Pulsatile flow culture of mammary celltypes for milk secretion

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

PULSATILE FLOW CULTURE OF MAMMARY CELL TYPES FOR MILK SECRETIONRELATED APPLICATIONS

[0001] This application claims the benefit of the priority of U.S. provisional application No.63 / 358,464, filed July 5, 2022, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

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

[0003] The demand for worldwide milk production continually increases in response to a range of factors, including global population growth and changing, i.e., westernization, of diets in Asian countries. Worldwide consumption of dairy is projected to increase by over $100B over the coming decade. This, in turn, is placing increasing pressure on natural resources, increasing animal welfare concerns, and leading to increased deforestation alongside greater production of greenhouse gases. Dairy consumption in the U.S. accounts for about 2% of the country’s greenhouse gas emissions. To meet the worldwide demand, around 270 million dairy cows are farmed every day. Every component in the milk has been associated with an array of both positive and negative health effects.

[0004] Milk is a complex colloidal matrix that contains milk fat, lactose, and a number of different components, including naturally occurring hormones. The sensory and functional characteristics of milk stem from its micronutrient profile that has proven costly and difficult to replicate. In general, the gross composition of cow's milk in the U.S. 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), breed (Holstein, Jersey), the animal's feed, and the stage of lactation.

[0005] Studies have shown that the fat and protein content of milk is based on the breed of cattle. Holsteins have the lowest fat and protein content, while Jersey and Guernsey breeds have the highest fat and protein content. Even within a single herd, the milk protein can range from 1.57% to 4.66%, with an average of 3.05%; while the milk fat ranges from 1.77% to 5.98%, with an average of 3.76%. The food source, temperature, humidity, and seasonality allplayed important roles to the variation of milk quality in milk production. To address variations in milk composition, a common practice is to blend the milk from different cows together in bulk tanks, producing a relatively consistent composition of milk year round in the U.S.

[0006] Another issue with milk quality is the presence of naturally occurring hormones found in milk. These hormones include Prolactin (15.4 ± 1 ng / mL), IGF-1 (4 ± 1 ng / mL), PGE2 (2.4 ± 0.3 ng / mL), PGF2a (2 ± 0.5 ng / mL), TXB2 (1 ± 0.5 ng / mL), Corticosteroids (14 ± 4 ng / mL), Testosterone (0.09 ± 0.03 ng / mL), 5a-esteroids (3 ± 1 ng / mL), Progesterone (12 ± 2 ng / mL), Esterone (0.13 ng / mL), 17P-estradiol (0.02 ng / mL), Esteriol (0.027 ± 0.01 ng / mL). Additionally, hormones and other foreign agents, such as antibiotics and pharmaceutics, are often present in industrial-scale milk arising from the need to treat an entire herd for infections during their milk production (i.e., chronic udder infections).

[0007] While there is little debate about the presence of physiologic concentrations of these hormones, the potential biological effects of such hormones on animals and humans may not be well understood and can be profound. Further, the presence of hormones can raise significant safety concerns about dairy foods, especially steroid hormones like estrogens. Some evidence suggests potential links with breast and prostate cancers. Accordingly, special attention is warranted, especially during major developmental periods, e.g., perinatal and pubertal periods.

[0008] To this end, and with respect to the considerable progress in developing of analytical methods and bioassays, it is important to clarify the potential impact of the presence of hormones, especially steroid hormones like estrogens, when they are a common component of diets and being consumed at a regular basis for years.

[0009] Dietary alternatives to dairy products are found in plant-based milk and milk products. Milk from plants such as almond, soy, cashew, and oat lack the naturally occurring hormones found in animal-based milk and are becoming increasingly popular as a good source of protein without raising the issues of dairy intolerance that many people experience. However, the plant-based milks from these alternative sources tend not to be as effective in providing the functionality of animal-based milk that translates to production of other dairy products such as cheese, butter, and yogurt. Furthermore, the increased popularity of plant-based milks has brought to light other sustainability concerns, for example, the widespread deforestation that has been employed to expand soy production, or the large amounts of water required for almond production in areas experiencing long-term drought.

[0010] Another area of interest within the dairy business is the alternative milk product called“A2 milk”. Cow’s milk generally contains two types of P-casein proteins: Al and A2 types, which differ in amino acid histidine or proline, respectively, at position 67 of the protein structure. The hypothesis is that traditional bovine milk that contains Al P-casein proteins may contribute to negative health effects, including digestive discomfort, intestinal inflammation, and milk allergies, whereas A2 P-casein does not appear to induce these effects.

[0011] Both the Al P-casein and the A2 P-casein proteins contain 209 amino acids (AA). However, upon digestion, the Al P-casein releases P-casomorphin-7 (BCM-7), which triggers a cascade of events that increase inflammation and gastrointestinal discomfort. Therefore, consumption of milk containing Al P-casein is hypothesized to be associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. A recent study confirmed that subjects consuming A1 / A2 P-casein milk presented greater digestive symptoms associated with lactose intolerance, whereas the consumption of A2 P-casein milk was not found to aggravate these symptoms. Inflammatory markers, such as IL-4, IgG, IgE, and IgGl, were significantly lower in A2 P- casein milk consumers. Currently, no economic solutions to this Al P-casein issue have been developed due to the close similarity of the two types of P-casein, Al and A2 types and as cows producing milk rich in the Al -protein tend to belong to the breeds that produce milk in high quantities (high-producing cows), for example Holstein cows.

[0012] Milk and dairy products, including cream, butter, yogurt, and cheese, constitute a significant component of the human diet, providing important sources of protein, vitamins, and minerals. For many people, dairy is the easiest way to obtain supplementary nutrients to keep the heart, muscles, and bones healthy and functioning properly. However, the milk quality related issues discussed above have plagued the dairy industry for many years and urgently need a solution.

[0013] The need for improvements to milk production technology is apparent, driven by several factors including the current unsustainable approach to producing milk through cattle farming (inefficient use of land and agricultural resources, greenhouse gas production, and more); the lack of comparable alternatives 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, IL)), incorporated herein by reference, discloses a method for in vitro milk production by employing an array of mammary organoids seeded on tertiary-branched, resilient duct scaffolding. In an exemplaryimplementation, the milk production system includes an array of vessels, each vessel comprising a plurality of mammary organoids (MO); a nutrient supply reservoir operable to feed each vessel and a milk collection module. The key component of the system is the MOs, which are the mammary epithelial cells forming multicellular three-dimensional structures (mammary organoids) embedded in matrix. Upon seeding of the scaffolding with the MO, estrogen and progesterone are instrumental in inducing growth and morphogenesis of epithelium via induction of paracrine signaling between mammary stroma and epithelium comprising the seeded MOs. The MOs then start secreting milk for a period of between 10-21 days in a medium containing prolactin, nutrients, and growth factors. While the Wilk patent discloses a method for in vitro production of milk, it does not provide a range of options for modifying the milk product in terms of cellular structure so as to enhance the nutritional content, reduce the reliance on hormones, select the preferable A2 P-casein, and other “designer milk” products, e.g., hypoallergenic milk, that will serve to provide a more sustainable and nutritional food source to meet growing worldwide demand.

[0015] In view of growing demand for dairy products, the present invention is directed to a more sustainable and versatile approach to milk production, to overcome the environmental and animal welfare challenges faced by the current dairy industry while providing a reliable, high quality source safe, clean milk.SUMMARY

[0016] According to embodiments of the invention, a system and method are provided for in vitro milk production using mammary cells cultured for enhanced cell proliferation and milk and milk-component secretion. Enhancement of cell proliferation is achieved at least in part through mechanical stimulation via the application of flow to the culture medium and alteration of flow through a variety of different methods, including, but not limited to, unidirectional laminar flow, turbulent flow, pulsatile flow, and oscillating flow. The constant mechanical stimulation provided by the inventive system enables the production of healthy epithelial cells (ECs) and production of milk that is substantially hormone-free (other than prolactin). The system and method disclosed herein are generally applicable to production of cellular proteins and dairy for food culture and for bioindustrial use.

[0017] In one aspect of the invention, an apparatus for in vitro milk production includes: a reactor assembly comprising: an elongated shell configured for retaining an extracellularmedium; one or more hollow tube disposed within the extracellular medium generally in longitudinal alignment within the shell, the one or more tube having an inlet end, an outlet end, and an inner surface configured for attaching a monolayer of lactating cells, wherein at least a portion of the the hollow tube is formed from a semipermeable material configured to permit diffusion of the extracellular medium into the one or more tube, wherein the extracellular medium comprises a nutrient solution for maintaining the lactating cells; a pump in fluid connection with the one or more tube, the pump configured to generate a pulsatile or oscillating flow of a lactating medium through the hollow tubes to induce a shear stress on the lactating cells, wherein the pump is controlled to alternate among different shear stress levels within a predetermined range, wherein variation of the shear stress stimulates the lactating cells to produce a milk product; a supply loop for supplying and circulating the extracellular medium through the shell; and a reservoir in fluid connection with the outlet end of the one or more tube, the reservoir configured for collecting the milk product. The apparatus may further include a feedback loop disposed near the outlet ends of the one or more tube for recirculating the milk product into the inlet end for further enrichment. In some embodiments, the predetermined range of shear stress levels is 2 to 75 dyn / cm2. The supply loop may include a reservoir configured for removing used extracellular medium and adding 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 may be extracted from healthy mammary 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, VIM, CD34 / CD39 / CD140b, and CD49e. In some embodiments, the lactating cells may be extracted from healthy mammary tissue selected from 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 may be genetically modified to induce hyperlactation, or to produce milk that is one or more of hypoallergenic, reduced lactose, and has increased A2 P-casein proteins. In some embodiments, each hollow tube may be coated with collagen. The one or more tube is formed from a material at least a portion of which is a semi-permeable capillarymembrane. In some embodiments, the inner surface of the one or more tube is first bound with a layer of a support matrix, wherein the lactating cells are attached to a non-bound surface of the support matrix, and wherein the lactating cells form a monolayer of the lactating cells on top of the support matrix.

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

[0020] An optional mechanical stimulation assembly may be included to apply a squeezing force to an exterior of the one or more tube in a direction from the inlet end toward the outlet end. An optional one or more light source may be disposed within the reactor assembly to expose the lactating cells to light stimulation. In some embodiments, the one or more light source is a light emitting diode (LED) that emits light at 450nm.

[0021] The lactating medium may include one or more of an EpiCult™ Plus medium, a Lab- grown FCS alternative medium, and a FCS free medium, and may further include prolactin.

[0022] In another aspect of the invention, a milk production facility may be constructed by interconnecting a plurality of the above-described apparatuses.

[0023] In still another aspect of the invention, a method of in vitro milk production in the above-described apparatus includes: supplying the one or more tube with the lactating medium, wherein the lactating medium comprises prolactin; forming a monolayer of lactating cells attached on the inner surface of the one or more tube; and controlling the pump to alternate among different shear stress levels within a predetermined range, wherein variation of the shear stress levels during a processing period stimulates the lactating cells to produce a milk product. The method may further include recirculating the milk product via a feedback loop into the one or more tube to enrich the milk product until the predetermined milk quality is achieved In some embodiments, the lactating cells are co-cultured with feeder cells. The feeder cells may be peripheral blood mononuclear cells. The lactating medium may be one or more of an EpiCult™ Plus medium, a Lab-grown FCS alternative medium, and a FCS free medium

[0024] The lactating cells may be isolated from the milk of healthy cows in some embodiments. In other embodiments, the lactating cells may be extracted from healthy mammary 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, VIM, CD34 / CD39 / CD140b, and CD49e.In other embodiments, the lactating cells may be extracted from healthy mammary tissue selected from 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.

[0025] The lactating cells may be genetically modified to induce hyperlactation and / or to produce milk that is one or more of hypoallergenic, reduced lactose, and has increased A2 P- casein proteins.DESCRIPTION OF THE DRAWINGS

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

[0027] FIG. 1A is a diagrammatic view of the basic components of the inventive bioreactor according to one embodiment; FIG. IB diagrammatically illustrates elements within a hollow tube that simulate the native environment for supporting a monolayer of co-cultured alveolar cells and feeder cells attached to the inner lumen of the tube; FIG. 1C is a diagrammatic view with tube cross-sections showing monolayers of lactating cells attached to the inner surface of the hollow fibers; FIG. ID illustrates laminar flow in the hollow fiber bioreactor; FIG. IE is a diagrammatic view of an alternative tube configuration.

[0028] FIGs. 2A-2C diagrammatically illustrate another embodiment of the inventive bioreactor, where FIGs. 2A and 2B provides a front and side views, respectively, of an embodiment of a bioreactor assembly; FIG. 2C shows a bottom view of the assembly with a cross-section of the cartridge and a 3-D image of the hollow tubes; FIG. 2D illustrates an exemplary layout of a bank of bioreactors in a milk production facility.

[0029] FIG. 3 diagrammatically illustrates a model of the formation of a monolayer of cocultured alveolar cells and feeder cells attached to the inner lumen of the hollow fiber or hollow tube bioreactor. The mammary epithelium consists of two differentiated cell types organized into two cell layers. An inner layer of luminal epithelial and an outer layer of myoepithelial cells in direct contact with the basal membrane. Prompted by the hormone prolactin, the alveoli take up nutrients from the blood supply, and produce breast milk.

[0030] FIG. 4 compares effects of different types of mechanical stimulation by the flow, Shear Stress vs Shear Strain, to the endothelial cells for healthy EC cells. The lower panelprovides SEM micrographs of EC cells with and without exposure to shear stress.

[0031] FIGs. 5A-5B are photomicrographs of mammary epithelial cells possessing an epithelial-like phenotype and mammary myoepithelial cells possessing a spindle-like phenotype, respectively; FIG. 5C shows growth rate curves of breast tissue cell lines IxlO4cells were seeded and measurements were subsequently taken on the 4 following days. Values for three independent cell samples for each day were compared by using unpaired t-tests. Significance between mammary epithelial cells (green) and mammary myoepithelial cells (red) cell growth rates was thereby determined for day 3 (p=0.007) and day 4 (p=0.0228). These values are highlighted in the diagram through asterisks; FIGs. 5D and 5E are plots showing co-culture PBMC -conditioned media growth rates.

[0032] FIG. 6 is a diagram of the induction of hyperlactation in cells by CRISPR.DETAILED DESCRIPTION OF EMBODIMENTS

[0033] To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:

[0034] As used herein , the term “amino acids” refers to the molecular basis for constructing and assembling proteins , such as enzymes. Peptide bonds ( i.e., polypeptides ) are formed between amino acids and assemble three - dimensionally (3-D) . The 3- D assembly can influence the properties , function , and conformational dynamics of the protein . Within biological systems, the protein may: (i) catalyze reactions as enzymes; (ii) transport vesicles , molecules , and other entities within cells as transporter entities; (iii) provide structure to cells and organisms as protein filaments; (iv) replicate deoxyribonucleic acid (DNA); and (v) coordinate actions of cells as cell signalers.

[0035] As used herein , the term “nucleotides” 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 . The specific purines are adenine (A) and guanine (G) . The specific pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA , whereas U is found in RNA . The genetic code defines the sequence of nucleotide triplets (i.e., codons) for specifying which amino acids are added during protein synthesis.

[0036] As used herein , the term "genes” refers to regions of DNA . Amino acid sequences in the proteins , as defined by the sequence of a gene , are encoded in the genetic code.

[0037] As used herein, a recombinant nucleic acid or protein is a nucleic acid or proteinproduced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012).

[0038] The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of 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, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intramolecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide. The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0039] As used herein, the term “bioreactor” refers to vessels or tanks in which whole cells or cell-free enzymes transform raw materials into biochemical products and / or less undesirable by-products. The bioreactor is designed and operated to provide the environment for product formation, in this case, a milk product. Industrial bioreactors may be operated as batch reactors or continuously, aerobically or anaerobically, and with pure or mixed cultures. In some bioreactors, three phases (gas, liquid, and solid) are present and mass transfer can be an important consideration. Biofilms and immobilized cells can be used to retain microbial biomass in a flow bioreactor. Sensors, instrumentation, and control systems are essential for industrial bioreactors.

[0040] As used herein, the term “Shear Stress,” often denoted by T (Greek: tau), refers to the component of stress coplanar with a material cross section. (See, e.g., FIG. 4.) It arises from the shear force, the component of force vector parallel to the material cross section. Any real fluids (liquids and gases included) moving along a solid boundary will incur a shear stress at that boundary. The no-slip condition dictates that the speed of the fluid at the boundary (relative to the boundary) is zero; although at some height from the boundary the flow speed must equalthat of the fluid. The region between these two points is named the boundary layer. For all Newtonian fluids in laminar flow, the shear stress is proportional to the strain rate in the fluid, where the viscosity is the constant of proportionality. For non-Newtonian fluids, the viscosity is not constant. The shear stress is imparted onto the boundary as a result of this loss of velocity.

[0041] As used herein, the term “Shear Strain” refers to the ratio of displacement to an object's original dimensions due to stress and is the amount of deformation perpendicular to a given line rather than parallel to it. Shear strain is a sideways force exerted on a medium and is measured as a change in angle between lines that were originally perpendicular. (See, e.g., FIG. 4 )

[0042] As used herein, the term “Pulsatile flow” refers to flow with a periodic pressure fluctuation wave traveling along the flow path. Pulsatile flow systems may mimic blood flow characteristics within the heart and vasculature system.

[0043] As used herein, the term “Effective amount” refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject’s condition, will have a desired biological effect, e.g., an amount that will cure, prevent, inhibit, or at least partially arrest or partially prevent a target reaction.

[0044] A number of acronyms and abbreviations are used throughout this disclosure. Table 1 below summarizes and provides a glossary of several of the frequently used acronyms and abbreviations:Table 1

[0045] The terms “hollow tube”, “hollow tubing”, “hollow fiber”, and “lumen” may be used interchangeably through the description to refer to an elongated hollow structure through whichfluid flows under pressure applied by a pumping system in fluid connection with the hollow structure. The hollow structure may be formed from a number of different materials which may be rigid, flexible, permeable, semipermeable, or non-permeable. Examples of materials include, but are not limited to, glass, acrylic, plastic, polymers, fibers, silicone, ceramic, and filter membranes. In some embodiments, a hollow structure may be compressible or contractable so as to simulate a peristaltic-like action, for example, by applying opposing rollers to an outer surface of the tubing to “squeeze” the fluid within the interior of the tubing from a first end to a second end of the tubing. (See, e.g., FIG. IB). In other embodiments, the tube may be a hybrid assembly of sections of tube made from different materials, some of which may be permeable or semi-permeable membranes, while others may be non-permeable.

[0046] Lactation Biology

[0047] Lactation is the process by which milk is synthesized and secreted from the mammary glands. Mammary glands are modified sweat glands, and are composed primarily of adipose and collagenous tissue, with mammary glands making up a very minor proportion of breast volume. The mammary gland is composed of milk-transporting lactiferous ducts, which expand and branch extensively in response to estrogen, growth hormone, cortisol, and prolactin. Moreover, in response to progesterone, clusters of breast alveoli bud from the ducts and expand outward toward the chest wall. Breast alveoli are balloon-like structures lined with milksecreting cuboidal cells, or lactocytes, that are surrounded by a net of contractile myoepithelial cells. Milk is secreted from the lactocytes, fills the alveoli, and is squeezed into the ducts. Clusters of alveoli that drain to a common duct are called lobules; the lactating female has I O lobules organized radially around the nipple. Milk drains from lactiferous ducts into lactiferous sinuses that meet at 4 to 18 perforations in the nipple, called nipple pores.

[0048] The pituitary hormone prolactin is instrumental in the establishment and maintenance of breast milk supply. Prolactin and other hormones prepare the breasts anatomically for the secretion of milk. When the infant suckles, sensory nerve fibers in the areola trigger a neuroendocrine reflex that results in milk secretion from lactocytes into the alveoli. The posterior pituitary releases oxytocin, which stimulates myoepithelial cells to squeeze milk from the alveoli so it can drain into the lactiferous ducts, collect in the lactiferous sinuses, and discharge through the nipple pores. It takes less than 1 minute from the time when an infant begins suckling (the latent period) until milk is secreted (the let-down). The prolactin-mediated synthesis of milk changes with time. Frequent milk removal by breastfeeding (or pumping)will maintain high circulating prolactin levels for several months. However, even with continued breastfeeding, baseline prolactin will decrease over time to its pre-pregnancy level. In addition to prolactin and oxytocin, growth hormone, cortisol, parathyroid hormone, and insulin contribute to lactation, in part by facilitating the transport of maternal amino acids, fatty acids, glucose, and calcium to breast milk.

[0049] Pulsatile Flow Culture of Mammary Cell Types for Milk Secretion

[0050] The inventive system is designed to simulate the native environment of mammary alveoli and ducts for healthy epithelial cells (ECs) and optimal milk production by inducing variable shear stresses on the cells.

[0051] Referring to FIGs. 1A-1E, embodiments of the inventive bioreactor are designed to mirror the native environment of mammary alveoli and ducts for healthy epithelial cells (ECs) and optimal milk production. FIG. 1A provides diagrammatic side (left) and cross-sectional end (right) views showing the basic components of an exemplary embodiment of a bioreactor flow system 100, which is configured to implement adaptable shear stress within an enclosed sterile system. The incubator 102 includes a fluid-tight housing or shell 120 that encloses one or more semipermeable hollow tube 104, a pump system 106, valves 108 for controlling extracellular media fluid flow, reservoirs 110, 112, and a feedback loop 118, with valve 116, for recirculating partially processed fluid (accumulation medium) through the reactor tubing for further processing / enrichment. Shell 120 may be formed from polycarbonate or similar polymer, glass, stainless steel, or any material considered suitable for food handling, i.e., sterilizable. Fluid extracellular medium (ECM) is introduced into and circulated through the bioreactor via reservoir 110, valves 108, and inlet-outlet pipes 128 and 130. (A pump for controlling 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 lactating / 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, e.g., applying pulsatile and / or continuous flow through tubes 104 and harvesting the milk. System controller 122 may also be configured to generate control signals to valves 108, 116, the ECM loop operation, and any additional valves and other operations within the system. In some embodiments, system controller 122 may include one or more computer processor and memory combination(s) for retaining and executing programming for automated operation of the system. In some embodiments, parameters for optimized operation of the system may be established throughthe use of learning machines within the system controller, with input including system sensor signals (flow rate, pressure, temperature, etc.) and quality control measurements obtained through testing of the milk product(s).

[0052] In some embodiments, pump 106 is controlled to generate continuous, unidirectional, oscillating, and pulsatile flow with adaptable flow rates to expose the cells in the tube to variable shear stresses. Generally, the range of shear stresses produced by pump 106 will be alternated among different settings selected within a range of about 2 dyn / cm2(considered “low”) and 75 dyn / cm2(considered “high”) through signals from system controller 122. Pump sequences generated by controller 122 may cause the pump flow to induce shear stresses alternating between different levels, e.g., low (within range -2-10 dyn / cm2) to medium (range -11-40 dyn / cm2), high (range -41-75 dyn / cm2) to medium, high to low, or high to low to medium and back, etc. It is the variation of shear stresses on the cells via the pulsatile / oscillating flow that helps stimulate lactation. As the lactating medium flows past the cells it accumulates milk components to become the “accumulation medium”, which is processed through hollow tube 104, to be collected downstream at reservoir 112. One or more access port (not shown) may be included at or near outlet 162 to allow the accumulation medium to be sampled to determine whether it has attained the desired qualities, e.g., density, appearance, consistency, fat content, chemical content, etc. for completed milk product. If testing indicates the product is complete, the milk may be harvested. If not, the accumulation medium may be returned via valve 116 and feedback loop 118 to reactor inlet 160 for further enrichment. Alternatively, testing may not need to be performed on a regular basis once an optimal processing duration has been determined for given operating conditions.

[0053] FIG. IB diagrammatically illustrates additional details of the inventive bioreactor structure and function, showing a section of an exemplary hollow tube 104, which, in some embodiments, may be one of a plurality of elongated tubes or lumens arranged in a parallel array extending longitudinally within a generally cylindrical outer shell 120 to collectively define an enclosed sterile bioreactor cartridge 102. The interior of tube 104 is referred to as the intracapillary or InC space 134 while the space outside of the tube (within shell 120) is referred to as the extracapillary or ExC space 132. In some embodiments, the inner surface of tube 104 may further be lined with a collagen membrane (see, e.g., FIG. 1C). Feeder (support) cells 138 and mEP cells 140 are coated onto the inner surface of hollow tube 104. It should be noted that, for convenience, primary cilia 142, which play an important role in milk production,are illustrated only for a small number of cells 140. As will be recognized by those in the art, healthy ECs should all have primary cilia. The mechanosensation resulting from the adaptable flow within tube 104 helps to keep the cells differentiated and healthy. Tube 104 may be formed from a semipermeable membrane material, e.g., polymer or similar material, through which nutrients contained within the ECM can be diffused to provide nutrition 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 inventive bioreactor, an ibidi™ peristaltic pump (ibidi GmbH, Graefelfing, DE) was used to provide the needed mechano-stimulation for milk production via pulsatile flow. For larger systems, one or more larger capacity pump is used to achieve the desired pulsatile flow. Milk can be harvested daily, as a batch, from the reperfusion reservoirs 112 located at the downstream end of the reactor. An optional filter 114 may be included, either at the upstream or downstream side of reservoir 112, to remove built up dead cells and other particulate materials that may be in the milk prior to harvesting.

[0054] While hollow tube 104 is described herein as “semi-permeable”, its construction is not limited to a semi-permeable material. Rather, the only requirement is that parts of the tube have sufficient permeability to allow an amount of extracellular medium into the InC to nourish the lactating cells. Thus, hollow tube 104 is still considered “semi-permeable” as a hybrid assembly of multiple sections, some of which are permeable or semi-permeable, and others of which are non-permeable. For example, a ceramic filter membrane may be used for sections of the tube to supply the cells with ECM while the remainder of the tube may be less-permeable or non-permeable. This hybrid approach allows custom control over how much ECM is available to the cells, which avoids providing too much ECM, which could potentially spoil the final product.

[0055] The embodiment illustrated in FIG. 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 tracks 152 positioned within shell 120 so that they are parallel to the tube. The rollers slightly compress the tube from multiple sides to gently “squeeze” and push the contents of the tube, i.e., the lactating medium and accumulation medium with milk components 144, from near the inlet end towards the outlet end of the reactor. The pressure applied by the rollers should be uniform and sufficiently strong 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 rollers provides an additional means for applyingadjustable shear stress. Once the rollers have completed their travel along the track, they will then be shifted apart to release pressure on the tube and moved back on the track to the starting position near the inlet end to repeat the pushing sequence. While two opposing rollers are shown, it will be readily apparent that different combinations of rails and rollers, or annular sphincters, may be used to achieve the desired inlet-to-outlet pushing action, which assists in freeing up the cells for continued milk production. Signals for control of the optional mechanical stimulation assembly will be provided by system controller 122.

[0056] FIG. 1C provides diagrammatic cross-sectional views of monolayers of lactating cells attached to the inner surfaces of each hollow fiber 104 within an array of fibers of a reactor cartridge 102. In the illustrated embodiment, the bioreactor is a modified re-perfusion reactor with multiple hollow fiber elements 104 fed by a peristaltic pump (not shown) to generate continuous, unidirectional, oscillating, and pulsatile flow with adaptable flow rates. Reactor cartridge 102 may include multi -tube bioreactors with inlets and outlets for inputting / outputting fluids to / from the intercapillary and extracapillary spaces. As illustrated, InC space inlet 160 feeds hollow tubes 104 which then output the milk (i.e., accumulation medium) to the outlet from InC space 162. Extracellular media (ECM) is fed (from reservoir 110) into ExC space via inlet 128 and then circulated back into reservoir 110 through 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, transverse cross-sections of five individual hollow fibers 104 are shown with the inner surface of the outer fiber wall lined or coated with a collagen membrane 156. Cells 140 (and feeder (support) cells) are grown on the inner lumen of the hollow fiber. The collagen membrane supports a monolayer of cells so that the milk can flow through the spacing (lumen) past the cell layer. The upper right panel shows a longitudinal cross-section of a single fiber, again with the collagen membrane “liner” and the monolayer of cells 140 with the milk 170 flowing through the lumen. In this example, the inner diameter of the fiber is indicated to be 2mm, however, as will be apparent to those in the art, the inner diameter of the fiber may be selected to support the appropriate flow parameters for generating the shear forces needed for mechanical stimulation of the cells on the inner surface of the tube.

[0057] The above-described implementation of the inventive bioreactor employs a peristaltic pump for the purpose of inducing a shear stress on the lactating cells with a continuous fluid flow within a sterile reactor environment. As will be recognized by those of skill in the art, alternative approaches for inducing controlled shear stress with continuous or variable fluidflow within a sterile environment include use of a spinning vortex, a magnetic vortex, e.g., a stirring bar, and a wave pump or valve pump. Such approaches will involve placing the vortex and / or pump within a sterile enclosure, which could negatively impact the scalability of the process relative to the above-described implementation.

[0058] As will be recognized by those of skill in the art, the number of hollow tubes within a bioreactor assembly may vary and the illustrated examples are not intended to be limiting. One example of a variation in the tube configuration is provided in FIG. IE, where a single long tube 304 is arranged within shell 320 in a zig-zag, looped, coiled, or meandering pattern. The general direction of flow is still parallel with the length of the shell, i.e., from inlet to outlet, and may be recirculated through a feedback loop similar to those in other embodiments. In this example, tube 304 is a hybrid assembly formed from multiple tubular sections of different materials, where tube sections 344 are a permeable or semi-permeable material to allow perfusion of the ECM into the interior of the tube through these sections only. Tube sections 346 are non-permeable. Pump 306 controls the pulsatile flow through the tube from inlet to outlet. It should be noted that this is an illustrative example only and is not intended to be limiting.

[0059] The bioreactor embodiments illustrated in FIGs. 1A-1C and described above are shown in a horizontal orientation, however, there is no requirement that any specific orientation needs to be employed. Referring to FIGs. 2A-2C, some embodiments of the bioreactor employ a vertical orientation, which may assist in scaling up the capacity of a processing facility to generate larger volumes of milk. Each cartridge 202 includes the same basic components as described above: a shell 220 enclosing one or more hollow tube reactor 204. Pump 206 feeds fluid, e.g., PBS, into the top of cartridge 202 at inlet 260 which feeds the fluid into tubes 204. Hollow tubes 204 have a monolayer of cells, specifically, ECs, coated on their inner surfaces, as shown in FIG. 2C. As in the previously-described embodiment, pump 206 is configured to induce an adaptable shear stress as fluid flows through the tubes to stimulate milk production by the cells. The vertical orientation of cartridges 202 provides gravitational assistance to the flow through the tubes. Pump 206 may recirculate partially processed milk (accumulation medium) through feedback loop 218 until the desired milk characteristics are achieved. While not shown, one or more test ports / valves may be provided at or near outlet 262 to allow testing of the processed milk to determine completion. A filter 214 may be located at outlet 262 to remove waste products. Valve 264 allows the completedmilk product to be diverted to a reservoir for harvesting. The right panel of FIG. 2A provides a diagrammatic cross-sectional view of cartridge 202 with hollow tubes 204 defining the ExC (outside the tubes) and InC (inside the tubes) spaces as described above. The ECM is fed into the ExC space within shell 220 via inlet 228 and removed from the shell at outlet 230. Valves 208 and one or more pump (not shown) provide control for the ECM flow. Additional details of tubes 204 are shown in FIG. 2C, where the lower panel provides a exemplary 3-D perspective view of the tubes.

[0060] In some embodiments of the inventive bioreactor, optional light sources 216, specifically light emitting diodes (LEDs) may be included to produce different effects. In one embodiment, LEDs emitting in the blue light range, e.g., around 450nm, have been shown to stimulate primary cilia. In a study by Prosseda, et al., blue light was used to stimulate primary ciliary regulation in cells and increase cellular 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 primary cilia of the ECs must be stimulated to produce milk, the addition of optical stimulation to the mechanical stimulation provided by the adaptable shear stress serves to increase the overall stimulation required to milk production. The optical stimulation may be further use intensity pulsing to create additional variable stimulation of the cells. Since the light is used to stimulate the ECs, the material of which tubes 204 are formed should be capable of transmission of the desired wavelength. For example, glass or clear tube sections may be used in close proximity to the LEDs. While the LEDs are shown in the lower portion of the cartridge, it will be readily apparent that they may be positioned at multiple locations throughout the interior of shell 220 to increase exposure of the ECs to the light.

[0061] In other embodiments, other light sources may be included in the cartridge or in the plumbing outside of the cartridge. For example, UV light sources (100-280 nm) may be used to enhance sterility of the product, however, care must be taken to avoid damage to the cells. Other light sources may be used to promote the health of the ECs. For example, lights sources that emit at wavelengths including 660, 700, 810 and 850 nm have been reported as promoting cell activity and growth. Control signals for operation of a light stimulation system may be provided by a system controller.

[0062] FIG. 2D diagrammatically illustrates a possible arrangement of a bank of multiple bioreactors within a production facility. Multiple such banks with any number of units, i.e.,cartridges and associated plumbing, can be mounted in support frames (not shown) within the facility which may preferably include a clean room. In one possible implementation, each bank may have its own dedicated reservoir 210 for providing extracellular media to the bioreactor units within the bank and a milk collection reservoir 212 that will collect the milk produced by units within the bank. In other implementations, multiple banks may receive media from one or more larger central reservoir and output milk to one or more large central tank. Various combinations of central and dedicated reservoirs may be used. To provide an illustrative example, in a large production facility, one or more banks of bioreactor units may be used to produce different types of milk, e.g., full fat, low fat, hypoallergenic, lactose-free, or any of a variety of “designer” milk products such as those described below.

[0063] The mammary epithelium consists of two differentiated cell types organized into two cell layers, an inner layer of luminal epithelial and an outer layer of myoepithelial cells in direct contact with the basement membrane. Mammary epithelial cells (MECs) can proliferate as monolayers on plastic. However, only when receiving signals from ECM proteins plus hormones (prolactin, growth factors), i.e., structures similar to those observed in vivo, and tissue-specific gene expression (e.g., casein genes), does milk production actually occur. In some implementations, the MECs can be harvested from fresh milk as shown in one of the examples.

[0064] Using the disclosure herein, those of ordinary skill in the art will be able to implement similar strategies to extract MECs from the fresh milk or mammary glands using other mammary epithelial cell biomarkers. Table 2 below provides a list of biomarkers in breast tissue (see, e.g., FIG. 3) that may be useful for extraction of MECs for use in the inventive method. An exemplary selection panel may include a combination of these biomarkers. For example, a healthy breast cell panel may be assembled from a combination of biomarkers from one or a combination of sources, e.g., all, all epithelium, luminal, etc. selected from those listed in the lower seven rows of the table.Table 2

[0065] Pulsatile Flow: Pulsatile flow is a periodic pressure fluctuation wave traveling along the flow path such as that created in the hollow tube(s) of the bioreactor. Pulsatile flow systems can closely mimic blood flow characteristics within the heart and vasculature system.

[0066] The Mammary Endothelial cells (MEC) adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured MEC exposed to a laminar steady-state flow-induced shear stress. However, in vivo MEC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actinmicrofilament localization in confluent bovine aortic endothelial cell (BAEC). These results demonstrate that EC can discriminate between different types of pulsatile flow environments. Furthermore, these experiments indicate the importance of engineering the cell culture environment so as to include pulsatile flow in investigations of endothelial cell biology.

[0067] Elimination of FCS from the Lactating Medium: Today, standard culture media rely on established yet outdated notions that blood serum from unborn calves (FCS) is required, which has served as the "gold standard" since the 1960s. For many years, problems with FCS, and the reasons it needs to be replaced with better alternatives, have become an area of considerable research interest. These problems with FCS include the fact that: (a) its production is a cruel undertaking, and (b) FCS cannot be used in clean milk production for human consumption due to reported serum contamination with viruses, along with other safety concerns in terms of endotoxins, mycoplasma, RNA contaminants, or prion proteins. Accordingly, the medium used for culturing the cells for use in the inventive bioreactor is preferably FCS- and hormone-free (except for prolactin) as well as being based on the latest scientific research and cell culturing techniques.

[0068] Serum-starvation in general can make cells unhealthy, however the adverse effect can be counteracted by flow modulation. Changes in the flow strength can induce cilia growth. Therefore, cilia need to be stimulated using flow to achieve milk-production.

[0069] Mechanical stimulation (Flow) for healthy EC cells to counteract elimination of FCS from the lactating medium: Hormonal and mechanical signals can both induce gene expression changes in mammary epithelial cells (MEC) that culminate in milk synthesis and secretion. Using serum-free media and a combination of hormonal and mechanical stimuli, bovine mammary epithelial cells (BMEC) were transitioned from primary multicellular organoids to coordinated 3 -dimensional ductal networks. See, for example, FIGs. 5A-5B showing mammary epithelial cells possessing an epithelial-like phenotype and mammary myoepithelial cells possessing a spindle-like phenotype, respectively. At the structural level there was evidence that mechanical signaling via gel release, prolactin, and the combination of the two resulted in changes in cellular morphology that were consistent with initiation of stage I lactogenesis.

[0070] Endothelial cells require mechanical stimulation to grow healthy. Studies have shown that a certain type of flow can provide shear mechanical stimulation to EC cells, resulting in increased cell performance. In some embodiments, constant mechanical stimulation forhealthy ECs resulted in an increased, hormone-free milk-production process. It is important to understand the nature of the stimulation applied to the cells, the principles of which are illustrated in FIG. 4. Mechanical strain occurs when a force acts directly against the cell. With high strain, the endothelial barrier can be loosened, resulting in cell death and high cell turnover. Epigenetic modifications and inflammation can occur. In contrast, mechanical shear involves parallel movement of the fluid overtop of the cells, which improves cell survival with low turnover. The goal is, thus, to avoid mechanical strain while increasing mechanical shear to stimulate cellular function. The lower panel of FIG. 4 provides a comparison of a cell layer after (left) and before (right) application of shear stress.

[0071] In some embodiments, lactating media and test cultures are fluidically driven with a peristaltic pump, for example, the ibidi™ pump system used in a smaller scale prototype bioreactor, or other pump capable of producing pulsatile flow that provides adaptable shear stress in an enclosed sterile system. In some embodiments, mechanical shear stress is assessed and constantly monitored.

[0072] Feeder Cells: Feeder or support cells 138 comprise a layer of cells that are unable to divide, but which provide extracellular secretions to promote proliferation of other cells, i.e., lactating cellsl40. Feeder cells differ from a co-culture system because only one cell type is able to proliferate.

[0073] In some embodiments, feeder cells are co-cultured with MECs and form a monolayer of lactating cells attached to the inner lumen of the elongated hollow fiber bioreactor. In some embodiments, feeder cells may be seeded in the extracapillary (ExC) space surrounding the hollow fibers.

[0074] Hollow Fiber Bioreactor: A hollow fiber bioreactor is a 3-dimensional cell culturing system based on hollow fibers, which, in some embodiments, are small, semi-permeable capillary membranes arranged in an array with a typical molecular weight cut-off (MWCO) range of 10-30 kDa. Exemplary implementations are described above with reference to FIGs. 1A-1C, 2A-2D. When multiple tubes are employed, the tubes may be bundled and housed within cylindrical shells of polycarbonate, glass, stainless steel, or other appropriate material, i.e., sterilizable, to create hollow fiber bioreactor cartridges. Within the cartridges, which are also fitted with inlet and outlet ports, are two compartments: the intracapillary (InC) space 134 within the hollow fibers, and the extracapillary (ExC) space 132 surrounding the hollow fibers.

[0075] Cells are seeded into InC space 134 (FIG. IB) of the hollow fiber bioreactor 104 andexpand therein to coat the inner surface of the hollow fiber 104 with a monolayer. Cell culture medium is pumped through ExC space 132 to deliver oxygen and nutrients to the cells via hollow fiber membrane perfusion. As the cells expand, their waste products and CO2 also perfuse the hollow fiber membranes and are carried away by the pumping of the milk accumulation medium through InC space 134. As waste products build up due to increased cell mass within the InC, the rate of medium flow may need to be increased so that cell growth is not inhibited by waste product toxicity. Waste products can be filtered out by a filter disposed within or near the reservoir after the accumulation medium has been fully enriched, i.e., milk production has been completed.

[0076] Supporting Extracellular Matrix: The mammary epithelium may include two differentiated cell types organized into two cell layers, an inner layer of luminal epithelial and an outer layer of myoepithelial cells in direct contact with the basement membrane. Mammary epithelial cells can proliferate as monolayers on plastic. However, only when receiving signals from ECM proteins plus hormones (prolactin, growth factors), structures similar to those observed in vivo, and tissue-specific gene expression (e.g., casein genes), does milk production occur. Thus, in order to simulate natural milk production, the bioreactor must re-create these structures and an environment with the same (or similar) stimuli as occur in an in vivo setting. In some embodiments, this can be achieved by adjusting the supporting extracellular matrix, the composition of culture media and by the co-culture of necessary cell types to achieve functional differentiation.

[0077] Other successful approaches may also stimulate milk genes. In some embodiments, cells were shown to reorganize and form 3D structures like those observed in vivo and exhibiting milk protein genes expression on collagen gels. In some embodiments, epithelial cells grown in laminin-rich gels underwent functional differentiation based on the expression of the casein genes. Natural and synthetic polymeric materials have been investigated as alternatives to ECM proteins.

[0078] In some embodiments, 3D bioprinting of mammary tissue may be used. In some embodiments, 3D culture procedures including lactogenic hormones (prolactin, insulin and hydrocortisone) are able to induce caseins synthesis from primary mouse epithelial cells embedded in floating collagen gels. In some embodiments, differentiation medium, complemented with oleic acid, pituitary extract, and dexamethasone, was shown to induce the synthesis of major milk constituents such as P-casein, triglycerides and lactose, in a 3D in vitromodel of bovine primary mammary epithelial cells grown on a cell culture insert coated with collagen.

[0079] Morphological and functional differentiation of cryopreserved lactating bovine mammary cells can be cultured on floating collagen gels. In some embodiments, bovine primary epithelial cells from lactating mammary tissue grown on floating collagen gel exhibited a polarized conformation with high differentiation status evidenced by the observation of apical microvilli, tight junctions, and fat droplets surrounded by caseincontaining secretory vesicles.

[0080] The use of collagen gel provides the advantage of mimicking in vivo conditions and allows relatively long-term studies.

[0081] The milk produced by the inventive bioreactor has the ability to match nutritional content, taste, and quality of milk obtained traditionally.

[0082] EXAMPLES

[0083] Aspects of the inventive system and method may 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.

[0084] Example 1 : Bioreactor

[0085] As described above, the inventive bioreactor is designed to mirror the native environment of mammary alveoli and ducts for healthy epithelial cells (ECs) and optimal milk production. The goal of the system and method is to employ process control conditions and cell biology to provide an FCS-free, or substantially FCS-free, culture of primary ECs. In some embodiments, constant mechanical stimulation for healthy ECs results in an increased, hormone-free milk-production process. (Noting that the hormone prolactin is still necessary.) In some embodiments, lactating media and test cultures are fluidically driven with a peristaltic pump to produce adaptable shear stress in an enclosed sterile system. In some embodiments, mechanical shear stress may be assessed and continuously or periodically monitored. In some embodiments, the bioreactor is a modified re-perfusion reactor with a hollow fiber module, controlled by peristaltic pump to generate continuous unidirectional, oscillating, and pulsatile flow with adaptable flow rates. In some embodiments, lactating cells are grown or coated on the inner surface of the hollow tube 104. The inlet of each hollow tube is connected to thepump to provide flow stimulation via pulsatile flow. Milk may be harvested in batches at regular intervals, from periods ranging from daily to weekly to monthly, depending on the scale (volume) of the overall bioreactor system. The system supplies an extracellular medium through permeable and / or semipermeable membranes in the tube to nourish the lactating cells inside the tube. The ECM may be replenished and / or replaced periodically through the processing period.

[0086] Example 2: Mammary Epithelial Cells (MECs) Harvest

[0087] Fresh bovine milk (i.e., from Guernsey or Galloway cows) was defatted by low-speed centrifugation at typical refrigeration temperature (~4°C). The skim 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 isolate the MECs from the total milk somatic cells and to remove the leukocytes, which were collected separately for further use. Immunomagnetic separation involved incubating the total milk cell suspension with magnetic beads (Dynabeads) coated with a primary monoclonal antibody directed 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. MECs were then cultured in complete medium for 1 week to ensure sufficient cellular proliferation. The complete medium included DMEM / F12 (alternatively EpiCult) supplemented with 2% FBS or FCS-replacement, 5 ug / ml insulin, 100 ng / ml hydrocortisone L-Glutamine, optionally supplemented with antibiotic.

[0088] Example 3: MEC Culture & Lactation

[0089] Seeding of mammary myoepithelial and epithelial cells (MEPs) in the complete medium was performed and maintained until a monolayer was established. Low shear stress was used for in-flow in complete medium for cellular proliferation (1 week). Shear stress was 0.007 dyn / cm2. Next, a sedimentation and attachment period was carried out in the hollow fiber reactor for ~4 hours. Physiological shear-stress at about 5 dyn / cm2, i.e., low shear stress, was performed for proliferation and monolayer formation over a period of several days. The media was changed every 3 days or fed-batch culture.

[0090] Next, serum withdrawal and supplementation with lactation media was performed to induce lactogenic differentiation. Pulsatile flow was initiated, with the pump being controlled to periodically change fluid flow to induce variable shear stresses alternating at different levelswithin a range from around 2 to 75 dyn / cm2over a period of at least one week, more preferably about 2-3 weeks. The total culturing time will vary depending on system size (volume), with a small scale system taking around 2 weeks and larger scale systems taking up to multiple months for recirculating the accumulation medium through the reactor for complete culturing. Over a multi-week period, lactation media changes were made every 3 days, or fed-batch culture. The product was then harvested after culturing was complete. Processing after harvest included preservation, by one or more of pasteurization, freezing, or dehydration. Quality control testing included evaluation of bacterial, mycoplasma, fungi, and principal components.

[0091] Example 4: Using Matrix to Replace Feeder Cells

[0092] Feeder cells are generally suitable for supporting stem cell co-culturing, however, when dealing with different species, this could create a potential for transfer of animal pathogens and / or unwanted immune response that could impact milk production. Matrigel matrix structures offer a suitable and functional support structure to cells requiring feeder cells.

[0093] For coating of Matrigel (BD Biosciences, Bedford, MA), 500 pL of BD Matrigel was transferred into each well of six wells plate on ice and spread by rocking gently. The plates were then incubated at 37 °C for 2 h to allow the BD Matrigel to polymerize into gel. Then, the suspensions of reprogrammed HDFs (human dermal fibroblasts) (2.4>< 104 / cm2) were transferred onto coated palates with BD Matrigel matrix in DMEM media containing 10 % FBS and the plate was returned into incubator. After 48 h the media was replaced by supplemented DMEM / F-12 (including supplemented factors as mentioned above) and was changed every day for formation of hiPSC colonies. The iPSCs culture was maintained and followed for 10 passages.

[0094] Example 5: Preparation of Feeder Cells with Conventional Method

[0095] CF-1 MEFs of passage 3 (P3) at 80-90% confluence were inactivated with 10 pg / ml of MMC (Hisun Pharmaceutical Company, China) for 0, 0.5, 1.0, 1.5, and 2.0 h at 37 °C. After the incubations, the cells were washed with PBS 6 times, trypsinized, centrifuged at 180 x g for 5 min, and re-suspended in MEF medium. Cells were counted and frozen for later use.

[0096] Example 6: Preparation of Feeder cells with Suspension-Adhesion Method

[0097] We prepared feeder cells by SAM according to Fig. 1. Briefly, CF-1 MEFs of P3 were cultured for four days, digested to single cells with 0.25% trypsin / EDTA (Dalian MeilunBiotech Co., Ltd, China), and collected in 50 ml-centrifuge tubes. The cells were seeded at 8 * 104-1.1 x 105 cells / cm2 in 10 cm-dishes. MMC (10 pg / ml) was added after 2.0-3.0 h at 37 °C. Medium containing MMC was discarded 0.5-3.5 h post-treatment. The cells were then washed with PBS 6 times, trypsinized, centrifuged at 180 * g for 5 min, and resuspended in MEF medium. Cells were counted and frozen for later use.

[0098] Example 7: Preparation of Feeder Cells with Three-Dimensional (3D) Suspension Method

[0099] After connecting the CELLSPIN System (5-75RPM, CELLSPIN System with glassball stirring pendulum, Integra Bio-Sciences, Switzerland) to the incubator, spinner flasks were sterilized by autoclaving. Feeder cells were prepared by 3DSM. Briefly, CF-1 MEFs of P3 growing for four days were digested to single cells by 0.25% trypsin / EDTA and collected into a 50 ml-centrifuge tube. Cells were transferred to spinner flasks with glass ball pendulum, which accommodate 25-1000 ml of volume, at a density of 0.5-1.3 x 106 cells / ml. MMC were added at 10 pg / ml. After incubation for 0.5, 1.0, 1.5, and 2.0 h at 37 °C, the cells were centrifuged at 180 x g for 5 min, washed with PBS 3 times, resuspended in MEF medium, counted, and cryopreserved for later use.

[0100] Example 8: Production feeder cells / or support matrix for bioreactor

[0101] 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 exclusively mouse or human-derived feeder cells. One of the goals of the inventive system and method is to produce custom feeder cells derived from cow fibroblasts. Potential feeder cells that may be used in embodiments of the inventive method include fibroblasts, mEP and myoepithelial cells, and mEP on fibrous extracellular matrix.

[0102] Alternative synthetic methods may be employed as support for milk producing cells. The following examples identify a few of these methods.

[0103] Example 9: Lab -grown FCS Alternative

[0104] Upon activation, PBMCs secrete a powerful cocktail of pro-survival factors that helps them to combat infections. These are secreted into the blood and become part of the serum-phase. Co-culture experiments show that the supplementation of cells with thesupernatant phase of cultivated PBMCs significantly prolongs their survival. In cell culture, this is used by the “tight packing” of cells to rely on the microenvironmental factors, i.e., suspensions at a high cell density to achieve healthy cell growth and recovery after freezing. Addition of Innate Immune cell supernatant provides a survival-factor, as strong as a comparable direct survival stimulation.

[0105] PBMCs were extracted from the peripheral blood of volunteers using Percoll gradients. After supplying them with a pro-activation stimulus, containing A) LPS B) opsonized beads C) Mechanical stimulation in an ibidi™ pump system, the cells were maintained in a stimulated state and the supernatant was enriched for 24 to 48 hours and then collected. The supernatant phase was separated from the cellular fraction and used for the supplementation of media. For future up-scaling, conditionally immortalized ER-Hoxb8 hematopoietic progenitor cells can be used and differentiated on demand into PBMCs, to provide an infinite supply of PBMCs.

[0106] Example 10: EpiCult™ Plus Mammary Epithelial Serum-Free Medium

[0107] Successful tests in mammary cells were performed using cell culturing techniques adapted from custom modifications of cell culturing methods of Epithelial cells (Mammary) from FCS-free Database (https: / / fcs-free.org / fcs-database) Animal free research Foundation.

[0100] FIGs. 5A-5E provide representative growth curves & images. Photographic images of mammary breast tissue cells were taken using live-cell microscopy with a Kodak Imaging System, where FIG. 5A shows mammary epithelial cells, possessing an epithelial-like phenotype and FIG. 5B shows mammary myoepithelial cells, possessing a spindle-like phenotype. FIG. 5C provides growth rate curves of breast tissue cell lines, lx 104cells were seeded and measurements were subsequently taken on the 4 following days. Values for three independent cell samples for each day were compared by using unpaired t-tests. Significance between mammary epithelial cells (green) and mammary myoepithelial cells (red) cell growth rates was thereby determined for day 3 (p=0.007) and day 4 (p=0.0228). These values were highlighted in the diagram through asterisks. FIGs. 5D-5E are plots showing that addition of 2% PBMCs (5D) or 1 :3 neutrophil conditioned media (i.e., supernatant) (5E) provides a survival stimulus comparable to the addition of pro-survival mediators dbcAMP or LPS and PGE2 in cultured immune cells.

[0101] In some implementations, a learning machine (Al)-guided approach may be used to optimize media composition and as well as optimizing and adjusting processing parameters.

[0102] Example 11 : In Vitro Milk Production Using Extracted Mammary Epithelial Progenitors From Fresh Bovine Milk

[0103] The bioreactor was seeded with mammary epithelial cells (MEPs) in complete medium supplemented with 2% FCS (FCS replacement), 5 ug / ml insulin, 100 ng / ml hydrocortisone L-Glutamine, optionally supplemented with antibiotic, IGF-I, or EGF. The complete medium can be selected from DMEM, F12 or EpiCult. MEPs were incubated until a monolayer was established. A low shear rate was applied for in-flow in complete medium to maintain cellular proliferation for 1 week. The sedimentation and attachment period in hollow fiber reactor was 4 hours. Medium to high shear-stress was maintained for proliferation and monolayer formation for 2 days. Full media changes were performed every 3 days or fed-batch cultured. Serum withdrawal and supplementation with lactation media was performed to induce lactogenic differentiation. The lactation medium included DMEM / F12 (alternatively EpiCult) supplemented with 2% FBS (or FCS-placement), L-Glutamine, 1-5 ug / ml prolactin (or shear stress as defined above). Pulsatile flow was induced by alternately switching the pump between different pump rates, e.g., high, medium, and / or low, in order to create shear stresses within a range of 2 to 75 dyn / cm2. This step was continued for a period of at least one week, more preferably about 2-3 weeks. As will be apparent to those in the art, the total processing time will depend on system size (volume). A small scale system may take as few as 2 weeks to produce fully cultured milk, while larger scale systems could take up to a few months for complete culturing. Lactation medium changes were performed every 3 days or fed-batch culture. The product was then harvested and processed as described in Example 3.

[0104] Example 12: Genetic Engineering to Induce Hyperlactation and Other Modifications

[0105] Hyperlactation can be induced in cells using CRISPR technology. Referring to the diagram in FIG. 6, the following protocol may be used: CSN2 polymorphisms (SNPs): His220, G->A in Glu223; LALBA Polymorphism: in the 5’LALBA promoter; PRLR SNPs: rs62355518, rsl0941235, rsl610218, rs34024951, rs9292575; PRL SNPs: rs849872; Jak2 gain of function mutation for hyperactivation: V617F. Additionally, pharmaceutical interventions may include use of Retinoid acid and ATRA to target CSN3 expression.

[0106] SNPs can be modified (via CRISPR) to decrease lactose content for diet, lactose-free milk: CSN2 (Glu340, Thrl74, Lysl4), B4GALT1 (T224A).

[0107] Hypoallergenic milk can be produced using a CRISPR cluster bomb to introduce point mutations in the base pairs 186 and 213 of the gene CSN1S1, corresponding to the sites CDS84-123 (CSN1S1) and CDS 87-123 (CSN1S2).

[0108] Table 3 below lists a number of potential modifications that can be achieved through genetic engineering of mEPs and mEP-derived cell lines.Table 3

[0109] Particularly desirable modifications that are enabled by the inventive methods include the production of A2 rich milk from cells.

[0110] The foregoing describes embodiments of a system and method for in vitro milk production . Although specific example embodiments have been described, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader scope of the invention. Accordingly, the detailed description is not to be taken in a limiting sense, and the scope of various embodiments is defined only by the appendedclaims, along with the full range of equivalents to which such claims are entitled.

[0111] References1. 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

CLAIMS:

1. An apparatus for in vitro milk production, the apparatus comprising: a reactor assembly comprising: an elongated shell configured for retaining an extracellular medium; one or more elongated hollow tube disposed within the extracellular medium in longitudinal alignment within the shell, the one or more tube having an inlet end, an outlet end, and an inner surface configured for attaching a monolayer of lactating cells, wherein at least a portion of the one or more tube 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 in fluid connection with the one or more tube, the pump configured to generate a pulsatile or oscillating flow of a lactating medium through the one or more tube to induce a shear stress on the lactating cells, wherein the pump is controlled to alternate among different shear stress levels within a predetermined range, wherein variation of the shear stress stimulates the lactating cells to create a milk product; a supply loop for supplying and circulating the extracellular medium through the shell; and a reservoir in fluid connection with the outlet end of the one or more tube, the reservoir configured for collecting the milk product.

2. The apparatus of claim 1, further comprising a feedback loop disposed near the outlet end the one or more tube for recirculating the milk product into the inlet end of the one or more tube for further enrichment.

3. The apparatus of claim 1, wherein the predetermined range of shear stress levels is 5 to 15 dyn / cm2.

4. The apparatus of claim 1, wherein the supply loop comprises a reservoir configured for removing used extracellular medium and adding fresh extracellular medium.

5. The apparatus of any one of claims 1 to 4, wherein the lactating cells are cocultured with feeder cells.

6. The apparatus of claim 5, wherein the feeder cells are peripheral bloodmononuclear cells.

7. The apparatus of any one of claims 1 to 4, wherein the lactating cells are isolated from the milk of healthy cows.

8. The apparatus of any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary 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, VIM, CD34 / CD39 / CD140b, and CD49e.

9. The apparatus of any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary tissue selected from 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.

10. The apparatus of any one of claims 1 to 4, wherein the lactating cells are genetically modified to induce hyperlactation.

11. The apparatus of any one of claims 1 to 4, wherein the lactating cells are genetically modified to produce milk that is one or more of hypoallergenic, reduced lactose, and has increased A2 P-casein proteins.

12. The apparatus of any one of claims 1 to 4, wherein the one or more tube is coated with collagen.

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

14. The apparatus of any one of claims 1 to 4, wherein the one or more tube is formed from a semi-permeable capillary membrane.

15. The apparatus of any one of claims 1 to 4, wherein the one or more tube has a hybrid construction comprising sections of non-permeable material and sections of permeable or semi-permeable material.

16. The apparatus of any one of claims 1 to 4, wherein the inner surface of the the one or more tube is first bound with a layer of a support matrix, wherein the lactating cells are attached to a non-bound surface of the support matrix, and wherein the lactating cells form amonolayer of the lactating cells on top of the support matrix.

17. The apparatus of any one of claims 1 to 4, further comprising a mechanical stimulation assembly configured to apply a squeezing force to an exterior of the one or more tube in a direction from the inlet end toward the outlet end.

18. The apparatus of any one of claims 1 to 4, further comprising one or more light source disposed within the reactor assembly configured to expose the lactating cells to light stimulation.

19. The apparatus of claim 18, wherein the one or more light source is a light emitting diode (LED) that emits light at 450nm.

20. The apparatus of any one of claims 1 to 4, wherein the lactating medium comprises one or more of an EpiCult™ Plus medium, a Lab-grown FCS alternative medium, and a FCS free medium.

21. The apparatus of any one of claims 1 to 4, wherein the lactating medium further comprises prolactin.

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

23. A method of in vitro milk production in the apparatus of any one of claims 1-4, the method comprising: supplying the one or more tube with the lactating medium, wherein the lactating medium comprises prolactin; forming a monolayer of lactating cells attached on the inner surface of the one or more tube; controlling the pump to alternate among different shear stress levels within a predetermined range, wherein variation of the shear stress stimulates the lactating cells to produce a milk product; and harvesting the milk product when a predetermined milk quality is achieved.

24. The method of claim 23, further comprising recirculating the milk product via a feedback loop into the one or more tube to enrich the milk product until the predetermined milk quality is achieved.

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

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 the lactating cells are isolated from the milk of healthy cows.

28. The method of any one of claims 23 to 26, wherein the lactating cells are extracted from healthy mammary 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, VIM, CD34 / CD39 / CD140b, and CD49e.

29. The method of any one of claims 23 to 26, wherein the lactating cells are extracted from healthy mammary tissue selected from 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.

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

31. The method of any one of claims 23 to 26, wherein the lactating cells are genetically modified to produce milk that is one or more of hypoallergenic, reduced lactose, and has increased A2 P-casein proteins.

32. The method of any one of claims 23 to 26, wherein the lactating medium comprises one or more of an EpiCult™ Plus medium, a Lab-grown FCS alternative medium, and a FCS free medium.

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