoding the JAK2 tyrosine kinase domain is linked to the 3' end of the polynucleotide encoding the STAT5 tyrosine kinase domain); and / or (ii) a prolactin receptor intracellular domain fused to a JAK2 tyrosine kinase domain; (e) a loss-of-function mutation introduced into the circadian rhythm-related gene PER2 (period circadian protein homolog 2); and / or (f) a polynucleotide encoding one or more glucose transporter genes GLUT1 and / or GLUT12, thereby increasing the rate of nutrient uptake at the basal surface of the monolayer.

[0085] bracket

[0086] In some embodiments, the living cell construct further comprises a scaffold having a top surface / exterior surface and a bottom surface / interior surface. In some embodiments, the scaffold is a two-dimensional surface or a three-dimensional surface (e.g., a three-dimensional micropatterned surface, and / or as a cylindrical structure assembled into a bundle). Non-limiting examples of two-dimensional surface scaffolds are Filter. In some embodiments, the support is a three-dimensional surface. Non-limiting examples of three-dimensional micro-patterned surfaces include microstructured bioreactors, decellularized tissues (e.g., decellularized mammary glands or decellularized plant tissues), micro-patterned supports made of biomaterials or biocompatible materials by casting or three-dimensional printing, and textured surfaces. In some embodiments, the support (e.g., hollow fiber bioreactor) is manufactured by electrospinning cellulose nanofibers and / or cylindrical structures that can be assembled into bundles. In some embodiments, the support is porous. In some embodiments, the support is a 3D support. In some embodiments, the three-dimensional support is any structure with a closed hollow interior / central cavity. In some embodiments, the three-dimensional support is connected to one or more surfaces to form a closed inner chamber / base compartment. For example, the support can be connected to one or more walls of the bioreactor to form an inner chamber / base compartment. In some embodiments, the support is a hollow fiber bioreactor. In some embodiments, the 3D support is a tube in which the central cavity is limited by the inner surface of the support. In some embodiments, the 3D support is a hollow sphere in which the central cavity is limited by the inner surface of the support.

[0087] For in vitro culture methods for intestinal absorption studies, two-dimensional surface scaffolds such as They have long been considered the standard because they provide both apical and basolateral spaces to mimic the gut-blood barrier and enable both active and passive transport of drugs and nutrients. However, cells seeded onto flat supports exhibit a phenotype that is significantly different from that of cells in vivo, in part due to the poor representation of the 3-D extracellular microenvironment.

[0088] Three-dimensional scaffolds allow mammary cells (e.g., MECs) to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, 3D cell culture allows in vitro cells to grow in all directions, approaching the in vivo mammary environment. In addition, 3D scaffolds allow for a larger surface area for cell culture and for metabolite and gas exchange, plus it achieves the necessary compartmentalization—enabling cultured milk products to be secreted into one compartment while the cell culture medium is in contact with mammary cells in another compartment. To date, a confluent monolayer with polarized separation of the basal and apical cell surfaces using mammary epithelial cells on a 3D surface has not been achieved (Sharfstein et al. 1992).

[0089] In some embodiments, the scaffold is porous. In some embodiments, the scaffold is permeable to cell culture medium, allowing the cell culture medium to contact the cells of the cell monolayer. In some embodiments, the scaffold is traversed by at least one pore, which allows the cell culture medium to contact the basal surface of the cells of the cell monolayer.

[0090] In some embodiments, the top surface / exterior surface of the scaffold is coated with a matrix material. In some embodiments, the matrix is ​​composed of one or more extracellular matrix proteins. Non-limiting examples of extracellular matrix proteins include collagen, laminin, nidogen, tenascin, and / or fibronectin. In some embodiments, the scaffold includes natural polymers, biocompatible synthetic polymers, synthetic peptides, and / or complexes derived from any combination thereof. In some embodiments, natural polymers that can be used in the present invention include, but are not limited to, collagen, chitosan, cellulose, agarose, alginate, gelatin, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, and / or hyaluronic acid. In some embodiments, biocompatible synthetic polymers that can be used in the present invention include, but are not limited to, cellulose, polysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate, polyvinyl alcohol, sodium polyacrylate, acrylate polymers, and / or polyethylene glycol. In some embodiments, the top of the scaffold is coated with laminin and collagen.

[0091] In some embodiments, the matrix material is porous. In some embodiments, the matrix material is permeable to cell culture medium, allowing the cell culture medium to contact the cells of the cell monolayer. In some embodiments, the matrix material is traversed by at least one pore, which allows the cell culture medium to contact the basal surface of the cells of the cell monolayer.

[0092] 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 μm. 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 μm. 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 at least about 1.5 μm. 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 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 1.8 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 1.9 μm. 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 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 2.4 μm. 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 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 2.7 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 2.8 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 2.9 μm. In some embodiments, the pore size of the scaffold and / or matrix material is at least about 3.0 μm.

[0093] In some embodiments, the living cell construct comprises: a scaffold having an apical / exterior surface and a basal / interior surface; and a continuous monolayer of (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) viable immortalized mammary epithelial cells on the apical surface of the scaffold, wherein the continuous monolayer of (a), (b), and / or (c) has an apical surface and a basal surface (e.g., the cells form a polarized and confluent monolayer of cells). : (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) immortalized mammary epithelial cells, wherein the construct comprises an apical compartment above and adjacent to the apical surface of a continuous monolayer of the following (a), (b) and / or (c), and a basal compartment below and adjacent to the bottom surface of the scaffold: (a) viable primary mammary epithelial cells, (b) a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or (c) immortalized mammary epithelial cells.

[0094] bioreactor

[0095] In certain embodiments, disclosed herein is a bioreactor comprising: (a) an apical compartment comprising a cultured dairy product; and (b) at least one living cell construct comprising: (i) a three-dimensional scaffold having an outer surface, an inner surface defining an inner cavity / basal compartment, and more than one pore extending from the inner surface to the outer surface; (ii) a matrix material disposed on the outer surface of the three-dimensional scaffold; (iii) a culture medium disposed within the inner cavity / basal compartment and in fluid contact with the inner surface; and (iv) a monolayer of polarized mammary cells at least 70% confluent disposed on the matrix material, wherein the mammary cells are selected from the group consisting of: viable primary mammary epithelial cells, viable mammary myoepithelial cells, viable mammary progenitor cells, viable immortalized mammary epithelial cells, viable immortalized mammary myoepithelial cells, and viable immortalized mammary progenitor cells; wherein the apical surface of the mammary cells is in fluid contact with the apical compartment.

[0096] In some embodiments, the bioreactor is a closed bioreactor.In some embodiments, the top chamber is substantially isolated from the lumen / base compartment.

[0097] Hollow fiber bioreactor is an exemplary bioreactor for the method disclosed herein. Hollow fiber bioreactor is a high-density continuous perfusion culture system that is very close to the in vivo cell growth environment. It consists of thousands of semi-permeable 3D scaffolds (i.e., hollow fibers) in a parallel array in a cartridge housing equipped with inlet and outlet ports. These fiber bundles are potted or sealed at each end so that any liquid entering the cartridge end must flow through the inside of the fiber. Cells are typically seeded outside the fibers in the cartridge, in the extracapillary space (ECS).

[0098] Three fundamental features distinguish hollow fiber cell culture from other methods: (1) cells adhere well to the porous matrix, as they do in vivo, rather than to plastic dishes, microcarriers, or other impermeable supports, (2) the molecular weight cutoff of the support matrix can be controlled, and (3) the extremely high surface area to volume ratio (150 cm per mL) is achieved. 2 or more), providing a large area for metabolite and gas exchange for the efficient growth of host cells.

[0099] The bioreactor structure provides a fiber matrix that allows the infiltration of nutrients, gases and other basic culture medium components and cell waste products, but does not allow the infiltration of cells, in which cells can be expanded. Hollow fiber bioreactor technology has been used to obtain high-density cell expansion by utilizing hollow fibers to create a semi-permeable barrier between the cell growth chamber and the culture medium flow. Because the surface area provided by this design is large, using this fiber as a culture substrate allows the production of large quantities of cells. The cells grown in the three-dimensional environment within the bioreactor are immersed in fresh culture medium when perfused through the hollow fibers.

[0100] In order to replicate the topography of the intestine, Costello et al. developed a 3-D printed bioreactor that can simultaneously contain porous villus scaffolds via micromolding (Costello et al. 2017 Scientific Reports 7(12515):1-10). This geometrically complex molded scaffold provides separation of the top space and the base-side space in a manner where fluid flow exposes intestinal epithelial cells to physiologically relevant shear stresses (Costello et al. 2017). Similarly, Morada et al. created long-term in vitro culture in a simulated intestinal environment using a hollow fiber bioreactor that allows two controlled, isolated environments (biphasic) to provide oxygen and nutrients to host cells from the base layer while allowing a hypoxic, nutrient-rich environment to form on the top surface (Morada et al. 2016 International Journal for Parasitology 26:21-29).

[0101] When configuring a hollow fiber bioreactor, design considerations and parameters can vary according to the goals associated with cell expansion. One such design consideration is the size of the holes in the fiber wall. This is typically designed to allow nutrients to pass through to enter the cells, take away waste, provide the cells with desired products (such as growth factors), remove the desired products from the cells, and exclude certain factors that may exist from reaching the cells. Therefore, the pore size of the fiber wall can vary, to adjust which components can pass through the wall. For example, the pore size can allow large protein molecules to pass through, including growth factors, including but not limited to epidermal growth factor and platelet-derived growth factor. One of ordinary skill in the art will understand how to change the pore size according to the component of the material that is desired to pass through the fiber wall to reach the cells or to carry from the cells.

[0102] In some embodiments, the pore size is about 0.2 μm. In some embodiments, the pore size is about 0.1 μm. In some embodiments, the pore size is about 0.2 μm. In some embodiments, the pore size is about 0.3 μm. In some embodiments, the pore size is about 0.4 μm. In some embodiments, the pore size is about 0.5 μm. In some embodiments, the pore size is about 0.6 μm. In some embodiments, the pore size is about 0.7 μm. In some embodiments, the pore size is about 0.8 μm. In some embodiments, the pore size is about 0.9 μm. In some embodiments, the pore size is about 1.0 μm. In some embodiments, the pore size is about 1.1 μm. In some embodiments, the pore size is about 1.2 μm. In some embodiments, the pore size is about 1.3 μm. In some embodiments, the pore size is about 1.4 μm. In some embodiments, the pore size is about 1.5 μm. In some embodiments, the pore size is about 1.6 μm. In some embodiments, the pore size is about 1.7 μm. In some embodiments, the pore size is about 1.8 μm. In some embodiments, the pore size is about 1.9 μm. In some embodiments, the pore size is about 2.0 μm. In some embodiments, the pore size is about 2.1 μm. In some embodiments, the pore size is about 2.2 μm. In some embodiments, the pore size is about 2.2 μm. In some embodiments, the pore size is about 2.3 μm. In some embodiments, the pore size is about 2.4 μm. In some embodiments, the pore size is about 2.5 μm. In some embodiments, the pore size is about 2.6 μm. In some embodiments, the pore size is about 2.7 μm. In some embodiments, the pore size is about 2.8 μm. In some embodiments, the pore size is about 2.9 μm. In some embodiments, the pore size is about 3.0 μm.

[0103] Methods for making living cell constructs

[0104] In certain embodiments, disclosed herein is a method for manufacturing a living cell construct for producing cultured dairy products. In some embodiments, method includes (a) separating primary mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells from mammary tissue (for example, mammary gland, breast, nipple tissue), biopsy sample or original breast milk, to produce separated mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells; (b) cultivating the primary mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells separated, to produce a mixed population of primary mammary epithelial cells, mammary myoepithelial cells and / or mammary progenitor cells; (c) cultivating the mixed population of (b) on a support with upper and lower surfaces, to produce the polarized monolayer of the primary mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells of a mixed population on the upper surface of the support, wherein the polarized monolayer includes top surface and base surface, so as to produce a living cell construct for producing cultured dairy products.

[0105] In some embodiments, the method comprises: a) isolating primary mammary epithelial cells, myoepithelial cells and / or mammary progenitor cells from a mammary explant, a biopsy sample, or raw breast milk from mammary tissue (e.g., mammary gland, breast, nipple tissue) to produce isolated mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells; (b) culturing the isolated primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells to produce primary mammary epithelial cells, mammary myoepithelial cells, and / or mammary progenitor cells; (c) sorting the mixed population of primary mammary epithelial cells, myoepithelial cells, and / or mammary progenitor cells (e.g., selecting primary mammary epithelial cells) to produce a population of primary mammary epithelial cells; and (d) culturing the population of primary mammary epithelial cells on a scaffold having an upper surface and a lower surface to produce a polarized monolayer of primary mammary epithelial cells on the upper surface of the scaffold, wherein the polarized monolayer comprises an apical surface and a basal surface, thereby producing a living cell construct for producing a cultured dairy product.

[0106] In some embodiments, the method includes (a) culturing immortalized mammary epithelial cells to produce an increased number of immortalized mammary epithelial cells; (b) culturing the immortalized mammary epithelial cells of (a) on a scaffold having an upper surface and a lower surface to produce a polarized monolayer of immortalized mammary epithelial cells on the upper surface of the scaffold, wherein the polarized monolayer includes an apical surface and a basal surface, thereby producing a living cell construct for producing a cultured dairy product.

[0107] In some embodiments, the culturing and / or incubation of breast cells used in the living cell constructs is carried out at a temperature of about 35° C. 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 thereof, e.g., about 35° C. to about 38° C., about 36° C. to about 39° C., about 36.5° C. to about 39° C., about 36.5° C. to about 37.5° C., or about 36.5° C. to about 38° C.). In some embodiments, the culturing and / or incubation is carried out at a temperature of about 37° C.

[0108] In some embodiments, culturing and / or cultivating of breast cells for use in living cell constructs is performed at an atmospheric concentration of CO2 of about 4% to about 6% (e.g., about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, or 6%, or any value or range thereof, such as 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, culturing and / or cultivating is performed at an atmospheric concentration of CO2 of about 5%.

[0109] In some embodiments, culturing and / or growing breast cells for use in a living cell construct comprises culturing and / or growing in culture medium that is changed from about every 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 therein, e.g., about every day to every 3 days, about every 3 days to every 10 days, about every 2 days to every 5 days). In some embodiments, culturing and / or cultivating further comprises culturing in a culture medium that is replaced about every day or about every few hours to about every 10 days (e.g., about every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours to about every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or any value or range therein). For example, in some embodiments, culturing and / or cultivating further comprises culturing and / or cultivating 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 about every 5 hours to about every 3 days.

[0110] In some embodiments, the living cell construct is stored in a freezer or in liquid nitrogen. Storage temperature depends on required storage length. For example, if the cell will be used within 6 months (for example, in 1 month, 2 months, 3 months, 4 months, 5 months, 6 months), a freezer temperature can be used (for example, at about 0 ° C to about -80 ° C or lower temperature storage, for example, about 0 ° C, -10 ° C, -20 ° C, -30 ° C, -40 ° C, -50 ° C, -60 ° C, -70 ° C, -80 ° C, -90 ° C, -100 ° C or any value or scope therein). For example, liquid nitrogen can be used for long-term storage (e.g., storage for 6 months or more, such as 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, or more years) (e.g., storage at -100°C or lower (e.g., -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, -200°C, or lower)).

[0111] In some embodiments, breast cells are isolated and sorted via fluorescence activated cell sorting, magnetic activated cell sorting, and / or microfluidic cell sorting.

[0112] Basal culture medium and lactation culture medium

[0113] In some embodiments, the culture medium comprises 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 carbon source, the chemical buffer system, the one or more essential amino acids, the one or more vitamins and / or cofactors, and / or the one or more inorganic salts are food grade.

[0114] In some embodiments, culture medium is lactation culture medium.In some embodiments, culture medium also comprises prolactin (for example mammal prolactin, for example human prolactin), linoleic acid and α-linoleic acid, estrogen and / or progesterone.For example, in some embodiments, culture medium comprises the prolactin (or prolactin is added with following amount) of following amount: about 20ng / mL to about 200ng / mL culture medium, for example, about 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 110ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL or 200ng / mL or any value or scope therein. In some embodiments, the culture medium comprises prolactin in an amount (or prolactin is added in an amount) of about 20 ng / mL to about 195 ng / mL, about 50 ng / mL to about 150 ng / mL, about 25 ng / mL to about 175 ng / mL, about 45 ng / mL to about 200 ng / mL, or about 75 ng / mL to about 190 ng / mL of culture medium. In some embodiments, the culture medium also comprises other factors that improve efficiency, including but not limited to insulin, epidermal growth factor, and / or hydrocortisone.

[0115] In some embodiments, the culture medium comprises a carbon source in an amount of about 1 g / L to about 15 g / L of culture medium (e.g., about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L, or about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L to about 7 g / L, 8 g / L, 9 g / L, or 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L of culture medium. Non-limiting examples of carbon sources include glucose and / or pyruvate. For example, in some embodiments, the culture medium comprises glucose in an amount of about 1 g / L to about 12 g / L culture medium, such as about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L, or any value or range therein. In some embodiments, the culture medium comprises glucose in an amount of about 1 g / L to about 6 g / L, about 4 g / L to about 12 g / L, about 2.5 g / L to about 10.5 g / L, about 1.5 g / L to about 11.5 g / L, or about 2 g / L to about 10 g / L culture medium. In some embodiments, the culture medium comprises glucose in an amount between about 1 g / L, 2 g / L, 3 g / L, or 4 g / L and about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L, or between about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L and about 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L. In some embodiments, the culture medium comprises pyruvate in an amount between about 5 g / L and about 15 g / L of culture medium, for example, about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L, or any value or range therein. In some embodiments, the culture medium comprises pyruvic acid in an amount of about 5 g / L to about 14.5 g / L, about 10 g / L to about 15 g / L, about 7.5 g / L to about 10.5 g / L, about 5.5 g / L to about 14.5 g / L, or about 8 g / L to about 10 g / L culture medium. In some embodiments, the culture medium comprises pyruvic acid in an amount of about 5 g / L, 6 g / L, 7 g / L, or 8 g / L to about 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L, or about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L to about 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L.

[0116] In some embodiments, the culture medium comprises a chemical buffer system in an amount of about 1 g / L to about 4 g / L of culture medium (e.g., about 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, or 4 g / L, or any value or range thereof) or about 10 mM to about 25 mM (e.g., about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or any value or range thereof). In some embodiments, the chemical buffer system includes, but is not limited to, sodium bicarbonate and / or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). For example, in some embodiments, the culture medium comprises sodium bicarbonate in an amount of about 1 g / L to about 4 g / L culture medium, such as about 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, or 4 g / L, or any value or range therein. In some embodiments, the culture medium comprises sodium bicarbonate in an amount of about 1 g / L to about 3.75 g / L, about 1.25 g / L to about 4 g / L, about 2.5 g / L to about 3 g / L, about 1.5 g / L to about 4 g / L, or about 2 g / L to about 3.5 g / L culture medium. In some embodiments, the culture medium comprises HEPES in an amount of about 10 mM to about 25 mM, such as about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or any value or range therein. In some embodiments, the culture medium comprises HEPES in an amount of about 11 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.

[0117] In some embodiments, the culture medium comprises one or more essential amino acids in an amount of about 0.5 mM to about 5 mM (e.g., about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any value or range therein), or about 0.5 mM, 1 mM, 1.5 mM, 2 mM to about 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM. In some embodiments, the one or more essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, and / or arginine. For example, in some embodiments, the culture medium comprises arginine in an amount of about 0.5 mM to about 5 mM, such as about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any value or range therein. In some embodiments, the culture medium comprises essential amino acids 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.

[0118] In some embodiments, the culture medium comprises one or more vitamins and / or cofactors in an amount of about 0.01 μM to about 50 μM (e.g., about 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 49.025 μM, 49.05 μM, 49.075 μM or 50 μM, or any value or range thereof, or about 0.01μM, 0.02μM, 0.03μM, 0.04μM, 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.6μM, 0. 7μM, 0.8μM or 0.9μM to about 1μM, 1.1μM, 1.2μM, 1.3μM, 1.4μM, 1.5μM, 1.6μM, 1.7μM, 1.8μM, 1.9μM, 2μM, 2.1μM, 2.2μM, 2.3μM, 2.4μM , 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM or about 0.02 μM, 0.025 μM, 0.05 μM, 0.075 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM to about 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 49.025 μM, 49.05 μM, 49.075 μM 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 comprises thiamine in an amount of about 0.025 μM to about 50 μM, e.g., about 0.025 μM, 0.05 μM, 0.075 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 49.025 μM, 49.05 μM, 49.075 μM, or 50 μM, or any value or range therein. In some embodiments, the culture medium comprises 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 about 45.5 μM. In some embodiments, the culture medium comprises riboflavin in an amount of about 0.01 μM to about 3 μM, for example, about 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM , 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM or 3 μM, or any value or range therein. In some embodiments, the culture medium comprises riboflavin in an amount of about 0.01 μM to about 2.05 μM, about 1 μM to about 2.95 μM, about 0.05 μM to about 3 μM, about 0.08 μM to about 1.55 μM, or about 0.05 μM to about 2.9 μM.

[0119] In some embodiments, the culture medium comprises one or more inorganic salts in an amount of about 100 mg / L to about 150 mg / L of culture medium (e.g., about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range thereof) or about 100 mg / L to about 150 mg / L of culture medium (e.g., about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range thereof). 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 comprises calcium in an amount of about 100 mg / L to about 150 mg / L culture medium, for example, about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range therein. In some embodiments, the culture medium comprises 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 comprises magnesium in an amount of about 0.01 mM to about 1 mM, for example, about 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 0.91 mM, 0.92 mM, 0.93 mM, 0.94 mM, 0.95 mM, 0.96 mM, 0.97 mM, 0.98 mM, 0.99 mM, or 1 mM, or any value or range therein. In some embodiments, the culture medium comprises magnesium in amounts 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.

[0120] In some embodiments, the culture medium comprises a carbon source in an amount of about 1 g / L to about 15 g / L culture medium (e.g., about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L, or about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L to about 7 g / L, 8 g / L, 9 g / L, or 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 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 comprises glucose in an amount of about 1 g / L to about 12 g / L culture medium, such as about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, or 12 g / L, or any value or range therein. In some embodiments, the culture medium comprises glucose in an amount of about 1 g / L to about 6 g / L, about 4 g / L to about 12 g / L, about 2.5 g / L to about 10.5 g / L, about 1.5 g / L to about 11.5 g / L, or about 2 g / L to about 10 g / L culture medium. In some embodiments, the culture medium comprises pyruvic acid in an amount of about 5 g / L to about 15 g / L culture medium, e.g., about 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L, or any value or range therein. In some embodiments, the culture medium comprises pyruvic acid in an amount of about 5 g / L to about 14.5 g / L, about 10 g / L to about 15 g / L, about 7.5 g / L to about 10.5 g / L, about 5.5 g / L to about 14.5 g / L, or about 8 g / L to about 10 g / L culture medium.

[0121] In some embodiments, the culture medium comprises a chemical buffer system in an amount of about 1 g / L to about 4 g / L of culture medium (e.g., about 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, or 4 g / L, or any value or range thereof) or about 10 mM to about 25 mM (e.g., about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or any value or range thereof). 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 comprises sodium bicarbonate in an amount of about 1 g / L to about 4 g / L culture medium, such as about 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, or 4 g / L, or any value or range therein. In some embodiments, the culture medium comprises sodium bicarbonate in an amount of about 1 g / L to about 3.75 g / L, about 1.25 g / L to about 4 g / L, about 2.5 g / L to about 3 g / L, about 1.5 g / L to about 4 g / L, or about 2 g / L to about 3.5 g / L culture medium. In some embodiments, the culture medium comprises HEPES in an amount of about 10 mM to about 25 mM, such as about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM, or any value or range therein. In some embodiments, the culture medium comprises 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.

[0122] In some embodiments, the culture medium comprises one or more essential amino acids in an amount of about 0.5 mM to about 5 mM (e.g., about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any value or range therein) or about 0.5 mM, 1 mM, 1.5 mM, 2 mM to about 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM. In some embodiments, the one or more essential amino acids are arginine and / or cysteine. For example, in some embodiments, the culture medium comprises arginine in an amount of about 0.5 mM to about 5 mM, e.g., about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any value or range therein. In some embodiments, the culture medium comprises 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 comprises cysteine ​​in an amount of about 0.5 mM to about 5 mM, such as about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any value or range therein. In some embodiments, the culture medium comprises cysteine ​​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.

[0123] In some embodiments, the culture medium comprises one or more vitamins and / or cofactors in an amount of about 0.01 μM to about 50 μM (e.g., about 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM). , 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 49.025 μM, 49.05 μM, 49.075 μM or 50 μM, or any value or range thereof) or About 0.01μM, 0.02μM, 0.03μM, 0.04μM, 0.05μM, 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, 0.6μM, 0 .7μM, 0.8μM or 0.9μM to about 1μM, 1.1μM, 1.2μM, 1.3μM, 1.4μM, 1.5μM, 1.6μM, 1.7μM, 1.8μM, 1.9μM, 2μM, 2.1μM, 2.2μM, 2.3μM, 2.4μ 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 comprises thiamine in an amount of about 0.025 μM to about 50 μM, e.g., 0.025 μM, 0.05 μM, 0.075 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 49.025 μM, 49.05 μM, 49.075 μM, or 50 μM, or any value or range therein. In some embodiments, the culture medium comprises 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 about 45.5 μM. In some embodiments, the culture medium comprises riboflavin in an amount of about 0.01 μM to about 3 μM, for example, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, In some embodiments, the culture medium comprises riboflavin in an amount of about 0.01 μM to about 2.05 μM, about 1 μM to about 2.95 μM, about 0.05 μM to about 3 μM, about 0.08 μM to about 1.55 μM, or about 0.05 μM to about 2.9 μM.

[0124] In some embodiments, the culture medium comprises one or more inorganic salts in an amount of about 100 mg / L to about 150 mg / L of culture medium (e.g., about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range thereof) or about 100 mg / L to about 150 mg / L of culture medium (e.g., about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range thereof). In some embodiments, exemplary one or more inorganic salts are calcium and / or magnesium. For example, in some embodiments, the culture medium comprises calcium in an amount of about 100 mg / L to about 150 mg / L culture medium, for example, about 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, or 150 mg / L, or any value or range therein. In some embodiments, the culture medium comprises 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 comprises magnesium in an amount of about 0.01 mM to about 1 mM, for example, about 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 0.91 mM, 0.92 mM, 0.93 mM, 0.94 mM, 0.95 mM, 0.96 mM, 0.97 mM, 0.98 mM, 0.99 mM, or 1 mM, or any value or range therein. In some embodiments, the culture medium comprises magnesium in amounts 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.

[0125] In some embodiments, the carbon source, the chemical buffer system, the one or more essential amino acids, the one or more vitamins and / or cofactors, and / or the one or more inorganic salts are food grade.

[0126] In some embodiments, culture medium is a lactogenic culture medium, for example, culture medium also includes prolactin (for example, mammal prolactin, for example, human prolactin).For example, in some embodiments, culture medium includes the prolactin of following amount (or prolactin is added with following amount): about 20ng / mL to about 200ng / L culture medium, for example, about 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 110ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL or 200ng / mL or any value or scope therein. In some embodiments, culture medium comprises the prolactin of following amount (or prolactin is added with following amount): about 20ng / mL to about 195ng / mL, about 50ng / mL to about 150ng / mL, about 25ng / mL to about 175ng / mL, about 45ng / mL to about 200ng / mL or about 75ng / mL to about 190ng / mL culture medium.In some embodiments, method also comprises adding prolactin to culture medium, thereby providing lactation culture medium.In some embodiments, prolactin is produced by the microbial cell and / or human cell of expressing recombinant prolactin (for example, the prolactin comprising the replacement (S179D) of the serine residue at position 179 of the prolactin gene with aspartic acid, for example, S179D-prolactin). In some embodiments, adding prolactin to the culture medium comprises conditioning the culture medium by culturing cells that express and secrete prolactin, and applying the conditioned culture medium comprising prolactin to the basal surface of a monolayer of primary mammary epithelial cells, the basal surface of a monolayer of a mixed population, or the basal surface of a monolayer of viable immortalized mammary epithelial cells.

[0127] In some embodiments, culture medium also comprises other factors that improve efficiency, include but not limited to insulin, epidermal growth factor and / or hydrocortisone.In some embodiments, method of the present invention also comprises adding other factors (for example, insulin, epidermal growth factor and / or hydrocortisone) to culture medium, for example, to improve efficiency.

[0128] Methods for producing cultured dairy products

[0129] Disclosed herein are methods of making cultured dairy products. In some embodiments, the methods include culturing a living cell construct disclosed herein in a bioreactor comprising a basal compartment and an apical compartment, wherein the basal compartment comprises culture medium and the mammary cells secrete the cultured dairy product into the apical compartment.

[0130] In some embodiments, the living cell construct comprises a scaffold comprising an upper surface and a lower surface and a polarized monolayer of living primary mammary epithelial cells, a continuous polarized monolayer of a mixed population of living primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or a continuous polarized monolayer of living immortalized mammary epithelial cells having an apical surface and a basal surface, wherein the continuous polarized monolayer of living primary mammary epithelial cells, the continuous polarized monolayer of a mixed population of living primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or the continuous polarized monolayer of living immortalized mammary epithelial cells are located on the upper surface of the scaffold.

[0131] In some embodiments, the lower surface of the scaffold is adjacent to the basal compartment. In some embodiments, the apical surface of the continuous polarized monolayer of viable primary mammary epithelial cells, the continuous polarized monolayer of a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, and / or the continuous polarized monolayer of viable immortalized mammary epithelial cells is adjacent to the apical compartment. In some embodiments, the continuous polarized monolayer of viable primary epithelial mammary cells, the continuous polarized monolayer of a mixed population of viable primary mammary epithelial cells, mammary myoepithelial cells, and mammary progenitor cells, or the continuous polarized monolayer of immortalized mammary epithelial cells secretes milk into the apical compartment through its apical surface, thereby producing milk in culture.

[0132] In some embodiments, a polarized monolayer of epithelial breast cells forms a barrier separating the apical and basal compartments, wherein the basal surfaces of the breast cells are attached to the scaffold and the apical surfaces are oriented toward the apical compartment.

[0133] In some embodiments, the base compartment is adjacent to the lower surface of the support. In some embodiments, the base compartment comprises a culture medium in fluid contact with the base surface of a polarized monolayer of mammary epithelial cells (e.g., a polarized monolayer of primary mammary epithelial cells, a polarized monolayer of a mixed population, or a polarized monolayer of living immortalized mammary epithelial cells).

[0134] In some embodiments, the culture medium comprises 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.

[0135] In some embodiments, the bioreactor comprises a top compartment adjacent to the top surface of the monolayer. In some embodiments, the top compartment is adjacent to the upper surface of the scaffold.

[0136] In some embodiments, the total cell density of breast cells in the bioreactor is at least 10 11 In some embodiments, the total cell density of the breast cells in the bioreactor is at least 10 12In some embodiments, the total cell density of the breast cells in the bioreactor is at least 10 13 breast cells.

[0137] In some embodiments, the total cell density of breast cells in the bioreactor is 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 20 to 55 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 20 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is 25 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 30 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 35 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 40 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 45 cells per 100 μm 2 In some embodiments, the total cell density of breast cells in the bioreactor is about 50 cells per 100 μm 2 About 55 cells.

[0138] In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 1.5 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 2 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 2.5 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 3 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 4 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 5 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 10 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 15 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 20 m 2In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 25 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 50 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 100 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 250 m 2 In some embodiments, the total surface area of ​​the breast cells within the bioreactor is at least about 500 m 2 .

[0139] In some embodiments, the bioreactor maintains a temperature of about 27°C to about 39°C (e.g., 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 about 39°C, or any value or range of temperatures therein, for example, about 27°C to about 38°C, about 36°C to about 39°C, about 36.5°C to about 39°C, about 36.5°C to about 37.5°C, or about 36.5°C to about 38°C). In some embodiments, the bioreactor maintains a temperature of about 37°C.

[0140] In some embodiments, the bioreactor has an atmospheric concentration of CO of about 4% to about 6%, for example, about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75% or 6%, or any value or range thereof, for example, 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, the bioreactor has an atmospheric concentration of CO of about 5%.

[0141] In some embodiments, the bioreactor has an atmospheric concentration of CO of about 4% to about 6%, for example, about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75% or 6%, or any value or range thereof, for example, 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, the bioreactor has an atmospheric concentration of CO of about 5%.

[0142] In some embodiments, the method includes monitoring the concentration of dissolved O2 and CO2. In some embodiments, the concentration of dissolved O2 is maintained between about 10% and about 25%, or any value or range therein (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 concentration of dissolved O2 is maintained between about 12% and about 25%, about 15% and about 22%, about 10% and about 20%, about 15%, about 20%, or about 22%. In some embodiments, the concentration of CO is maintained at between about 4% and about 6%, for example, about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75% or 6%, or any value or range of CO therein, for example, 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, the concentration of CO is maintained at about 5%.

[0143] In some embodiments, the culture medium is replaced from about every 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 thereof, for example, from about every day to every 3 days, from about every 3 days to every 10 days, from about every 2 days to every 5 days). In some embodiments, the culture medium is replaced from about every day or from about every few hours to about every 10 days, for example, from about every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours to about every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or any value or range thereof. For example, in some embodiments, the culture medium is changed from about every 12 hours to about every 10 days, from about every 10 hours to about every 5 days, or from about every 5 hours to about every 3 days.

[0144] In some embodiments, the method comprises monitoring the glucose concentration and / or glucose consumption rate in the culture medium and / or in the lactogenic medium. In some embodiments, prolactin is added when the glucose consumption rate in the culture medium is at steady state.

[0145] In some embodiments, the method further comprises applying transepithelial electrical resistance (TEER) to measure the maintenance of the epithelial cell monolayer. TEER measures the voltage difference between fluids (e.g., culture medium) in two compartments (e.g., between the apical compartment and the basal compartment), wherein the fluids in the two compartments can mix if the barrier between the compartments loses integrity. When there is fluid mixing, the voltage difference decreases or disappears; the voltage difference indicates that the barrier is intact. In some embodiments, when the voltage loss is detected by TEER, the scaffold (e.g., filters, microstructured bioreactors, decellularized tissues, hollow fiber bioreactors, etc.) are reseeded with additional cells and allowed time to re-establish the barrier (e.g., monolayer) before resuming production of the cultured dairy product (e.g., milk production).

[0146] In some embodiments, the method further comprises collecting the cultured dairy product from the top compartment to produce the collected cultured dairy product. In some embodiments, the cultured dairy product is collected via a port, via gravity, and / or via a vacuum. In some embodiments, a vacuum is attached to the port.

[0147] In some embodiments, the method further comprises freezing the collected cultured dairy product to produce a frozen cultured dairy product, and / or freeze-drying the collected cultured dairy product to produce a freeze-dried cultured dairy product.

[0148] In some embodiments, the method further comprises packaging the collected cultured dairy product, the frozen cultured dairy product, and / or the freeze-dried cultured dairy product into containers.

[0149] In some embodiments, method also comprises extracting one or more components from the culture dairy product of collection.Non-limiting examples from the component of the culture dairy product of collection include milk protein, lipid, carbohydrate, vitamin and / or mineral content.In some embodiments, the component from the culture dairy product of collection is freeze-dried and / or concentrated to produce freeze-dried or concentrated culture dairy component product.In some embodiments, by such as membrane filtration and / or reverse osmosis concentration from the component of the culture dairy product of collection.In some embodiments, freeze-dried or concentrated culture dairy component product is packaged in a container, optionally wherein container is aseptic and / or food grade container.In some embodiments, container is vacuum sealed.In some embodiments, container is jar, cylinder, bottle, bag, box or pouch (pouch).

[0150] cultured dairy products

[0151] In certain embodiments, cultured dairy products are disclosed herein. In some embodiments, the cultured dairy products are standardized, sterile cultured dairy products. In some embodiments, the cultured dairy products are used for nutritional purposes.

[0152] In some embodiments, the cultured dairy product is produced by any of the methods disclosed herein.

[0153] Breast milk contains low but measurable concentrations of environmental pollutants, hazardous chemicals from industry and manufactured products that are widely disseminated in the environment. Environmental pollutants are partially secreted in breast milk. The pollutant levels in breast milk reflect the pollutant levels in the mother's body and are therefore ideal for monitoring exposure levels. Toxic environmental pollutants can be passed from the mother to the baby through breastfeeding. Persistent organic pollutants (POPs) are a class of lipophilic stable chemicals that bioaccumulate in adipose tissue and produce a lasting toxic body burden. Breastfeeding is an important source of exposure to POPs in the early stages of human life, and its impact is unknown.

[0154] In some embodiments, the culture dairy product does not include or is substantially free of one or more environmental pollutants. In some embodiments, the culture dairy product does not include or is substantially free of persistent organic pollutants (POPs). In some embodiments, the culture dairy product does not include or is substantially free of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs) and pesticides such as DDT.

[0155] Heavy metals, such as mercury, lead, arsenic, cadmium, nickel, chromium, cobalt, zinc and other potential toxic metals dispersed throughout the environment, also have the known bioaccumulation characteristics accumulated in human milk, and are therefore worrying for nursing infants. The metals in breast milk derive from exogenous sources, i.e., are taken in via polluted air, food and drinking water, and are released endogenously together with essential trace elements. For example, lead and mercury spread equally in the human food chain, and their impact on fetal development is determined to a great extent by the mother's diet and nutritional status. Being exposed to toxic metals has a significant public health impact, and even at low concentrations and under acute exposure, these metals are still toxic to the human race. Nursing infants may be exposed to toxic metals during the most susceptible period. Nursing infants may be exposed to the heavy metals that exceed the amount they should contact through breast milk, and exposure may have a health impact on infants. Especially for infants, these exposures may have a negative impact on the developing central nervous system, leaving lifelong defects to their cognitive abilities.

[0156] In some embodiments, the cultured dairy product does not contain or is 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 does not contain or is substantially free of arsenic. In some embodiments, the cultured dairy product does not contain or is substantially free of lead. In some embodiments, the cultured dairy product does not contain or is substantially free of cadmium. In some embodiments, the cultured dairy product does not contain or is substantially free of nickel. In some embodiments, the cultured dairy product does not contain or is substantially free of mercury. In some embodiments, the cultured dairy product does not contain or is substantially free of chromium. In some embodiments, the cultured dairy product does not contain or is substantially free of cobalt. In some embodiments, the cultured dairy product does not contain or is substantially free of zinc. In some embodiments, the cultured dairy product does not contain or is substantially free of arsenic, lead, cadmium, nickel, mercury, chromium, cobalt, and zinc.

[0157] Foreign allergenic proteins can be difficult to distinguish from endogenous human milk proteins. Food proteins with allergenic potential that have been detected in human milk include egg and peanut proteins. In the United States, there are eight major food allergens, known as the Big 8, which are responsible for the majority of severe food allergic reactions. The Big 8 list includes milk allergens, egg allergens, fish allergens, crustacean shellfish allergens, tree nut allergens, peanut allergens, wheat allergens, and soy allergens. Proteins known to cause egg allergy include ovomucoids, ovalbumin, and conalbumin. Peanut proteins include arachin 6, arachin 3, conarachin, the major allergen Arah1, and arachin 2. As an example of maternal dietary protein transport into milk, it has been shown that consuming one egg daily results in higher concentrations of the egg allergen ovalbumin (OVA) in human milk compared to mothers who do not consume eggs.

[0158] In some embodiments, the cultured dairy product contains no or substantially no one or more food allergens. In some embodiments, the cultured dairy product contains no or substantially no egg allergens, fish allergens, crustacean shellfish allergens, tree nut allergens, peanut allergens, wheat allergens, and soy allergens. In some embodiments, the cultured dairy product contains no or substantially no 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.

[0159] In some embodiments, the cultured dairy product contains no or substantially no arachidin-6, arachidin-3, conarachidin, Arah1, and Arah2.

[0160] In some embodiments, the cultured dairy product contains no or substantially no ovalbumin (OVA).

[0161] Having described the present invention, 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 present invention. Example

[0162] Example 1:

[0163] The cell culture system that is designed for collecting milk should support the compartmentalized secretion of product so that milk is not exposed to the culture medium that provides nutrients to the cells. In vivo, the epithelial cells lining the inner surface of the mammary gland are continuous monolayer. Monolayer is oriented so that the base surface is attached to the basement membrane below, and milk is secreted from the top surface and stored in the luminal compartment (luminal compartment) of the gland or follicle until it is taken out during milking or feeding. Tight connection along the lateral surface of the cell ensures a barrier between the tissue below and the milk in the follicle compartment. Therefore, in vivo, the tissue of the mammary gland is arranged so that milk secretion is compartmentalized, and the mammary epithelial cells themselves set up an interface and maintain directional nutrient absorption and milk secretion.

[0164] The present disclosure describes a cell culture device that recapitulates the compartmentalization capability of the mammary gland for collecting milk from mammary epithelial cells grown outside the body. Such a device may include a scaffold to support the proliferation of mammary cells at the interface between the two compartments, such that the epithelial monolayer provides a physical boundary between the nutrient medium and the secreted milk. In addition to providing a surface for growth, the scaffold also provides spatial signals that guide the polarization of the cells and ensures directionality of absorption and secretion. The present disclosure describes the preparation, culture, and stimulation of mammary epithelial cells in a compartmentalized cell culture device for the production and collection of milk for nutritional purposes (see, e.g., Figure 1 ).

[0165] Preparation of mammary epithelial cells. Mammary epithelial cells are obtained from surgical explants, biopsy samples or raw breast milk of dissected mammary tissue (e.g., mammary gland, breast, nipple). Typically, after surgical dissection of mammary tissue, any fat or stromal tissue is manually removed under sterile conditions, and the remaining tissue of the mammary gland is enzymatically digested with collagenase and / or hyaluronidase prepared in a chemically defined nutrient medium, which should be composed of "generally recognized as safe" (GRAS) components. The sample is maintained at 37°C with gentle stirring. After digestion, a suspension of single cells or organoids is collected 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 an appropriate extracellular matrix component (e.g., collagen, laminin, fibronectin).

[0166] Alternatively, the explant sample can be processed into small pieces, for example by mincing with a sterile scalpel.The tissue pieces are plated onto a suitable surface, such as gelatin sponge or plastic tissue culture plates coated with an appropriate extracellular matrix.

[0167] The plated cells were maintained at 37° C. in a humidified incubator with a 5% CO 2 atmosphere. During the incubation period, the culture medium was changed approximately every 1 to 3 days, and the cells were subcultured until a sufficient number of viable cells was achieved for subsequent processing, which included preparation for storage in liquid nitrogen; development of immortalized cell lines by stable transfection of genes such as SV40, TERT, or other genes associated with senescence; isolation of mammary epithelial cells, myoepithelial cells, and stem / progenitor cell types by, for example, fluorescence-activated cell sorting; and / or introduction into compartmentalized tissue culture devices for production and collection of milk for human consumption.

[0168] For milk Production Culture of mammary epithelial cells. Milk for nutritional use is produced by mammary epithelial cells isolated as described above and cultured in a manner that supports compartmentalized secretion, thereby maintaining separation between nutrient medium and product. The system relies on the ability of mammary epithelial cells to establish a continuous monolayer with appropriate apical-basal polarity when seeded onto an appropriate scaffold positioned at the interface between the apical compartment, into which milk is secreted, and the nutrient medium provided by the basal compartment (see, e.g., Figure 2 For example, Filters, as well as bioreactors based on hollow fibers or microstructured scaffolds, are used to support these features.

[0169] After the separation and amplification of mammary epithelial cells, the cells are suspended in a chemically defined nutrient medium composed of food grade components and inoculated into a culture device pre-coated with a mixture of extracellular matrix proteins (such as collagen, laminin and / or fibronectin). A cell culture device is any design that allows compartmentalized absorption of nutrients and secretory products from polarized, confluent epithelial monolayers. Examples include hollow fiber and microstructured scaffold bioreactors (see, e.g., Figure 3 and Figure 4 Alternatives include other three-dimensional tissue culture approaches, such as preparing decellularized mammary glands as scaffolds that can be repopulated with stem cells to generate functional organs in vitro, or harvesting milk from the lumen of mammary epithelial cell organoids, or “mammospheres,” grown in a hydrogel matrix or suspension.

[0170] The device includes a sealed enclosure that maintains a temperature of approximately 37°C in a humidified atmosphere of approximately 5% CO2. As the cells proliferate within the bioreactor, glucose uptake is monitored to assess the growth of the culture. Stabilization of glucose consumption indicates that the cells have reached a confluent, contact-inhibited state. Transepithelial electrical resistance is used to ensure the integrity of the monolayer. Sensors monitor the concentrations of dissolved O2 and CO2 in the culture medium at multiple locations. A computerized pump circulates the culture medium through the bioreactor at a rate that balances the delivery of nutrients with the removal of metabolic waste products such as ammonia and lactate. After removing waste using lactate supplementation and adaptation techniques (Freund et al. 2018 Int J Mol Sci. 19(2)) or by passing through a chamber filled with zeolite, the culture medium can be recycled by the system.

[0171] Stimulation of milk production. In vivo and in cultured mammary epithelial cells, the production and secretion of milk are stimulated by prolactin. In culture, prolactin can be supplied from an exogenous source in a nutrient medium at a concentration close to those observed in vivo during lactation, for example, from about 20 ng / mL to about 200 ng / mL. Purified prolactin can be obtained commercially; however, alternative methods for providing prolactin or stimulating lactation are used, including expressing and purifying recombinant proteins from microorganisms or mammalian cell cultures. Alternatively, conditioned medium prepared by culturing cells expressing and secreting prolactin can be applied to mammary epithelial cell cultures to stimulate lactation. Bioreactors can be arranged in series so that the culture medium of the cells expressing prolactin or other key culture medium supplements is regulated before being exposed to the mammary cells grown in the compartmentalized culture device as described.

[0172] Other approaches to upregulating milk production and / or avoiding the use of exogenous prolactin include molecular manipulation of signaling pathways regulated by the binding of prolactin to its receptor on the surface of mammary epithelial cells, such as the following: (a) expression of constructs that target post-translational modification of prolactin; (b) expression of alternative isoforms of the prolactin receptor; (c) expression of chimeric prolactin receptors in which the extracellular domain is exchanged with the binding site for a different ligand; (d) introduction of genes encoding modified forms of the constitutively or conditionally active prolactin receptor or its downstream effectors, such as STAT5 or Akt; (e) knockout or modification of the PER2 circadian rhythm gene; and / or (f) molecular approaches aimed at increasing the rate of nutrient uptake at the basal surface of the mammary epithelial monolayer.

[0173] Collection of milk. Secreted milk is collected continuously or intermittently, for example, through a port installed in the top chamber of the culture device. A vacuum is applied to the port to facilitate collection and also help stimulate further production. The collected milk is packaged into sterile containers and sealed for distribution, frozen or freeze-dried for storage, or processed to extract specific components.

[0174] The present invention provides mammary epithelial cell cultures for producing milk for nutritional purposes. In addition to human breast milk, this method can also 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 in vitro, this technology may generate new business opportunities in addition to providing alternative production models for existing products. The social and economic effects of the commercial development of this technology are broad and far-reaching. Producing human breast milk from cultured cells can provide a means to address infant malnutrition in food-scarce communities, provide essential nutrients for premature infants who cannot be breastfed, and provide mothers with a new option for feeding their babies that provides optimal nutrition with the convenience of infant formula. The production of cow's or goat's milk provides an opportunity to reduce the impact of animal husbandry on the environment, society and animal welfare. The methods described here address an important gap in the emerging field of cellular agriculture and introduce an opportunity to significantly update the human food supply without compromising our biological and cultural attachment to our most basic sources of nutrition.

[0175] The foregoing examples illustrate the invention and should not be construed as limiting thereof.While the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the appended claims.

Claims

1. A method for producing an isolated cultured dairy product from mammary cells, the method comprising: (a) cultivating a living cell construct in a bioreactor under conditions to produce the cultured dairy product, the living cell construct comprising: (i) a three-dimensional scaffold having an outer surface, an inner surface defining an inner lumen / basal chamber, and more than one pore extending from the inner surface to the outer surface; (ii) a matrix material disposed on an outer surface of the three-dimensional scaffold; (iii) a culture medium disposed within the lumen / chamber and in fluid contact with the inner surface; and (iv) a confluent monolayer of polarized mammary cells disposed on a matrix material, wherein the mammary cells are selected from the group consisting of: viable primary mammary epithelial cells, viable mammary myoepithelial cells, viable mammary progenitor cells, and viable immortalized mammary epithelial cells, and wherein the polarized mammary cells comprise an apical surface and a basal surface, the cultured dairy product being secreted from the apical surface; and (b) isolating the cultured dairy product secreted from the apical surface of the mammary cells into the apical compartment.

2. The method of claim 1, wherein the mammary cells are selected from the group consisting of living immortalized mammary myoepithelial cells and living immortalized mammary progenitor cells.

3. The method of claim 1 or 2, wherein the basal surface of the breast cells is in fluid contact with the culture medium.

4. The method according to claim 1 or 2, wherein the bioreactor is a closed bioreactor.

5. The method of claim 1 or 2, wherein the bioreactor comprises an apical compartment in fluid contact with the apical surface of the breast cells, separated from the lumen / basal compartment of the living cell construct, and free of cell culture medium. The method of claim 5 , wherein the apical compartment is in fluid contact with the apical surface of the breast cells. The method of claim 6 , wherein the cultured dairy product is secreted from the apical surface of the mammary cells into the apical compartment. The method of claim 5 , wherein the culture medium does not contact the cultured dairy product.

9. The method of claim 1 or 2, wherein the total cell density of breast cells in the bioreactor is at least 10 11 .

10. The method of claim 1 or 2, wherein the total surface area of ​​the breast cells within the bioreactor is at least 1.5 m 2 .

11. The method of claim 1 or 2, wherein the culture medium comprises a carbon source, a chemical buffer system, one or more essential amino acids, one or more vitamins or cofactors, and one or more inorganic salts.

12. The method of claim 1 or 2, wherein the matrix material comprises one or more extracellular matrix proteins.

13. The method of claim 1 or 2, wherein the scaffold comprises a natural polymer, a biocompatible synthetic polymer, a synthetic peptide, or any combination thereof.

14. The method according to claim 13, wherein the natural polymer is collagen, chitosan, cellulose, agarose, alginate, gelatin, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate and / or hyaluronic acid.

15. The method of claim 13, wherein the biocompatible synthetic polymer is polysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate, polyvinyl alcohol, sodium polyacrylate, an acrylate polymer, and / or polyethylene glycol.

16. The method according to claim 1 or 2, wherein the culturing is performed at a temperature of 27°C to 39°C. The method according to claim 16 , wherein the culturing is performed at a temperature of 30° C. to 37° C.

18. The method according to claim 1 or 2, wherein the culturing is carried out at an atmospheric concentration of CO2 of 4% to 6%.

19. The method according to claim 18, wherein the culturing is carried out at an atmospheric concentration of 5% CO2.

20. A bioreactor, comprising: (a) an apical compartment comprising a cultured dairy product; and (b) at least one living cell construct comprising: (i) a three-dimensional scaffold having an outer surface, an inner surface defining an inner lumen / basal chamber, and more than one pore extending from the inner surface to the outer surface; (ii) a matrix material disposed on an outer surface of the three-dimensional scaffold; (iii) a culture medium disposed within the lumen / chamber and in fluid contact with the inner surface; and (iv) a confluent monolayer of polarized mammary cells disposed on a matrix material, wherein the mammary cells are selected from the group consisting of: viable primary mammary epithelial cells, viable mammary myoepithelial cells, viable mammary progenitor cells, and viable immortalized mammary epithelial cells, and wherein the polarized mammary cells comprise an apical surface and a basal surface, the cultured dairy product being secreted from the apical surface; wherein the apical surface of the mammary cells is in fluid contact with the apical compartment.

21. The bioreactor of claim 20, wherein the mammary cells are selected from the group consisting of living immortalized mammary myoepithelial cells and living immortalized mammary progenitor cells.

22. The bioreactor of claim 20 or 21, wherein the total cell density of breast cells within the bioreactor is at least 10 11 .

23. The bioreactor of claim 20 or 21, wherein the total surface area of ​​the breast cells within the bioreactor is at least 1.5 m 2 .

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