TW202409265A - Pulsatile flow culture of mammary cell types for milk secretion - Google Patents

Abstract

An apparatus and method for in vitro milk production includes a reactor assembly with an elongated shell that encloses one or more hollow semipermeable tube within an extracellular nutritional medium. A monolayer of lactating cells is formed on the tube’s inner surface. A pump connected to the tube generates a pulsatile flow of a lactating medium to induce a shear stress on the lactating cells. A variable pump rate subjects the lactating cells to different shear stress levels to stimulate milk production. The lactating medium is continuously recirculated through the tube until a predetermined milk quality is achieved, after which it is harvested. The lactating cells may be extracted from milk or breast tissue and may be genetically modified to produce milk with designer features such as hypoallergenic or reduced lactose.

Description

Pulsating flow culture of milk-secreting breast cell types

The present invention relates to an apparatus and method for in vitro emulsion production, including emulsions with enhanced health effects. Related applications

This application claims the benefit of U.S. Provisional Application No. 63/358,464, filed on July 5, 2022; the disclosure of which is incorporated herein by reference in its entirety.

The global demand for milk production is increasing due to a number of factors, including global population growth and changes in diets in Asian countries (i.e., Westernization). Global dairy consumption is expected to increase by more than $100 billion over the next decade. This in turn has put increasing pressure on natural resources, raised concerns about animal welfare, and led to increased deforestation and greenhouse gas emissions. Dairy consumption in the United States accounts for approximately 2% of the country's greenhouse gas emissions. To meet worldwide demand, approximately 270 million dairy cows are raised every day. Each component of milk is associated with a range of positive and negative health effects.

Milk is a complex colloidal matrix containing milk fat, lactose, and a variety of different components, including naturally occurring hormones. The sensory and functional properties of milk are derived from its micronutrient profile, which has proven to be costly and difficult to replicate. In general, in the United States, the total composition of cow's milk is 87.7% water, 4.9% lactose (carbohydrate), 3.4% fat, 3.3% protein, and 0.7% minerals. Milk composition varies depending on the species (cow, goat, sheep), the breed (Holstein, Jersey), the animal's diet, and the stage of lactation.

Research has shown that the fat and protein content of emulsions are based on the breed of cow. Haostan has the lowest fat and protein content, while Juanshan and Gengsai varieties have the highest fat and protein content. Even within a single herd, milk protein can range from 1.57% to 4.66%, with an average of 3.05%, while milk fat can range from 1.77% to 5.98%, with an average of 3.76%. Food sources, temperature, humidity and seasonality all play an important role in variations in emulsion quality during emulsion production. To account for variations in emulsion composition, a common practice is to blend emulsions from different cows in bulk tanks to produce an emulsion of relatively consistent composition throughout the year in the United States.

Another issue with lotion quality is the presence of naturally occurring hormones found in lotions. 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), corticosteroids (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). Furthermore, due to the need to treat the entire herd for infection during its milk production (i.e. chronic udder infection), hormones and other exogenous agents such as antibiotics and drugs are often present in industrial scale milks.

Although there is little controversy regarding the existence of physiological concentrations of these hormones, the potential biological effects of such hormones in animals and humans may not be well understood and may be profound. In addition, the presence of hormones, particularly steroid hormones (e.g. estrogen), can cause significant safety concerns in dairy products. Some evidence suggests a potential link to breast and prostate cancer. Therefore, special attention is required, especially during major developmental periods such as the perinatal period and adolescence.

For this reason, and in view of the considerable progress that has been made in the development of analytical methods and bioassays, it is important to clarify the potential effects of the presence of hormones, especially steroid hormones such as estrogens, when they are a common component of the diet and consumed regularly over the year.

Meal alternatives to dairy products are found in plant-based lotions and lotion products. Plant-based lotions such as almond, soy, cashew and oat do not contain the naturally occurring hormones found in animal-based lotions and are increasingly popular as good sources of protein without causing the dairy intolerance that many people experience. problem. However, these alternative sources of plant-based emulsions are often not as capable as animal-based emulsions in providing the functionality of animal-based emulsions, which can be converted into the production of other dairy products such as cheese, butter, and yogurt. In addition, the growing popularity of plant-based lotions raises other sustainability issues, such as widespread deforestation to expand soy production or the large amounts of water required to produce almonds in chronically dry regions.

Another related area within the dairy industry is the alternative milk product known as "A2 milk". Cow's milk normally contains two types of beta-casein: A1 and A2, which differ in that the 67th amino acid in the protein structure is either histidine or proline. The hypothesis is that conventional cow's milk containing A1 beta-casein may have negative health effects, including digestive discomfort, intestinal inflammation and milk allergies, while A2 beta-casein does not seem to cause these effects.

Both A1 β-casein and A2 β-casein contain 209 amino acids (AA). However, upon digestion, A1 beta-casein releases beta-casomorphin-7 (BCM-7), which triggers a cascade of events that increases inflammation and gastrointestinal discomfort. It is hypothesized that consumption of lotions containing A1 beta-casein is associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and reduced cognitive processing speed and accuracy. Recent studies have confirmed that individuals consuming A1/A2 beta-casein emulsion exhibit more severe digestive symptoms associated with lactose intolerance, while consuming A2 beta-casein emulsion was not found to worsen these symptoms. Inflammatory markers such as IL-4, IgG, IgE and IgG1 were significantly lower among A2 beta-casein emulsion consumers. Currently, because the two types of beta-casein (type A1 and type A2) are very similar, and cows that produce latex rich in A1 protein tend to belong to breeds with high milk production (high-yielding cows), such as Holstein cows, No economical solution to the A1 beta-casein problem has yet been developed.

Milk and dairy products, including cream, butter, yogurt and cheese, are an important part of the human diet, providing an important source of protein, vitamins and minerals. For many people, dairy products are the easiest way to obtain nutrients to keep the heart, muscles and bones healthy and functioning properly. However, milk quality-related issues discussed above have plagued the dairy industry for many years and need to be resolved urgently.

The need for improved emulsion production technology is clear and is driven by a variety of factors, including the current unsustainable method of producing emulsion through cattle farming (inefficient use of land and agricultural resources, greenhouse gas production, etc.); the lack of similar emulsion alternatives; And the sources of stable and high-quality lotions are limited.

U.S. Patent No. 11,236,299 (assigned to Biomilk Ltd. (Rehovot, IL)), incorporated herein by reference, discloses a method for producing breast organoids by using a series of seeded on a three-stage branching, elastic milk duct scaffold. Methods for producing emulsions in vitro. In an exemplary implementation, an emulsion production system includes an array of containers, each container containing a plurality of mammary organoids (MOs), a nutrient supply reservoir operable to feed each container, and an emulsion collection module. The key components of the system are MOs, which are breast epithelial cells that form multicellular three-dimensional structures (breast organoids) embedded in the matrix. After seeding scaffolds with MO, estrogen and progesterone help induce epithelial growth and morphogenesis by inducing paracrine signaling between the breast stroma and the epithelium containing seeded MO. Subsequently, MO begins to secrete milk in a medium containing prolactin, nutrients and growth factors, which lasts for 10-21 days. While the Wilk patent discloses a method for producing emulsions in vitro, it does not provide a range of options for modifying the emulsion product in terms of cellular structure to enhance nutritional content, reduce dependence on hormones, select for optimal A2 beta-casein, and others "Designed lotion" products, such as hypoallergenic lotions, will provide more sustainable and nutritious food sources to meet growing global demand.

In view of the growing demand for dairy products, the present invention aims to provide a more sustainable and versatile method for producing emulsions to overcome the environmental and animal welfare challenges currently faced by the dairy industry, while providing a reliable, high-quality source of safe and clean emulsions.

According to embodiments of the present invention, a system and method for in vitro emulsion production using breast cells cultured to enhance cell proliferation and secretion of emulsion and emulsion components are provided. Enhanced cell proliferation is achieved at least in part through mechanical stimulation by applying flow to the culture medium and changing the flow through a variety of different methods, including, but not limited to, unidirectional laminar flow, turbulent flow, pulsatile flow, and oscillatory flow. The constant mechanical stimulation provided by the system of the invention enables the generation of healthy epithelial cells (EC) and the production of an emulsion that is substantially free of hormones (other than prolactin). The systems and methods disclosed herein are generally applicable to the production of cellular proteins and dairy products for food culture and bioindustrial applications.

In one aspect of the invention, an apparatus for in vitro emulsion production includes: a reactor assembly comprising: an elongated shell configured to retain an extracellular culture medium; one or more hollows Tubes disposed within the extracellular medium in generally longitudinal alignment within the housing, the one or more tubes having an inlet end, an outlet end, and an interior surface configured for attachment of a monolayer of lactating cells, wherein at least a portion of the hollow tube is formed from a semipermeable material configured to permit diffusion of the extracellular medium into the one or more tubes, wherein the extracellular medium includes a nutrient solution for maintaining the lactation cells ; a pump in fluid connection with the one or more tubes, the pump configured to generate a pulsatile or oscillating flow of lactation medium through the hollow tube to induce shear stress on the lactation cells, wherein the pump is controlled alternating between different shear stress levels within a predetermined range, wherein changes in the shear stress stimulate the lactation cells to produce an emulsion product; a supply circuit for supplying the extracellular medium and circulating it through the housing; and a reservoir fluidly connected to the outlet end of the one or more tubes, the reservoir configured to collect the emulsion product. The apparatus may further include a feedback loop disposed proximate the outlet end of the one or more tubes for recycling the emulsion product for further enrichment. In some embodiments, the predetermined range of shear stress levels is 2 to 75 dyn/cm ² . The supply loop may include a reservoir configured for removing used extracellular medium and adding fresh extracellular medium.

In some embodiments, lactating cells are co-cultured with feeding cells. Feeder cells can be peripheral blood mononuclear cells. In some embodiments, lactation cells can be isolated from the milk of healthy cows. In other embodiments, the lactation cells can be extracted from healthy breast tissue identified using a panel 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 lactation-producing cells can be selected from the group consisting of all breast cells, all breast epithelial cells, breast luminal cells, breast luminal precursor cells, mature breast luminal cells, breast myoepithelial cells, and breast stromal cells of healthy breasts. extracted from the tissue. The lactating cells can be genetically modified to induce hyperlactation or to produce an emulsion that is one or more of hypoallergenic, reduced lactose, and increased A2 beta-casein. In some embodiments, each hollow tube can be coated with collagen. One or more tubes are formed from a material that is at least partially a semipermeable capillary membrane. In some embodiments, the inner surface of the one or more tubes is first bonded to a layer of support matrix, wherein the lactation cells are attached to an unbound surface of the support matrix, and wherein the lactation cells are on the support matrix. A monolayer of lactating cells forms on top.

In some embodiments, the device may further include a system controller configured to generate control signals to the pump and supply circuit.

Optionally, a mechanical stimulation assembly may be included to apply extrusion pressure to the exterior of one or more tubes in a direction from the inlet end toward the outlet end. Optionally, 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 are light emitting diodes (LEDs) that emit light at 450 nm.

The lactation medium may include one or more of EpiCult™ Plus medium, laboratory-grown FCS replacement medium, and FCS-free medium, and may further include prolactin.

In another scope of the present invention, an emulsion production facility can be constructed by interconnecting a plurality of the above-mentioned devices.

In yet another aspect of the present invention, the in vitro emulsion production method in the above device includes: supplying the lactation medium to the one or more tubes, wherein the lactation medium includes prolactin; forming a lactation medium attached to the one or more tubes a monolayer of lactation cells on the inner surface; and controlling the pump to alternate between different shear stress levels within a predetermined range, wherein changes in the shear stress stimulate the lactation cells to produce an emulsion product. The method may further include recycling the emulsion product to the one or more tubes via a feedback loop to enrich the emulsion product until the predetermined emulsion quality is achieved. In some embodiments, lactating cells are co-cultured with feeding cells. Feeder cells can be peripheral blood mononuclear cells. The lactation medium includes one or more of an EpiCult™ Plus medium, a laboratory-grown FCS replacement medium, and an FCS-free medium.

In some embodiments, lactating cells can be isolated from the milk of healthy cows. In other embodiments, the lactating cells can be extracted from healthy breast tissue identified using a panel of biomarkers selected from the following: 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 can be selected from healthy breast tissues including all breast cells, all breast epithelial cells, breast luminal cells, breast luminal progenitor cells, mature breast luminal cells, breast myoepithelial cells and breast stromal cells.

The lactating cells can be genetically modified to induce hyperlactation and/or to produce an emulsion with one or more of hypoallergenic, reduced lactose, and increased A2 beta-casein.

To facilitate understanding of the present invention, various terms and abbreviations used herein are defined as follows:

As used herein, the term "amino acid" refers to the molecular building blocks used to build and assemble proteins, such as enzymes. Peptide bonds (ie, polypeptides) are formed between amino acids and assemble in three dimensions (3-D). 3-D assemblies can influence protein properties, function, and conformational dynamics. Within biological systems, proteins can: (i) catalyze reactions as enzymes; (ii) transport vesicles, molecules, and other entities within cells as transport entities; (iii) provide structure to cells and organisms as proteins Filaments; (iv) replication of deoxyribonucleic acid (DNA); and (v) coordination of cells as cell signatures.

As used herein, the term "nucleotide" refers to the molecular building blocks used in the construction and assembly of nucleic acids, such as DNA and ribonucleic acid (RNA). There are two types of nucleotides - purines and pyrimidines. Specific purines are adenine (A) and guanine (G). The specific pyrimidines are cytosine (C), uracil (U) and thymine (T). T is found in DNA and U is found in RNA. The genetic code defines a sequence of nucleotide triplets, or codons, that specify which amino acids are added during protein synthesis.

As used herein, the term "gene" refers to a region of DNA. As defined by the gene sequence, the amino acid sequence in a protein is encoded by the gene code.

As used herein, a recombinant nucleic acid or protein is a nucleic acid or protein produced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012).

The terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to an amino acid chain in which amino acid residues are linked by peptide bonds or modified peptide bonds. An "amino acid chain" may have any length greater than two amino acids. Unless otherwise specified, the terms "polypeptide", "protein" and "peptide" also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristylated forms, palmitylated forms, ribosylated forms, acetylated forms and the like. Modifications also include intramolecular cross-linking and covalent linkage of various moieties such as lipids, flavonoids, biotin, polyethylene glycol or its derivatives and the like. In addition, modifications may include protein cyclization, amino acid chain branching, and protein cross-linking. In addition, amino acids other than the known twenty amino acids encoded by the gene may be included in the polypeptide. The term "protein" or "polypeptide" may also encompass a "purified" polypeptide (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants) that is substantially separated from other polypeptides in the cell or organism in which the polypeptide is naturally produced.

As used herein, the term "bioreactor" refers to a container or tank in which whole cells or cell-free enzymes convert feedstock into biochemical products and/or less desirable byproducts. Bioreactors are designed and operated to provide an environment for product formation, in this case an emulsion product. Industrial bioreactors can be operated as batch reactors or continuously, aerobically or anaerobically, and can use pure cultures or mixed cultures. In some bioreactors, there are three phases (gas, liquid, and solid) and mass transfer can be an important consideration. Biofilms and immobilized cells can be used to retain microbial biomass in flow bioreactors. Sensors, instrumentation, and control systems are essential for industrial bioreactors.

As used herein, the term shear stress (generally represented by τ (Greek: tau)) refers to the stress component that is coplanar with the cross-section of the material. (See, for example, Figure 4. ) It is caused by shear stress, with the component of the force vector being parallel to the material cross-section. Any real fluid (including liquids and gases) moving along a solid boundary will induce shear stress at that boundary. The no-slip condition stipulates that the velocity of the fluid at the boundary (relative to the boundary) is zero; but at a certain height from the boundary, the flow velocity must be equal to the flow velocity of the fluid. The area between these two points is named the boundary layer. For all Newtonian fluids in laminar flow, shear stress is proportional to the strain rate in the fluid, where viscosity is the constant of proportionality. For non-Newtonian fluids, the viscosity is not constant. Shear stress is imposed on the boundary due to this velocity loss.

As used herein, the term "shear strain" refers to the ratio of displacement due to stress to the initial dimensions of the object and is the amount of deformation perpendicular to a given line rather than parallel thereto. Shear strain is the lateral force exerted on the culture medium and is measured as the change in angle between initially vertical lines. (See, for example, Figure 4. )

As used herein, the term "pulsating flow" refers to a flow having periodic pressure fluctuation waves that travel along the flow path. Pulsatile flow systems simulate blood flow characteristics within the heart and vasculature.

As used herein, the term "effective amount" refers to an amount that, when administered to a particular individual, will have the desired biological effect, e.g., an amount that will cure, prevent, inhibit or at least partially arrest or partially prevent the target response, in view of the nature and severity of that individual's condition.

Various abbreviations and abbreviations are used throughout this disclosure. Table 1 below summarizes and provides a glossary of some commonly used acronyms and abbreviations: abbreviation definition EC epithelial cells mEP or MEP Breast myoepithelial and epithelial cells MEC breast epithelial cells ECM extracellular matrix or culture medium HDF human dermal fibroblasts InC between capillaries ExC outside capillary Table 1

The terms "hollow tube", "hollow tube", "hollow fiber" and "lumen" may be used interchangeably throughout this specification to refer to an elongated hollow structure that flows under pressure through a pump fluidically connected to the hollow structure. The siphon system flows through the elongated hollow structure. Hollow structures can be formed from a variety of different materials, which can be rigid, flexible, permeable, semipermeable or impermeable. Examples of materials include, but are not limited to, glass, acrylic, plastic, polymers, fibers, silicones, ceramics, and filter membranes. In some embodiments, the hollow structures may be compressible or may be paired to squeeze fluid within the interior of the conduit from a first end of the conduit to a second end, such as by applying opposing rollers to the outer surface of the conduit. end to simulate a peristaltic motion. (See e.g. Figure 1B ). In other embodiments, the tube may be a mixed assembly of tube sections made of different materials, some of which may be permeable or semipermeable membranes and others which may be impermeable.

lactation biology

Lactation is the process of making and secreting milk from the mammary glands. The mammary glands are modified sweat glands and are composed primarily of adipose and collagenous tissue, with the mammary glands constituting a very small proportion of the breast volume. The mammary glands consist of milk-carrying ducts, which dilate and branch extensively in response to estrogen, growth hormone, cortisol, and prolactin. In addition, in response to progesterone, clusters of alveoli sprout from the ducts and expand outward toward the chest wall. The alveoli are balloon-like structures lined with milk-secreting cuboidal cells, or milk cells, and surrounded by a network of contractile myoepithelial cells. Milk is secreted by the milk cells, fills the alveoli, and is squeezed into the ducts. The clusters of alveoli that drain into the common ducts are called lobules; there are 12-20 lobules arranged radially around the nipple of a lactating female. Milk flows from the milk ducts into the lactiferous sinuses, which meet at a point where there are 4 to 18 holes in the nipple called foramina.

The pituitary hormone prolactin plays a role in the establishment and maintenance of breast milk supply. Prolactin and other hormones prepare the breast anatomically for milk secretion. When the infant sucks, sensory nerve fibers in the areola trigger a neuroendocrine reflex that causes milk to be secreted from the milk cells into the alveoli. The posterior pituitary releases oxytocin, which stimulates the myoepithelial cells to squeeze milk from the alveoli, draining it into the lactiferous ducts, where it collects in the lactiferous sinuses and is discharged through the nipple orifice. It takes less than 1 minute from the beginning of sucking (latent phase) to milk secretion (lactation). Prolactin-mediated milk synthesis varies over time. Frequent milk discharge through breastfeeding (or pumping) will maintain high circulating prolactin levels for several months. However, even with continued breastfeeding, baseline prolactin will decline over time to pre-pregnancy levels. In addition to prolactin and oxytocin, growth hormone, cortisol, parathyroid hormone, and insulin also contribute to lactation, in part by promoting the transport of maternal amino acids, fatty acids, glucose, and calcium into breast milk.

Pulsating flow culture of milk-secreting breast cell types

The system of the present invention is designed to simulate the native environment of breast alveoli and ducts for healthy epithelial cells (ECs) and optimal milk production by inducing variable shear stress on the cells.

1A - 1E , embodiments of the bioreactor of the present invention are designed to mirror the natural environment of breast alveoli and tubes for healthy epithelial cells (EC) and optimal milk production. FIG. 1A provides a diagrammatic side (left) and cross-sectional end (right) view showing the basic assembly of an exemplary embodiment of a bioreactor flow system 100 configured to implement adjustable shear stress within a closed sterile system. The culture vessel 102 includes a fluid-impermeable housing or shell 120 enclosing one or more semipermeable hollow tubes 104, a pump system 106, a valve 108 for controlling the flow of extracellular medium fluid, reservoirs 110, 112 and a feedback loop 118 and a valve 116 for recirculating partially processed fluid (accumulated medium) through the reactor tubes for further processing/enrichment. The shell 120 can be formed of polycarbonate or similar polymers, glass, stainless steel, or any material deemed suitable for food handling, i.e., sterilizable. Fluid extracellular matrix (ECM) is introduced into the bioreactor via reservoir 110, valve 108, and inlet and outlet conduits 128 and 130 and circulated through the bioreactor. (The pump used to control this ECM loop is not shown.) Flow through tube 104 is configured to apply mechanical shear stress to the cells within the tube as the lactation/accumulation medium flows under pressure from the pump. System controller 122 provides control signals to pump 106 to perform various operations, including draining the system, such as applying pulsating flow and/or continuous flow through tube 104, and collecting milk. System controller 122 can also be configured to generate control signals to valves 108, 116, ECM loop operations, and any additional valves and other operations within the system. In some embodiments, the system controller 122 may include one or more computer processor and memory combinations for storing and executing programs for automated operation of the system. In some embodiments, the parameters for optimal operation of the system may be established through the use of a learning machine within the system controller, where inputs include system sensor signals (flow rate, pressure, temperature, etc.) and quality control measurements obtained by testing emulsion products.

In some embodiments, the pump 106 is controlled to generate continuous, unidirectional, oscillatory, and pulsating flows with adjustable flow rates to expose cells in the tube to variable shear stress. Generally, the range of shear stress generated by the pump 106 will alternate between different settings selected via a signal from the system controller 122 within the range of approximately 2 dyn/cm ² (considered "lower") and 75 dyn/cm ² (considered "higher"). The pump sequence generated by the controller 122 can cause the pump flow to induce shear stress alternating between different levels, such as low (in the range of ~2-10 dyn/cm ² ) to medium (range ~11-40 dyn/cm ² ), high (range ~41-75 dyn/cm ² ) to medium, high to low, or high to low to medium and the like. It is to change the shear stress on the cells through pulsating/oscillating flow, which helps stimulate lactation. As the lactation medium flows through the cells, it accumulates the emulsion components to become an accumulation medium, which is processed through the hollow tube 104 and collected downstream in the reservoir 112. One or more access ports (not shown) may be included at or near the outlet 162 to allow the accumulation medium to be sampled to determine whether it has achieved the desired quality, such as density, appearance, consistency, fat content, chemical content, etc. for the finished emulsion product. If the test indicates that the product is complete, the emulsion can be collected. If not, the accumulation medium can be returned to the reactor inlet 160 via valve 116 and feedback loop 118 for further enrichment. Alternatively, once the optimal treatment duration has been determined for given operating conditions, regular testing may not be required.

Figure IB illustrates additional details of the structure and function of the bioreactor of the present invention, showing portions of an exemplary hollow tube 104, which in some embodiments may be arranged in a parallel array extending longitudinally within a generally cylindrical housing 120. One of a plurality of elongated tubes or lumens collectively defines an enveloped sterile bioreactor barrel 102 . The interior of the tube 104 is referred to as the intracapillary or InC space 134, and the space outside the tube (within the housing 120) is referred to as the extracapillary or ExC space 132. In some embodiments, the inner surface of tube 104 may be further lined with a collagen membrane (see, eg, Figure 1C ). Feeder (support) cells 138 and mEP cells 140 are applied to the inner surface of the hollow tube 104 . It should be noted that for convenience, only the primary cilia 142, which play an important role in emulsion production, are shown for a few cells 140. Those skilled in the art will recognize that healthy ECs should all have primary cilia. The mechanical sensation created by the adjustable flow within tube 104 helps maintain cell differentiation and health. Tube 104 may be formed from a semipermeable membrane material, such as a polymer or similar material, through which nutrients contained within the ECM may diffuse to provide nutrients to the cells. The flow inlet for tube 104 (at the downstream end) is connected to pump system 106 . In a small-scale prototype implementation of the bioreactor of the present invention, an ibidi ^™ peristaltic pump (ibidi GmbH, Graefelfing, DE) was used to provide the required mechanical stimulation to generate the emulsion via pulsatile flow. For larger systems, one or more larger capacity pumps are used to achieve the required pulsating flow. The emulsion can be collected daily in batch form from the reperfusion reservoir 112 located at the downstream end of the reactor. A filter 114 may be included on the upstream or downstream side of the reservoir 112 as appropriate to remove accumulated dead cells and other particulate material that may be in the emulsion prior to collection.

Although the hollow tube 104 is described herein as "semipermeable," its construction is not limited to semipermeable materials. Rather, the only requirement is that the portion of the tube be permeable enough to allow a certain amount of extracellular medium to pass into the InC to enrich the lactating cells. Therefore, the hollow tube 104 is still considered semipermeable because some of the portions are permeable or semipermeable and others of the portions are impermeable. For example, ceramic filter membranes can be used in the portion of the tube to supply cells with ECM, while the remainder of the tube can be less permeable or impermeable. This hybrid approach allows customized control of how much ECM is available to the cells, which avoids providing too much ECM, which could potentially damage the final product.

The embodiment illustrated in Figure IB includes an optional implementation in which additional mechanical stimulation is provided in the form of an assembly of opposing cylindrical rollers 150 that run along a track 152 disposed within the housing 120 such that it is parallel to the tube. The rollers slightly compress the tube from multiple sides to gently "squeeze" and push the contents of the tube, namely the lactation medium with emulsion component 144 and the accumulation medium near the inlet end toward the outlet end of the reactor. The pressure exerted by the roller should be uniform and strong enough to move the fluid, but gentle enough to avoid damaging the cells or dislodging them from the inner surface of the tube. Varying the speed of the drum provides an additional means for applying adjustable shear stress. Once the roller has completed its travel along the track, it will then separate to release pressure on the tube and move back on the track to its starting position near the inlet end to repeat the pushing sequence. Although two opposing rollers are shown, it will be readily apparent that different combinations of rails and rollers or annular sphincter can be used to achieve the required inlet to outlet pushing action which helps release cells to continue emulsion production. Signals for controlling the optional mechanical stimulation assembly will be provided by system controller 122.

1C provides a diagrammatic cross-sectional view of a monolayer of lactation cells attached to the inner surface of each hollow fiber 104 within the fiber array of reactor barrel 102. In the illustrated embodiment, the bioreactor is a modified reperfusion reactor with multiple hollow fiber elements 104 fed by a peristaltic pump (not shown) to create a continuous, Unidirectional, oscillatory and pulsating flow. Reactor barrel 102 may include a multi-tube bioreactor with inlets and outlets for fluid input/output into the intercapillary and extracapillary spaces. As illustrated, the InC space inlet 160 feeds the hollow tube 104, which then exports emulsion (ie, accumulated culture medium) to the outlet 162 from the InC space. Extracellular medium (ECM) is fed (from reservoir 110 ) into the ExC space via inlet 128 and then circulated back into reservoir 110 via outlet 130 . The flow 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, a transverse cross-section of five individual hollow fibers 104 is shown with an inner surface lined or coated with an outer fiber wall 156 of collagen membrane. Cells 140 (and feeder (support) cells) grow on the lumen of the hollow fiber. The collagen membrane supports the monolayer of cells so that emulsion can flow through the compartments (lumens) beyond the cell layer. The upper right image shows a longitudinal cross-section of a single fiber, again with a collagen membrane backing and a single layer of cells 140 with emulsion 170 flowing through the lumen. In this example, the inner diameter of the fiber is indicated as 2 mm, however, as will be apparent to those skilled in the art, the inner diameter of the fiber can be selected to support the shear required to produce the mechanical stimulation of cells on the inner surface of the tube. Appropriate flow parameters for shear stress.

The above implementation of the bioreactor of the present invention employs a peristaltic pump for the purpose of inducing shear stress on the lactating cells by means of a continuous fluid flow in a sterile reactor environment. As will be appreciated by those skilled in the art, alternative methods of inducing controlled shear stress by means of a continuous or variable fluid flow in a sterile environment include the use of swirl, magnetic vortex (e.g., stirring bars), and wave or valve pumps. Such methods would involve placing the vortex and/or pump within a sterile enclosure, which may negatively impact the scalability of the process relative to the above implementation.

As will be appreciated by those skilled in the art, the number of hollow tubes within a bioreactor assembly may vary and the illustrated example is not intended to be limiting. One example of a variation of the tube configuration is provided in FIG . 1E , where a single length of tube 304 is arranged within a housing 320 in a Z-shaped, annular, coiled, or serpentine pattern. The general direction of flow remains parallel to the length of the housing, i.e., from inlet to outlet, and may be recirculated via a feedback loop similar to those in other embodiments. In this example, tube 304 is a hybrid assembly formed of multiple tubular sections of different materials, wherein tube section 344 is a permeable or semi-permeable material that allows ECM to be perfused into the interior of the tube through these sections and only through these sections. Tube section 346 is impermeable. Pump 306 controls the pulsating flow from the inlet to the outlet through the tube. It should be noted that this is only an illustrative example and is not intended to be limiting.

The bioreactor embodiments shown in Figures 1A to 1C and described above are shown in a horizontal orientation, however , no particular orientation is required. Referring to Figures 2A- 2C , some embodiments of the bioreactor adopt a vertical orientation, which can help to scale up the capacity of the treatment facility to produce larger volumes of emulsions. Each filter cartridge 202 includes the same basic components as described above: a housing 220 enclosing one or more hollow tube reactors 204. A pump 206 feeds a fluid (e.g., PBS) into the top of the filter cartridge 202 at an inlet 260 that feeds the fluid into the tube 204. The hollow tube 204 has a monolayer of cells, particularly ECs, coated on its inner surface, as shown in Figure 2C. As in the previously described embodiments, pump 206 is configured to induce an adaptive shear stress as fluid flows through the tube to stimulate emulsion production by cells. The vertical orientation of the filter cartridge 202 provides gravity assistance for flow through the tube. Pump 206 can recirculate a portion of the processed emulsion (accumulated medium) via a feedback loop 218 until the desired emulsion characteristics are achieved. Although not shown, one or more test ports/valves may be located at or near outlet 262 to allow testing of the processed emulsion to determine completion. Filter 214 may be located at outlet 262 to remove waste products. Valve 264 allows the completed emulsion product to be transferred to a reservoir for collection. The right side of FIG. 2A provides a diagrammatic cross-sectional view of the filter cartridge 202, wherein the hollow tube 204 defines the ExC (outside the cannula) and InC (inside the cannula) spaces as described above. ECM is fed into the ExC space within the housing 220 via the inlet 228 and removed from the housing at the outlet 230. A valve 208 and one or more pumps (not shown) provide control of the ECM flow. Other details of the conduit 204 are shown in FIG. 2C , wherein the lower figure provides an exemplary 3-D perspective view of the tube.

In some embodiments of the bioreactor of the present invention, optional light sources 216, specifically light emitting diodes (LEDs), may be included to produce different effects. In one embodiment, LEDs emitting light in the blue range (eg, about 450 nm) have been shown to stimulate primary cilia. In the study by Prosseda et al., blue light was used to stimulate primary ciliary accommodation in cells and increase cell contraction. ("Optogenetic stimulation of phosphoinositides reveals a critical role for primary cilia in intraocular pressure regulation", Science Advances , April 29, 2020, Volume 6, Issue 18, DOI: 10.1126/sciadv.aay8699, Incorporated herein by reference.) Since the primary cilia of ECs must be stimulated to produce emulsion, adding optical stimulation to the mechanical stimulation provided by tunable shear stress serves to increase the overall stimulation required for emulsion production. Optical stimulation can further use intensity pulses to produce additional variable stimulation of cells. Since light is used to stimulate EC, the material forming tube 204 should be able to transmit the desired wavelength. For example, glass or transparent tube sections may be used in close proximity to the LEDs. Although the LED is shown in the lower portion of the box, it will be readily apparent that it can be positioned at various locations throughout the interior of housing 220 to increase the exposure of the EC to light.

In other embodiments, other light sources may be included in the filter cartridge or in a conduit external to the filter cartridge. For example, UV light sources (100-280 nm) can be used to enhance the sterility of products, however, care must be taken to avoid damaging the cells. Other light sources can be used to promote EC health. For example, light sources emitting at wavelengths including 660, 700, 810, and 850 have been reported to promote cell activity and growth. Control signals for operation of the light stimulation system may be provided by the system controller.

Figure 2D illustrates a possible configuration of a bank of multiple bioreactors within a production facility. Multiple such groups with any number of units (ie, filter cartridges and associated piping) can be mounted in a support frame (not shown) within the facility, which may preferably include a clean room. In one possible implementation, each group may have its own dedicated reservoir 210 for providing extracellular medium to the bioreactor unit within the group; and an emulsion collection reservoir 212 that collects the emulsion from the group within the group. The emulsion produced by the unit. In other implementations, multiple groups may receive media from one or more larger central reservoirs and output emulsion to one or more larger central tanks. Various combinations of central and dedicated reservoirs are available. To provide an illustrative example, in a large production facility, one or more sets of biosensor units can be used to produce different types of emulsions, such as full-fat, low-fat, hypoallergenic, lactose-free, or various "designer" emulsions. Any of the products, such as those described below.

Breast epithelial cells consist of two differentiated cell types organized into two cell layers, an inner layer of luminal epithelium and an outer layer of myoepithelial cells in direct contact with the basement membrane. Breast epithelial cells (MECs) can proliferate as a monolayer on plastic. However, milk production actually occurs only upon receipt of signals from ECM proteins plus hormones (prolactin, growth factors), i.e., structural and tissue-specific gene expression similar to that observed in vivo (e.g., casein genes). In some embodiments, as shown in one of the examples, MECs can be harvested from fresh milk.

Using the disclosure herein, one of ordinary skill in the art will be able to use other breast epithelial cell biomarkers to extract MECs from fresh milk or mammary glands using similar strategies. Table 2 below provides a list of biomarkers in breast tissue (see, e.g., FIG. 3 ) that may be suitable for extracting MECs for use in the methods of the present invention. Exemplary selection groups may include combinations of such biomarkers. For example, a healthy breast cell group may be assembled from a combination of biomarkers from one source or multiple sources, such as all, all epithelial, luminal, etc., of those listed in the following seven rows of the table. Source Biomarkers Human mammary epithelial cells MUC1, EpCAM, CD49f, CD24, CD29, CD133, Thy1, CD10, ALDH Mouse mammary epithelial cells CD24, CD29, CD49f, EpCAM, CD49b, Sca1, Prominin-1, CD61 Bovine mammary epithelial cells CD49f high, CD24-, CD10, KRT14, vimentin, PROCR (basal), CD49flow (luminal: CD24 negative [ducts], CD24+ [alveoli]), breast stem cells: CD49f high CD24+ (CD10, KRT14, KRT7 subsets have unknown functions) Basal/myoepithelial breast epithelial cells CD44, CD90, CD49f, cytokeratin 14, CD10, SMA, cytokeratin 5, vimentin, CD271, connexin 43, integrin α9β1, p63, calcineurin Luminal breast epithelial cells EpCAM+, CD49flo (non-cellular population), CD49f, EpCAM/ CD326, cytokeratin 7, cytokeratin 8, cytokeratin 10, cytokeratin 18, cytokeratin 19, Muc1/ CD227, CD24, CD133/ PROM1, Her2, CD117, CD166, CD282, CD340, claudin 4, C-MET, ER, PR, VDR Luminal (cell colony) breast epithelial cells EpCAM+, CD49fhi, ALDH+, Other breast cells (healthy tissue) BRCA1, CD49b, CD66, EGFR, CD321 Stroma is absent in epithelial cells (negative selection) CD26, CD34, CD39, CD45, CD61, CD73, CD140b, HPC Variable performance (healthy tissue) Erα, PR, SMA, EGFR, CD90, CD10, CD44, CD54/I-CAM Healthy Breast Cell Group All breast cells CD9, CD47, CD54, CD59, CD95, CD164 All epithelial cells CD49b, CD66 Lumen CD24, CK8/18, CK19, MUC1, GATA3, EPCAM Luminal progenitor cells CD13、CD15s/CD73/CLA Mature lumen + CD166/CD227/CD340 Myoepithelial CD10, CD90, CD200, CD29/CD142/CD271, CK5, CK14, CK17, SMA, V1M Base CD34/CD39/CD140b、CD49e Table 2

Pulsating Flow : Pulsating flow is a periodic pressure wave that travels along a flow path generated in a hollow tube such as a bioreactor. Pulsating flow systems can closely simulate the blood flow characteristics in the heart and vascular system.

Mammary endothelial cells (MECs) adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate similar changes in cultured MECs exposed to shear stress induced by laminar steady-state flow. However, MEC are exposed to a pulsatile flow environment in vivo; therefore, in this study, the effects of pulsatile flow on cell shape and orientation and actin microfilament localization in confluent bovine aortic endothelial cells (BAEC) were examined. These results demonstrate that EC can distinguish between different types of pulsatile flow environments. Furthermore, these experiments indicate the importance of engineering cell culture environments to include pulsatile flow in studies of endothelial cell biology.

Eliminating FCS from Lactation Media : Today, standard media rely on the established but outdated concept of requiring serum from unborn calves (FCS), which has been the "gold standard" since the 1960s. Over the years, the problems with FCS and the reasons why it needs to be replaced with a better alternative have been an area of considerable research attention. These issues with FCS include the fact that (a) its production is a cruel undertaking, and (b) FCS cannot be used in the production of clean milk for human consumption due to reports of viral contamination of the serum, as well as other safety issues such as endotoxins, molds, RNA contaminants, or prion proteins. Therefore, the culture medium used to culture cells used in the bioreactor of the present invention is preferably free of FCS and hormones (except prolactin) and based on the latest scientific research and cell culture technology.

In general, serum starvation makes cells unhealthy, but this adverse effect can be counteracted by flow regulation. Changes in flow intensity can induce cilia growth. Therefore, flow stimulation of cilia is required to achieve emulsion production.

Mechanical stimulation ( flow ) of healthy EC cells to counteract the elimination of FCS from lactation media : Both hormonal and mechanical signals can induce changes in gene expression in mammary epithelial cells (MECs) that ultimately occur in lactation synthesis and secretion. Using serum-free medium and a combination of hormonal and mechanical stimulation, bovine mammary epithelial cells (BMECs) are transferred from initial multicellular organic receptors to a coordinated 3-dimensional ductal network. See, for example, Figures 5A - 5B , which show breast epithelial cells with an epithelial-like phenotype and breast myoepithelial cells with an axial-like phenotype, respectively. At a structural level, there is evidence that mechanical signaling via gel release, prolactin, and a combination of the two induces changes in cell morphology consistent with the onset of phase I milk synthesis.

Endothelial cells require mechanical stimulation for healthy growth. Studies have shown that certain types of flow can provide shearing mechanical stimulation to EC cells, resulting in increased cell performance. In some embodiments, continued mechanical stimulation of healthy ECs results in increased hormone-free emulsion production. It is important to understand the nature of the stimulation applied to the cells, the principle of which is illustrated in Figure 4 . Mechanical strain occurs when a force acts directly on the cell. Under high strains, the endothelial barrier can loosen, leading to cell death and higher cell turnover. Epigenetic modifications and inflammation can occur. In contrast, mechanical shear involves parallel movement of fluid over the top of the cell, which improves cell survival at low switching conditions. Therefore, the goal is to avoid mechanical strain while increasing mechanical shear to stimulate cell function. The lower panel of Figure 4 provides a comparison of the cell layer after application of shear stress (left) and before application (right).

In some embodiments, the lactation medium and the test culture are driven by a peristaltic pump fluid, such as an ibidi™ pump system used in a smaller scale prototype bioreactor or other pump capable of generating a pulsating flow providing adjustable shear stress in a closed sterile system. In some embodiments, mechanical shear stress is assessed and continuously monitored.

Feeder Cells : Feeder or support cells 138 comprise a layer of cells that cannot divide but provide extracellular secretions to promote proliferation of other cells (ie, lactation cells 140). Feeder cells differ from co-culture systems in that only one cell type is able to proliferate.

In some embodiments, feeder cells are co-cultured with MECs and form a monolayer of lactating cells connected to the lumen of an elongated hollow fiber bioreactor. In some embodiments, feeder cells can be seeded in the extracapillary (ExC) space surrounding the hollow fibers.

Hollow Fiber Bioreactors : Hollow fiber bioreactors are hollow fiber-based 3-dimensional cell culture systems that, in some embodiments, are smaller configured in arrays with typical molecular weight cutoff (MWCO) ranges of 10-30 kDa. Semipermeable capillary membrane. Exemplary implementations are described above with reference to Figures 1A-1C , 2A-2D . When multiple tubes are used, the tubes can be bundled and contained within a cylindrical shell of polycarbonate, glass, stainless steel, or other suitable (ie, sterilizable) material to form a hollow fiber bioreactor cartridge. There are two compartments within the filter cartridge, which is also equipped with an inlet port and an outlet port: the intracapillary (InC) space 134 within the hollow fiber and the extracapillary (ExC) space 132 surrounding the hollow fiber.

Cells are seeded into the InC space 134 of the hollow fiber bioreactor 104 ( FIG. 1B ) and expanded therein to coat the inner surface of the hollow fiber 104 with a single layer. Cell culture medium is pumped through the ExC space 132 to deliver oxygen and nutrients to the cells through the hollow fiber membrane perfusion. As the cells swell, their waste products and _CO2 also permeate the hollow fiber membrane and are carried away by pumping out the emulsion accumulation medium through the InC space 134. As waste products accumulate due to increased cell mass within the InC, it may be necessary to increase the medium flow rate so that cell growth is not inhibited by waste product toxicity. After the accumulation medium has been completely enriched, that is, emulsion production has been completed, the waste product can be filtered out by a filter placed in or near the reservoir.

Supporting extracellular matrix : Breast epithelial cells can include two differentiated cell types organized into two cell layers, an inner layer of luminal epithelium and an outer layer of myoepithelial cells in direct contact with the basement membrane. Breast epithelial cells can proliferate in a monolayer on plastic. However, emulsion production occurs only when receiving signals from ECM proteins plus hormones (prolactin, growth factors) similar to the structural and tissue-specific gene expression observed in vivo (eg, casein genes). Therefore, to simulate natural emulsion production, bioreactors must recreate these structures and present the same (or similar) stimulus environment as would occur in an in vivo setting. In some embodiments, this can be accomplished by modulating the composition of the supporting extracellular matrix, culture medium, and achieving functional differentiation through co-culture of the desired cell types.

Other successful approaches can also stimulate emulsion genes. In some embodiments, cells show reorganization and form 3D structures similar to those observed in vivo and exhibit emulsion gene expression on collagen gels. In some embodiments, epithelial cells grown in laminin-rich gels undergo functional differentiation based on the expression of casein genes. Natural and synthetic polymeric materials have been studied as substitutes for ECM proteins.

In some embodiments, 3D bioprinting of breast tissue may be used. In some embodiments, a 3D culture procedure including lactation hormones (prolactin, insulin, and cortisol) can induce casein synthesis from primary mouse epithelial cells embedded in floating collagen gels. In some embodiments, differentiation medium supplemented with oleic acid, pituitary extract, and dexamethasone is shown to induce the synthesis of primary emulsions in a 3D in vitro model of bovine primary mammary epithelial cells grown on collagen-coated cell culture inserts. Components such as beta-casein, triglycerides and lactose.

Morphological and functional differentiation of cryopreserved bovine udder cells can be cultured on floating collagen gels. In some embodiments, bovine primary epithelial cells from lactating udder tissue grown on floating collagen gels exhibit a polarized architecture with a highly differentiated state, as evidenced by the observation of apical microvilli surrounded by casein-containing secretory vesicles, tight junctions, and fat droplets.

The use of collagen gels offers the advantage of mimicking in vivo conditions and allowing for relatively long-term studies.

The emulsion produced by the bioreactor of the present invention has the ability to match the nutritional content, taste and quality of emulsions obtained traditionally.

Examples

The scope of the system and method of the present invention can be further understood based on the following examples, which should not be understood as limiting the scope of the teachings of the present invention in any way.

Examples 1 : Bioreactor

As described above, the bioreactor of the present invention is designed to reflect the natural environment of breast alveoli and ducts to achieve healthy epithelial cells (EC) and optimal emulsion production. The goal of the systems and methods is to provide FCS-free or substantially FCS-free cultures of primary EC using programmed control of conditions and cell biology. In some embodiments, sustained mechanical stimulation of healthy ECs results in an increase in the hormone-free emulsion production process. (Note that the hormone prolactin is still necessary.) In some embodiments, a peristaltic pump is used to fluidly drive the lactation medium and test culture to create adaptable shear stress in a closed sterile system. In some embodiments, mechanical shear stress can be assessed and monitored continuously or periodically. In some embodiments, the bioreactor is a modified reperfusion reactor with a hollow fiber module, controlled by a peristaltic pump to generate continuous unidirectional, oscillating and pulsatile flow with adjustable flow rates. In some embodiments, lactation cells grow or coat the interior surface of hollow tube 104. The inlet of each hollow tube was connected to a pump to provide flow stimulation via pulsatile flow. Depending on the scale (volume) of the entire bioreactor system, the emulsion can be collected in batches at regular intervals, ranging from daily to weekly to monthly. The system supplies extracellular medium through a permeable and/or semipermeable membrane in the cannula to enrich lactation cells inside the tube. The ECM may be periodically replenished and/or replaced through processing sessions.

Example 2 : breast epithelial cells (MEC) Collection

Fresh milk (i.e., from Genset or Galloway cows) was defatted by low-speed centrifugation at typical refrigeration temperature (~4°C). The defatted milk was removed and the remaining total cell pellet was washed multiple times in phosphate-buffered saline (PBS) and resuspended in PBS. Immunomagnetic separation was used to separate MECs from total milk cells and remove leukocytes, which were separately collected for further use. Immunomagnetic separation involved incubating the total milk cell suspension with magnetic beads (Dynabeads) coated with primary monoclonal antibodies against cytokeratin 8 (K8.13) (Boutinaud et al., 2008). The resulting antibody-bound MECs are then washed and the purified MECs are resuspended in PBS. MECs are then cultured for 1 week in complete medium to ensure adequate cell proliferation. Complete medium includes DMEM/F12 (alternatively EpiCult) supplemented with 2% FBS or FCS replacement, 5 μg/ml insulin, 100 ng/ml corticosteroid L-glutamine, and antibiotics as appropriate.

Examples 3 : MEC Cultivation and lactation

Inoculation of breast myoepithelial and epithelial cells (MEP) was performed in complete medium and maintained until a monolayer was established. Low shear stress was used for influx in complete medium for cell proliferation (1 week). Shear stress was 0.007 dyn/cm ² . Subsequently, settling and attachment periods were performed in hollow fiber reactors for about 4 hours. Physical shear stress of about 5 dyn/cm ² (i.e. low shear stress) was applied over a period of several days for proliferation and monolayer formation. Medium was changed every 3 days or fed-batch culture.

Subsequently, serum extraction is performed and lactation medium is replenished to induce lactogenic differentiation. Pulsating flow is initiated, wherein the pump is controlled to periodically change the fluid flow to induce variable shear stress at different levels in the range of about 2 to 75 dyn/cm ² over a period of at least one week, preferably about 2 to 3 weeks. The total culture time will vary depending on the size (volume) of the system, with smaller scale systems taking about 2 weeks and larger scale systems taking multiple months to recirculate the accumulated medium through the reactor for complete culture. Over a multi-week period, lactation medium changes are performed every 3 days or each batch of culture. The product is then harvested after the culture is completed. Post-harvest processing includes preservation by one or more of pasteurization, freezing, or dehydration. Quality control testing includes evaluation of bacteria, molds, fungi, and major components.

Example 4 : Use matrix to replace feeder cells

However, feeder cells are generally suitable for supporting stem cell co-cultures, which can create the possibility of transferring animal pathogens and/or undesirable immune responses that can affect the production of the emulsion when dealing with different species. The matrix gel matrix structure provides a suitable and functional support structure for cells that require feeder cells.

For the application of Matrigel (BD Biosciences, Bedford, MA), 500 μL of BD Matrigel was transferred to each well of a six-well plate on ice and spread by gentle shaking. The plates were then incubated at 37°C for 2 hours to allow the BD Matrigel to polymerize into a gel. Subsequently, a suspension of reprogrammed HDFs (human dermal fibroblasts) (2.4×10 ⁴ /cm ² ) was transferred to the plates with BD Matrigel matrix application in DMEM medium containing 10% FBS and the plates were returned to the incubator. After 48 hours, the medium was replaced with supplemented DMEM/F-12 (including supplementary factors as mentioned above) and changed daily to form hiPSC colonies. iPSC cultures were maintained and tracked for 10 passages.

Example 5 : Preparation of feeder cells by conventional methods

CF-1 MEFs at passage 3 (P3) at 80-90% confluence were inactivated with 10 μg/ml MMC (Hisun Pharmaceutical Company, China) at 37°C for 0, 0.5, 1.0, 1.5, and 2.0 hours. After incubation, cells were washed 6 times with PBS, trypsinized, centrifuged at 180×g for 5 minutes, and resuspended in MEF medium. Cells were counted and frozen for later use.

Examples 6 : Preparation of feeder cells using the suspension attachment method

We prepared feeder cells by SAM according to Figure 1. Briefly, P3 CF-1 MEFs were cultured for four days, digested into single cells with 0.25% trypsin/EDTA (Dalian Meilun Biotech Co., Ltd, China), and collected in 50 ml centrifuge tubes. Cells were seeded in 10 cm culture dishes at 8 × 10 ⁴ -1.1 × 10 ⁵ cells/cm ² . MMC (10 μg/ml) was added after 2.0-3.0 h at 37°C. Discard the MMC-containing medium 0.5-3.5 hours after treatment. Cells were then washed 6 times with PBS, trypsinized, centrifuged at 180×g for 5 min, and resuspended in MEF medium. Cells were counted and frozen for later use.

Example 7 : Use 3D (3D) Preparation of feeder cells by suspension method

After the CELLSPIN system (5-75 RPM, CELLSPIN system with glass ball stirring pendulum, Integra Bio-Sciences, Switzerland) was connected to the incubator, the tubes were rotated by autoclaving. Feeder cells were prepared by 3DSM. Briefly, four-day-old P3 CF-1 MEFs were digested into single cells by 0.25% trypsin/EDTA and collected into 50 ml centrifuge tubes. Cells were transferred at a density of 0.5-1.3 × ¹⁰⁶ cells/ml to spinner flasks with glass ball stirring pendulums, these flasks accommodate volumes of 25-1000 ml. Add MMC at 10 μg/ml. After incubation 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 three times with PBS, resuspended in MEF medium, counted, and cryopreserved for subsequent use.

Example 8 : Produces feeder cells / or support matrix for bioreactors

Bovine embryonic stem cells (ESCs) are powerful tools for agricultural and biomedical applications and have been successfully cultured and differentiated using supporting feeder cells ( Cell Reprogram . 2012 Dec;14(6):520-9. doi: 10.1089/cell.2012.0038). Currently commercially available feeder cells are only mouse or human derived feeder cells. One goal of the present systems and methods is to generate customized feeder cells derived from bovine fibroblasts. Potential feeder cells that can be used in embodiments of the present methods include fibroblasts, mEPs, and myoepithelial cells and mEPs on fibroblast extracellular matrix.

Alternative synthetic methods can be used as carriers for emulsion-producing cells. The following examples identify several of these methods.

Example 9 : Laboratory grown type FCS Alternative

Upon activation, PBMCs secrete a potent cocktail of pro-survival factors that help them fight infection. These are secreted into the blood and become part of the serum phase. Co-culture experiments have shown that supplementing cells with the supernatant phase of cultured PBMCs significantly prolongs their survival. In cell culture, this is achieved by "tight packaging" the cells to be dependent on microenvironmental factors, i.e., suspension at higher cell density to achieve healthy cell growth and recovery after freezing. Addition of innate immune cell supernatant provides a survival factor as strong as an equivalent direct survival stimulus.

PBMC were extracted from peripheral blood of volunteers using Percoll gradient. After supplying cells containing A) LPS, B) opsonized beads, C) mechanical stimulation in an ibidi ^™ pump system, the cells were maintained in the stimulated state and the supernatants were enriched for 24 to 48 hours and subsequently collected. The supernatant phase was separated from the cell fraction and used to replenish the culture medium. For future augmentations, conditionally immortalized ER-Hoxb8 hematopoietic precursor cells can be used and differentiated into PBMCs as needed to provide an unlimited supply of PBMCs.

Examples 10 : EpiCult™ plus breast epithelial serum-free medium

Successfully tested in breast cells using cell culture techniques adapted from routine modifications of epithelial cell culture methods from the FCS-free database (https://fcs-free.org/fcs-database).

Figures 5A-5E provide representative growth curves and images. Photographic images of breast tissue cells were collected using a viable cell microscope with a Kodak imaging system, wherein Figure 5A shows breast epithelial cells with an epithelial-like phenotype, and Figure 5B shows breast myoepithelial cells with an axonal-like phenotype. Figure 5C provides growth rate curves for breast tissue cell lines. 1x10 ⁴ cells were inoculated and then measurements were obtained 4 days later. The values of three independent cell samples per day were compared by using an unpaired t-test. The significance between the cell growth rates of breast epithelial cells (green) and breast myoepithelial cells (red) was determined on day 3 (p=0.007) and day 4 (p=0.0228). These values are highlighted in the graphs by asterisks. Figures 5D-5E are graphs showing that the addition of 2% PBMCs ( 5D ) or 1:3 neutrophil conditioned medium (ie, supernatant) ( 5E ) provides comparable survival stimulation as the addition of pro-survival mediators dbcAMP or LPS and PGE2 to cultured immune cells.

In some implementations, methods guided by learning machines (AI) can be used to optimize culture medium composition and optimize and adjust processing parameters.

Example 11 : Performed using breast epithelial precursor cells extracted from fresh milk In vitro emulsion production

Bioreactors were inoculated with udders in complete medium supplemented with 2% FCS (FCS replacement), 5 μg/ml insulin, 100 ng/ml cortisol L-glutamine, antibiotics, IGF-I or EGF as appropriate. epithelial cells (MEP). Complete media can be selected from DMEM, F12 or EpiCult. MEPs were grown until a monolayer was established. A lower shear rate was applied in complete medium to maintain cell proliferation for 1 week. The settling and connecting time period in the hollow fiber reactor is 4 hours. The medium was maintained at higher shear stress for proliferation and monolayer formation for 2 days. Perform a complete medium change or batch feed culture every 3 days. Serum draws were performed and lactation medium was supplemented to induce lactational differentiation. Lactation medium consists of DMEM/F12 (alternatively EpiCult) supplemented with 2% FBS (or FCS replacement), L-glutamine, 1-5 μg/ml prolactin (or shear stress as defined above) . Pulsating flow is induced by alternately switching the pump between different pump rates (eg, high, medium, and/or low) to produce shear stresses in the range of ² to 75 dyn/cm. This step lasts for at least a week, and preferably a period of about 2 to 3 weeks. As will be apparent to those skilled in the art, the total processing time will depend on the system size (volume). Small-scale systems can take as little as 2 weeks to produce a fully cultured emulsion, while larger-scale systems can take up to several months for full culture. Change the lactation medium every 3 days or feed the culture in batches. The product was then harvested and processed as described in Example 3.

Example 12 : Genetic engineering to induce hyperlactation and other modifications

CRISPR technology can be used to induce hyperlactation in cells. Referring to the diagram in Figure 6 , the following scheme can be used: CSN2 polymorphisms (SNPs): His220, G->A in Glu223; LALBA polymorphism: in the 5'LALBA promoter; PRLR SNPs: rs62355518, rs10941235, rs1610218, rs34024951, rs9292575; PRL SNPs: rs849872; Jak2 overactivated gain-of-function mutation: V617F. In addition, pharmaceutical interventions may include the use of retinoids and ATRA to target CSN3 expression.

SNPs can be modified (via CRISPR) to reduce the lactose-free content of the diet, lactose-free emulsions: CSN2 (Glu340, Thr174, Lys14), B4GALT1 (T224A) .

A hypoallergenic lotion can be produced by introducing point mutations at base pairs 186 and 213 of the CSN1S1 gene (corresponding to CDS 84-123 (CSN1S1) and CDS 87-123 (CSN1S2) sites) using CRISPR cluster bombs.

Table 3 below lists many of the potential modifications that can be achieved through genetic engineering of mEPs and mEP-derived cell lines. Gene Polymorphism Location result CSN2 Point mutations, SNPs G->A in His220, Glu223 Excessive lactation CSN2 Point mutations, SNPs Glu340, Thr174, Lys14 Reduced lactose content in diet, lactose-free emulsions CSN1S1 Point mutations; SNPs bp186 and 213; CDS 84-123 Hypoallergenic CSN1S2 Point mutations; SNPs CDS 87-123 Hypoallergenic CSN3 - Retinoids and derivatives or ATRA agonists Increased CSN3 content B4GALT1 Point mutations, SNPs T224A Reduced lactose content in diet, lactose-free emulsions LALBA Point mutations; SNPs 5'LALBA promoter Excessive lactation PRLR Point mutations, SNPs rs62355518, rs10941235, rs1610218, rs34024951, rs9292575 Excessive lactation PRL Point mutations; SNPs rs849872 Excessive lactation Jak2 mutation V617F Jak2 overactivation ; excessive lactation CSN1S1 CRISPR CSN1S2 replaces CSN1S1 Hypoallergenic table 3

Particularly desirable modifications achieved by the methods of the present invention include the production of A2-enriched emulsions from cells.

The foregoing description describes embodiments of systems and methods for in vitro emulsion production. Although specific exemplary embodiments have been described, it will be apparent that various modifications and changes can be made to these embodiments without departing from the broader scope of the invention. This detailed description is therefore not to be taken in a limiting sense, and the scope of the various embodiments is defined only by the appended claims and the full scope of equivalents to which such claims are entitled.

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.

without

Those skilled in the art should understand that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way.

[ Fig. 1A] is a diagrammatic view of the alkaline assembly of the bioreactor of the present invention according to one embodiment; [ Fig. 1B] Illustration of elements within a hollow tube simulating a support attached to the lumen of the tube A monolayer of cultured milk vesicle cells and feeder cells in their native environment; [ Figure 1C] is a diagrammatic view from a cross-section of a tube showing a monolayer of lactation cells attached to the inner surface of a hollow fiber; [ Figure 1D] Description Laminar flow in a hollow fiber bioreactor; [ Figure 1E] is a diagrammatic view of an alternative tube configuration.

[ Figures 2A-2C] Schematically illustrate another embodiment of the bioreactor of the present invention, wherein Figures 2A and 2B respectively provide a front view and a side view of the embodiment of the bioreactor assembly; Figure 2C shows a horizontal view with a filter cartridge Bottom view of the assembly in cross-section and 3-D image of the hollow tube; [ Figure 2D] illustrates an exemplary layout of a set of bioreactors in an emulsion production facility.

[ Figure 3] A model illustrating the formation of a monolayer of co-cultured milk vesicle cells and feeder cells connected to the lumen of a hollow fiber or hollow tube bioreactor. Breast epithelial cells are composed of two differentiated cell types organized into two cell layers. The inner layer of luminal epithelial cells and the outer layer of myoepithelial cells are in direct contact with the basal membrane. Provided by the hormone prolactin, the milk vesicles absorb nutrients from the blood supply and produce breast milk.

[ Figure 4] Comparison of the effects of different types of flow mechanical stimulation (shear stress and shear strain) on healthy EC cells and endothelial cells. The lower panel provides SEM micrographs of EC cells exposed to shear stress and not exposed to shear stress.

[ Figures 5A-5B] are micrographs of mammary epithelial cells with an epithelial-like phenotype and mammary epithelial cells with an axonal-like phenotype, respectively; [ Figure 5C] shows the growth rate curve of 1×10 ⁴ cells inoculated with a mammary tissue cell line and then measured 4 days later. The values of three independent cell samples per day were compared by using an unpaired t-test. The significance between the cell growth rates of mammary epithelial cells (green) and mammary myoepithelial cells (red) was determined on day 3 (p=0.007) and day 4 (p=0.0228). These values are highlighted by asterisks in the graph; [ Figures 5D and 5E ] are graphs showing the growth rate of the conditioned medium of co-cultured PBMCs.

[ Figure 6] is a diagram of inducing hyperlactation in cells using CRISPR.

100: Bioreactor flow system

102: Culture vessel/tube

104: tube

106:Pump

108:Valve

110: Storage device

112:Reservoir

116:Valve

118:Loop

120: Shell

122:Controller

128: Inlet/Inlet Pipeline

130:Exit/Exit Pipe

160:Entrance

162:Export

Claims (32)

An apparatus for in vitro emulsion production, the apparatus comprising: A reactor assembly comprising: An elongated shell configured to retain an extracellular medium; One or more elongated hollow tubes disposed in longitudinal alignment within the extracellular medium within the shell, the one or more tubes having an inlet end, an outlet end, and an inner surface configured for attachment of a monolayer of lactating cells, wherein at least a portion of the one or more tubes comprises a semipermeable material configured to permit diffusion of the extracellular medium into the hollow tubes, wherein the extracellular medium comprises a nutrient solution for maintaining the lactating cells; a pump fluidically connected to the one or more tubes, the pump being configured to generate a pulsating or oscillating flow of a lactation medium through the one or more tubes to induce shear stress on the lactating cells, wherein the pump is controlled to alternate between different shear stress levels within a predetermined range, wherein the variation in shear stress stimulates the lactating cells to produce a milk product; a supply loop for supplying the extracellular medium and circulating the extracellular medium through the housing; and a reservoir fluidically connected to the outlet end of the one or more tubes, the reservoir being configured to collect the milk product. The apparatus of claim 1, further comprising a feedback loop disposed proximate the outlet end of the one or more tubes, the feedback loop being used to recirculate the emulsion product to the inlet end of the one or more tubes for further enrichment. The device of claim 1, wherein the predetermined range of shear stress level is 5 to 15 dyn/cm ² . The apparatus of claim 1, wherein the supply loop comprises a reservoir configured to remove used extracellular medium and add fresh extracellular medium. The apparatus of any one of claims 1 to 4, wherein the lactating cells are co-cultured with feeder cells. For example, the device of claim 5, wherein the feeder cells are peripheral blood mononuclear cells. An apparatus as claimed in any one of claims 1 to 4, wherein the lactating cells are isolated from milk of healthy cows. The apparatus of any one of claims 1 to 4, wherein the lactating cells are extracted from healthy breast tissue identified using a panel of biomarkers selected from the group consisting of 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. Such as requesting the device of any one of items 1 to 4, wherein the lactation-producing cells are selected from the group consisting of all breast cells, all breast epithelial cells, breast luminal cells, breast luminal precursor cells, mature breast luminal cells, and breast myoepithelial cells. Cells and breast stromal cells are extracted from healthy breast tissue. The device of any one of claims 1 to 4, wherein the lactation cells are genetically modified to induce hyperlactation. The device of any one of claims 1 to 4, wherein the lactation cells are genetically modified to produce an emulsion with one or more of hypoallergenicity, reduced lactose, and increased A2 beta-casein. The device of any one of claims 1 to 4, wherein the one or more tubes are coated with collagen. An apparatus as claimed in any one of claims 1 to 4, further comprising a system controller configured to generate control signals to the pump and the supply loop. A device as claimed in any one of claims 1 to 4, wherein the one or more tubes are formed by a semipermeable capillary membrane. An apparatus as claimed in any one of claims 1 to 4, wherein the one or more tubes have a mixed structure comprising portions of impermeable material and portions of permeable or semipermeable material. A device as in any one of claims 1 to 4, wherein the inner surface of the one or more tubes is first bound to a layer of supporting matrix, wherein the lactating cells attach to the unbound surface of the supporting matrix, and wherein the lactating cells form a monolayer of the lactating cells on top of the supporting matrix. The device of any one of claims 1 to 4, further comprising a mechanical stimulation assembly configured to apply a squeezing force to the one in a direction from the inlet end toward the outlet end. or outside of multiple tubes. The apparatus of any one of claims 1 to 4, further comprising one or more light sources disposed within the reactor assembly, the reactor assembly being configured to expose the lactation cells to light stimulation. The device of claim 18, wherein the one or more light sources are light emitting diodes (LEDs) that emit light at 450 nm. The equipment of any one of claims 1 to 4, wherein the lactation medium includes one or more of EpiCult™ Plus medium, laboratory growth FCS replacement medium, and FCS-free medium. The device of any one of claims 1 to 4, wherein the lactation medium further contains prolactin. An emulsion production facility comprising a plurality of interconnected devices as described in any one of claims 1 to 4. A method for producing milk in vitro in an apparatus as claimed in any one of claims 1 to 4, the method comprising: supplying the lactation medium to the one or more tubes, wherein the lactation medium comprises prolactin; forming a monolayer of lactating cells attached to the inner surface of the one or more tubes; controlling the pump to alternate between different shear stress levels within a predetermined range, wherein the variation in shear stress stimulates the lactating cells to produce milk product; and collecting the milk product when a predetermined milk quality is achieved. The method of claim 23, further comprising recirculating the emulsion product to the one or more tubes via a feedback loop to enrich the emulsion product until the predetermined emulsion quality is achieved. The method of claim 23, wherein the lactating cells are co-cultured with feeder cells. The method of claim 25, wherein the feeder cells are peripheral blood mononuclear cells. The method of any one of claims 23 to 26, wherein the lactation cells are isolated from the milk of healthy cows. The method of any one of claims 23 to 26, wherein the lactating cells are extracted from healthy breast tissue identified using a panel of biomarkers selected from the following: 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. The method of claim 23 to 26, wherein the lactation cell lines are selected from the group consisting of all breast cells, all breast epithelial cells, breast luminal cells, breast luminal precursor cells, mature breast luminal cells, and breast myoepithelial cells. Cells and breast stromal cells are extracted from healthy breast tissue. The method of any one of claims 23 to 26, wherein the lactation cells are genetically modified to induce hyperlactation. The method of any one of claims 23 to 26, wherein the lactating cells are genetically modified to produce milk having one or more of hypoallergenicity, reduced lactose, and increased A2 β-casein. The method of any of claims 23 to 26, wherein the lactation medium comprises one or more of EpiCult™ Plus medium, a laboratory-grown FCS replacement medium, and a medium without FCS.