Pulsatile flow culture of mammary cell types for milk secretion

Abstract

Apparatus and methods for in vitro milk production include a reactor assembly having an elongated shell that encloses one or more hollow semi-permeable tubes within an extracellular nutrient medium. A monolayer of lactating cells is formed on the inner surface of the tube. A pump connected to the tube generates a pulsatile flow of the lactation medium to induce shear stress on the lactating cells. By varying the pump speed, the lactating cells are exposed to different shear stress levels and milk production is enhanced. The lactation medium is continuously recycled through the tube until a predetermined milk quality is achieved and then harvested. The lactating cells may be extracted from milk or mammary tissue and genetically engineered to produce milk with designer functions such as low allergenicity or low lactose.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 358,464, filed on July 5, 2022, and the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an apparatus and method for in vitro milk production, including milk with enhanced health effects.

Background Art

[0003] In response to various factors such as the global population increase and the Westernization of diets in Asian countries, the demand for raw milk production worldwide has been on the rise. The global consumption of dairy products is predicted to increase by over $100 billion in the next decade. As a result, it increases the burden on natural resources, raises concerns about animal welfare, and leads to deforestation and increased greenhouse gas emissions. Dairy consumption in the United States accounts for approximately 2% of the country's greenhouse gas emissions. To meet the global demand, approximately 270 million dairy cows are raised every day. All components contained in milk can have both beneficial and adverse effects on health.

[0004] Milk is a complex colloidal matrix containing various components including milk fat, lactose, and various naturally occurring hormones. The sensory and functional properties of milk are derived from its micronutrient profile, and it has been proven to be costly and difficult to reproduce. Generally, the gross composition of cow's milk in the United States is 87.7% water, 4.9% lactose (carbohydrates), 3.4% fat, 3.3% protein, and 0.7% minerals. The components of milk vary depending on the species (cow, goat, sheep), breed (Holstein, Jersey), feed, and lactation period.

[0005] There are research results indicating that the fat and protein content in milk vary depending on the breed of cows. The Holstein breed has the lowest fat and protein content, while the Jersey and Guernsey breeds have the highest fat and protein content. Even within the same group, the milk protein content ranges from 1.57% to 4.66%, with an average of 3.05%, and the milk fat ranges from 1.77% to 5.98%, with an average of 3.76%. Feed source, temperature, humidity, and seasonality all play important roles in the variation of milk quality in milk production. To address the variation in milk components, as a common method, milk obtained from different dairy cows is mixed in a bulk tank, and this enables the production of relatively consistent milk components throughout the year in the United States.

[0006] Another issue regarding milk quality is the presence of naturally occurring hormones in milk. These hormones include prolactin (15.4 ± 1 ng / mL), IGF-1 (4 ± 1 ng / mL), PGE2 (2.4 ± 0.3 ng / mL), PGF2α (2 ± 0.5 ng / mL), TXB2 (1 ± 0.5 ng / mL), corticosteroid (14 ± 4 ng / mL), testosterone (0.09 ± 0.03 ng / mL), 5α-steroid (3 ± 1 ng / mL), progesterone (12 ± 2 ng / mL), estrone (0.13 ng / mL), 17β-estradiol (0.02 ng / mL), estriol (0.027 ± 0.01 ng / mL). Additionally, hormones and other foreign substances such as antibiotics and pharmaceuticals are often contained in industrially produced milk due to the need to treat infections (i.e., chronic mastitis) in the entire herd during milk production.

[0007] There is little debate about the presence of these hormones at physiological concentrations, but the potential biological effects they have on animals and humans are not well understood and could be significant. Furthermore, the presence of hormones, particularly steroid hormones such as estrogen, can raise serious concerns about the safety of dairy products. Some evidence suggests a potential link to breast and prostate cancer. For this reason, special attention is required, particularly during important periods of development such as the perinatal period or puberty.

[0008] For this purpose, and in relation to significant advances in analytical methods and the development of bioassays, it is important to clarify the potential effects of the presence of hormones, particularly steroid hormones such as estrogen, when they are a common component of the diet and are regularly consumed over many years.

[0009] Dietary alternatives to dairy products include plant-based milks and milk products. Milks derived from plants such as almonds, soybeans, cashew nuts, and oats do not contain naturally occurring hormones found in animal milk and are becoming increasingly popular as a good source of protein without causing problems for many people who suffer from allergies to dairy products. However, plant-based milks obtained from these alternative sources tend not to be as effective in providing the functionality of animal milk that leads to the production of other dairy products such as cheese, butter, and yogurt. Furthermore, the growing popularity of plant-based milks has highlighted other sustainability concerns such as the extensive deforestation carried out for, for example, the expansion of soybean production, and the use of large amounts of water required for almond production in regions facing long-term drought.

[0010] Another area of interest in the dairy industry is an alternative dairy product called "A2 milk". Cow's milk generally contains two types of β-casein proteins, namely A1 type and A2 type, which differ in the amino acid histidine or proline at the 67th position of the protein structure. In this hypothesis, conventional cow's milk containing A1-type β-casein protein may contribute to adverse health effects such as indigestion, intestinal inflammation, and milk allergies, while A2-type β-casein is thought not to induce these effects.

[0011] Both A1-type β-casein and A2-type β-casein proteins contain 209 amino acids (AA). However, when digested, A1-type β-casein releases β-casomorphin-7 (BCM-7), which causes a series of phenomena that increase inflammation and gastrointestinal discomfort. Therefore, cow's milk containing A1-type β-casein is hypothesized to be associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed passage, and decreased cognitive processing speed and accuracy. Recent studies have confirmed that subjects who consumed cow's milk containing A1 / A2-type β-casein presented with more digestive symptoms related to lactose intolerance, while these symptoms did not worsen with the consumption of cow's milk containing A2-type β-casein. Inflammatory markers such as IL-4, IgG, IgE, IgG1, etc. were significantly lower in consumers of cow's milk containing A2-type β-casein. Since the two types of β-casein, A1 and A2, are very similar, and cows that produce milk with a high content of A1 protein tend to belong to breeds that produce a large amount of milk, such as Holstein cows (high-producing cows), there is currently no economic solution developed for the A1-type β-casein problem.

[0012] Milk and dairy products, including cream, butter, yogurt, and cheese, are important components of human diets and are important sources of protein, vitamins, and minerals. For many people, dairy products are the most convenient way to obtain essential nutrients for maintaining the health and proper functioning of the heart, muscles, and bones. However, the aforementioned problems regarding the quality of milk have plagued the dairy industry for many years and an urgent solution is sought.

[0013] The need to improve milk production technology is clear, and the background includes the current unsustainable approach to milk production by livestock (such as inefficient use of land and agricultural resources, generation of greenhouse gases, etc.), the lack of alternatives comparable to milk, and the limited sources of consistent quality and high-quality milk.

[0014] U.S. Patent No. 11,236,299 (assigned to Biomilk Ltd., Rehovot, Israel), which is incorporated herein by reference, discloses a method for producing milk in vitro using an array of mammary organoids seeded on a scaffold structure with a three-branched structure. In an exemplary embodiment, this milk production system includes an array of containers each containing a plurality of mammary organoids (MOs), a nutrient supply reservoir for supplying nutrients to each container, and a milk collection module. The main components of this system are the MOs, which are mammary epithelial cells that form a multicellular three-dimensional structure (mammary organoids) and are embedded in a matrix. When the MOs are seeded on the scaffold structure, estrogen and progesterone contribute to inducing epithelial growth and morphogenesis by inducing paracrine signaling between the mammary stroma and epithelium containing the seeded MOs. Subsequently, the MOs begin to secrete milk over a period of 10 - 21 days in a medium containing prolactin, nutrients, and growth factors. Wilk's patent discloses a method for producing milk in vitro, but does not provide a wide range of options for modifying dairy products from a cellular structure perspective to meet the increasing global demand in order to provide a more sustainable and nutritious food source that improves nutritional value, reduces hormone dependence, selects for the preferred A2 type β-casein, and produces other "designer milk" products such as low-allergy milk.

[0015] Given the increasing demand for dairy products, the present invention is directed towards a more sustainable and versatile approach to milk production for the stable supply of safe, clean, and high-quality milk while overcoming the environmental and animal welfare issues faced by the current dairy industry. SUMMARY OF THE INVENTION

[0016] According to an embodiment of the present invention, there is provided a system and method for in vitro milk production using mammary gland cells cultured to promote cell growth and the secretion of milk and milk components. The promotion of cell growth is achieved, at least in part, by mechanical stimulation through the application of a flow to the culture medium and changes in the flow by various different methods including, but not limited to, unidirectional laminar flow, turbulent flow, pulsatile flow, oscillatory flow, etc. The continuous mechanical stimulation provided by the system of the present invention enables the production of healthy epithelial cells (EC) and milk substantially free of hormones (other than prolactin). The systems and methods disclosed herein are generally applicable to the production of cell proteins and dairy products for food culture and bioprocessing applications.

[0017] In one aspect of the present invention, an apparatus for in vitro milk production includes an elongated shell configured to hold an extracellular medium, one or more hollow tubes having an inlet end, an outlet end, and an inner surface configured to attach a monolayer of lactating cells, wherein at least a portion of the hollow tube is formed of a semipermeable membrane material configured to allow diffusion of the extracellular medium into one or more tubes, the extracellular medium containing a nutrient solution for maintaining the lactating cells, one or more hollow tubes generally longitudinally aligned and disposed within the extracellular medium in the shell; a pump fluidly connected to the one or more tubes and configured to generate a pulsatile or oscillating flow of a lactation medium through the hollow tubes to induce shear stress on the lactating cells, wherein the pump is controlled to alternately switch between different shear stress levels within a predetermined range, and the change in shear stress stimulates the lactating cells to produce dairy products; a supply loop for supplying and circulating the extracellular medium through the shell; and a reservoir fluidly connected to the outlet end of the one or more tubes and configured to collect dairy products, including a reactor assembly. The apparatus may further include a feedback loop disposed near the outlet end of the one or more tubes for recirculating the dairy product to the inlet end to further concentrate the dairy product. In some embodiments, the predetermined range of shear stress levels is 2-75 dyn / cm 2 2. The supply loop can include a reservoir configured to remove used extracellular medium and add fresh extracellular medium.

[0018] In some embodiments, the lactating cells are co-cultured with feeder cells. The feeder cells may be peripheral blood mononuclear cells. In some embodiments, the lactating cells may be isolated from the milk of healthy cows. In other embodiments, the lactating cells are extracted from healthy mammary tissue identified using a series of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EPCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e. In some embodiments, the lactating cells may be extracted from healthy mammary tissue selected from whole mammary cells, whole mammary epithelial cells, intra-mammary cavity cells, intra-mammary cavity progenitor cells, mature intra-mammary cavity cells, mammary myoepithelial cells, and mammary stromal cells. The lactating cells may be genetically engineered to induce hyperlactation or to produce milk having any one or more of reduced allergenicity, reduced lactose, and increased A2-type β-casein protein. In some embodiments, each hollow tube may be coated with collagen. One or more tubes are formed from a material that is at least partially a semi-permeable capillary membrane. In some embodiments, on the inner surface of one or more tubes, there is first a layer of a support matrix to which the lactating cells attach on the non-attached surface of the support matrix, and the lactating cells form a monolayer of lactating cells on the support matrix.

[0019] In some embodiments further, the device may include a system controller configured to generate control signals for a pump and a supply loop.

[0020] An additional mechanical stimulation assembly may be included and may be capable of applying a pressing force in a direction from an inlet end to an outlet end on the outside of one or more tubes. Additionally, one or more light sources may be disposed within the reactor assembly to expose the lactating cells to light stimulation. In some embodiments, the one or more light sources may be light-emitting diodes (LEDs) that emit light at 450 nm.

[0021] The lactation medium may include one or more of EpiCult™ Plus medium, an artificial culture FCS replacement medium, and a FCS-free medium, and may further include prolactin.

[0022] In another aspect of the present invention, a milk production facility can be constructed by interconnecting the plurality of devices described above.

[0023] In yet another aspect of the present invention, the in vitro milk production method in the above-described device comprises supplying to one or more tubes, wherein the lactation medium contains prolactin; forming a monolayer of lactating cells adhered on the inner surface of the one or more tubes; and controlling a pump to alternately switch between different shear stress levels within a predetermined range, wherein the change in the shear stress level during the treatment period stimulates the lactating cells to produce dairy products. This method may further include recycling the dairy products to the one or more tubes via a feedback loop and concentrating the dairy products until a predetermined dairy quality is achieved. In some embodiments, the lactating cells may be co-cultured with feeder cells. The feeder cells may be peripheral blood mononuclear cells. The lactation medium may be one or more of EpiCult™ Plus medium, an artificial culture FCS replacement medium, and a FCS-free medium.

[0024] In some embodiments, the lactating cells may be isolated from the milk of healthy cows. In other embodiments, the lactating cells may be extracted from healthy mammary tissue identified using a set of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EPCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e. In other embodiments, the lactating cells may be extracted from healthy breast tissue selected from whole breast cells, whole breast epithelial cells, intra-mammary cavity cells, intra-mammary cavity progenitor cells, mature intra-mammary cavity cells, mammary myoepithelial cells, and mammary stromal cells.

[0025] The lactating cells may be genetically engineered to induce hyperlactation and / or to produce milk having one or more of reduced allergenicity, reduced lactose, and increased A2-type beta-casein protein.

[0026] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. These drawings are not intended to limit the scope of the present teachings in any way.

Brief Description of the Drawings

[0027]

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Modes for Carrying Out the Invention

[0028] To facilitate the understanding of the present invention, some terms and abbreviations used in this specification are defined as follows.

[0029] As used herein, the term "amino acid" refers to the molecular basis for constructing and assembling proteins such as enzymes. Peptide bonds (i.e., polypeptides) are formed between amino acids and assembled three-dimensionally (3D). This 3D assembly can affect the properties, functions, and structural dynamics of proteins. In biological systems, proteins can play the following roles. (i) Catalyze reactions as enzymes, (ii) Transport vesicles, molecules, and other substances within cells as transporters, (iii) Provide structure to cells and organisms as protein filaments, (iv) Replicate deoxyribonucleic acid (DNA), and (v) Regulate the actions of cells as cell signaling factors.

[0030] As used herein, the term "nucleotide" refers to the molecular basis for constructing and assembling nucleic acids such as DNA and ribonucleic acid (RNA). There are two types of nucleotides: purines and pyrimidines. Specifically, the purines are adenine (A) and guanine (G). Specifically, the pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA, and U is found in RNA. The genetic code defines the sequence of nucleotide triplets (i.e., codons) that specify which amino acids are added during protein synthesis.

[0031] As used herein, the term "gene" refers to a region of DNA. The amino acid sequence within a protein defined by the gene sequence is encoded within the genetic code.

[0032] As used herein, a recombinant nucleic acid or recombinant protein is a nucleic acid or protein produced by recombinant DNA technology, as described, for example, in Green and Sambrook (2012).

[0033] As used herein, the terms "polypeptide", "protein", and "peptide" are used interchangeably to refer to a chain of amino acid residues joined by peptide bonds or modified peptide bonds. The amino acid chain can be of any length greater than two amino acids. Unless otherwise specified, the terms "polypeptide", "protein", and "peptide" include their various modified forms. These modified forms can be naturally occurring modifications or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, etc. Modifications include intramolecular cross-linking and covalent attachment of various moieties such as lipids, flavins, biotin, polyethylene glycol or its derivatives. Furthermore, modifications can include cyclization of proteins, branching of amino acid chains, and cross-linking of proteins. Additionally, a polypeptide may contain amino acids other than the usual 20 amino acids encoded by genes. The term "protein" or "polypeptide" may also include a "purified" polypeptide substantially separated from other polypeptides within a cell or organism in which the polypeptide naturally occurs (e.g., free of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% contaminants).

[0034] As used herein, the term "bioreactor" refers to a vessel or tank in which whole cells or cell-free enzymes convert raw materials into biochemical products and / or less desirable by-products. The bioreactor is designed and operated to provide an environment for producing a product, here a dairy product. Industrial bioreactors can operate as batch reactors or in continuous operation, under aerobic or anaerobic conditions, and in pure or mixed cultures. In some bioreactors, three phases (gas phase, liquid phase, and solid phase) are present, and mass transfer can be an important consideration. In a fluidized bioreactor, microbial biomass can be retained using biofilms or immobilized cells. Industrial bioreactors are indispensable with sensors, measuring instruments, and control systems.

[0035] As used herein, the term "shear stress" is usually represented by the Greek letter τ (tau) and refers to the component of stress that is in the same plane as the cross-section of the material. (See, for example, FIG. 4.) This results from the shear force, which is the component of the force vector parallel to the cross-section of the material. When a real fluid (including liquids and gases) moves along a solid boundary, shear stress is generated at that boundary. Due to the no-slip condition, the velocity of the fluid at the boundary (relative velocity with respect to the boundary) is required to be zero, but at a certain height from the boundary, the flow velocity becomes equal to the velocity of the fluid itself. The region between these two points is called the boundary layer. In a Newtonian fluid in laminar flow, the shear stress is proportional to the strain rate in the fluid, and the proportionality constant is the viscosity. In the case of a non-Newtonian fluid, the viscosity is not constant. The shear stress is imparted to the interface as a result of this velocity loss.

[0036] As used herein, the term "shear strain" refers to the ratio of the displacement of an object from its original dimensions due to stress, and is the amount of deformation in a direction perpendicular rather than parallel to a given line. Shear strain is a lateral force applied to a medium and is measured as the change in the angle between lines that were originally perpendicular. (See, for example, FIG. 4.)

[0037] As used herein, the term "pulsatile flow" refers to a flow accompanied by periodic pressure fluctuation waves that move along a flow path. A pulsatile flow system can mimic the blood flow characteristics within the heart and vascular system.

[0038] As used herein, the term "effective amount" refers to an amount that, when administered to a particular subject taking into account the nature and severity of the subject's condition, results in a desired biological effect, e.g., an amount having the effect of curing, preventing, suppressing, or at least partially halting or partially preventing a targeted reaction.

[0039] Throughout the present disclosure, several abbreviations and shorthand notations are used. Table 1 below provides a glossary summarizing several frequently used abbreviations and shorthand notations. [Table 1]

[0040] The terms "hollow tube", "hollow conduit", "hollow fiber", and "lumen" may be used interchangeably throughout this specification to refer to an elongated hollow structure through which fluid flows while being pressurized by a pumping system. The hollow structure can be made of various materials, which can be rigid or flexible and can have permeability, semi-permeability, or non-permeability. Examples of materials include, but are not limited to, glass, acrylic, plastic, polymer, fiber, silicone, ceramic, and filter membranes. In some embodiments, the hollow structure can be compressible or contractible, and for example, by applying rollers opposing the outer surface of a tube to "squeeze out" the fluid inside the tube from a first end to a second end, an operation similar to a peristaltic motion can be mimicked. (See, e.g., FIG. 1B). In other embodiments, the tube is a hybrid assembly of sections made of different materials, some of these materials being permeable or semi-permeable membranes while other materials can be non-permeable.

[0041] Lactation Biology Lactation is the process by which milk is synthesized and secreted from the mammary glands. The mammary glands are modified sweat glands, mainly composed of adipose tissue and collagenous tissue, and the proportion of the mammary glands in the breast volume is very small. The mammary glands are composed of milk ducts that transport milk, and they expand and branch greatly in response to estrogen, growth hormone, cortisol, and prolactin. Furthermore, in response to progesterone, aggregates of mammary alveoli sprout from the milk ducts and expand outward toward the chest wall. The mammary gland lobules are balloon-like structures lined with cuboidal cells that secrete milk, i.e., milk cells, and are surrounded by a network of contractile myoepithelial cells. Milk is secreted from the milk cells, fills the alveoli, and is squeezed into the milk ducts. An aggregate of alveoli that drains into a common milk duct is called a lobule, and in lactating women, 12 to 20 lobules are radially organized around the nipple. Milk is drained from the milk ducts into the lactiferous sinuses, which communicate with openings called 4 to 18 lactiferous ducts in the nipple.

[0042] Prolactin, a pituitary hormone, plays an important role in the establishment and maintenance of breast milk supply. Hormones including prolactin anatomically prepare the breasts to promote milk secretion. When an infant sucks milk, sensory nerve fibers in the areola trigger a neuroendocrine reflex, as a result of which milk is secreted from the milk cells into the alveoli. The posterior pituitary releases oxytocin, which stimulates the myoepithelial cells to squeeze milk out of the alveoli, into the milk ducts, where it collects in the lactiferous sinuses and is discharged through the lactiferous ducts. The time (latency period) from when the infant begins sucking until milk is secreted (ejection) is less than 1 minute. Milk synthesis via prolactin changes over time. Frequent removal of milk by breastfeeding (or milk pumping) maintains high circulating prolactin levels for several months. However, even if breastfeeding continues, basal prolactin declines to pre-pregnancy levels over time. Furthermore, in addition to prolactin and oxytocin, growth hormone, cortisol, parathyroid hormone, and insulin also contribute to lactation by transporting maternal amino acids, fatty acids, glucose, and calcium into breast milk.

[0043] Pulsatile Flow Culture of Mammary Cell Types for Milk Secretion The system of the present invention is designed to mimic the natural environment of mammary alveoli and mammary ducts for healthy epithelial cells (EC) and optimal milk secretion by inducing variable shear stress in cells.

[0044] Referring to FIGS. 1A - 1E, embodiments of the bioreactor of the present invention are designed to reproduce the native environment of mammary alveoli and milk ducts for healthy epithelial cells (EC) and optimal milk secretion. FIG. 1A provides a schematic side view (left) and cross - sectional view (right) showing the basic configuration of an exemplary bioreactor flow system 100 configured to achieve shear stress adapted within a sealed sterile system. The incubator 102 includes a fluid - tight housing or shell 120, within which are housed one or more semi - permeable hollow tubes 104, a pump system 106, a valve 108 for controlling the flow of extracellular medium fluid, storage tanks 110 and 112, and a feedback loop 118 with a valve 116 for recirculating partially processed fluid (accumulation medium) through the tubes of the reactor for further processing / concentration. The shell 120 may be formed of polycarbonate or similar polymers, glass, stainless steel, or a material considered suitable for food handling, i.e., a sterilizable material. Fluid - like extracellular medium (ECM) is introduced and circulated through the bioreactor via storage tank 110, valve 108, and inlet / outlet tubes 128 and 130. (The pump controlling this ECM loop is not shown). The flow through tube 104 is configured to impart mechanical shear stress to the cells within the tube as the lactation medium / accumulation medium flows under pressure from the pump. The system controller 122 sends control signals to the pump 106 and executes various operations such as system discharge, e.g., application of pulsatile flow and / or continuous flow through tube 104 and milk collection. The system controller 122 may further be configured to generate control signals for valve 108, 116, operation of the ECM loop, and any additional valves and other operations within the system. In some embodiments, the system controller 122 may include a combination (s) of one or more computer processors and memory for holding and executing programs for automated operation of the system. In some embodiments, parameters for optimized operation of the system may be established using inputs including system sensor signals (flow rate, pressure, temperature, etc.) and quality control measurements obtained from tests of milk products (s) through the use of a learning machine within the system controller.

[0045] In some embodiments, pump 106 is controlled to produce continuous flow, unidirectional flow, oscillatory flow, and pulsatile flow at a flow rate adaptable to expose cells in the tube to varying shear stress. Generally, the range of shear stress generated by pump 106 is selected from about 2 dyn / cm 2 (considered "low") to 75 dyn / cm 2 (considered "high") and switched alternately between different settings. The pump sequence generated by controller 122 can induce shear stress to alternate between different levels, such as from low (in the range of about 2 - 10 dyn / cm 2 up to) to medium (in the range of about 11 - 40 dyn / cm 2 up to), high (in the range of about 41 - 75 dyn / cm 2 up to), from high to medium, from high to low, or from high to low and back. Such variations in shear stress via pulsatile / oscillatory flow can help promote lactation. As the lactation medium flows around the cells, milk components accumulate and become the "accumulated medium", which is processed through the hollow tube 104 and collected in the downstream storage tank 112. One or more access ports (not shown) may be provided near or at the outlet 162 to enable sampling to determine whether the accumulated medium has achieved the desired quality, such as density, appearance, viscosity, fat content, chemical composition content, etc., suitable for the finished dairy product. If the product is determined to be complete through inspection, milk can be collected. Otherwise, the accumulated medium can be returned to the reactor inlet 160 via valve 116 and feedback loop 118 for further concentration. Alternatively, if the optimal processing time has been determined under certain operating conditions, it may not be necessary to perform inspections regularly.

[0046] Figure 1B graphically shows additional details regarding the structure and function of the bioreactor of the present invention, depicting a cross-section of an exemplary hollow tube 104. In some embodiments, these hollow tubes may be one of a plurality of elongated tubes or lumens arranged in parallel and extending longitudinally within a generally cylindrical outer shell 120, collectively defining a sterile bioreactor cartridge 102. The interior of the tube 104 is referred to as the capillary inner space or InC space 134, and the space outside the tube (within the shell 120) is called the capillary outer space or ExC space 132. In some embodiments, the inner surface of the tube 104 may be further lined with a collagen membrane (see, for example, FIG. 1C). Feeder (indicator) cells 138 and mEP cells 140 are coated on the inner surface of the hollow tube 104. For convenience, note that the primary cilia 142, which play an important role in milk secretion, are shown only for a few cells 140. As will be recognized by those skilled in the art, all healthy ECs should have primary cilia. The mechanical sensing resulting from the adaptable flow within the tube 104 helps to differentiate the cells and maintain their health. The tube 104 is formed from a semi-permeable membrane material such as a polymer or similar material and can supply nutrients to the cells by diffusing the nutrients contained within the ECM. The inlet (downstream end) of the tube 104 is connected to a pump system 106. In a small-scale prototype embodiment of the bioreactor of the present invention, an ibidi (trademark) peristaltic pump (ibidi GmbH, Graefelfing, DE) was used to provide the mechanical stimulation required via pulsatile flow for milk secretion. In larger-scale systems, one or more larger-capacity pumps are used to achieve the desired pulsatile flow. Milk can be collected daily in batch units from a re-perfusion reservoir 112 located at the downstream end of the reactor. Optionally, a filter 114 may be placed upstream or downstream of the reservoir 112 to remove accumulated dead cells and other particulates present in the milk before collection.

[0047] The hollow tube 104 is described herein as "semipermeable", but its structure is not limited to semipermeable materials. Rather, the only requirement is that a portion of the tube has sufficient permeability such that extracellular medium can flow into the InC and supply nutrients to the lactating cells. Thus, the hollow tube 104 can be a hybrid assembly consisting of multiple sections, and is considered "semipermeable" even if some parts are permeable or semipermeable and other parts are non-permeable. For example, a ceramic filter membrane for supplying ECM to cells can be used in a section of the tube, and the remaining part of the tube can be made low-permeable or non-permeable. This hybrid approach allows for custom control of the amount of ECM available to the cells and avoids the supply of excessive ECM that could potentially damage the final product.

[0048] The embodiment shown in FIG. 1B includes an optional implementation that provides additional mechanical stimulation, provided in the form of an assembly of opposing cylindrical rollers 150 that are disposed within the shell 120 and travel along a track 152 so as to be positioned parallel to the tube. These rollers slightly compress the tube from multiple directions and gently "squeeze out" the accumulated medium within the tube, i.e., the milk secretion medium and the milk components 144, from near the inlet end of the reactor towards the outlet end. The pressure applied by the rollers should be uniform and strong enough to move the fluid, yet gentle enough not to damage the cells or cause them to peel off from the inner surface of the tube. By varying the speed of the rollers, additional means are provided for applying adjustable shear stress. Once the rollers have completed their movement along the track, the rollers are released to return to the track to relieve the pressure on the tube and move back to the starting position near the inlet end to repeat the extrusion sequence. Although two opposing rollers are shown, it will also be readily apparent that different combinations of rails and rollers, or an annular sphincter device, can be used to achieve the desired extrusion motion from the inlet end to the outlet end that helps to release the cells and continue milk production. The signals for controlling this optional mechanical stimulation assembly are provided by the system controller 122.

[0049] Figure 1C provides a cross-sectional view according to a diagram showing a monolayer of lactating cells attached to the inner surface of each hollow fiber 104 within the fiber bundle of the reactor cartridge 102. In the illustrated embodiment, the bioreactor comprises a plurality of hollow fiber elements 104 and is a recirculation reactor improved to be supplied by a peristaltic pump (not shown) and generate continuous, unidirectional, oscillatory, and pulsatile flows at adjustable flow rates. The reactor cartridge 102 may include a multi-tubular bioreactor with inlets and outlets for fluid to flow into and out of the intercapillary space and the extracapillary space. As shown, the InC space inlet 160 supplies the hollow tube 104, and then milk (i.e., the storage medium) flows out through the InC space outlet 162. The extracellular medium (ECM) is supplied to the ExC space (from the storage tank 110) through the inlet 128 and then circulated back to the storage tank 110 through the outlet 130. The inlet 160 of the hollow fiber is connected to a pump for flow stimulation via pulsatile flow. In the upper left panel of the figure, the cross-sections of five hollow fibers 104 are shown, and the inner surface of the outer fiber wall is lined or coated with a collagen membrane 156. Cells 140 (and feeder (support) cells) grow within the lumen of the hollow fiber. The collagen membrane supports a monolayer of cells and allows milk to flow through the cell layer and through the space (lumen). In the upper right panel, a longitudinal section of a single fiber is shown, also showing the "lining" of the collagen membrane and the monolayer of cells 140 through which milk 170 flows through the lumen. In this example, the inner diameter of the fiber is shown to be 2 mm, but as will be apparent to those skilled in the art, the inner diameter of the fiber can be selected to support appropriate flow rate parameters to generate the shear force required for mechanical stimulation of the cells on the inner surface of the tube.

[0050] In the above-described embodiments of the bioreactor of the present invention, a peristaltic pump is used for the purpose of inducing shear stress on lactating cells using a continuous fluid flow within a sterile reactor environment. As will be recognized by those skilled in the art, alternative approaches for inducing shear stress controlled by a continuous or fluctuating fluid flow within a sterile environment include the use of rotational vortices, magnetic vortices, such as stirring bars, and wave pumps or valve pumps. In such approaches, the vortices and / or pumps would be placed within a sterile sealed container, which could, compared to the above-described embodiments, have an adverse effect on the scalability of the process.

[0051] As will be recognized by those skilled in the art, the number of hollow tubes within a bioreactor assembly can vary, and the example shown is not intended to be limiting. One example of a modified tube configuration is shown in FIG. 1E, which is an example where a single long tube 304 is arranged in a zigzag, loop, coiled, or serpentine pattern within the shell 320. The general flow direction is parallel to the length of the shell, i.e., from the inlet to the outlet, and can be recirculated through a feedback loop similar to that found in other embodiments. In this example, the tube 304 is a hybrid assembly formed from a plurality of tube sections of different materials, the tube section 344 is formed of a permeable or semi-permeable material, and ECM can only be perfused into the interior of the tube through these sections. The tube section 346 is non-permeable. The pump 306 controls the pulsatile flow through the tube from the inlet to the outlet. It should be noted that this example is illustrative only and not intended to be limiting.

[0052] The embodiments of the bioreactor shown in FIGS. 1A - 1C and described above are illustrated in a horizontal orientation, but it is not necessary to adopt a specific orientation. Referring to FIGS. 2A - 2C, some embodiments of the bioreactor adopt a vertical orientation, which can expand the scale of the processing device and enhance the ability to produce a larger amount of milk. Each cartridge 202 includes the basic configuration described above, i.e., a shell 220 surrounding one or more hollow tube reactors 204. A pump 206 supplies a fluid such as PBS to an inlet 260 at the top of the cartridge 202, from which the fluid is supplied into the tube 204. The hollow tube 204 is coated on the inner surface of a single layer of cells, specifically EC, as shown in FIG. 2C. Similar to the previous embodiments, the pump 206 is configured to flow the fluid through the tube and induce an adjustable shear stress to stimulate milk production by the cells. The vertical orientation of the cartridge 202 provides gravity assistance to the flow through the tube. The pump 206 can recirculate the partially processed milk (accumulation medium) through a feedback loop 218 until the desired milk characteristics are achieved. Although not shown, one or more test ports / valves may be provided near the outlet 262 to enable testing to determine the degree of completion of the processed milk. A filter 214 may be disposed at the outlet 262 to remove waste. A valve 264 can transfer the finished milk product to a storage tank for storage. The right panel of FIG. 2A provides a schematic cross - sectional view of the cartridge 202 having a hollow tube 204 that defines an ExC (outside the tube) space and an InC (inside the tube) space as described above. ECM is supplied to the ExC space within the shell 220 through an inlet 228 and removed outside the shell at an outlet 230. A valve 208 and one or more pumps (not shown) control the flow of ECM. Additional details of the tube 204 are shown in FIG. 2C, and the lower panel provides a 3D perspective view of the tube.

[0053] In some embodiments of the bioreactor of the present invention, optionally, a light source 216, specifically, a light-emitting diode (LED) may be included to produce different effects. In one embodiment, an LED that emits light in the range of blue light, for example, light of about 450 nm, has been shown to stimulate primary cilia. In a study by Prosseda et al., blue light was used to stimulate the regulation of primary cilia in cells and increase cell contraction. (「Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation」, Science Advances, 29 Apr. 2020, Vol. 6, Issue 18, DOI: 10.1126 / sciadv.aay8699, incorporated herein by reference.) Since it is necessary to stimulate the primary cilia of ECs to produce milk, by adding light stimulation to the mechanical stimulation provided by adjustable shear stress, the overall stimulation required to secrete milk is increased. The light stimulation may further provide additional variable stimulation to the cells using the intensity of the pulsation. Since light is used to stimulate ECs, the material forming the tube 204 must be able to transmit the desired wavelength. For example, glass or a transparent portion of the tube may be used at a position close to the LED. Although the LED is shown at the bottom of the cartridge, it will be readily apparent to those skilled in the art that it may be disposed at multiple positions throughout the interior of the shell 220 to increase the exposure of ECs to light.

[0054] In other embodiments, other light sources may be included in the piping inside or outside the cartridge. For example, an ultraviolet light source (100 - 280 nm) can be used to enhance the sterility of the product, but care must be taken not to damage the cells. Other light sources may be used to promote the health of ECs. For example, light sources that emit light at wavelengths including 660 nm, 700 nm, 810 nm, and 850 nm have been reported to promote cell activity and growth. A control signal for the operation of the light stimulation system may be provided by the system controller.

[0055] Figure 2D graphically shows possible arrangements of multiple groups of bioreactors within a production facility. Such multiple groups, each with any number of units, i.e., cartridges and associated piping, can be attached to a support frame (not shown) within a facility that can preferably include a cleanroom. In one possible embodiment, each group may have its own dedicated storage tank 210 for supplying extracellular medium to the bioreactor units within the group and a milk collection storage tank 212 for collecting milk produced by the units within the group. In other embodiments, multiple groups may receive medium from one or more large central storage tanks and discharge milk into one or more large central tanks. Various combinations of central storage tanks and dedicated storage tanks may be used. By way of example, in a large-scale production facility, one or more groups of bioreactor units can be used to produce whole milk, low-fat milk, low-allergenic milk, lactose-free milk, or various "designer" dairy products as described below.

[0056] The mammary epithelium is composed of two types of differentiated cell types that are organized into two cell layers, an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells that are in direct contact with the basement membrane. Mammary epithelial cells (MECs) can grow as a monolayer on plastic. However, actual milk secretion occurs only when signals are obtained from ECM proteins and hormones (prolactin, growth factors), i.e., structures similar to those observed in vivo, and tissue-specific gene expression (e.g., the casein gene). In some embodiments, as shown in one of the examples, MECs can be harvested from fresh milk.

[0057] By using the disclosure of this specification, those skilled in the art will be able to implement a similar strategy for extracting MECs from fresh milk or mammary glands using other mammary epithelial cell biomarkers. Table 2 below provides a list of biomarkers in breast tissue (see Figure 3) that may be useful when extracting MECs for use in the methods of the present invention. Exemplary selection panels may include combinations of these biomarkers. For example, a healthy breast cell panel may be assembled by combining biomarkers selected from those listed in the bottom 7 rows of the table, from one or a combination of sources, such as all, all epithelial, luminal, etc.

Table 2

[0058] Pulsatile flow: Pulsatile flow refers to a periodic pressure fluctuation wave that moves along a flow path, such as that generated within the hollow tube(s) of a bioreactor. A pulsatile flow system can very accurately mimic the blood flow characteristics within the heart and vascular system.

[0059] Mammary endothelial cells (MECs) adapt their morphology and function to the hemodynamic environment of the living body in which they exist. In in vitro experiments, it has also been shown that similar changes occur in cultured MECs exposed to shear stress induced by steady flow due to laminar flow. However, MECs in vivo are exposed to a pulsatile flow environment, and thus, in this study, the effect of pulsatile flow on cell shape and orientation, and on the localization of actin microfilaments in confluent bovine aortic endothelial cells (BAECs) was investigated. These results indicate that ECs can distinguish between different types of pulsatile flow environments. Furthermore, these experiments demonstrate the importance of designing cell culture environments to include pulsatile flow in the study of endothelial cell biology.

[0060] Removal of FCS from the lactation medium: Currently, standard culture media rely on an established but outdated concept that fetal calf serum (FCS), which has functioned as the "gold standard" since the 1960s, is required. For many years, the problems with FCS and the need to replace it with better alternatives have been a major area of research. The problems with FCS include the following facts: namely, (a) its production is a cruel act, and (b) FCS has been reported to be contaminated with viruses, and there are also safety concerns related to endotoxins, mycoplasmas, RNA contaminants, or prion proteins, so it cannot be used in the production of clean dairy products for human consumption. Therefore, the medium used for culturing cells in the bioreactor of the present invention is preferably based on the latest scientific research and cell culture techniques and does not contain FCS and hormones (except prolactin).

[0061] Generally, serum starvation can potentially damage cell health, but its adverse effects can be offset by flow regulation. Changes in the intensity of the flow can potentially induce cilia growth. Therefore, in order to achieve milk secretion, it is necessary to use the flow to stimulate the cilia.

[0062] Mechanical stimulation (flow) to healthy EC cells to counteract the removal of FCS from the lactation medium: Both hormonal and mechanical signals can induce changes in gene expression in mammary epithelial cells (MEC), which ultimately leads to milk synthesis and secretion. By using a serum-free medium and combining hormonal stimulation and mechanical stimulation, bovine mammary epithelial cells (BMEC) were transformed from primary multicellular organoids into a coordinated three-dimensional tubular network. For example, Figures 5A - 5B show mammary epithelial cells with an epithelial-like phenotype and mammary myoepithelial cells with a spindle-shaped phenotype, respectively. At the structural level, there is evidence that mechanical signaling via gel release, prolactin, and combinations of the two caused changes in cell morphology consistent with the onset of the first stage of milk secretion.

[0063] For endothelial cells to grow healthily, mechanical stimuli are required. According to research, a certain type of flow has been shown to impart shear mechanical stimuli to EC cells, thereby improving cell performance. In some embodiments, continuous mechanical stimuli are applied to healthy ECs, increasing the hormone-free milk secretion process. Understanding the nature of the stimuli applied to the cells is important, and the principle is shown in Figure 4. Mechanical strain occurs when a force acts directly on the cells. When the strain is large, the endothelial barrier may loosen, leading to cell death and high cell turnover. Epigenetic modifications and inflammation may occur. On the other hand, mechanical shear is involved in the parallel movement of the fluid over the cells and improves cell viability with low turnover. Therefore, the goal is to stimulate cell function by increasing mechanical shear while avoiding mechanical strain. The lower panel of Figure 4 shows a comparison of the cell layers before (right) and after (left) the application of shear stress.

[0064] In some embodiments, the lactation medium and test cultures are fluidically driven by a peristaltic pump, such as an ibidi (trademark) pump system used within a small-scale prototype bioreactor, or other pumps capable of generating a pulsatile flow that provides adjustable shear stress within a closed sterile system. In some embodiments, the mechanical shear stress is evaluated and continuously monitored.

[0065] Feeder cells: Feeder cells or support cells 138 are a cell layer that cannot divide but provides extracellular secretions to promote the growth of other cells, namely lactating cells 140. Feeder cells are different from co-culture systems because only one type of cell can proliferate.

[0066] In some embodiments, the feeder cells are co-cultured with MECs to form a monolayer of lactating cells attached to the lumen of an elongated hollow fiber bioreactor. In some embodiments, the feeder cells may be seeded in the extra-capillary (ExC) space surrounding the hollow fibers.

[0067] Hollow Fiber Bioreactor: A hollow fiber bioreactor is a three-dimensional cell culture system based on hollow fibers. In some embodiments, the hollow fibers are small semi-permeable capillary membranes arranged in an array with a typical molecular weight cut-off (MWCO) range of 10-30 kDa. Exemplary embodiments are described in connection with FIGS. 1A-1C and FIGS. 2A-2D. When using multiple tubes, these tubes can be bundled and housed within a cylindrical shell of polycarbonate, glass, stainless steel, or other suitable material (i.e., a sterilizable material) to form a hollow fiber bioreactor cartridge. The cartridge is equipped with an inlet port and an outlet port, and there are two compartments: the intrafiber (InC) space 134 within the hollow fibers and the extrafiber (ExC) space 132 surrounding the hollow fibers.

[0068] Cells are seeded in the InC space 134 (FIG. 1B) of the hollow fiber bioreactor 104 and expand therein to cover the inner surface of the hollow fibers 104 in a monolayer. The cell culture medium is pumped through the ExC space 132 to supply oxygen and nutrients to the cells via perfusion of the hollow fiber membrane. As the cells expand, cellular waste products and CO2 also perfuse through the hollow fiber membrane and are removed by the pumping of the spent culture medium through the InC space 134. As waste products accumulate with an increase in cell mass within the InC, it may be necessary to increase the flow rate of the medium so that cell growth is not inhibited by the toxicity of the waste products. The waste products can be filtered by a filter placed within or near a storage tank after the spent culture medium has been completely concentrated, i.e., after the production of dairy products has been completed.

[0069] Supporting extracellular matrix: Mammary epithelium may contain two differentiated cell types organized into two cell layers, an inner layer of luminal epithelium in direct contact with the basement membrane and an outer layer of myoepithelial cells. Mammary epithelial cells can grow as a monolayer on plastic. However, milk secretion occurs only in response to signals from ECM proteins and hormones (prolactin, growth factors), as well as structures similar to those observed in vivo and tissue-specific gene expression (e.g., the casein gene). Therefore, to mimic natural milk secretion, a bioreactor must reproduce these structures and the same (or similar) stimuli that occur in the in vivo environment. In some embodiments, functional differentiation can be achieved by adjusting the composition of the supporting extracellular matrix, the culture medium, and co-culturing the necessary cell types.

[0070] Other successful approaches may also stimulate milk component genes. In some embodiments, cells have been reconstituted to form 3D structures as observed in vivo and shown to express milk protein genes on collagen gels. In some embodiments, epithelial cells grown in laminin-rich gels have achieved functional differentiation based on the expression of the casein gene. Natural and synthetic polymeric materials are being investigated as alternatives to ECM proteins.

[0071] In some embodiments, 3D bioprinting of mammary tissue may be used. In some embodiments, 3D culture techniques containing mammary stimulating hormones (prolactin, insulin, and hydrocortisone) can induce casein synthesis from primary mouse-derived epithelial cells embedded in floating collagen gels. In some embodiments, a differentiation medium supplemented with oleic acid, pituitary extract, and dexamethasone has been shown to induce the synthesis of major milk components such as β-casein, triglycerides, and lactose in a 3D in vivo model of primary bovine mammary epithelial cells grown on collagen-coated cell culture inserts.

[0072] The morphological and functional differentiation of cryopreserved lactating bovine mammary cells can be cultured on floating collagen gels. In some embodiments, bovine primary epithelial cells derived from lactating mammary tissue grown on floating collagen gels have shown a polarized structure with a high degree of differentiation, as observed by apical microvilli, tight junctions, and lipid droplets surrounded by secretory vesicles containing casein.

[0073] By using collagen gels, it is possible to mimic in vivo conditions, which has the advantage of enabling relatively long-term studies.

[0074] The milk produced by the bioreactor of the present invention has the ability to rival the nutritional components, taste, and quality of milk obtained by conventional methods.

Examples

[0075] Aspects of the systems and methods of the present invention can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

[0076] Example 1: Bioreactor As described above, the bioreactor of the present invention is designed to mimic the native environment of mammary alveoli and ducts for healthy epithelial cells (ECs) and optimal milk secretion. The aim of this system and method is to utilize process control conditions and cell biology to provide a culture of primary ECs that is FCS-free or nearly FCS-free. In some embodiments, continuous mechanical stimulation of healthy ECs results in an improved hormone-free milk production process. (However, the hormone prolactin is still required.) In some embodiments, the lactation medium and test cultures are fluidically driven by a peristaltic pump to generate adaptable shear stress within a closed sterile system. In some embodiments, mechanical shear stress can be evaluated and monitored continuously or periodically. In some embodiments, the bioreactor is a modified perfusion reactor equipped with a hollow fiber module and is controlled by a peristaltic pump to generate continuous unidirectional flow, oscillatory flow, and pulsatile flow at an adjustable flow rate. In some embodiments, the lactating cells grow or coat on the inner surface of the hollow tube 104. The inlet of each hollow tube is connected to a pump to provide a flow stimulus via pulsatile flow. Milk can be harvested at regular intervals, daily, weekly, monthly, etc., depending on the scale (volume) of the overall bioreactor system, batch by batch. This system supplies extracellular medium through a permeable and / or semipermeable membrane within the tube to nourish the lactating cells within the tube. During the treatment period, the ECM can be replenished and / or replaced periodically.

[0077] Example 2: Collection of Mammary Epithelial Cells (MECs) Fresh milk (e.g., milk from Guernsey or Galloway cows) was skimmed by low-speed centrifugation at normal refrigeration temperature (~about 4°C). The skim milk was removed, and the remaining total cell pellet was washed multiple times with phosphate-buffered saline (PBS) and resuspended in PBS. Using immunomagnetic separation, MECs were isolated from the somatic cells of the whole milk, and leukocytes were removed. These leukocytes were collected separately for reuse. In immunomagnetic separation, the cell suspension of the whole milk was cultured with magnetic beads (Dynabeads) coated with a primary monoclonal antibody against cytokeratin 8 (K8.13) (Boutinaud et al., 2008). The resulting antibody-bound MECs were then washed, and the purified MECs were resuspended in PBS. Thereafter, to ensure sufficient cell growth, the MECs were cultured in complete medium for 1 week. The complete medium contained DMEM / F12 (alternatively, EpiCult) supplemented with 2% FBS or FCS substitute, 5 μg / ml insulin, 100 ng / ml hydrocortisone, L-glutamine, and optionally antibiotics.

[0078] Example 3: Culture and lactation of MECs Seeding of mammary myoepithelial cells and epithelial cells (MEP) in complete medium was performed and maintained until a monolayer was established. Low shear stress was used for the inflow within the complete medium for cell growth (1 week). The shear stress was 0.007 dyn / cm 2 . Next, as the sedimentation and attachment period, it took about 4 hours in the hollow fiber reactor. At a physiological shear stress of about 5 dyn / cm 2 , i.e., low shear stress, growth and monolayer formation were carried out over several days. The medium was changed every 3 days or with each fed-batch.

[0079] Next, the serum was removed and the lactation medium was supplemented to induce lactation differentiation. The pump was run at about 2 - 75 dyn / cm for at least 1 week, more preferably for a period of about 2 - 3 weeks. 2To induce variable shear stresses of different levels alternately within a certain range, the fluid flow was periodically controlled to change. The total required time for culturing varies depending on the size (volume) of the system. It takes about two weeks for a small-scale system and several months for a larger-scale system to circulate the culture medium through the reactor to complete the culturing. During a period of several weeks, the lactation medium was changed every three days or fed-batch culture was performed. After the culturing was completed, the product was harvested. The post-harvest processing included preservation by any one or more of pasteurization, freezing, or dehydration. The quality control tests included the evaluation of bacteria, mycoplasma, fungi, and the main components.

[0080] Example 4: Use of a matrix for replacing feeder cells Feeder cells are usually suitable for supporting the co-culture of stem cells. However, when dealing with different species, this can cause the possibility of the transfer of animal pathogens and / or undesirable immune reactions that may affect milk production. The Matrigel matrix structure provides an appropriate and functional support structure for cells that require feeder cells.

[0081] To coat Matrigel (BD Biosciences, Bedford, MA), 500 μL of BD Matrigel was transferred to each well of a six-well plate on ice and gently shaken to spread. Next, the plate was cultured at 37 °C for 2 hours to polymerize the BD Matrigel into a gel state. Next, a suspension of reprogrammed HDF (human dermal fibroblasts) (2.4×10 4 / cm 2 ) was transferred onto a pallet coated with a BD Matrigel matrix in DMEM medium containing 10% FBS, and the plate was returned to the incubator. After 48 hours, the medium was replaced with DMEM / F-12 supplemented (containing the supplementation factors described above), and the medium was changed daily to form hiPSC colonies. The culturing of iPSCs was maintained and tracked over 10 passages.

[0082] Example 5: Preparation of feeder cells by the conventional method CF-1 MEFs at passage 3 (P3) with 80 - 90% confluence were inactivated at 37°C for 0, 0.5, 1.0, 1.5, and 2.0 hours using 10 μg / ml of MMC (Hisun Pharmaceutical Company, China). After incubation, the cells were washed 6 times with PBS, trypsinized, centrifuged at 180×g for 5 minutes, and resuspended in MEF medium. The cells were counted and cryopreserved for later use.

[0083] Example 6: Preparation of feeder cells by the suspension - adhesion method According to Figure 1, feeder cells were prepared using the SAM suspension - adhesion method. Briefly, P3 CF - 1 MEFs were cultured for 4 days, digested into single cells with 0.25% trypsin / EDTA (Dalian Meilun Biotech Co., Ltd, China), and collected in 50 mL centrifuge tubes. The cells were seeded in a 10 cm dish at 8×10 4 ~1.1×10 5 cells / cm 2 . After 2.0 - 3.0 hours, MMC (10 μg / mL) was added at 37°C. The medium containing MMC was discarded 0.5 - 3.5 hours after treatment. Next, the cells were washed 6 times with PBS, trypsinized, centrifuged at 180×g for 5 minutes, and resuspended in MEF medium. The cells were counted and frozen for later use.

[0084] Example 7: Preparation of feeder cells by the three - dimensional (3D) suspension method After connecting the CELLSPIN system (5 - 75 RPM, CELLSPIN system with a glass - ball stirring shaker, Integra Bio - Sciences, Switzerland) to the incubator, the spinner flask was sterilized by autoclaving. Feeder cells were prepared by 3DSM. Briefly, P3 CF - 1 MEFs grown for 4 days were digested into single cells with 0.25% trypsin / EDTA and collected in 50 mL centrifuge tubes. The cells were placed in a 25 - 1000 mL volume with a glass - ball shaker at 0.5 - 1.3×10 6The cells were transferred to a spinner flask at a cell density of cells / mL. MMC was added at 10 μg / mL. After culturing at 37 °C for 0.5, 1.0, 1.5, and 2.0 hours, the cells were centrifuged at 180×g for 5 minutes, washed 3 times with PBS, resuspended in MEF medium, counted, and cryopreserved for later use.

[0085] Example 8: Production of feeder cells / or support matrix for bioreactor Bovine embryonic stem cells (ESCs) are powerful tools in agricultural and biomedical applications and have been successfully cultured and differentiated using supportive feeder cells (Cell Reprogram. 2012 Dec;14(6):520 - 9.doi:10.1089 / cell.2012.0038). Currently, commercially available feeder cells are exclusively mouse - or human - derived feeder cells. One of the goals of the systems and methods of the present invention is to produce custom feeder cells derived from bovine fibroblasts. Feeder cells that can be used in embodiments of the methods of the present invention include fibroblasts, mEPs and myoepithelial cells, and mEPs on a fibrous extracellular matrix.

[0086] Alternative synthetic methods may be used to support milk - secreting cells. The following examples illustrate some of these methods.

[0087] Example 9: Artificial FCS substitutes When activated, PBMCs secrete a mixture of powerful survival - promoting factors that help fight infection. These are secreted into the blood and become part of the serum phase. In co - culture experiments, it has been shown that supplementing cells with the supernatant phase of cultured PBMCs significantly extends their survival period. In cell culture, this is used to "crowd" the cells and achieve healthy cell growth and recovery after freezing, depending on microenvironmental factors, i.e., suspension at high cell density. Adding the supernatant of natural immune cells provides survival factors as powerful as direct survival stimulation.

[0088] PBMCs were extracted from the peripheral blood of volunteers using a Percoll gradient. After PBMCs were supplied with activation-promoting stimuli including A) LPS, B) opsonized beads, and C) mechanical stimulation by an ibidi™ pumping system, the cells were maintained in a stimulated state, and the supernatant was concentrated and then collected over 24 - 48 hours. The supernatant phase was separated from the cell fraction and used for medium supplementation. For future scale-up, conditionally immortalized ER-Hoxb8 hematopoietic progenitor cells can be used and differentiated into PBMCs as needed to provide an unlimited source of PBMCs.

[0089] Example 10: EpiCult™ Plus Serum-Free Medium for Mammary Epithelium Successful tests of mammary cells were carried out using a cell culture technique that custom-modified the cell culture method of epithelial cells (mammary gland) from the Animal free research Foundation's FCS-free database (https: / / fcs-free.org / fcs-database).

[0090] Figures 5A - 5E show representative growth curves and images. Photographic images of mammary gland tissue cells were taken using live cell microscopy with a Kodak Imaging System. Here, Figure 5A shows mammary epithelial cells with an epithelial-like phenotype, and Figure 5B shows mammary myoepithelial cells with a spindle-shaped phenotype. Figure 5C shows the growth rate curve of a mammary tissue cell line. 1×10 4 cells were seeded and then measured over 4 days. Values of three independent cell samples per day were compared using an unpaired t-test. A significant difference was determined between the cell growth rates of mammary epithelial cells (green) and mammary myoepithelial cells (red) on day 3 (p = 0.007) and day 4 (p = 0.0228). These values are highlighted by asterisks in the figure. Figures 5D - 5E are plots showing that the addition of 2% PBMCs (5D) or 1:3 neutrophil-conditioned medium (i.e., supernatant) (5E) provides a survival stimulus equivalent to the addition of the survival-promoting mediators dbcAMP or LPS and PGE2 in cultured immune cells.

[0091] In some embodiments, in addition to optimizing the medium composition, an approach using a learning machine (AI) may be used to optimize and adjust the processing parameters.

[0092] Example 11: In vitro milk production using mammary epithelial progenitor cells extracted from fresh bovine milk The bioreactor was seeded with mammary epithelial cells (MEP) in a complete medium supplemented with 2% FCS (FCS substitute), 5 μg / mL insulin, 100 ng / mL hydrocortisone, L-glutamine, optionally antibiotics, IGF-I, or EGF. The complete medium can be selected from DMEM, F12, or EpiCult. The MEP were cultured until a monolayer was established. To maintain cell growth for one week, a low shear rate inflow was applied in the complete medium. The sedimentation and attachment period in the hollow fiber reactor was 4 hours. For growth and monolayer formation, medium to high shear stress was maintained for 2 days. The complete medium was exchanged every 3 days or fed-batch culture was performed. To induce mammary differentiation, serum was removed and the mammary medium was supplemented. The mammary medium contained DMEM / F12 (alternatively EpiCult) supplemented with 2% FBS (or FCS substitute), L-glutamine, 1 - 5 μg / mL prolactin (or the shear stress defined above). To generate shear stress within the range of 2 - 75 dyn / cm 2 a pulsatile flow was induced by switching the pump alternately at different flow rates, such as high, medium, and / or low. This step continued for at least one week, more preferably about 2 - 3 weeks. As will be apparent to those skilled in the art, the overall processing time depends on the scale (volume) of the system. In a small-scale system, the time required to produce fully cultured milk is as few as 2 weeks, but in a larger-scale system, full culturing may take several months. The exchange of the milk secretion medium was performed every 3 days or whenever fed-batch culture was carried out. Thereafter, the product was collected and processed as described in Example 3.

[0093] Example 12: Genetic engineering and other modifications to induce hyperlactation By using CRISPR technology, it is possible to induce hyperlactation in cells. Referring to the figure in Figure 6, the following protocol can be used. CSN2 polymorphisms (SNPs): G->A at His220, Glu223; LALBA polymorphism: within the 5' LALBA promoter; PRLR SNPs: rs62355518, rs10941235, rs1610218, rs34024951, rs9292575; PRL SNP: rs849872; Jak2 functional mutation for overreaction: V617F. Additionally, the use of retinoic acid and ATRA targeting CSN3 expression may also be included in the pharmaceutical intervention.

[0094] By modifying SNPs (via CRISPR) to reduce lactose content, lactose-free milk for dietary use can be produced: CSN2 (Glu340, Thr174, Lys14), B4GALT1 (T224A).

[0095] Using the CRISPR cluster bomb, hypoallergenic milk can be produced by introducing point mutations at sites corresponding to CDS 84-123 (CSN1S1) and CDS 87-123 (CSN1S2) within base pairs 186 and 213 of the CSN1S1 gene.

[0096] Table 3 below shows a list of numerous modifications achievable through genetic engineering of mEP and mEP-derived cell lines.

Table 3

[0097] Particularly desirable modifications made possible by the method of the present invention include the production of A2-rich milk from cells.

[0098] The embodiments of the system and method for milk production in vitro have been described above. Although specific exemplary embodiments have been described, it will be apparent that various modifications and changes may be made to these embodiments without departing from the broad scope of the invention. Accordingly, the detailed description should not be taken in a limiting sense, and the scope of the various embodiments is defined only by the appended claims, which are defined together with the full scope of equivalents to which such claims are entitled.

[0099] References 1. Quaglino, A., et al., Mechanical strain induces involution-associated events in mammary epithelial cells. BMC Cell Biol 10, 55 (2009). https: / / doi.org / 10.1186 / 1471-2121-10-55.

Claims

Claim 1 An apparatus for in vitro milk production, the apparatus comprising: a reactor assembly including an elongated shell configured to hold an extracellular medium; one or more elongated hollow tubes disposed longitudinally aligned within the extracellular medium within the shell, the one or more tubes having an inlet end, an outlet end, and an inner surface configured to attach a monolayer of milk-producing cells, at least a portion of the one or more tubes including a semipermeable material configured to allow diffusion of the extracellular medium into the hollow tubes, the extracellular medium including a nutrient solution for maintaining the milk-producing cells, the hollow tubes; a pump fluidly connected to the one or more tubes, the pump configured to generate a pulsatile or oscillating flow of a milk-producing medium through the one or more tubes to induce shear stress on the milk-producing cells, the pump being controlled to alternately switch between different shear stress levels within a predetermined range, the change in shear stress stimulating the milk-producing cells to produce dairy products, the pump; a supply loop for supplying and circulating the extracellular medium through the shell; a storage tank fluidly connected to the outlet end of the one or more hollow tubes and configured to collect the dairy products. The apparatus. Claim 2 The apparatus according to claim 1, further comprising a feedback loop disposed near the outlet end of the one or more tubes and configured to recirculate the dairy products to the inlet end of the one or more tubes for further concentration. Claim 3 The predetermined range of the shear stress level is 5 to 15 dyn / cm ２ The apparatus according to claim 1, wherein the range is as defined above. Claim 4 The apparatus according to claim 1, wherein the supply loop includes a storage tank configured to remove used extracellular medium and add fresh extracellular medium. Claim 5 The apparatus according to any one of claims 1 to 4, wherein the milk-producing cells are co-cultured with feeder cells. Claim 6 The apparatus according to claim 5, wherein the feeder cells are peripheral blood mononuclear cells. Claim 7 The apparatus according to any one of claims 1 to 4, wherein the milk-producing cells are isolated from the milk of healthy cows. Claim 8 The device according to any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary tissue identified using a series of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EpCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, VIM, CD34 / CD39 / CD140b, and CD49e.

9. The device according to any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary tissue selected from whole breast cells, whole breast epithelial cells, intra-mammary cavity cells, intra-mammary cavity progenitor cells, mature intra-mammary cavity cells, mammary myoepithelial cells, and mammary stromal cells.

10. The device according to any one of claims 1 to 4, wherein the lactating cells are genetically engineered to induce hyperlactation.

11. The device according to any one of claims 1 to 4, wherein the lactating cells are genetically engineered to produce milk having one or more of low allergenicity, low lactose, and increased A2-type β-casein protein.

12. The device according to any one of claims 1 to 4, wherein the one or more tubes are coated with collagen.

13. The device according to any one of claims 1 to 4, further comprising a system controller configured to generate control signals for the pump and the supply loop.

14. The device according to any one of claims 1 to 4, wherein the one or more tubes are formed of a semipermeable capillary membrane.

15. The device according to any one of claims 1 to 4, wherein the one or more tubes have a hybrid structure including a portion of an impermeable material and a portion of a permeable or semipermeable material.

16. The device according to any one of claims 1 to 4, wherein the inner surface of the one or more tubes is first bonded with a layer of a support matrix, the lactating cells adhere to the unbonded surface of the support matrix, and the lactating cells form a monolayer of lactating cells on the support matrix.

17. 5. The apparatus of claim 1, further comprising a mechanical stimulation assembly configured to apply a clamping force to an exterior of the one or more tubes in a direction from the inlet end toward the outlet end.

18. 5. The apparatus of claim 1, further comprising one or more light sources disposed within the reactor assembly configured to expose the lactating milk cells to a light stimulus.

19. 20. The apparatus of claim 18, wherein the one or more light sources are light emitting diodes (LEDs) that emit light at 450 nm.

20. The device according to any one of claims 1 to 4, wherein the lactation medium comprises one or more of EpiCult™ Plus medium, an artificial cultured FCS replacement medium, and an FCS-free medium.

21. The device according to any one of claims 1 to 4, wherein the lactation medium further comprises prolactin.

22. A milk production facility comprising a plurality of interconnected devices according to any one of claims 1 to 4.

23. A method for in vitro milk production in a device according to any one of claims 1 to 4, said method comprising the steps of: providing said one or more ducts with a lactation medium containing said prolactin; forming a monolayer of milk-producing cells attached to the interior surface of said one or more ducts; controlling the pump to alternate between different shear stress levels within a predetermined range, the changes in shear stress stimulating the milk-producing cells to produce a milk product; and recovering the dairy product once a predetermined milk quality is achieved.

24. 24. The method of claim 23, further comprising recirculating the dairy product through the one or more lines via a feedback loop to concentrate the dairy product until the predetermined milk quality is achieved.

25. 24. The method of claim 23, wherein the milk producing cells are co-cultured with feeder cells.

26. 26. The method of claim 25, wherein the feeder cells are peripheral blood mononuclear cells.

27. The method of any one of claims 23 to 26, wherein said milk producing cells are isolated from the milk of said healthy cow.

28. The method according to any one of claims 23 to 26, wherein the lactating cells are extracted from healthy mammary tissue identified using a series of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EpCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e.

29. The method according to any one of claims 23 to 26, wherein the lactating cells are extracted from healthy mammary tissue selected from total mammary cells, total mammary epithelial cells, intra-mammary cavity cells, intra-mammary cavity progenitor cells, mature intra-mammary cavity cells, mammary myoepithelial cells, and mammary stromal cells.

30. The method according to any one of claims 23 to 26, wherein the lactating cells are genetically engineered to induce hyperlactation.

31. The method according to any one of claims 23 to 26, wherein the lactating cells are genetically engineered to produce milk that is one or more of hypoallergenic, low lactose, and increased A2 type β-casein protein.

32. The method according to any one of claims 23 to 26, wherein the lactation medium comprises one or more of EpiCult (trademark) Plus medium, artificial culture FCS replacement medium, and FCS-free medium.

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