Pulsating flow culture of mammary gland cell types for milk secretion

Abstract

An apparatus and method for in vitro milk production includes a reactor assembly having an elongate housing enclosing one or more hollow semi-permeable tubes within an extracellular nutrient medium. A monolayer of lactating cells is formed on the inner surface of the tube. A pump connected to the tube produces a pulsating flow of the lactating medium to induce shear stress on the lactating cells. Variable pump rates subject the lactating cells to different shear stress levels to stimulate milk production. The lactation medium is continuously recycled through the tube until a predetermined milk quality is reached, after which it is harvested. The lactating cells may be extracted from milk or mammary tissue and may be genetically modified to produce milk with design characteristics such as low allergenicity or reduced lactose.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 358,464, filed on July 5, 2022, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present invention relates to devices and methods for in vitro milk production, including milk having enhanced health effects. BACKGROUND ART

[0004] The demand for global milk production is increasing to address a range of factors, including global population growth and changes in diet in Asian countries (i.e., westernization). Over the next decade, global dairy consumption is expected to increase by more than $100B. This in turn places increasing pressure on natural resources, raises concerns about animal welfare, and results in increased deforestation and more greenhouse gas generation. Dairy consumption in the United States accounts for approximately 2% of the country's greenhouse gas emissions. To meet global demand, approximately 270 million dairy cows are raised every day. Each component in milk is associated with both a range of positive and negative health effects.

[0005] Milk is a complex colloidal matrix that contains milk fat, lactose, and many different components, including naturally occurring hormones. The sensory and functional characteristics of milk stem from its micronutrient profile, which has proven costly and difficult to replicate. Generally, the total composition of milk from U.S. dairy cows is 87.7% water, 4.9% lactose (carbohydrates), 3.4% fat, 3.3% protein, and 0.7% minerals. Milk composition varies according to species (cow, goat, sheep), breed (Holstein, Jersey), the animal's diet, and lactation stage.

[0006] Studies have shown that the fat and protein content of milk is based on the breed of cow. Holstein cows have the lowest fat and protein content, while Jersey and Guernsey breeds 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 ranges from 1.77% to 5.98%, with an average of 3.76%. In milk production, food source, temperature, humidity, and season all play important roles in the variation of milk quality. To address the variation in milk composition, a common practice is to blend milk from different dairy cows in a bulk tank to produce a relatively consistent milk composition throughout the year in the United States.

[0007] 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), 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-steroids (3 ± 1 ng / mL), progesterone (12 ± 2 ng / mL), androsterone (0.13 ng / mL), 17β-estradiol (0.02 ng / mL), estriol (0.027 ± 0.01 ng / mL). Additionally, due to the need to treat infections in the entire herd during their milk production (i.e., chronic mastitis), hormones and other foreign substances such as antibiotics and drugs are commonly present in industrially produced milk.

[0008] While there is little controversy regarding the presence of these hormones at physiological concentrations, their potential biological effects on animals and humans may not be well understood and could be profound. Additionally, the presence of hormones can raise significant safety concerns regarding dairy foods, especially steroid hormones such as estrogen. Some evidence suggests a potential link to breast and prostate cancer. Therefore, special attention is needed, especially during major developmental periods (e.g., perinatal and adolescent).

[0009] For this reason, and given the significant progress made in the development of analytical methods and bioassays, it is important to clarify the potential impact of the presence of hormones (especially steroid hormones such as estrogen) when they are common components of the diet and have been consumed regularly over the years.

[0010] Dietary alternatives to dairy products are found in plant-based milks and dairy products. Milks from plants such as almonds, soybeans, cashews, and oats lack the naturally occurring hormones found in animal-based milk and are becoming increasingly popular as a good source of protein without causing the dairy intolerance issues experienced by many people. However, plant-based milks from these alternative sources are often less effective in providing the functionality of animal-based milk for the production of other dairy products such as cheese, butter, and yogurt. Additionally, the increasing popularity of plant-based milks has also raised other sustainability issues, such as the extensive deforestation that has been adopted to expand soybean production or the large amounts of water required to produce almonds in regions experiencing long-term drought.

[0011] Another area of interest in the dairy industry is an alternative milk product known as "A2 milk". Cow's milk typically contains two types of beta-casein proteins: A1 and A2, which differ in having the amino acid histidine or proline at position 67 of the protein structure, respectively. The hypothesis is that conventional cow's milk containing A1 beta-casein protein may cause negative health effects, including digestive discomfort, gut inflammation, and milk allergy, while A2 beta-casein does not seem to cause these effects.

[0012] Both A1 beta-casein and A2 beta-casein proteins contain 209 amino acids (AA). However, upon digestion, A1 beta-casein releases beta-casomorphin-7 (BCM-7), which triggers a series of events that increase inflammation and gastrointestinal discomfort. Thus, it is hypothesized that consumption of milk containing A1 beta-casein is associated with increased gut inflammation, worsening of PD3 symptoms, transit delays, and reduced cognitive processing speed and accuracy. Recent studies have confirmed that subjects consuming A1 / A2 beta-casein milk presented with more severe digestive symptoms associated with lactose intolerance, while consumption of A2 beta-casein milk was not found to exacerbate these symptoms. Inflammatory markers, such as IL-4, IgG, IgE, and IgG1, were significantly lower in consumers of A2 beta-casein milk. Currently, since the two types of beta-casein, A1 and A2, are very similar, and since cows producing milk rich in A1-protein tend to be of breeds that produce large amounts of milk (high-yield cows), such as Holstein cows, no economical solution to this A1 beta-casein problem has been developed.

[0013] Milk and dairy products, including cream, butter, yogurt, and cheese, constitute an important component of the human diet, providing an important source of protein, vitamins, and minerals. For many people, dairy products are the easiest way to obtain supplementary nutrients to maintain the health and proper functioning of the heart, muscles, and bones. However, the milk quality-related issues discussed above have plagued the dairy industry for many years, and there is an urgent need for solutions.

[0014] The need for improved milk production technologies is evident, driven by several factors, including the current unsustainable methods of milk production through cattle farming (inefficient use of land and agricultural resources, generation of greenhouse gases, etc.); the lack of comparable milk alternatives; and the limited sources of consistent and high-quality milk.

[0015] U.S. Patent No. 11,236,299, which is incorporated herein by reference and assigned to Biomilk Ltd. (Rehovot, IL), discloses a method for in vitro milk production by employing a series of mammary organoids inoculated on a three - branched, flexible catheter scaffold. In an exemplary embodiment, the milk production system includes an array of containers, each container including a plurality of mammary organoids (MOs); a nutrient supply reservoir operable to supply each container, and a milk collection module. The key component of the system is the MO, which is mammary epithelial cells that form a multicellular three - dimensional structure (mammary organoid) embedded in a matrix. After inoculating the scaffold with MOs, estrogen and progesterone contribute to inducing the growth and morphogenesis of the epithelium via paracrine signaling between the mammary stroma and the epithelium containing the inoculated MOs. Then, the MOs begin to secrete milk in a culture medium containing prolactin, nutrients, and growth factors for a period of time between 10 - 21 days. While the Wilk patent discloses a method for in vitro milk production, it does not provide a series of options for modifying milk products in terms of cellular structure to increase nutritional content, reduce hormone dependence, select preferred A2β - casein, and other "specialty milk" products (such as hypoallergenic milk), which would help provide a more sustainable and nutritious food source to meet the growing global demand.

[0016] In view of the growing demand for dairy products, the present invention relates to a more sustainable and versatile method of milk production to overcome the environmental and animal welfare challenges faced by the current dairy industry while providing a reliable, high - quality source of safe, clean milk. Summary of the Invention

[0017] According to embodiments of the present invention, there are provided systems and methods for in vitro milk production using mammary cells that are cultured for enhanced cell proliferation and milk and milk component secretion. The enhancement of cell proliferation is achieved at least in part by applying flow to the culture medium and by varying the mechanical stimuli of the flow by various different methods, including but not limited to unidirectional laminar flow, turbulent flow, pulsatile flow, and oscillatory flow. The continuous mechanical stimuli provided by the system of the present invention can generate healthy epithelial cells (ECs) and produce milk that is substantially hormone - free (except for prolactin). The systems and methods disclosed herein are generally applicable to the production of cell proteins and dairy products for food cultivation and for biotechnological industrial use.

[0018] In one aspect of the present invention, an apparatus for in vitro milk production includes: a reactor assembly comprising: an elongated housing configured to retain an extracellular medium; one or more elongated hollow tubes longitudinally aligned within the housing and disposed within the extracellular medium, the one or more tubes having an inlet end, an outlet end, and an inner surface configured to adhere a monolayer of lactating cells, wherein at least a portion of the one or more tubes is formed of 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 lactating cells; a pump fluidly connected to the one or more tubes and configured to generate a pulsatile or oscillatory flow of a milking medium through the hollow 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 and circulating the extracellular medium through the housing; and a reservoir fluidly connected to the outlet end of the one or more tubes and configured to collect the milk product. The apparatus further includes a feedback loop disposed near the outlet end of the one or more tubes for recycling 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 / cm 2 . The supply loop may include a reservoir configured to remove spent extracellular medium and add fresh extracellular medium.

[0019] In some embodiments, the lactating cells are co-cultured with feeder cells. The feeder cells can be peripheral blood mononuclear cells. In some embodiments, the lactating cells can be isolated from the milk of healthy cows. In other embodiments, the lactating cells can be extracted from healthy mammary tissue, which is identified using a set of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EPCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e. In some embodiments, the lactating cells can be extracted from healthy mammary tissue selected from all mammary cells, all mammary epithelial cells, mammary luminal cells, mammary luminal progenitor cells, mature mammary luminal cells, mammary myoepithelial cells, and mammary stromal cells. The lactating cells can be genetically modified to induce hyperlactation, or produce milk that is hypoallergenic, reduced in lactose, and has increased A2β-casein protein. In some embodiments, each hollow tube can be coated with collagen. The one or more tubes are formed of a material at least a portion of which is a semi-permeable capillary membrane. In some embodiments, the inner surface of the one or more tubes is first bound to a layer of a support matrix, wherein the lactating cells attach to the unbound surface of the support matrix, and wherein the lactating cells form a monolayer of lactating cells on top of the support matrix.

[0020] In some embodiments, the device further includes a system controller configured to generate control signals to the pump and the supply loop.

[0021] An optional mechanical stimulation assembly can be included to apply a squeezing pressure to the exterior of the one or more tubes in a direction from the inlet end towards the outlet end. One or more optional light sources can 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.

[0022] The lactation medium can include EpiCult TM Plus medium, a laboratory-grown FCS alternative medium, and / or a serum-free medium, and can further include prolactin.

[0023] In another aspect of the present invention, a milk production facility can be constructed by interconnecting a plurality of the above-described devices.

[0024] In yet another aspect of the present invention, a method for in vitro milk production in the above-described device includes: supplying a lactation medium to the one or more tubes, wherein the lactation medium contains prolactin; forming a monolayer of lactating cells attached to the inner surface of the one or more tubes; and controlling a pump to alternate between different shear stress levels within a predetermined range, wherein the change in the shear stress level during the processing period stimulates the lactating cells to produce milk products. The method may further include recycling the milk products into the one or more tubes via a feedback loop to enrich the milk products until a 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 lactation medium may be EpiCult TM Plus medium, a laboratory-grown FCS alternative medium, and / or one or more of the serum-free media.

[0025] In some embodiments, the lactating cells can be isolated from the milk of healthy cows. In other embodiments, the lactating cells can be extracted from healthy mammary tissue, which is identified using a set of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EPCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e. In other embodiments, the lactating cells can be extracted from healthy mammary tissue selected from all mammary cells, all mammary epithelial cells, mammary luminal cells, mammary luminal progenitor cells, mature mammary luminal cells, mammary myoepithelial cells, and mammary stromal cells.

[0026] The lactating cells can be genetically modified to induce hyperlactation and / or to produce milk that is hypoallergenic, low in lactose, and has increased A2β-casein protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Those skilled 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.

[0028] Figure 1A is a diagrammatic view of the basic components of a bioreactor of the present invention according to one embodiment; Figure 1B diagrammatically shows the elements within a hollow tube that mimics the natural environment for supporting a monolayer of acinar cells and feeder cells co-cultured attached to the inner lumen of the tube; Figure 1Cis a schematic view of a tube cross-section showing a monolayer of lactating cells attached to the inner surface of a hollow fiber; Figure 1D shows laminar flow in a hollow fiber bioreactor; Figure 1E is a schematic view of an alternative tube configuration.

[0029] Figures 2A - 2C Schematically shows another embodiment of the bioreactor of the present invention, where Figure 2A and Figure 2B provide a front view and a side view of an embodiment of a bioreactor assembly, respectively; Figure 2C shows a bottom view of the assembly having a cross-section of a cartridge and a 3-D image of a hollow tube; Figure 2D shows an exemplary layout of a group of bioreactors in a milk production facility.

[0030] Figure 3 Schematically shows a formation model of a monolayer of co-cultured alveolar cells and feeder cells attached to the inner lumen of a 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 epithelium and an outer layer of myoepithelial cells that are in direct contact with the basement membrane. Driven by the prolactin hormone, the alveoli absorb nutrients from the blood supply and produce breast milk.

[0031] Figure 4 Compares the effects of different types of mechanical stimulation (shear stress and shear strain) by flow on endothelial cells of healthy EC cells. The following figure provides SEM micrographs of EC cells with and without exposure to shear stress.

[0032] Figures 5A - 5B are photomicrographs of mammary epithelial cells with an epithelial-like phenotype and mammary myoepithelial cells with a spindle-like phenotype, respectively; Figure 5C shows the growth rate curve of a mammary tissue cell line inoculated with 1x10 4 cells and then measured over the next 4 days. Values of three independent cell samples per day were compared using an unpaired t-test. Significance between the 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 figure; Figure 5D and Figure 5E is a graph showing the growth rate of co-cultured PBMC conditioned medium.

[0033] Figure 6 is a graph of inducing hyperlactation in cells by CRISPR. Detailed Description

[0034] For the sake of understanding the present invention, some terms and abbreviations used herein are defined as follows:

[0035] As used herein, the term "amino acid" refers to the molecular basis for constructing and assembling proteins such as enzymes. Peptide bonds (i.e., polypeptides) form between amino acids and undergo three-dimensional (3-D) assembly. The 3-D assembly can affect the properties, functions, and conformational dynamics of proteins. In biological systems, proteins can: (i) catalyze reactions as enzymes; (ii) transport vesicles, molecules, and other entities within cells as transporter entities; (iii) provide structure as protein filaments for cells and organisms; (iv) replicate deoxyribonucleic acid (DNA); and (v) coordinate the actions of cells as cell signalers.

[0036] As used herein, the term "nucleotide" refers to the molecular basis for constructing and assembling nucleic acids such as DNA and ribonucleic acid (RNA). There are two types of nucleotides - purines and pyrimidines. Specific purines are adenine (A) and guanine (G). Specific pyrimidines are cytosine (C), uracil (U), and thymine (T). T is present in DNA, while U is present in RNA. The genetic code defines the sequence of nucleotide triplets (i.e., codons) for specifying which amino acids are added during protein synthesis.

[0037] As used herein, the term "gene" refers to a region of DNA. The amino acid sequence in a protein defined by the sequence of a gene is encoded by the genetic code.

[0038] As used herein, a recombinant nucleic acid or protein is a nucleic acid or protein produced by recombinant DNA technology, for example, as described in Green and Sambrook (2012).

[0039] The terms "polypeptide", "protein", and "peptide" are used interchangeably herein and refer to an amino acid chain in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chain can be of any length greater than two amino acids. Unless otherwise specified, the terms "polypeptide", "protein", and "peptide" also encompass their various modified forms. Such modified forms can 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, etc. Modifications also include intramolecular crosslinking and covalent attachment of various moieties, such as lipids, flavins, biotin, polyethylene glycol, or derivatives thereof. In addition, modifications can include protein cyclization, branching of the amino acid chain, and crosslinking of proteins. Further, amino acids other than the conventional twenty amino acids encoded by genes can also be included in the polypeptide. The term "protein" or "polypeptide" can also encompass a "purified" polypeptide that is substantially separated from other polypeptides in the 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).

[0040] As used herein, the term "bioreactor" refers to a vessel or tank in which whole cells or cell-free enzymes convert raw materials into biochemical products and / or less undesirable by-products. Bioreactors are designed and operated to provide an environment for product formation, in this case dairy products. Industrial bioreactors can operate as batch reactors or continuously, aerobically or anaerobically, and in the case of pure cultures or mixed cultures. In some bioreactors, there are three phases (gas phase, liquid phase, and solid phase), 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 crucial for industrial bioreactors.

[0041] As used herein, the term "shear stress", which is typically denoted by τ (Greek: tau), refers to the component of stress that is coplanar with the cross-section of a material. (See, for example, Figure 4。) It is generated by shear stress, which is the component of the force vector parallel to the cross-section of the material. Any real fluid (including liquids and gases) moving along the solid boundary will generate 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; although at a certain height from the boundary, the flow velocity must be equal to the flow velocity of the fluid. The region between these two points is called the boundary layer. For all Newtonian fluids in laminar flow, the shear stress is proportional to the strain rate in the fluid, where viscosity is the proportionality constant. For non-Newtonian fluids, the viscosity is not constant. Due to this velocity loss, shear stress is applied to the boundary.

[0042] As used herein, the term "shear strain" refers to the ratio of the displacement due to stress to the original dimensions of the object, and is the amount of deformation perpendicular to a given line rather than parallel to a given line. Shear strain is the lateral force applied to the medium and is measured as the change in the angle between initially perpendicular lines. (See, for example, Figure 4 。)

[0043] As used herein, the term "pulsatile flow" refers to a flow having periodic pressure fluctuation waves traveling along the flow path. A pulsatile flow system can mimic the blood flow characteristics within the heart and vasculature.

[0044] As used herein, the term "effective amount" refers to those amounts that, when administered to a particular subject taking into account the nature and severity of the subject's condition, will have the desired biological effect, such as the amount that will cure, prevent, inhibit, or at least partially arrest or partially prevent the target response.

[0045] Throughout this disclosure, a number of acronyms and abbreviations are used. Table 1 below summarizes and provides a glossary of some commonly used acronyms and abbreviations:

[0046] Acronyms Definitions EC Epithelial Cells mEP or MEP Mammary Myoepithelial and Epithelial Cells MEC Mammary Epithelial Cells ECM Extracellular Matrix or Culture Medium HDF Human Dermal Fibroblasts InC Intercapillary ExC Extracapillary

[0047] Table 1

[0048] The terms "hollow tube", "hollow conduit", "hollow fiber", and "lumen" may be used interchangeably throughout the description to refer to an elongated hollow structure through which fluid flows under pressure applied by a pumping system fluidly connected to the hollow structure. The hollow structure can be formed of many different materials, which can be rigid, flexible, permeable, semi-permeable, or non-permeable. Examples of materials include, but are not limited to, glass, acrylic, plastic, polymer, fiber, silicone, ceramic, and filter membranes. In some embodiments, the hollow structure can be compressible or contractible to mimic a peristaltic-like action, such as by applying opposing rollers to the outer surface of the conduit to "squeeze" the fluid inside the conduit from a first end of the conduit to a second end. (See, for example,Figure 1B )。In other embodiments, the tube can be a hybrid assembly of segments of tubes made of different materials, some of which can be permeable membranes or semi-permeable membranes, while others can be non-permeable.

[0049] Lactation Biology

[0050] Lactation is the process of synthesizing and secreting milk from the mammary glands. The mammary glands are modified sweat glands and are mainly composed of adipose and collagenous tissue, where the mammary glands constitute a very small part of the breast volume. The mammary glands are composed of lactiferous ducts that transport milk, and the lactiferous ducts expand and branch extensively in response to estrogen, growth hormone, cortisol, and prolactin. In addition, in response to progesterone, clusters of mammary alveoli sprout from the ducts and expand outward toward the chest wall. Mammary alveoli are balloon-like structures lined with cuboidal or milk-secreting cells surrounded by a network of contractile myoepithelial cells. Milk is secreted by the milk-secreting cells, fills the alveoli, and is squeezed into the ducts. Clusters of alveoli that drain into a common duct are called lobules; lactating females have 12 - 20 radially arranged lobules around the nipple. Milk drains from the lactiferous ducts into the lactiferous sinuses, which converge at 4 to 18 perforations (called nipple pores) in the nipple.

[0051] The pituitary hormone prolactin helps to establish and maintain the breast milk supply. Prolactin and other hormones prepare the breasts anatomically for milk secretion. When an infant suckles, sensory nerve fibers in the areola trigger a neuroendocrine reflex that causes milk to be secreted from the milk-secreting cells into the alveoli. The posterior pituitary releases oxytocin, which stimulates the myoepithelial cells to squeeze the milk out of the alveoli so that it can drain into the lactiferous ducts, be collected in the lactiferous sinuses, and be expelled through the nipple pores. The time from when the infant starts suckling (latency) until milk secretion (lactation) takes less than 1 minute. Prolactin-mediated milk synthesis changes over time. Frequent removal of milk by breastfeeding (or pumping) will maintain high circulating prolactin levels for several months. However, even with continued breastfeeding, baseline prolactin will decline over time to its pre-pregnancy level. In addition to prolactin and oxytocin, growth hormone, cortisol, parathyroid hormone, and insulin also contribute to lactation, partly by promoting the transport of maternal amino acids, fatty acids, glucose, and calcium into breast milk.

[0052] Pulsatile Flow Cultivation of Mammary Cell Types for Milk Secretion

[0053] The system of the present invention is designed to simulate the natural environment of healthy epithelial cells (ECs) and mammary alveoli and ducts for optimal milk production by inducing variable shear stress on the cells.

[0054] Reference Figures 1A - 1E, embodiments of the bioreactor of the present invention are designed to reflect the natural environment of healthy epithelial cells (ECs) and mammary alveoli and ducts for optimal milk production. Figure 1A A diagrammatic side view (left) and cross-sectional end (right) view showing the basic components of an exemplary embodiment of a bioreactor flow system 100 are provided, which is configured to achieve adaptive shear stress within a closed sterile system. The incubator 102 includes: a liquid-sealed housing or casing 120 enclosing one or more semi-permeable hollow tubes 104; a pump system 106; a valve 108 for controlling the flow of extracellular culture medium fluid; reservoirs 110, 112; and a feedback loop 118 with a valve 116 for recycling a portion of the processed fluid (accumulated culture medium) through the reactor pipes for further processing / enrichment. The housing 120 can be formed of polycarbonate or a similar polymer, glass, stainless steel, or any material considered suitable for food handling (i.e., sterilizable). The fluid extracellular culture medium (ECM) is introduced into and circulated through the bioreactor via reservoirs 110, valve 108, and inlet-outlet pipes 128 and 130. (A pump for controlling this ECM loop is not shown.) The flow through tube 104 is configured to apply mechanical shear stress to the cells within the tube as the lactation / accumulated culture medium flows under pressure from the pump. The system controller 122 provides control signals to the pump 106 to perform various operations, including emptying the system, applying pulsatile and / or continuous flow through tube 104, for example, and harvesting milk. The system controller 122 can also be configured to generate control signals for valves 108, 116, ECM loop operation, and any additional valves and other operations within the system. In some embodiments, the system controller 122 can include a combination of one or more computer processors and memory for storing and executing programming for automated operation of the system. In some embodiments, parameters for optimized operation of the system can be established by using a learning machine within the system controller, where the inputs include system sensor signals (flow rate, pressure, temperature, etc.) and quality control measurements obtained through testing of the milk product.

[0055] In some embodiments, the pump 106 is controlled to produce a continuous, unidirectional, oscillating, and pulsatile flow with an adaptive flow rate to expose the 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 within a range from about 2 dyn / cm 2 (considered "low") and 75 dyn / cm 2 (considered "high") by a signal from the system controller 122. The pump sequence generated by the controller 122 can cause the pump flow to induce shear stress that alternates between different levels, for example, low (at about 2 - 10 dyn / cm 2within a range of) to medium (range of about 11 - 40 dyn / cm 2 ) and high (range of about 41 - 75 dyn / cm 2 ) to medium, high to low, or high to low to medium and then to back, etc. It is the change in shear stress applied to the cells via the pulsatile / oscillatory flow that helps stimulate lactation. When the lactation medium flows through the cells, it accumulates milk components to become the "accumulated medium", which is processed through the hollow tube 104 and collected in the reservoir 112 downstream. One or more inlet ports (not shown) may be included at or near the outlet 162 to allow sampling of the accumulated medium to determine whether it has reached the desired quality of the finished milk product, such as density, appearance, consistency, fat content, chemical content, etc. If the test indicates that the product is complete, the milk can be harvested. If not, the accumulated medium can be returned to the reactor inlet 160 via the valve 116 and the feedback loop 118 for further enrichment. Alternatively, once the optimal processing duration has been determined for a given operating condition, it may not be necessary to perform the test regularly.

[0056] Figure 1B Additional details of the bioreactor structure and function of the present invention are schematically shown, showing a section of an exemplary hollow tube 104, which in some embodiments can be one of a plurality of elongated tubes or lumens arranged in a parallel array that longitudinally extends within a generally cylindrical outer housing 120 to jointly define a closed sterile bioreactor chamber 102. The interior of the tube 104 is referred to as the capillary inner space or InC space 134, while the space outside the tube (within the housing 120) is referred to as the capillary outer space or ExC space 132. In some embodiments, the inner surface of the tube 104 can be further lined with a collagen membrane (see, for example, Figure 1C ). Feeder (support) cells 138 and mEP cells 140 are coated onto the inner surface of the hollow tube 104. It should be noted that for convenience, only a few cells 140 are shown with primary cilia 142 that play an important role in milk production. As those skilled in the art will recognize, healthy ECs should all have primary cilia. The mechanical sensing generated by the adaptive flow within the tube 104 helps maintain cell differentiation and health. The tube 104 can be formed of a semi-permeable membrane material (such as a polymer or similar material) through which nutrients contained within the ECM can diffuse to provide nutrients to the cells. The inlet of the tube 104 (at the downstream end) is connected to the pump system 106. In a small-scale prototype implementation of the bioreactor of the present invention, ibidi TMA peristaltic pump (ibidi GmbH, Graefelfing, DE) provides the mechanical stimulation required for milk production via a pulsatile flow. For larger systems, one or more pumps with larger capacities are used to achieve the required pulsatile flow. Milk can be harvested in batches daily from a reperfusion reservoir 112 located at the downstream end of the reactor. An optional filter 114 can be included on the upstream or downstream side of the reservoir 112 to remove dead cells and other particulate material that may accumulate in the milk prior to harvesting.

[0057] While the hollow tube 104 is described herein as "semi-permeable", its construction is not limited to semi-permeable materials. Instead, the only requirement is that portions of the tube have sufficient permeability to allow a certain amount of extracellular medium to enter the InC to nourish the lactating cells. Thus, as a hybrid assembly of multiple segments, the hollow tube 104 is still considered "semi-permeable", some of these segments being permeable or semi-permeable while others are non-permeable. For example, a ceramic filter membrane can be used for segments of the tube to supply ECM to the cells, while the remainder of the tube can be less permeable or non-permeable. This hybrid approach allows for customized control of how much ECM is available to the cells, which avoids providing too much ECM, which could potentially disrupt the final product.

[0058] Figure 1B The embodiments shown include optional configurations in which additional mechanical stimulation is provided in the form of a assembly of opposing cylindrical rollers 150 that travel along tracks 152 positioned within the housing 120 such that they are parallel to the tube. The rollers gently compress the tube from multiple sides to gently "squeeze" and push the contents of the tube, i.e., the lactation medium and the accumulating medium with milk components 144, from near the inlet end of the reactor towards the outlet end. The pressure applied by the rollers should be uniform and strong enough to move the fluid, 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 applying adjustable shear stress. Once the rollers have completed their travel along the tracks, they are moved away to release the pressure on the tube and are moved back on the tracks to their 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 tracks and rollers or annular sphincters can be used to achieve the desired inlet-to-outlet pushing action, which aids in releasing the cells for continued milk production. The signals for controlling the optional mechanical stimulation assembly will be provided by the system controller 122.

[0059] Figure 1CA graphical cross-sectional view of a monolayer of lactating cells attached to the inner surface of each hollow fiber 104 within an array of fibers attached to reactor cylinder 102 is provided. In the illustrated embodiment, the bioreactor is a modified reperfusion reactor having a plurality of hollow fiber elements 104 supplied by a peristaltic pump (not shown) to produce a continuous, unidirectional, oscillatory, and pulsatile flow with an adaptable flow rate. Reactor cylinder 102 may comprise a multi-tube bioreactor having inlets and outlets for inputting fluid to / from the inter-capillary space and the extra-capillary space. As shown, the InC space inlet 160 supplies the hollow tubes 104, which then output milk (i.e., accumulated culture medium) from the InC space 162 to the outlet. The extracellular culture medium (ECM) is supplied into the ExC space via inlet 128 (from reservoir 110) and then recycled back to 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 portion of the figure, a transverse cross-section of five individual hollow fibers 104 is shown, wherein the inner surface of the outer fiber wall is lined or coated with a collagen membrane 156. Cells 140 (and feeder (support) cells) grow on the inner lumen of the hollow fiber. The collagen membrane supports the monolayer of cells so that milk can flow through the interstices (lumens) of the cell layer. The upper right portion of the figure shows a longitudinal cross-section of a single fiber, also having a collagen membrane "lining" and a monolayer of cells 140, with milk 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 appropriate flow parameters for generating the shear forces required to mechanically stimulate the cells on the inner surface of the tube.

[0060] The above-described embodiment of the bioreactor of the present invention employs a peristaltic pump for the purpose of inducing shear stress on lactating cells with a continuous fluid flow within a sterile reactor environment. As will be recognized by those skilled in the art, alternative methods for inducing controlled shear stress with a continuous or variable fluid flow within a sterile environment include using rotational vortices, magnetic vortices, such as stir bars and wave pumps or valve pumps. Such methods would involve placing the vortices and / or pumps within a sterile enclosure, which, relative to the above-described embodiment, could negatively impact the scalability of the process.

[0061] As will be recognized by those skilled in the art, the number of hollow tubes within the bioreactor assembly can vary and the illustrated example is not intended to be limiting. Figure 1EAn example of a variation in the tube configuration is provided, where a single long tube 304 is arranged in a zigzag, looped, coiled, or serpentine pattern within the housing 320. The overall direction of flow remains parallel to the length of the housing, i.e., from the inlet to the outlet, and recirculation can occur through a feedback loop similar to the feedback loops in other embodiments. In this example, the tube 304 is a hybrid assembly formed from multiple tubular segments of different materials, where the tube segment 344 is a permeable or semi-permeable material to allow the ECM to be perfused only through these segments into the interior of the tube. The tube segment 346 is non-permeable. The pump 306 controls the pulsatile flow through the tube from the inlet to the outlet. It should be noted that this is only an illustrative example and is not intended to be limiting.

[0062] Figures 1A - 1C The bioreactor embodiments shown and described above are presented in a horizontal orientation; however, no particular orientation is required. Referring to Figures 2A - 2C , some embodiments of the bioreactor employ a vertical orientation, which can assist in scaling up the capacity of the processing facility to produce larger volumes of milk. Each cartridge 202 includes the same basic components as described above: a housing 220 that encloses one or more hollow tube reactors 204. The pump 206 supplies a fluid (e.g., PBS) to the top of the cartridge 202 at the inlet 260, which feeds the fluid into the tube 204. The hollow tube 204 has a monolayer of cells, particularly ECs, coated on its inner surface, as Figure 2C shown. As in the previously described embodiments, the pump 206 is configured to induce an adaptive shear stress as the fluid flows through the tube to stimulate milk production by the cells. The vertical orientation of the cartridge 202 provides gravity assistance for the flow through the tube. The pump 206 can recirculate a portion of the partially processed milk (accumulated culture medium) through the feedback loop 218 until the desired milk characteristics are achieved. Although not shown, one or more test ports / valves can be provided at or near the outlet 262 to allow testing of the processed milk to determine completion. A filter 214 can be located at the outlet 262 to remove waste products. The valve 264 allows the delivery of the finished milk product to a reservoir for harvesting. Figure 2A The right figure of provides a diagrammatic cross-sectional view of the cartridge 202 with the hollow tube 204, where the hollow tube defines the ExC (external to the tube) space and the InC (internal to the tube) space as described above. The ECM is supplied into the ExC space within the housing 220 via the inlet 228 and removed from the housing at the outlet 230. The valve 208 and one or more pumps (not shown) provide control of the ECM flow. In Figure 2C additional details of the tube 204 are shown, where the lower figure provides an exemplary 3-D perspective view of the tube.

[0063] In some embodiments of the bioreactor of the present invention, an optional light source 216, particularly a light-emitting diode (LED), may be included to produce different effects. In one embodiment, LEDs emitting in the blue light range (e.g., about 450 nm) have been shown to stimulate primary cilia. In a study by Prosseda et al., blue light was used to stimulate primary cilia in cells to regulate and increase cell contraction. (“Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation”, Science Advances, April 29, 2020, Vol. 6, No. 18, DOI: 10.1126 / sciadv.aay8699, which is incorporated herein by reference.) Since the primary cilia of ECs must be stimulated to produce milk, optical stimulation is added to the mechanical stimulation provided by adaptive shear stress to increase the overall stimulation required for milk production. The optical stimulation can further use intensity pulses to produce additional variable stimulation of the cells. Since light is used to stimulate ECs, the material forming the tube 204 should be able to transmit the desired wavelength. For example, glass or transparent tube sections can be used near the LED. Although the LEDs are shown in the lower part of the cylinder, it will be readily apparent that they can be positioned at multiple locations throughout the interior of the housing 220 to increase the exposure of ECs to light.

[0064] In other embodiments, other light sources may be included in the cylinder or in the plumbing outside the cylinder. For example, a UV light source (100 - 280 nm) can be used to enhance the sterility of the product; however, care must be taken to avoid damaging the cells. Other light sources can be used to promote the health of ECs. For example, light sources emitting wavelengths including 660 nm, 700 nm, 810 nm, and 850 nm have been reported to promote cell activity and growth. The control signal for the operation of the light stimulation system can be provided by the system controller.

[0065] Figure 2DIllustrates diagrammatically a possible arrangement of a plurality of bioreactors within a production facility. Multiple such groups with any number of units (i.e., cartridges and associated piping systems) can be installed in a support frame (not shown) within the facility, which can preferably include a clean room. In one possible embodiment, each group can have its own dedicated reservoir 210 for supplying extracellular medium to the bioreactor units within the group; and a milk collection reservoir 212 for collecting the milk produced by the units within the group. In other embodiments, multiple groups can receive the medium from one or more larger central reservoirs and output the milk to one or more large central tanks. Various combinations of central and dedicated reservoirs can be used. For illustrative examples, in a large production facility, one or more groups of bioreactor units can be used to produce different types of milk, such as whole milk, low-fat milk, hypoallergenic milk, lactose-free milk, or any of a variety of "specialty" milk products, such as those described below.

[0066] The mammary epithelium consists of two differentiated cell types organized into two cell layers, an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells that are in direct contact with the basement membrane. Mammary epithelial cells (MECs) can proliferate as a monolayer on plastic. However, milk production actually occurs only when signals are received from ECM proteins plus hormones (prolactin, growth factors) (i.e., a structure similar to that observed in vivo) and tissue-specific gene expression (e.g., the casein gene). In some embodiments, MECs can be harvested from fresh milk, as shown in one of the examples.

[0067] Using the disclosure herein, one of ordinary skill in the art will be able to implement similar strategies to extract MECs from fresh milk or mammary glands using other mammary epithelial cell biomarkers. Table 2 below provides a list of biomarkers in mammary tissue (see, for example, Figure 3 ), which can be used to extract MECs for the methods of the present invention. Exemplary selection groups can include combinations of these biomarkers. For example, a group of healthy mammary cells can be assembled from a combination of biomarkers from one or a combination of sources, such as all, all epithelial, luminal, etc. selected from those listed in the bottom seven rows of the table.

[0068]

[0069]

[0070] Table 2

[0071] Pulsatile Flow:Pulsatile flow is a wave of periodic pressure fluctuations traveling along a flow path, such as the pressure fluctuation wave generated in the hollow tubes of a bioreactor. A pulsatile flow system can closely mimic the blood flow characteristics within the heart and vascular system.

[0072] Mammary endothelial cells (MECs) adapt their morphology and function to the hemodynamic environment in vivo where they are located. In vitro experiments have shown that similar changes occur in cultured MECs exposed to shear stress induced by laminar steady flow. However, in vivo MECs are exposed to a pulsatile flow environment; thus, in this study, the effects of pulsatile flow on cell shape and orientation and actin microfilament localization in confluent bovine aortic endothelial cells (BAECs). These results suggest that ECs can distinguish between different types of pulsatile flow environments. Additionally, these experiments demonstrate the importance of engineering the cell culture environment to include pulsatile flow in the study of endothelial cell biology.

[0073] Eliminating FCS from Lactation Medium: Today, standard media rely on the established but outdated concept that serum from unborn calves (FCS) is required, which has been used as the "gold standard" since the 1960s. Over the years, the problems with FCS and the reasons for the need to replace it with a better alternative have become a field of considerable research interest. These problems with FCS include the fact that (a) its production is a cruel enterprise, and (b) due to reports of viral contamination of serum, as well as other safety issues with endotoxins, mycoplasma, RNA contaminants, or prion proteins, FCS cannot be used in the production of clean milk for human consumption. Thus, the media used to culture the cells for use in the bioreactors of the present invention are preferably FCS-free and hormone-free (except for prolactin) and are based on the latest scientific research and cell culture techniques.

[0074] Generally, serum starvation makes cells unhealthy; however, this adverse effect can be offset by flow regulation. Changes in flow intensity can induce cilia growth. Thus, flow stimulation is needed to grow cilia for milk production.

[0075] Mechanical Stimulation (Flow) of Healthy EC Cells to Counteract FCS Elimination from Lactation Medium: Both hormones and mechanical signals can induce changes in gene expression in mammary epithelial cells (MECs), which ultimately lead to milk synthesis and secretion. Using a serum-free medium and a combination of hormonal and mechanical stimuli, bovine mammary epithelial cells (BMECs) transform from primary multicellular organoids into a coordinated 3D ductal network. See, for example, Figures 5A - 5B , which respectively show mammary epithelial cells with an epithelial-like phenotype and mammary myoepithelial cells with a spindle-like phenotype. At the structural level, there is evidence that mechanical signaling via gel release, prolactin, and a combination of both results in changes in cell morphology that coincide with the onset of stage I lactogenesis.

[0076] Endothelial cells require mechanical stimulation for healthy growth. Studies have shown that a certain type of flow can provide shear mechanical stimulation to EC cells, thereby improving cell performance. In some embodiments, continuous mechanical stimulation of healthy ECs results in an increased, hormone-free milk production process. It is important to understand the nature of the stimulation applied to the cells, the principle of which is shown in Figure 4 . When force acts directly on the cells, mechanical strain occurs. At high strain, the endothelial barrier may relax, leading to cell death and a high cell turnover rate. Epigenetic modifications and inflammation may occur. In contrast, mechanical shear involves the parallel movement of fluid over the top of the cells, which increases cell survival and has a low turnover rate. Therefore, the goal is to avoid mechanical strain while increasing mechanical shear to stimulate cell function. Figure 4 The following figure of

[0077] provides a comparison of the cell layer after (left) and before (right) the application of shear stress. TM In some embodiments, the lactation medium and test cultures are fluidly driven by a peristaltic pump, such as the ibidi

[0078] Feeder Cells: Feeder cells or support cells 138 comprise a layer of cells that cannot divide, but which provide extracellular secretions to promote the proliferation of other cells (i.e., lactating cells 140). Feeder cells are different from co-culture systems in that only one cell type is capable of proliferation.

[0079] In some embodiments, the feeder cells are co-cultured with MECs and form a monolayer of lactating cells attached to the inner lumen of an elongated hollow fiber bioreactor. In some embodiments, the feeder cells can be inoculated in the capillary outer (ExC) space surrounding the hollow fiber.

[0080] Hollow Fiber Bioreactor: A hollow fiber bioreactor is a three-dimensional cell culture system based on hollow fibers. In some embodiments, the hollow fibers are small semi-permeable capillary membranes arranged in an array, with a typical molecular weight cut-off (MWCO) range of 10 - 30 kDa. Exemplary embodiments are described above with reference to Figures 1A - 1C 、 Figures 2A - 2D . When multiple tubes are employed, the tubes can be bundled and housed within a cylindrical housing of polycarbonate, glass, stainless steel, or other suitable material (i.e., sterilizable) to produce a hollow fiber bioreactor cartridge. Inside the cartridge, which is also fitted with an inlet port and an outlet port, are two compartments: the capillary inner (InC) space 134 within the hollow fibers and the capillary outer (ExC) space 132 surrounding the hollow fibers.

[0081] Cells are seeded into the InC space 134 of the hollow fiber bioreactor 104( Figure 1B ) and expanded therein to coat the inner surface of the hollow fibers 104 with a monolayer. Cell culture medium is pumped through the ExC space 132 to deliver oxygen and nutrients to the cells via perfusion through the hollow fiber membrane. As the cells expand, their waste products and CO 2 also perfuse the hollow fiber membrane and are removed by pumping of the accumulated culture medium via the InC space 134. As waste products accumulate due to the increased cell mass within the InC, it may be necessary to increase the flow rate of the culture medium so that cell growth is not inhibited by waste product toxicity. After the accumulated culture medium has been fully enriched, i.e., milk production has been completed, the waste products can be filtered out by a filter disposed within or near the reservoir.

[0082] Supporting Extracellular Matrix: The mammary epithelium can comprise two differentiated cell types organized into two cell layers, an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells, which are in direct contact with the basement membrane. Mammary epithelial cells can proliferate as a monolayer on plastic. However, milk production occurs only when signals are received from ECM proteins plus hormones (prolactin, growth factors), structures similar to those observed in vivo, and tissue-specific gene expression (such as the casein gene). Thus, to mimic natural milk production, the bioreactor must recreate these structures and an environment having the same (or similar) stimuli as occur in the in vivo environment. In some embodiments, this can be achieved by modulating the supporting extracellular matrix, the composition of the culture medium, and by co-culturing the necessary cell types to achieve functional differentiation.

[0083] Other successful methods can also stimulate milk genes. In some embodiments, the cells show reorganization and formation of 3D structures such as those observed in vivo and exhibiting milk protein gene expression on collagen gels. In some embodiments, epithelial cells grown in gels rich in laminin undergo functional differentiation based on the expression of the casein gene. Natural and synthetic polymeric materials have been investigated as alternatives to ECM proteins.

[0084] In some embodiments, 3D bioprinting of mammary tissue can be used. In some embodiments, 3D culture procedures including prolactin (prolactin, insulin, and hydrocortisone) are capable of inducing casein synthesis from primary murine epithelial cells embedded in floating collagen gels. In some embodiments, in a 3D in vitro model of bovine primary mammary epithelial cells grown on collagen-coated cell culture inserts, a differentiation medium supplemented with oleic acid, pituitary extract, and dexamethasone shows induction of the synthesis of major milk components such as β-casein, triglycerides, and lactose.

[0085] The morphological and functional differentiation of cryopreserved lactating bovine mammary cells can be cultured on floating collagen gels. In some embodiments, bovine primary epithelial cells from lactating mammary tissue grown on floating collagen gels exhibit a polarized conformation with a highly differentiated state, as demonstrated by the observation of apical microvilli, tight junctions, and lipid droplets surrounded by casein-containing secretory vesicles.

[0086] The use of collagen gels provides the advantage of mimicking in vivo conditions and allows for relatively long-term studies.

[0087] The milk produced by the bioreactor of the present invention has the ability to match the nutritional content, taste, and quality of conventionally obtained milk.

[0088] Examples

[0089] Aspects of the systems and methods of the present invention can be further understood from the following examples, which should not be construed as limiting the scope of the teachings of the present invention in any way.

[0090] Example 1: Bioreactor

[0091] As described above, the bioreactor of the present invention is designed to reflect the natural environment of healthy epithelial cells (ECs) and the mammary alveoli and ducts for optimal milk production. The goal of the system and method is to utilize process control conditions and cell biology to provide primary EC cultures that are FCS-free or substantially FCS-free. In some embodiments, continuous mechanical stimulation of healthy ECs results in an increased, hormone-free milk production process. (It should be noted that the prolactin hormone is still necessary.) In some embodiments, the lactation medium and test cultures are fluidically driven by a peristaltic pump to generate adaptive shear stress in a closed sterile system. In some embodiments, mechanical shear stress can be evaluated and continuously or periodically monitored. In some embodiments, the bioreactor is a modified reperfusion reactor with a hollow fiber module, which is controlled by a peristaltic pump to generate a continuous, unidirectional, oscillating, and pulsatile flow with an adaptive flow rate. In some embodiments, lactating cells grow or are coated on the inner surface of the hollow tube 104. The inlet of each hollow tube is connected to a pump to provide flow stimulation via pulsatile flow. Milk can be harvested in batches at prescribed intervals, from a time period ranging from daily to weekly to monthly, depending on the scale (volume) of the entire bioreactor system. The system supplies extracellular medium through a permeable and / or semi-permeable membrane in the tube to nourish the lactating cells inside the tube. ECM can be periodically supplemented and / or replaced throughout the processing period.

[0092] Example 2: Mammary Epithelial Cell (MEC) Harvest

[0093] Fresh raw milk (i.e., from Guernsey or Galloway cows) is skimmed by low-speed centrifugation at typical refrigeration temperatures (about 4 °C). The skim milk is removed, and the remaining total cell pellet is washed several times in phosphate-buffered saline (PBS) and resuspended in PBS. Immunomagnetic separation is used to isolate MEC from total milk somatic cells and remove white blood cells, which are collected separately for further use. Immunomagnetic separation involves incubating the total milk cell suspension with magnetic beads (Dynabeads) coated with a primary monoclonal antibody against cytokeratin 8 (K8.13) (Boutinaud et al., 2008). The resulting antibody-bound MEC are then washed, and the purified MEC are resuspended in PBS. The MEC are then cultured in complete medium for 1 week to ensure sufficient cell proliferation. Complete medium consists of DMEM / F12 (alternatively EpiCult), which is supplemented with 2% FBS or FCS substitute, 5 μg / ml insulin, 100 ng / ml hydrocortisone, L-glutamine, and optionally supplemented with antibiotics.

[0094] Example 3: MEC Culture and Lactation

[0095] Mammary myoepithelial and epithelial cells (MEP) are seeded in complete medium and maintained until a monolayer is established. Low shear stress is used for flow-in in complete medium for cell proliferation (1 week). The shear stress is 0.007 dyn / cm 2 . Next, a settling and attachment period of about 4 hours is carried out in a hollow fiber reactor. A physiological shear stress of about 5 dyn / cm 2 is applied over a period of several days for proliferation and monolayer formation. The medium is changed every 3 days or fed-batch cultured.

[0096] Next, serum withdrawal is performed and lactation medium is supplemented to induce lactogenic differentiation. Pulsatile flow is initiated, where the pump is controlled to periodically change the fluid flow to induce variable shear stress alternating at different levels in the range of about 2 to 75 dyn / cm 2 over a period of at least one week, more preferably about 2 - 3 weeks. The total culture time will vary depending on the system size (volume), where small-scale systems require about 2 weeks, while larger-scale systems require up to several months for the medium to accumulate through reactor recirculation to complete the culture. Over a multi-week period, the lactation medium is changed every 3 days or fed-batch cultured. After the culture is completed, the product is harvested. Processing after harvest includes preservation by one or more of pasteurization, freezing, or dehydration. Quality control testing includes assessment of bacteria, mycoplasma, fungi, and major components.

[0097] Example 4: Using Matrix to Replace Feeder Cells

[0098] Feeder cells are generally suitable for supporting stem cell co - culture; however, when dealing with different species, this may create the possibility of transfer of animal pathogens and / or unwanted immune responses that may affect milk production. The Matrigel matrix structure provides a suitable and functional support structure for cells that require feeder cells.

[0099] To coat Matrigel (BD Biosciences, Bedford, MA), 500 μL of BD Matrigel was transferred to each well of a six - well plate on ice and spread by gentle shaking. Then the plate was incubated at 37 °C for 2 h to allow the BD Matrigel to polymerize into a gel. Then, a suspension of reprogrammed HDF (human dermal fibroblasts) (2.4×104 / cm 2 ) was transferred onto the coated plate with BD Matrigel matrix in DMEM medium containing 10% FBS, and the plate was returned to the incubator. After 48 h, the medium was replaced with supplemented DMEM / F - 12 (including the supplemented factors as described above), and the medium was changed daily to form hiPSC colonies. The iPSC culture was maintained and passaged 10 times.

[0100] Example 5: Preparation of Feeder Cells by Conventional Method

[0101] At 37 °C, the 3rd passage (P3) CF - 1 MEFs at 80%–90% confluence were inactivated with 10 μg / ml of MMC (Hisun Pharmaceutical Company, China) for 0 h, 0.5 h, 1.0 h, 1.5 h, and 2.0 h. After incubation, the cells were washed 6 times with PBS, digested with trypsin, centrifuged at 180×g for 5 min, and resuspended in MEF medium. The cells were counted and frozen for later use.

[0102] Example 6: Preparation of Feeder Cells by Suspension - Adhesion Method

[0103] We prepared feeder cells by SAM according to Figure 1. Briefly, the 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 a 50 - ml centrifuge tube. The cells were seeded at 8×104–1.1×105 cells / cm2 in a 10 - cm culture dish. MMC (10 μg / ml) was added after 2.0–3.0 h at 37 °C. The medium containing MMC was discarded 0.5 - 3.5 h after treatment. Then the cells were washed 6 times with PBS, digested with trypsin, centrifuged at 180×g for 5 min, and resuspended in MEF medium. The cells were counted and frozen for later use.

[0104] Example 7: Preparation of Feeder Cells by Three - Dimensional (3D) Suspension Method

[0105] After connecting the CELLSPIN system (5–75 RPM, CELLSPIN system with glass bead agitator, Integra Bio-Sciences, Switzerland) to the incubator, the rotating flask was sterilized by autoclaving. Feeder cells were prepared by 3DSM. Briefly, P3 CF-1 MEF grown for four days were digested into single cells with 0.25% trypsin / EDTA and collected into 50 ml centrifuge tubes. The cells were transferred to a rotating flask with a glass bead agitator that accommodates a volume of 25–1000 ml at a density of 0.5–1.3×106 cells / ml. MMC was added at 10 μg / ml. After incubation at 37 °C for 0.5 h, 1.0 h, 1.5 h, and 2.0 h, the cells were centrifuged at 180×g for 5 min, washed 3 times with PBS, resuspended in MEF medium, counted, and cryopreserved for later use.

[0106] Example 8: Production of Feeder Cells / or Supporting Matrix for Bioreactor

[0107] Bovine embryonic stem cells (ESCs) are powerful tools for agricultural and biomedical applications and have been successfully cultured and differentiated using feeder cells (Cell Reprogram. December 2012;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 systems and methods of the present invention is to generate customized feeder cells derived from bovine fibroblasts. Potential feeder cells that can be used in embodiments of the methods of the present invention include fibroblasts, mEPs and myoepithelial cells, and mEPs on fibroblast extracellular matrix.

[0108] Alternative synthetic methods can be employed as supports for milk-producing cells. The following examples identify some of these methods.

[0109] Example 9: Laboratory - Grown FCS Substitute

[0110] After activation, PBMCs secrete a powerful mixture of pro-survival factors that help them fight infection. They are secreted into the blood and become part of the serum phase. Co-culture experiments have shown that supplementing cells with the supernatant of cultured PBMCs significantly prolongs their survival. In cell culture, this is exploited by the "tight packing" of cells to rely on microenvironmental factors, namely a suspension of high cell density, to achieve healthy cell growth and recovery after freezing. Adding innate immune cell supernatant provides survival factors as strong as comparable direct survival stimulation.

[0111] PBMCs were extracted from the peripheral blood of volunteers using a Percoll gradient. After supplying them with an activating stimulus, the activating stimulus contains A) LPS, B) opsonized beads, C) mechanical stimulation in an ibidi TM pump system, the cells were kept in a stimulated state, and the supernatant was enriched for 24 to 48 hours and then collected. The supernatant was separated from the cell fraction and used to supplement the medium. For future scale-up, conditionally immortalized ER-Hoxb8 hematopoietic progenitor cells can be used and differentiated into PBMCs as needed to provide an unlimited supply of PBMCs.

[0112] Example 10: EpiCult TM Plus Serum-Free Medium for Mammary Epithelium

[0113] Successful tests were conducted in mammary gland cells using cell culture techniques adapted from a customized modification of the cell culture method for epithelial cells (mammary gland) from the FCS-free database (https: / / fcs-free.org / fcs-database) of the Foundation for Animal-Free Research.

[0114] Figures 5A - 5E Representative growth curves and images were provided. Photographic images of mammary gland tissue cells were taken using a live cell microscope with a Kodak imaging system, where Figure 5A mammary gland epithelial cells with an epithelial-like phenotype were shown, and Figure 5B mammary gland myoepithelial cells with a spindle-like phenotype were shown. Figure 5C The growth rate curve of the mammary gland tissue cell line was provided. 1x10 4 cells were seeded and then measured over the next 4 days. Values of three independent cell samples per day were compared using an unpaired t-test. The significance between the cell growth rates of mammary gland epithelial cells (green) and mammary gland myoepithelial cells (red) on day 3 (p = 0.007) and day 4 (p = 0.0228) was thus determined. These values were highlighted by asterisks in the figure. Figures 5D - 5E is a figure showing that adding 2% PBMCs (5D) or 1:3 neutrophil-conditioned medium (i.e., supernatant) (5E) provides a survival stimulus equivalent to adding the prosurvival mediator dbcAMP or LPS and PGE2 in cultured immune cells.

[0115] In some embodiments, an AI-guided method can be used to optimize the medium composition and optimize and adjust the processing parameters.

[0116] Example 11: In Vitro Milk Production Using Extracted Mammary Epithelial Progenitor Cells from Fresh Cow Milk

[0117] In a complete medium supplemented with 2% FCS (FCS substitute), 5 μg / ml insulin, 100 ng / ml hydrocortisone, L-glutamine, and optionally supplemented with antibiotics, IGF-I or EGF, a bioreactor is inoculated with mammary epithelial cells (MEP). The complete medium can be selected from DMEM, F12 or EpiCult. Incubate the MEP until a monolayer is established. Apply a low shear rate for inflow in the complete medium to maintain cell proliferation for 1 week. The sedimentation and attachment period in the hollow fiber reactor is 4 hours. Maintain medium to high shear stress for proliferation and monolayer formation for 2 days. Perform a complete medium change or fed-batch culture every 3 days. Perform serum withdrawal and supplement with lactation medium to induce lactogenic differentiation. The lactation medium comprises DMEM / F12 (alternatively EpiCult), which is supplemented with 2% FBS (or FCS-substitute), L-glutamine, 1 - 5 μg / ml prolactin (or shear stress as defined above). Induce pulsatile flow by alternately switching the pump between different pump rates (e.g., high, medium and / or low) in order to generate a shear stress in the range of 2 to 75 dyn / cm 2 . This step continues for a period of at least one week, more preferably about 2 - 3 weeks. As will be apparent to those skilled in the art, the total processing time will depend on the system size (volume). A small-scale system may only require 2 weeks to produce fully cultured milk, while a larger-scale system may require up to several months to complete the culture. Perform a lactation medium change or fed-batch culture every 3 days. Then harvest and process the product as described in Example 3.

[0118] Example 12: Genetic Engineering and Other Modifications for Inducing Hyperlactation

[0119] Hyperlactation can be induced in cells using CRISPR technology. Referring to Figure 6 the figures in, the following protocols can be used: CSN2 polymorphisms (SNPs): G->A in His220, Glu223; LALBA polymorphism: in the 5' LALBA promoter; PRLR SNPs: rs62355518, rs10941235, rs1610218, rs34024951, rs9292575; PRL SNPs: rs849872; Gain-of-function mutations with Jak2 overactivation: V617F. In addition, drug intervention can include the use of retinoic acid and ATRA to target CSN3 expression.

[0120] SNPs can be modified (via CRISPR) to reduce the lactose content in dietetic, lactose-free milk: CSN2 (Glu340, Thr174, Lys14), B4GALT1 (T224A).

[0121] A low-allergenic milk can be produced by introducing point mutations in base pairs 186 and 213 of the gene CSN1S1 using a CRISPR cluster bomb, which correspond to positions CDS 84-123 (CSN1S1) and CDS 87-123 (CSN1S2).

[0122] Table 3 below lists many potential modifications that can be achieved by genetic engineering of mEP and mEP-derived cell lines.

[0123]

[0124]

[0125] Table 3

[0126] Particularly desirable modifications achieved by the methods of the present invention include the production of A2-rich milk from cells.

[0127] Embodiments of systems and methods for in vitro milk production have been described above. Although specific exemplary embodiments have been described, it is apparent that various modifications and changes can be made to these embodiments without departing from the broader scope of the invention. Accordingly, the detailed description should not be understood 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 such claims.

[0128] References

[0129] 1. Quaglino, A. et al., Mechanical strain induces involution-associated events in mammary epithelial cells. BMC Cell Biol 10, 55 (2009). https: / / doi.org / 10.1186 / 1471-2121-10-55.

Claims

1. An apparatus for in vitro milk production, the apparatus comprising: a reactor assembly, the reactor assembly comprising: an elongate housing configured to retain an extracellular medium; one or more elongate hollow tubes longitudinally aligned within the housing and disposed within the extracellular medium, the one or more tubes having an inlet end, an outlet end, and an inner surface configured to attach a monolayer of lactating cells, wherein at least a portion of the one or more tubes comprises a semi-permeable material configured to allow the extracellular medium to diffuse into the hollow tube, wherein the extracellular medium comprises a nutrient solution for maintaining the lactating cells; a pump fluidly connected to the one or more tubes and configured to generate a pulsating or oscillating flow of milking 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 milk products; a supply loop for supplying and circulating the extracellular medium through the housing; and a reservoir fluidly connected to the outlet end of the one or more tubes and configured to collect the milk products.

2. The apparatus according to claim 1, further comprising a feedback loop disposed near the outlet end of the one or more tubes for recycling the milk products into the inlet end of the one or more tubes for further enrichment.

3. The device according to claim 1, wherein the predetermined range of the shear stress level is from 5 to 15 dyn / cm 2 .

4. The apparatus according to claim 1, wherein the supply loop comprises a reservoir configured to remove spent extracellular medium and add fresh extracellular medium.

5. The apparatus according to any one of claims 1 to 4, wherein the lactating cells are co-cultured with feeder cells.

6. The apparatus according to claim 5, wherein the feeder cells are peripheral blood mononuclear cells.

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

8. The apparatus according to any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary tissue identified using a set of biomarkers selected from CD9, CD47, CD54, CD59, CD95, CD164, CD49b, CD66, CD24, CK8 / 18, CK19, MUC1, GATA3, EPCAM, CD13, CD15s / CD73 / CLA, +CD166 / CD227 / CD340, CD10, CD90, CD200, CD29 / CD142 / CD271, CK5, CK14, CK17, SMA, V1M, CD34 / CD39 / CD140b, and CD49e.

9. The device according to any one of claims 1 to 4, wherein the lactating cells are extracted from healthy mammary tissue, and the healthy mammary tissue is selected from all mammary cells, all mammary epithelial cells, mammary luminal cells, mammary luminal progenitor cells, mature mammary luminal cells, mammary myoepithelial cells, and mammary stromal cells.

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

11. The device according to 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 in lactose, and has increased A2β-casein protein.

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

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

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

15. The device according to any one of claims 1 to 4, wherein the one or more tubes have a hybrid structure that includes a section of non-permeable material and a section of permeable or semi-permeable material.

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

17. The device according to any one of claims 1 to 4, further comprising a mechanical stimulation assembly configured to apply a squeezing pressure to the outside of the one or more tubes in a direction from the inlet end towards the outlet end.

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

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

20. The device according to any one of claims 1 to 4, wherein the lactation medium comprises one or more of EpiCult TM Plus medium, a laboratory-grown FCS alternative medium, and a serum-free medium.

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

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

23. A method for in vitro milk production in a device according to any one of claims 1 to 4, 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 products; and Harvest the dairy product when a predetermined milk quality is reached.

24. The method according to claim 23, further comprising recycling the dairy product into the one or more tubes via a feedback loop to enrich the dairy product until the predetermined milk quality is reached.

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

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

27. The method according to any one of claims 23 to 26, wherein the lactating cells are isolated from milk of healthy dairy cows.

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

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

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

31. The method according to any one of claims 23 to 26, wherein the lactating cells are genetically modified to produce milk that is hypoallergenic, reduced in lactose, or both, and has increased A2β-casein protein.

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

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