Lipid compositions and methods of producing same

Abstract

A method of increasing lipid synthesis or accumulation in and optionally secretion from a mammary epithelial cell (MEC) culture is provided. The method contacts the MEC with an LXR agonist in the presence of a fatty acid to increase lipid synthesis or accumulation and optionally increase lipid secretion. Also provided is a method of producing synthetic fat globules, the method comprising: (a) isolating lipid droplets from cultured cells; (b) encapsulating the droplets with a lipid bilayer, thereby producing synthetic fat globules. Compositions thus produced are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 339,466, filed on May 8, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing Statement

[0004] An xml sequence listing named 96064.xml, created on May 8, 2023 and filed concurrently with the present invention, comprises 28,672 bytes, and is incorporated herein by reference.

[0005] Technical Field and Background Art

[0006] The present invention, in some embodiments thereof, relates to lipid compositions and methods of producing the same.

[0007] The global dairy market, which includes the processing and harvesting of animal milk for human consumption, typically from cattle, goats, buffalo, camels, and sheep, reached a value of $718.9 billion in 2019. With widespread demand for dairy products and its active role in the global food industry, dairy plays a vital role in economic growth around the world.

[0008] Existing dairy alternatives, such as soy, almond, rice or coconut milk, fall short in terms of flavor and functionality; moreover, much of dairy’s industrial and cultural significance stems from its role in derivative products such as cheese, yogurt, cream or butter. Non-dairy plant-based milks, while addressing environmental and health concerns (and providing adequate flavor for a small segment of the population), are almost universally incapable of forming such derivative products when subjected to the same processes used for dairy milk.

[0009] Additionally, a recent IATP report found that emissions from 13 top dairy companies increased 11% in 2017 compared to 2015, which is equivalent to an increase of 32.3 million metric tons of greenhouse gases, an amount equal to the emissions released by adding 6.9 million cars on the road for a year.

[0010] Mammary epithelial cells (MEC) can be cultured in a given culture medium to synthesize and secrete milk components. Commonly used commercial growth media usually include amino acids, essential fatty acids and glucose or pyruvate to provide cells with the nutrients required to produce milk components (and milk). See, for example, Nan et al., Physiol Genomics 46: 268-275, 2014. However, compared to the amount in vivo, especially compared to the amount of milk protein, milk fat and lactose, its secretion capacity is quite low.

[0011] Additional background technologies include:

[0012] Cohen et al., 2015 PLoS ONE 10(3):e0121645

[0013] Cohen et al., 2017 Journal of Mammary Gland Biology and Neoplasia (2017) 22: 235-249

[0014] Hadaya, O et al., "Pistacia lentiscus extract enhances mammary epithelialcells'productivity by modulating their oxidative status." Scientific reports10.1(2020):1-16). Summary of the invention

[0015] According to one aspect of the present invention, there is provided a method of increasing lipid synthesis or accumulation in a mammary epithelial cell (MEC) culture and optionally secretion therefrom, the method comprising contacting the MEC with an LXR agonist in the presence of fatty acids, thereby increasing lipid synthesis or accumulation and optionally increasing secretion of lipids.

[0016] According to some embodiments, the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.

[0017] According to some embodiments, the fatty acid is selected from the group consisting of oleic acid and palmitic acid.

[0018] According to some embodiments, the fatty acid is oleic acid.

[0019] According to some embodiments, the contacting is performed in the presence of at least one of a phenolic composition, insulin, hydrocortisone, prolactin, and β-hydroxybutirate (BHBA).

[0020] According to some embodiments, the method further comprises harvesting lipids from the MECs.

[0021] According to some embodiments, the method further comprises harvesting lipid from the culture medium of the MECs.

[0022] According to some embodiments, the LXR agonist is GW3965 or T0901317.

[0023] According to some embodiments, the LXR agonist is 9-cis-13,14-dihydroretinoic acid.

[0024] According to some embodiments, the LXR agonist is at least one nucleic acid agent that upregulates LXR, its activators and / or effectors.

[0025] According to some embodiments, the nucleic acid agent encodes a LXR.

[0026] According to some embodiments, the at least one nucleic acid agent encodes LXR and PRLR.

[0027] According to some embodiments, the at least one nucleic acid agent introduces a nucleic acid alteration in the genomic sequence of an LXR, an activator and / or an effector thereof.

[0028] According to some embodiments, the genomic sequence is a coding sequence.

[0029] According to some embodiments, the genomic sequence is a non-coding sequence.

[0030] According to some embodiments, the nucleic acid alteration renders the LXR, its activators and / or effectors constitutively active.

[0031] According to some embodiments, the nucleic acid alteration causes LXR, its activators and / or effectors to be regulated in an inducible manner.

[0032] According to some embodiments, the nucleic acid alteration renders the LXR response element (LXRE) constitutively active.

[0033] According to some embodiments, the nucleic acid alteration renders the LXR response element (LXRE) inducible.

[0034] According to some embodiments, the culture comprises an LXR agonist, a fatty acid, and a phenolic composition.

[0035] According to some embodiments, the phenolic composition is selected from the group consisting of flavonols, flavanols, flavones, flavanones and anthocyanidins.

[0036] According to some embodiments, the phenolic composition is selected from the group consisting of gallic acid or myricetin or derivatives thereof.

[0037] According to some embodiments, the method is performed in the presence of albumin.

[0038] According to some embodiments, the albumin is bovine serum albumin (BSA).

[0039] According to some embodiments, the albumin is BSA or plant albumin.

[0040] According to some embodiments, the MEC is a human or bovine MEC.

[0041] According to one aspect of the present invention, there is provided a composition comprising a cellular or secretory proteome or a fraction of a cellular or secretory proteome obtainable according to the methods described herein.

[0042] According to one aspect of the present invention, there is provided a method for producing synthetic fat globules, the method comprising:

[0043] (a) Isolation of lipid droplets from cultured cells;

[0044] (b) Encapsulation of droplets with a lipid bilayer to generate synthetic fat globules.

[0045] According to some embodiments, the cell is a mammary epithelial cell (MEC).

[0046] According to some embodiments, the lipid bilayer is a cell membrane.

[0047] According to some embodiments, the cell membrane is of a mammary epithelial cell (MEC).

[0048] According to some embodiments, the lipid bilayer is a synthetic membrane.

[0049] According to some embodiments, the lipid bilayer comprises a peptide.

[0050] According to some embodiments, the membrane and the lipid droplet contents are from different sources.

[0051] According to some embodiments, one of the different sources comprises a MEC.

[0052] According to some embodiments, the membrane and the lipid droplet contents are derived from the same source.

[0053] According to some embodiments, the source is mammary epithelial cells (MEC).

[0054] According to some embodiments, the method further comprises subjecting the cells to a lipid synthesis induction regimen prior to step (a).

[0055] According to some embodiments, the lipid induction protocol is as described herein.

[0056] According to one aspect of the present invention, there is provided a composition comprising synthetic fat globules obtainable as described herein.

[0057] According to one aspect of the present invention, there is provided a composition comprising a synthetic fat globule comprising a lipid droplet core coated with a lipid bilayer.

[0058] According to some embodiments, the synthetic fat globules have a size range of 100 nm-15 μm.

[0059] According to one aspect of the present invention, there is provided a nutritional product comprising a composition as described herein.

[0060] Unless otherwise limited, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification (including limitations) shall prevail. In addition, these materials, methods and embodiments are only illustrative and are not meant to be restrictive.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Some embodiments of the present invention are described herein by way of example with reference to the accompanying drawings. With specific reference now to the accompanying drawings in detail, it should be emphasized that the details shown are exemplary and are for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description made in conjunction with the accompanying drawings makes it clear to those skilled in the art how to implement embodiments of the present invention.

[0063] In the attached picture:

[0064] Figure 1 It is shown that LXR agonists enhance lipid metabolism pathways via activation of SREBP1 and FASN in mammary epithelial cells (MECs). After overnight incubation with LXR agonists, the expression of lipid metabolism-related genes increased. LXR1 and LXR2 are two different LXR agonists, as described in the Examples section. IM and GM are controls for induction medium and growth medium, respectively.

[0065] Figure 2 are images showing that LXRa improves the negative effects of oleic acid (OA) treatment on cells. The effects of LXRa treatment (1 μM / ml, TO901317) on cell viability and morphology are demonstrated.

[0066] Figure 3 is a graphical representation showing that intracellular delta triglyceride (TG) levels are similar in the presence or absence of LXRa.

[0067] Figure 4 The effect of treatment with different combinations of fatty acids on the lipid profile distribution, as obtained by chromatograms (HPLC) of extracted lipid samples, is shown. Cells were treated with an induction step and then with palmitic acid or oleic acid. Green: bovine milk control. Black: untreated induced control. Blue: treatment with oleic acid. Pink: treatment with palmitic acid. Brown: infant formula.

[0068] Figure 5 Spheroids in suspension are shown. The cells produce round spherical structures that are hollow inside, allowing for cell polarization.

[0069] Figure 6 It was shown that mammary epithelial cells (MEC) can be grown on suspended microbeads. The cells adhered to the surface of the microbeads, which floated in suspension, allowing the cultivation of high cell masses using liquid bioreactors. DETAILED DESCRIPTION

[0070] The present invention, in some embodiments thereof, relates to lipid compositions and methods of producing the same.

[0071] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or shown by the embodiments. The present invention can have other embodiments or can be practiced or implemented in various ways.

[0072] Mammary epithelial cells can be cultured in culture medium to synthesize and secrete milk components. Commonly used commercial growth mediums usually include amino acids, essential fatty acids and glucose or pyruvate to provide cells with the nutrients required for the production of milk components. However, compared with in vivo production and secretion, especially compared with the production and secretion of milk fat, its secretory capacity is quite low.

[0073] While conceiving and reducing embodiments of the present invention to practice, the present inventors have identified a synergistic combination that enhances the production and even secretion of milk lipids (ie, triglycerides).

[0074] Specifically, the inventors have found that LXR agonists can induce lipid metabolism via expression of the LXR effectors SREBP1 and FASN in mammary epithelial cells (MECs). Activation of this pathway is evident in the absence of fatty acids such as oleic acid (OA), see Figure 1 However, LXR agonists can reduce the deleterious effects of fatty acids, such as high doses of OA, which itself is a lipogenic substance that often leads to cell death (see Figure 2 ). Overall, LXR agonists, together with fatty acids, increase the ability of MECs to accumulate and secrete lipids (see Figure 3 ), making this combination valuable for producing milk components in culture.

[0075] Thus, according to one aspect of the invention, there is provided a method of increasing lipid synthesis or accumulation and optionally secretion from a culture of mammary epithelial cells (MECs), the method comprising contacting the MECs with an LXR agonist in the presence of fatty acids, thereby increasing lipid synthesis or accumulation and optionally increasing secretion of lipids.

[0076] According to an additional or alternative aspect of the invention, there is provided a method of increasing lipid synthesis or accumulation in a culture of mammary epithelial cells (MECs), and optionally secretion therefrom, the method comprising contacting the MECs with an LXR agonist in the presence of at least one of a fatty acid and a phenolic composition (meaning in the presence of a fatty acid and / or a phenolic composition), thereby increasing lipid synthesis or accumulation and optionally increasing secretion of lipids.

[0077] According to an additional or alternative aspect of the invention, there is provided a method of increasing lipid synthesis or accumulation in a culture of mammary epithelial cells (MECs), and optionally secretion therefrom, the method comprising contacting the MECs with an LXR agonist in the presence of at least one of oleic acid (OA) and a phenolic composition (meaning in the presence of a fatty acid and / or a phenolic composition), thereby increasing lipid synthesis or accumulation and optionally increasing secretion of lipids.

[0078] As used herein, "increasing" or "increase" refers to an increase in lipid synthesis (de novo), accumulation and / or secretion by at least 10%, 20%, 30%, 40%, 50%, 70%, 90% or more, for example, by 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold or more, compared to MECs cultured under control conditions, wherein the control condition is the absence of the agent, i.e., at least one of an LXR agonist and a fatty acid, such as oleic acid (OA) and a phenolic composition (meaning in the presence of a fatty acid and / or a phenolic composition), but otherwise identical conditions.

[0079] As used herein, "lipid accumulation" refers to any milk lipid, such as triglycerides, polar lipids, glycerophospholipids, sphingolipids, ceramides, gangliosides, cholesterol, lysophosphatidyl choline, lysophospholipids and / or free fatty acids. According to a specific embodiment, lipid accumulation is mainly due to TG accumulation caused by the TG synthesis pathway.

[0080] The present teachings result in increased lipid synthesis, which can accumulate in cells and even be secreted from cells.

[0081] As used herein, "secretion" refers to the secretion of desired components (e.g., lipids) into the culture medium. In some cases, this may be referred to as "conditioned medium." However, in other cases, the nutrient medium in which the cells survive (culture) is separated from the medium in which the cells secrete milk components, thereby allowing the milk secretion (secretome) to be harvested, as further described below.

[0082] As previously published (see, e.g., Cohen et al., PLoS One.2015;10(3):e0121645) and reiterated herein, OA can increase lipid synthesis in MECs, however the effects of OA are deleterious to the cells, leading to cell death. LXR agonists may inhibit the cytotoxic effects of OA by allowing lipid droplets to disperse in the cell, alternatively or additionally, allowing accumulated lipids to be secreted from the cell.

[0083] As used herein, "tissue culture" refers to the growth of cells in vitro.

[0084] The cells can be isolated single cells, cell clusters (eg, organoids), or contained within tissues that typically contain more than one cell type, such as mammary epithelial cells and mammary stromal cells (primarily mammary fibroblasts and progenitor cells).

[0085] According to one specific embodiment, the methods and systems described herein are "Generally Recognized as Safe" (GRAS).

[0086] As used herein, "mammary epithelial cells (MEC)" refers to luminal cells, basal cells and alveolar cells. Typical markers include cytokeratin 18, cytokeratin 14, EpCAM, progesterone receptor, estrogen receptor, prolactin receptor, Elf5 and CD24.

[0087] Mammary cells can be human or any mammalian cell, but preferably dairy animals such as cows (cows), buffaloes (buffalo), goats, sheep and camels, as well as less commonly mammals such as yaks, water buffalos (water buffalo), horses, donkeys or even reindeer. Mice and rats for research use are also contemplated herein.

[0088] According to a specific embodiment, the MECs are of bovine or human origin.

[0089] The cell may be a primary cell or a cell line, such as an immortalized cell line, eg, genetically transformed / infected to express an immortalizing gene, such as MCF12A or TERT.

[0090] For example, mammary epithelial cells can be obtained from surgical explants of dissected mammary tissue (e.g., chest, breast, nipple). Typically, after surgical dissection of mammary tissue, any fat or stromal tissue is manually removed under sterile conditions, and the remaining tissue of the mammary gland is enzymatically digested with collagenase and / or hyaluronidase, wherein the collagenase and / or hyaluronidase are prepared in a chemically defined nutrient medium, which is generally composed of GRAS components. The sample is maintained at 37 ° C under gentle agitation. After digestion, a suspension of single cells or organoids is collected by centrifugation or by pouring the sample into a sterile nylon cell strainer. The cell suspension is then transferred to a tissue culture plate coated with an appropriate extracellular matrix component (e.g., collagen, laminin, fibronectin).

[0091] Alternatively, the explant sample can be processed into small pieces, for example by mincing with a sterile scalpel. The tissue pieces are spread on a suitable surface, such as gelatin sponge or a plastic tissue culture plate coated with a suitable extracellular matrix.

[0092] The plated cells were maintained in a humidified incubator at 37° C. with a 5% CO 2 atmosphere. During the incubation period, the culture medium was changed every approximately 1 to 3 days, and the cells were subcultured until sufficient viable cell numbers were obtained for subsequent processing, which may include preparation and storage in liquid nitrogen; development of immortalized cell lines by stable transfection of genes such as SV40, TERT (as described above), or other genes associated with senescence; and separation of mammary epithelial cells, myoepithelial cells, and stem / progenitor cell types by, for example, fluorescence activated cell sorting.

[0093] For example, the inventors used primary cultures of bovine mammary epithelial cells isolated from lactating Holstein cows according to established protocols (Cohen et al., 2015, supra).

[0094] Specifically, the primary culture of MEC is isolated from mammary biopsy tissue. In short, tissue is collected from lactating cows and immediately immersed in ice-cold growth medium, wherein antibiotics and heparin are supplemented to prevent cell coagulation. The tissue is minced and digested by shaking in a growth medium supplemented with enzymes such as collagenase and hyaluronidase. After incubation, the suspension is filtered through a mesh (e.g., 250 μm), and the filtrate is centrifuged. The precipitate is treated with trypsin-EDTA and DNase. The cells are then washed with a growth medium supplemented with heparin, and treated separately with DNase, filtered through a cell filter, and washed with growth medium.

[0095] According to some embodiments of the invention, MECs are allowed to proliferate, typically to a confluence of at least 80% in a 2D environment, or to a confluence of at least about 1×10 6According to some embodiments, this is followed by an induction (also called differentiation or maturation) step.

[0096] Thus, according to some embodiments, MECs are grown in a medium supplemented with insulin, hydrocortisone and / or prolactin to allow them to mature, ie, secrete milk components into the medium.

[0097] According to a specific embodiment, the induction is performed when primary cells (eg, MECs) or ex vivo differentiated pluripotent stem cells (ESCs or iPSCs) are used.

[0098] According to a particular embodiment, when immortalized cells are used, the method does not comprise an induction step.

[0099] According to one embodiment, prolactin is added in the induction step. According to one embodiment, for cell proliferation, the culture medium does not contain prolactin. In other embodiments, prolactin is present in both the expansion and induction steps, although the same or higher amount is used in the induction step compared to the proliferation (expansion) step.

[0100] The following are some exemplary non-limiting MECs that can be used in accordance with the present teachings.

[0101] Immortalized human mammary epithelial cells-SV40 is an immortalized mammary epithelial cell line that is not tumorigenic and does not exhibit anchorage-independent growth. Cells in G1, S and G2 / M phases are normally distributed, and they express normal mammary epithelial markers (E-cadherin, CK7 / 18 and CK5 / 14).

[0102] Bovine mammary epithelial cell cultures are described in Jedrzejczak et al., In Vitro Cell Dev Biol Anim. 2014; 50(5): 389-398.

[0103] The HC11 mammary epithelial cell line is available from ATCC.

[0104] Anand et al. describe the establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line wwdoi(.)org / 10(.)1371 / journal(.)pone(.)0040469.

[0105] According to some embodiments of the present invention, MECs include ex vivo differentiated MECs. The differentiation of MECs may be from stem cells, such as pluripotent stem cells or induced pluripotent stem (iPS) cells.

[0106] Hassiotou et al., in Stem Cells. 2012 October; 30(10): 2164-74. doi: 10.1002 / stem.1188, showed that human breast milk contains stem cells (hBSCs) with multi-lineage properties. Breast milk cells from different donors show variable expression of pluripotency genes that are usually present in human embryonic stem cells (hESCs). These genes include transcription factors (TFs) OCT4, SOX2, NANOG, which are known to constitute the core self-renewal circuit of hESCs. The methods described in this publication represent a protocol for generating human mammary-like cells and / or organoids from hBSCs.

[0107] WO2021219634 and WO2021219635 each describe a method for differentiating and culturing mammary epithelial cells in suspension. For example, by i) in a suitable culture medium (e.g., MammoCult culture medium, optionally supplemented with antibiotic-antimicrobial solution and fungicide (fungizone)) hBSC is cultured, and the mammary balls (mammosphere) formed are collected after one week; and ii) in a suitable system (e.g., mammary differentiation culture medium, it includes, for example, culture medium RPMI (Roswell Park Memorial Institute) 1640 and L-glutamine, optionally supplemented with fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, antibiotic-antimicrobial solution and fungicide) the mammary balls are grown for at least 1 week, for example, 2 to 4 weeks, to produce milk cells (lactocyte). In one embodiment of these publications, generating mammary cells comprises: i) accumulating and culturing hBSCs in an appropriate culture medium (e.g., MammoCult medium) under non-adherent conditions to form mammospheres; and ii) culturing such mammospheres in a 3D appropriate system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen, or a suspension culture in a non-adherent plate) for at least 10 days to generate mammary cells. In one embodiment, mammary commitment is obtained by applying a conditioned medium (e.g., EpiCultB) supplemented with specific factors (e.g., parathyroid hormone (pTHrP), hydrocortisone, insulin, FGF10, and HGF). Mammary-like organoids can be generated by culturing cells under conditions selected from the group consisting of: 2D monolayer cells, 2D cells with EBs attached, suspended in a non-adherent plate, and in a mixed floating gel. Other protocols for generating monolayers and organoids are provided in these publications and are incorporated herein by reference.

[0108] According to some embodiments, MECs include MEC monolayers. Cell monolayers are typically grown under adherent conditions using a basal adherent matrix or feeder layer (matrix). However, suspension cultures are also included herein (e.g., mammospheres).

[0109] Examples 5 and 6 below show the growth of MECs in the form of spheres (without any scaffold) or microbeads (ie, scaffolds).

[0110] Other examples of growth in suspension include, but are not limited to, Soboleaska et al., European Journal of Cell Biology 90 (2011) 854-864; and Qu et al., PLOS ONE | DOI: 10.1371 / journal.pone.0131285, each of which is incorporated herein by reference in its entirety.

[0111] It is believed that it is advantageous for cells to grow in a monolayer to achieve polarity, which enables cells to be fed at one location and collect the secretome from another location, i.e., a polarity system, see for example WO2021142241, further described below.

[0112] The cell may be a natural or transgenic cell (see Kuan et al., infra), eg, modified to express a recombinant protein, such as an antibody.

[0113] Regardless of the method used, once the MECs are obtained, they are mixed (contacted or incubated) with a culture medium composition.

[0114] As used herein, "culture medium" refers to an artificial or synthetic culture medium with a defined chemical structure.

[0115] Commonly used commercial growth media usually include amino acids, essential fatty acids and either glucose or pyruvate to provide the cells with the nutrients they need to produce milk components.

[0116] The cells can be expanded in vitro (ex vivo) until sufficient cells are obtained.

[0117] According to a specific embodiment, the cells are incubated in an induction medium designed to direct the cells to differentiate and produce milk components (eg, lipids, carbohydrates and / or proteins).

[0118] The culture medium may be supplemented with lactogenic factors such as prolactin, hydrocortisone and insulin.

[0119] For example, the culture medium can be a basal medium (e.g., DMEM / F12), or a complex medium (e.g., RPMI-1640, IMDM) supplemented with bovine serum albumin (BSA, e.g., 0.15% (v / v)) and insulin (e.g., 1 μg / ml), hydrocortisone (e.g., 0.5 μg / ml), and prolactin (e.g., 1 μg / ml) for a sufficient period of time, e.g., 48 hours, to induce a lactogenic response. Exemplary ranges are provided below: BSA (0.001-5%); insulin (0.001-1 μg / ml); hydrocortisone (0.05-5 μg / ml); prolactin (0.01-10 μg / ml).

[0120] According to a specific embodiment, the culture medium can be a plant-based culture medium composed of plant-based recombinant hormones and plant-derived albumin. Examples of such culture medium compositions include, but are not limited to, ExCellerate iPSC Expansion Medium, KM-acf medium (ScienCell), Cnt-Prime iPS Epithelial Differentiation Medium (Zenbio), EpiCM-A-PRF media (Sciencell), or their equivalents.

[0121] Regardless of the presence or absence of an induction step, cells are exposed to LXR and fatty acid and / or phenolic compositions to improve secretion of milk components.

[0122] As described above, the nutrient medium is mixed (supplemented) with biologically effective concentrations of an LXR agonist and a fatty acid.

[0123] In the context of the present disclosure, the term "biologically effective amount" or "biologically effective concentration" refers to the amount or (weight, volume, v / v or w / v or w / w) concentration of an active agent or composition required to affect a desired biological outcome (usually a beneficial one). The amount of agent used in the culture medium is the amount necessary to deliver a biologically effective amount of the agent to obtain the desired biological outcome.

[0124] As used herein, "at least one" means 1, 2, 3, 4, 5 or more, but not more than 10 agents.

[0125] According to some embodiments of the invention, the agent promotes the accumulation and / or secretion of lipids, which may be referred to as a "galactotropic response."

[0126] As used herein, "fatty acids" refers to fatty acids (FAs), which are one of the most important substances in milk and are essential for the correct development of children during the prenatal, postnatal and infant stages. FAs play a vital role in the energy processes occurring in cells, are the main building materials for cell membranes, and are precursors for important metabolic compounds.

[0127] In milk, most unsaturated fatty acids are MUFAs, with oleic acid (18:1) being the most important. Long-chain polyunsaturated fatty acids (LCPUFAs), including essential fatty acids (EFAs) such as linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3n-3), are also essential for the normal development of the nervous system, retina, and other structures. LCPUFAs form biologically important long-chain polyunsaturated derivatives such as gamma-linolenic acid (C18:3n-6), arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), omega-3, and docosahexaenoic acid (C22:6n-3).

[0128] According to a specific embodiment, the fatty acids belong to long chain fatty acids (LCFA) having an aliphatic tail of 13 to 33 carbons.

[0129] According to a specific embodiment, the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.

[0130] According to a particular embodiment, the fatty acid is chosen from the group consisting of oleic acid and palmitic acid.

[0131] According to a specific embodiment, the fatty acid is oleic acid.

[0132] The term refers to one or more (eg, 2-5, 2-3, 2-4) fatty acids.

[0133] As used herein, "oleic acid" refers to a fatty acid having a cis-9 lipid with a lipid number of 18: 1. Oleic acid is commercially available from Merck and Avanti. Food grade oleic acid is available from Univar solution.

[0134] The effects of fatty acids on lipid accumulation or secretion can be determined as described in the Examples section below, or specifically, using the Cobas assay for determining triglycerides (TG) by indirect glycerol content in the culture medium or another assay for determining TG in cell lysates. Specific fluorescence and bright field microscopy of TG-accumulating lipid droplets can be performed using Nile red and oil red staining, respectively.

[0135] For example, to analyze lipid secretion, total lipids are extracted from the collected culture medium. When analyzing intracellular lipids, the same operation can be performed on lysed cells. However, it is understood that the intracellular lipid composition may be different from the secreted lipids. The culture medium or cells are diluted in Folch reagent (chloroform-methanol 2:1). After incubation in the cold extract at 4°C overnight, the upper phase is removed and the lower phase is filtered through glass wool. The sample is then evaporated under a stream of nitrogen at 65°C, diluted in chloroform:methanol (97:3, v / v) and stored at -20°C until injection for HPLC analysis. Polar and neutral lipids are separated on a silica column using an evaporative light scattering detector. The specific operating conditions are described in the literature or in the subsequent Examples section. An external standard is used to identify the separated lipids. Quantification can be performed based on the external standard curve and expressed as μg / 10 6 The number of live cells can be expressed as % by weight of the total amount of phospholipids (μg) in the sample. The number of live cells can be determined using a hemocytometer using the trypan blue staining method.

[0136] According to a specific embodiment, the effective concentration of the fatty acid is about 50-1000 μM, 100-1000 μM, 100-500 μM, 50-500 μM or 50-360 μM.

[0137] According to a specific embodiment, the effective concentration of oleic acid is about 50-1000 μM, 100-1000 μM, 100-500 μM, 50-500 μM or 50-360 μM.

[0138] To reduce the toxic effects of fatty acids (e.g., oleic acid), oleic acid is preferably provided in the presence of albumin (e.g., BSA or plant albumin). Albumin prevents the deleterious effects of high levels of fatty acids (e.g., oleic acid, e.g., greater than 100 μM), such as cytotoxicity, oxidative stress, apoptosis, and necrosis. Examples of plant albumins are well known in the art, such as FuturMeat chickpea and any other 2S albumin, see, e.g., doi: 10.1016 / j.ijbiomac. 2020.09.04, which is incorporated herein by reference in its entirety.

[0139] Thus, according to some embodiments of the invention, the concentration of albumin (eg, BSA or plant-based) when provided with a fatty acid, such as oleic acid, is about 0.5-32 mg BSA / ml culture medium.

[0140] According to other embodiments, the biologically effective concentration is at least one of: a fatty acid (e.g., oleic acid) between about 50 μM and about 360 μM; an albumin (e.g., BSA or plant albumin) concentration between about 0.5 mg albumin (e.g., BSA or plant albumin) / ml culture medium and about 32 mg / ml.

[0141] Liver X receptors, also known as "LXRs", are members of the nuclear receptor family of transcription factors and are closely related to nuclear receptors such as PPAR, FXR, and RXR. Liver X receptors (LXRs) are important regulators of cholesterol, fatty acid, and glucose homeostasis. Endogenous oxysterols act as their ligands and are therefore physiological agonists.

[0142] Two isoforms of LXR have been identified, termed LXRα and LXRβ. Liver X receptors are classified as subfamily 1 (thyroid hormone receptor-like) of the nuclear receptor superfamily and are given the nuclear receptor nomenclature symbols NR1H3 (LXRα) and NR1H2 (LXRβ), respectively, and are activated by each of the following agonists: 25-hydroxycholesterol, GW3965, and TO901317.

[0143] Liver X receptor alpha is also referred to herein as an abbreviation for LXR agonist.

[0144] According to a specific embodiment, the activity of LXR is to trigger the expression of target genes, such as ABC, ApoE, CETP, ChREBP, CYP7A1, FAS, LPL, LXRa, LXRb, SREBP-1c, or specifically as analyzed in the Examples section:

[0145] FASN- (type I fatty acid synthase) is a member of the FAS family.

[0146] SREBP-1c-sterol regulatory element binding protein 1c or any one of ABCA1, ABCG1, LPCAT3, Arg1, Arg2, IL-10.

[0147] Methods for analyzing gene expression (eg, increased gene expression) include, but are not limited to, analyzing gene expression at the mRNA level (eg, RT-PCR, Northern blot, etc.) or at the protein level (eg, Western blotting, ELISA, etc.).

[0148] Agonists of LXRs can serve as their physiological ligands.

[0149] Examples include, but are not limited to, endogenous oxysterols.

[0150] Examples of oxysterols that may be used include, but are not limited to, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, or cholesteryl esters.

[0151] Synthetic LXR agonists are well known in the art and include, but are not limited to, GW3965 and T0901317 (referred to as LXR1 and LXR2, respectively, in the Examples section below), which are available from commercial suppliers such as Sigma. Other agonists include, but are not limited to, 25-hydroxycholesterol, SR9238, IMB-808, SR9243, LG100754, ethoxycarbonylmethyl, DMHCA, triethyl 2-phosphonobutyrate, indole-7-carboxaldehyde, desmodur (D6) ester, SR1078, 27-hydroxycholesterol, GSK4112, 27-hydroxycholesterol-d6.

[0152] According to a specific embodiment, the compound is part of the vitamin A cluster, together with the precursors 9-cis retinol, 9-cis retinyl esters, 9-cis-13,14-dihydroretinol, 9-cis-13,14-dihydroretinyl esters, which activate LXR signaling by binding to LXR-RXR heterodimers. Thus, according to some embodiments, the compound can be vitamin A or a derivative, the precursor 9-cis retinol, 9-cis retinyl esters, 9-cis-13,14-dihydroretinol or 9-cis-13,14-dihydroretinyl esters.

[0153] According to some embodiments, the LXR agonist is 9-cis-13,14-dihydroretinoic acid.

[0154] According to some embodiments, the LXR agonist is at least one nucleic acid agent that upregulates LXR, its activators and / or effectors.

[0155] According to some embodiments, the nucleic acid agent encodes an LXR.

[0156] According to some embodiments, the at least one nucleic acid agent encodes an LXR and a PRLR, which exhibit a synergistic effect on lipid synthesis or secretion.

[0157] According to some embodiments, the at least one nucleic acid agent introduces a nucleic acid alteration in the genomic sequence of the LXR, activator and / or effector thereof.

[0158] According to some embodiments, the genomic sequence is in a coding sequence. For example, as in LXRα, such as H383E, LXRE387Q, LXR H390E (Bedi et al.: Nucl Receptor Res. 2017; 4: 101302).

[0159] According to some embodiments, the genomic sequence is a non-coding sequence.

[0160] Association studies of LXRa and LXRb SNP analysis revealed that LXR activity is affected by SNPs in non-coding regions, as rs12221497 was associated with higher triglyceride levels (see, e.g., Zhang et al., Steroids Vol. 185, September 2022, 109057).

[0161] According to some embodiments, the nucleic acid alteration renders the LXR, the activator thereof and / or the effector thereof constitutively active.

[0162] According to some embodiments, the nucleic acid alteration causes said LXR, said activator thereof and / or its effector to be regulated in an inducible manner.

[0163] According to some embodiments, the nucleic acid alteration renders the LXR response element (LXRE) constitutively active.

[0164] According to another embodiment, constitutive activation is achieved by removal of LXRE and SER inhibition by FoxO1 (ganti et al., PNAS 114(6) E951-E960).

[0165] According to some embodiments, the nucleic acid alteration renders the LXR response element (LXRE) inducible, such as VP16-LXRα (e.g., Hong et al., J Lipid Res. 2011Mar;52(3):531–539).

[0166] According to some embodiments, an LXR agonist is at least one nucleic acid agent that upregulates LXR, its activators and / or effectors, collectively members of the LXR pathway.

[0167] According to one embodiment, the STAT5 pathway activates LXR. STAT5 is a transcription factor that plays a role in regulating a variety of biological processes, including cell proliferation, differentiation, and survival. It has been shown to activate LXRα by directly binding to the enhancer region in the LXRα promoter. According to one embodiment, the activator is a component of the Stat5 pathway.

[0168] This activation of LXRα by Stat5 results in increased expression of genes involved in lipid metabolism and cholesterol transport, including ABCA1 and ABCG1. The mechanism by which Stat5 activates LXRα is thought to involve the recruitment of coactivators and chromatin remodeling complexes to the promoter region, leading to increased transcriptional activity. It is also possible that Stat5 activates RXR expression, and FoxO1 degradation via Akt activation can eliminate the repression of LXRE & SRE elements (www(.)pubs(.)acs(.)org / doi / 10.1021 / acs.jafc.0c05237).

[0169] According to some embodiments of the present invention, LXR effector genes include but are not limited to: ABC, ApoE, CETP, ChREBP, CYP7A1, FAS, LPL, LXRa, LXRb, SREBP-1c.

[0170] According to such embodiments, the contacting involves a transformation, infection or transduction protocol.

[0171] Upregulation of at least one nucleic acid agent that upregulates an LXR, an activator thereof and / or an effector may be achieved at the genomic level (i.e., activation of transcription via promoters, enhancers, regulatory elements), the transcriptional level (i.e., correct splicing, polyadenylation, activation of translation), or the protein level (i.e., post-translational modification, interaction with substrates, etc.).

[0172] The following is a list of agents that are capable of upregulating the expression level and / or activity of at least one nucleic acid agent that upregulates an LXR, an activator and / or effector thereof.

[0173] The agent capable of upregulating the expression level and / or activity of at least one nucleic acid agent (which upregulates LXR, its activator and / or effector) can be an exogenous polynucleotide sequence designed and constructed to express at least one functional portion of LXR, its activator and / or effector. Thus, the exogenous polynucleotide sequence can be a DNA or RNA sequence encoding at least one nucleic acid agent that upregulates LXR, its activator and / or effector molecule, capable of activating the LXR pathway.

[0174] As used herein, the phrase "functional portion" refers to a portion of an LXR, an activator thereof and / or an effector protein (i.e., a polypeptide) that exhibits the functional properties of an enzyme, such as binding to a substrate. According to preferred embodiments of some embodiments of the present invention, the functional portion of an LXR, an activator thereof and / or an effector protein is a polypeptide sequence or a DNA or RNA encoding the same.

[0175] LXRs, their activators and / or effector proteins have been cloned from mammals (e.g., lactating mammals or humans). Therefore, the coding sequence information of LXRs, their activators and / or effector proteins is available from several databases, including www.ncbi(.)nlm(.)nlm(.)nih(.)gov / GenBank database available.

[0176] In order to express exogenous LXR, its activator and / or effector protein in mammalian cells, it is preferred to connect the polynucleotide sequence encoding LXR, its activator and / or effector protein to a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing the transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0177] It should be understood that the nucleic acid constructs of some embodiments of the present invention can also utilize homologs of LXR, its activator and / or effector proteins that exhibit the desired activity (i.e., increase lipid accumulation or secretion). Such homologs can, for example, be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the human or bovine sequence of LXR, its activator and / or effector protein, DNA or mRNA encoding the same, as determined using the Smith and Waterman algorithm using the BestFit software of the Wisconsin Sequence Analysis Package, wherein the gap weight is equal to 50, the length weight is equal to 3, the average match is equal to 10, and the average mismatch is equal to -9.

[0178] Constitutive promoters suitable for use in some embodiments of the present invention are promoter sequences that are active under most environmental conditions and most types of cells such as cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use in some embodiments of the present invention include tetracycline inducible promoters (Zabala M, et al., Cancer Res. 2004, 64 (8): 2799-804).

[0179] According to a specific embodiment, the upregulation is the upregulation of more than one gene in the LXR pathway.As shown in Example 4, when LXR and PRLR were overexpressed in human mammary epithelial cells from the mammary gland, a synergistic effect on TAG metabolism was observed.

[0180] LXR upregulation can be added as a preparation for the cells at any time prior to fatty acid treatment, or added simultaneously with the fatty acids. According to some embodiments, the fatty acids are added after the cells reach a predetermined confluence level, such as above 50%, 60%, 70%, 80%, 90% or 95%.

[0181] Typical incubation periods using LXR agonists (e.g., small molecules) and fatty acids (e.g., oleic acid) can range from 20 minutes to 36 hours, such as 12-24 hours. In addition to the fatty acid, the phenolic composition can be added at the same time as the fatty acid, or as an alternative to its presence.

[0182] When the LXR is a nucleic acid agent and constitutive expression is contemplated, typical culture periods using phenolic compositions or fatty acids (eg, oleic acid) can range from 20 minutes to 36 hours, such as 12-24 hours.

[0183] Cultures can be supplemented with phenolic compositions, which can also increase lipid accumulation (see Shalev et al., Heliyon Vol. 8, No. 3, March 2022, e09025).

[0184] As used herein, "phenolic composition" refers to a non-toxic (for human or domestic animal consumption) chemical comprising at least one phenolic group.

[0185] According to a specific embodiment, the phenolic composition is a derivative of gallic acid, or a glycosylated form of gallic acid or a flavanol, flavonoid, flavonoid, anthocyanin, or a compound known as a hydrolyzable compound or a condensed tannin. Such compounds include polymers of high molecular weight flavonoids and polyesters of gallic acid or tannic acid combined with different sugars. According to a specific embodiment, the composition is a polyphenol.

[0186] As used herein, "polyphenols" refers to a family of organic compounds characterized by multiple phenolic groups, which are typically found naturally in plants.

[0187] According to a particular embodiment, the phenolic composition is selected from flavonols, flavanols, flavones, flavanones and anthocyanidins.

[0188] According to a specific embodiment, the phenolic composition is glycosylated.

[0189] Exemplary phenolic compositions that can be used according to the present teachings include, but are not limited to:

[0190]

[0191] According to a specific embodiment, the phenolic composition is oleuropein, chlorogenic acid, catechin or myricetin, such as available from Cayman Chemicals (Ann Arbor, MI, USA). According to a specific embodiment, the phenolic composition is a galloyl derivative, a flavonol glucoside or a catechin.

[0192] According to a specific embodiment, the phenolic composition is a dietary phenolic compound typically derived from a food source of plant origin, typically rich in secondary metabolites.

[0193] According to one embodiment, the phenolic composition is synthetic.

[0194] According to one embodiment, the phenolic composition is purified from a natural source, such as a plant.

[0195] According to one embodiment, the phenolic composition is contained in a plant extract, for example, an ethanol extract, a chloroform extract, a hexane extract, an ethyl acetate extract. According to one embodiment, the plant extract is a chloroform extract or a hexane extract.

[0196] Examples of plants from which phenolic compositions may be extracted / purified include, but are not limited to, Pistacia lentiscus, Buckwheat, Sophora japonica, Eucalyptus, Quebracho caryophyllacea.

[0197] According to a specific embodiment, the phenolic composition is gallic acid or its derivatives (eg, polyesters formed by gallic acid or tannic acid combined with different sugars, such as epigallocatechin gallate (EGCG)), catechin.

[0198] Combinations of phenolic compositions may synergistically promote secretion of milk components.

[0199] The present inventors have demonstrated that the choice of phenolic composition can influence the accumulated / secreted milk components.

[0200] For example, studies have shown that polyphenolic compounds such as glycosylated polyphenols, e.g., rutin and myricetin, or their derivatives, e.g., EGCG, catechins, can increase protein, lactose, and triglyceride secretion, whereas gallic acid increases lipid droplet accumulation as well as lactose secretion.

[0201] Without being bound by theory, it is suggested that the phenolic compositions act as antioxidants like pyruvate, leading to glucose sparing and ultimately oligosaccharide synthesis and secretion.

[0202] It is suggested that the composition of milk components may be determined by the phenolic composition selected. Thus, for example, glycosylated polyphenols such as myricetin may lead to protein, lactose and triglyceride secretion, whereas gallic acid may lead to intracellular lipid accumulation, lipid secretion and lactose secretion.

[0203] According to one embodiment, the phenolic composition is purified from a natural source, such as a plant extract or a fraction thereof (not a whole extract).

[0204] According to a specific embodiment, the phenolic composition is gallic acid or a derivative thereof, such as methyl gallate, ethyl gallate, catechin and Epi-catechin.

[0205] According to one specific embodiment, the biologically effective concentration of gallic acid is about 1 ppm to 3 ppm.

[0206] Cells obtainable according to the present teachings are contemplated herein.

[0207] According to a specific embodiment, cells treated with LXRa and OA are more viable than cells treated with OA alone, as determined by trypan blue staining.

[0208] According to a specific embodiment, the cells are not goat cells.

[0209] Culturing MECs in culture as described herein can be performed using methods well known in the art.

[0210] For example:

[0211] Sharfstein et al., Biotechnology and Bioengineering, 40, pp. 672-680, 1992, describe basic methods for culturing MEC in extended-batch and hollow fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, with the base and apex exposed to the culture medium. Protein production was induced in batch and hollow fiber cultures in hormone-supplemented culture medium, for example.

[0212] Kuan et al. described the culture of mammary gland-like structures (ducts, lateral buds and alveoli) in Anim. Sci. Vol. 88, E-Suppl. 2 / J. Dairy Sci. No. 93, E-Suppl. 1 / Poult. Sci. No. 89, E-Suppl. 1. Hollow fiber bioreactors are used to culture mammalian cells on a large scale to produce milk components, such as recombinant proteins. Hollow fibers provide a culture system with a high surface area to volume ratio. The system allows for efficient exchange of nutrients and waste on the fiber wall.

[0213] WO20211142241 describes a cell culture system designed for collecting milk. The system is designed to support the compartmentalized secretion of products (i.e., secretory groups) so that milk or secretory groups are not exposed to the culture medium that provides nutrients to cells. In vivo, the epithelial cells of milk production are arranged on the inner surface of the mammary gland as a continuous monolayer. The monolayer is oriented so that its basal surface is attached to the basement membrane below, and milk is secreted from the top surface and stored in the chamber of the gland or acinus until it is removed during milking or feeding. The tight connection along the cell side surface ensures a barrier between the lower tissue and the milk in the acinus chamber. Therefore, in vivo, mammary tissue is arranged so that milk secretions are compartmentalized, and mammary epithelial cells themselves establish an interface and maintain the directional absorption of nutrients and the secretion of milk.

[0214] According to this embodiment, a cell culture device is provided that can reproduce the compartmentalization ability of the mammary gland and can be used to collect milk from mammary epithelial cells grown in vitro. Such a device can include a scaffold to support the proliferation of mammary cells at the interface between the two compartments, so that the epithelial monolayer provides a physical boundary between the nutrient medium and the secreted milk. In addition to providing a surface for growth, the scaffold also provides spatial cues to guide cell polarization and ensure directionality of absorption and secretion.

[0215] After isolation and expansion of mammary epithelial cells, the cells are suspended in a nutrient medium and inoculated into a culture device pre-coated with a mixture of extracellular matrix proteins (e.g., collagen, laminin, and / or fibronectin). The cell culture device can be of any design that allows absorption of nutrients and secreted products from polarized, confluent epithelial monolayer compartments. Examples include hollow fiber and microstructured scaffold bioreactors (e.g., see WO20211142241). Figure 3 and 4 ). Alternative approaches include other methods of three-dimensional tissue culture, such as preparing decellularized mammary glands as scaffolds and repopulating them with stem cells to generate functional organs in vitro, or harvesting milk from the lumen of mammary epithelial cell organoids or “mammospheres” grown in hydrogel matrices or suspensions.

[0216] The apparatus comprises a sealed enclosure that maintains a humidified atmosphere of about 5% CO2 at a temperature of about 37°C. Glucose uptake is monitored to assess the growth of the culture as the cells proliferate within the bioreactor. A steady state of glucose consumption indicates that the cells have reached a confluent, contact-inhibited state. Transepithelial electrical resistance is used to ensure the integrity of the monolayer. Sensors monitor the concentrations of dissolved O2 and CO2 at multiple locations in the culture medium. A computerized pump circulates the culture medium through the bioreactor at a rate that balances the delivery of nutrients with the removal of metabolic waste products such as ammonia and lactate.

[0217] The culture medium can be recirculated through the system using lactate supplementation and adaptation techniques (Freund et al., 20\SInt J Mol Sci. 19(2)) or after removal of waste through zeolite-filled chambers.

[0218] The culture medium may be supplemented with the agents described herein, either simultaneously or sequentially; or the culture medium may be replaced entirely.

[0219] According to a specific embodiment, cells can be cultured in suspension or in two-dimensional culture (2D).

[0220] According to one embodiment, cells may be cultured in three-dimensional (3D) conditions.

[0221] According to one embodiment, the cells may be cultured under adherent conditions.

[0222] The Examples section below provides embodiments of 2D and 3D cultures as monolayers or in suspension.

[0223] According to one embodiment, cells may be cultured directly on the bioreactor membrane.

[0224] Starting from 12 hours post-induction, after a predetermined time in culture, the lipid / milk fraction, also referred to herein as the "secretome", was harvested.

[0225] According to one aspect of the invention, compositions comprising cells or secretomes obtainable as described herein (e.g., complete unfractionated conditioned medium) or fractions of said cells or secretomes (e.g., lipid fractions, protein fractions, carbohydrate fractions, or combinations thereof) are contemplated herein.

[0226] Such compositions may be used in the food or pharmaceutical industries, as described below.

[0227] It will be appreciated that since cells are rich in lipids (TG), in particular lipid droplets, as determined by lipid staining (eg Nile Red), this fraction can be extracted from lysed cells.

[0228] Alternatively or additionally, lipid droplets can be obtained from the culture medium into which they are secreted, typically in the form of fat globules FG.

[0229] As used herein, "lipid droplets" refer to organelles found in most mammalian cells as well as various plant tissues and yeast. They consist of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol (i.e., micellar structure), in which specific proteins are embedded.

[0230] The lipid core of the core is mainly composed of triglycerides or cholesterol esters.

[0231] According to a specific embodiment, the size of the lipid droplets may range from 200 nm to 15 μm, such as 200 nm to 1 μm, 200-800 nm, 200 nm-5 μm, 200 nm-5 μm.

[0232] According to one embodiment, the separation of lipid droplets depends on the buoyancy density of the lipid droplets, which is <1 g / cm 3 . Two low-speed centrifugation steps and one ultracentrifugation step using a discontinuous density gradient will collect more than 95% of the lipid droplets from the cell lysate. More specifically, the nuclei are removed by low-speed centrifugation (e.g., 800-2000 × g), and the density of the post-nuclear supernatant is adjusted with sucrose, followed by the flotation of the lipid droplets through a single discontinuous sucrose gradient. The lipid droplet fraction is characterized by immunoblot analysis of the component proteins. For detailed information on the isolation of lipid droplets from cells or culture medium, please refer to Brasaemle Curr Protoc Cell Biol. 2016 Sep 1; 72: 3.15.1-3.15.13 , which is further outlined below.

[0233] Isolation of lipid droplets by centrifugation is a relatively simple method. Very low protein to lipid ratios make lipid droplets more buoyant than all other subcellular structures; lipid droplets can be separated from denser subcellular compartments using discontinuous density gradients. The following should be considered: Cell disruption methods that keep droplets intact, especially when dealing with cells containing very large lipid droplets. Therefore, according to a specific embodiment, mechanical disruption of cells is performed in the presence of liquid nitrogen and a Teflon pestle homogenizer. Gentle cell disruption is required to maintain lipid droplet structure. Gentle homogenization is performed using a manual homogenizer with a loose fit.

[0234] The buoyant density of lipid droplets facilitates flotation during centrifugation of any aqueous medium. Using electrolyte-free solutions may reduce the aggregation of lipid droplets with other subcellular organelles; using hypotonic solutions does not compromise the integrity of lipid droplets because they do not have an aqueous compartment. Since endoplasmic reticulum fragments and a large number of mitochondria are usually tightly fitted to droplets in intact cells, the isolated lipid droplet fraction contains low levels of these membranes. By layering the density-adjusted cell lysate before centrifugation so that it is located below one or two layers of decreasing density in a discontinuous gradient, the contamination of these membranes by lipid droplets can be minimized. The movement of lipid droplets upward through the gradient layers reduces the adhesion of contaminant membranes to the droplets and separates the droplets from soluble proteins. Contaminating membranes can be further removed by flotation of the separated lipid droplets by additional discontinuous density gradients, which can include solutions containing low concentrations of glycerol, 100mM to 1M sodium chloride, or 100mM sodium carbonate, pH 11.5 (Fujiki et al., 1982). However, the use of sodium carbonate wash solutions may remove loosely adhered proteins that are normally located on the lipid droplet outer membrane monolayer and may denature the proteins. It is recommended to use a swinging-bucket rotor to tightly constrain the floating lipid droplets to the top of the tube; the rotor should be allowed to coast to a stop during the final deceleration to minimize disruption of the lipid droplet layer.

[0235] After centrifugation, the lipid droplets appear as a milky layer at the top of the tube. Gentle homogenization with fewer strokes and slower pestle insertion will reduce disruption of the lipid droplets. There will be a pellet containing a dense membrane at the bottom of the tube with one or two translucent membrane bands at the interface of the dense phase.

[0236] According to a specific embodiment, most of the lipid droplet fractions of small volume are collected by using Beckman tube slicer in combination with thin-walled polyallomer or polycarbonate tubes. The tube is inserted into the tube slicer to position the cutting blade a few millimeters below the lipid droplet layer. The blade is stably and firmly inserted into the tube, and the lipid droplet layer can be isolated in a small amount of top gradient solution at the top of the tube slice. The rubber ring in the tube slicer forms a seal with the blade to prevent leakage of the top solution. Using the tube slicer is conducive to the most effective collection of lipid droplets with the smallest volume, and it allows the surface of the blade and the tube to be rinsed with additional solution to collect fractions more completely. In other embodiments, a pipetting device or a syringe with a wide bore needle can be used.

[0237] According to one embodiment, collection from the culture medium is also envisaged and can be performed using a similar method: concentrating the culture medium using a speed-vac centrifuge and separating lipids from the concentrated culture medium using gradient centrifugation. Alternatively, the unfractionated culture medium is collected.

[0238] Regardless of the method used, once the droplets are obtained, according to some embodiments, they can be used in a process to generate fat globules.

[0239] Therefore, according to one aspect of the present invention, there is provided a method for producing synthetic fat globules, the method comprising:

[0240] (a) isolating lipid droplets from cells in culture (as described above);

[0241] (b) Encapsulating the droplets with a lipid bilayer, thereby generating synthetic fat globules.

[0242] As used herein, "synthetic" refers to artificial fat globules involving a synthesis process, not just isolation.

[0243] Specifically, the core micelle structure is heterologous to the bilayer that encapsulates it. Since FGs are synthetic, they are also referred to as FG-like structures, or non-naturally occurring FGs.

[0244] As used herein, "fat globule" abbreviated as "FG" refers to a bilayer structure encapsulating a micellar structure having a lipid core content.

[0245] "Milk fat globules" consist of a core of triglycerides, cholesterol and retinol esters, which are surrounded by a biological membrane, the milk fat globule membrane, which is structured as a triple layer of polar lipids (phospholipids, sphingolipids), cholesterol and proteins (glycoproteins and enzymes). MFGM is found in cow's milk.

[0246] According to a specific embodiment, the cells from which the lipid droplets are isolated are mammalian cells.

[0247] According to a specific embodiment, the cell is a MEC as described above.

[0248] According to a specific embodiment, the cell is an epithelial stem cell, such as a mammal (as described above).

[0249] The cells can be freshly isolated, primary cultured, or cell lines.

[0250] The lipid droplets can be encapsulated in the lipid retarder using methods known to those skilled in the art.

[0251] According to a specific embodiment, the lipid bilayer is a cell membrane.

[0252] According to one embodiment, the lipid droplets and the cell membrane are of different origin.

[0253] According to a specific embodiment, at least one of the lipid droplet and the cell membrane is from a MEC.

[0254] According to one specific embodiment, both the lipid droplets and the cell membranes are derived from MECs.

[0255] According to a specific embodiment, the lipid droplets are derived from MECs.

[0256] According to a specific embodiment, the cell membrane is from MEC.

[0257] The membrane and lipid droplet fractions are used to generate thin lipid membrane layers as a step to generate lipid micelles (H. Zhang, 2017; W. Zhang et al., 2018) and as the first step in the production of MFGL (MFG-like) structures. The mixture of membrane and lipids in the thin lipid layer generates liposome-like structures, containing membrane and lipid droplet elements.

[0258] Another method of producing MFGL is by 30 cycles of 2 minutes heating-3 minutes cooling each cycle with vortexing during cooling, which also provides MFGL.

[0259] To further reduce the size of MFGL and make the population more homogeneous, MFGL can be homogenized by sonication (Mendez & Banerjee, 2017) or extrusion (Gven et al., 2009). Both methods can homogenize MFGL to a size of <20 μM. Homogenization of the final product improves its stability and increases the membrane fraction by increasing the (membrane / lipid droplet size) ratio.

[0260] An exemplary method for lipid extraction is as follows:

[0261] Use rotary evaporator to dry sample (cell, culture medium) in a newly weighed glass tube. Grind the dried sample into powder in the tube and weigh. Add ethanol to the dried ground sample. Vortex the combined group and extract twice (for example, at 45-800 ℃ for 20-200 minutes, at room temperature for 2-40 minutes and vortex). Calculate the ethanol volume by diluting the dried ground sample (mg) to, for example, 10mg. At the end of the extraction, the sample is centrifuged at 4-250 ℃ for 30 minutes. Use a new glass pipette to move the organic phase to a new glass tube. Wash the material. Then hexane is added to the ethanol in the tube (according to calculation, 1-6 volumes of hexane are added per 1 volume of ethanol), and vortexed for 5-60 seconds. After 20 minutes at room temperature, centrifuge at 4-250 ℃, for example, 400-5000 × G, for 30 minutes. The clear liquid was transferred to a new glass tube using a glass pipette, the residue was washed with a small amount of hexane, centrifuged, and the solvent was transferred to the tube containing the previous hexane-ethanol mixture. The extracted hexane-ethanol mixture was evaporated using a rotary evaporator. The oil was dissolved in a small amount of hexane and transferred to a newly weighed glass vial using a glass pipette and evaporated using a rotary evaporator.

[0262] According to one embodiment, the droplets and membranes are separated and extruded and / or sonicated to form particles ranging in size from 100 nm to 10 μm, which particles contain a coating cell membrane and a micelle core corresponding to the lipid droplets.

[0263] According to a specific embodiment, the membrane bilayer comprises peptides, proteins and / or carbohydrates.

[0264] For example, the structure may differ from native FG in terms of protein content, for example, reduced protein content or a different distribution of proteins or peptides in the coated bilayer structure.

[0265] Alternatively, the lipid bilayer is a synthetic membrane, such as a liposome having at least one phospholipid bilayer membrane encapsulating a hydrophobic core.

[0266] For example, pure phospholipids from egg yolk or soybean are dissolved and gently evaporated in a rotary evaporator to produce a thin layer of phospholipids. After the solvent has evaporated completely, preheated lipids (to 50°C) are added to form micelles, and vortexing and ultrasonication are used to enhance the process. In the final step, the micelles are homogenized using an Avanti extruder.

[0267] Other examples of producing FGs from synthetic bilayers are provided in the references below.

[0268] Zhang,H.(2017).Thin-film hydration followed by extrusion method forliposome preparation.In Methods in Molecular Biology(Vol.1522,pp.17–22).Humana Press Inc.www(.)doi(.)org / 10.1007 / 978-1-4939-6591-5_2

[0269] Zhang, W., Li, C., Jin, Y., Liu, X., Wang, Z., Shaw, JP, Baguley, BC, Wu, Z., & Liu, J. (2018). Multiseed liposomal drug delivery system using micelle gradientas driving force to improve amphiphilic drug retention and its anti-tumorefficacy. Drug Delivery,25(1),611–622.www(.)doi(.)org / 10.1080 / 10717544.2018.1440669

[0270] Bruce et al. Ann. Rev. Physiol. 1984. 46: 417-33

[0271] Keenan et al. Analytical Biochemistry 177, 194-1% (1989) (isolation of plasma membranes from mammary glands).

[0272] It is noteworthy that FGs are smaller than cells. They do not have complete cells or organelles.

[0273] Once FGs are obtained, they can be used in a variety of nutritional products.

[0274] Thus, according to one aspect of the present invention, there is provided a composition comprising a synthetic fat globule structure.

[0275] According to a specific embodiment, the composition is obtainable according to the method described herein.

[0276] According to a specific embodiment, the composition is a food or a beverage.

[0277] Also provided is a method of producing a nutritional product, the method comprising combining a food or beverage with a composition as described herein.

[0278] The composition comprising the secretome or a portion thereof, MFG, cells can be used as such or combined with components of the food, feed or beverage industry, pharmaceutical industry for human or veterinary use.

[0279] Therefore, according to one aspect of the present invention, there is provided a nutritional composition, which may be a food or feed comprising FG, cells, secretome or a portion thereof (eg, lipid droplets).

[0280] Foods can be vegan, vegetarian, dairy, or can include meat.

[0281] As used herein, "food" refers to food (for human consumption), feed (for animal consumption), liquid (beverage), solid or semisolid.

[0282] Thus, according to one aspect of the present invention, there is provided a method of producing a food or feed, the method comprising combining a composition comprising cells, lipid droplets, secretome or a fraction thereof, or FG during food production.

[0283] The method of producing the food may include fermenting, kneading, extruding, molding, shaping, steaming, boiling, baking, roasting, frying and any combination thereof.

[0284] Also provided is a method of providing nutrition to a subject in need thereof. The method comprises providing a food as described herein to the subject.

[0285] According to a specific embodiment, the subject is at risk for a nutritional deficiency.

[0286] According to one embodiment, the subject is a healthy subject (eg, not suffering from a nutrition / absorption related disease).

[0287] According to a specific embodiment, food used herein is "infant formula milk powder" refers to a nutritional composition, intended for infants and as defined in Codex Alimentarius, (Codex STAN 72-1981) and as defined in Codex Alimentarius (Codex STAN 72-1981) Special food for infants (including special medical use food). The invention further relates to a special nutritional food for infants to use in the first few months after birth, which itself meets the nutritional needs of this type of people (Article 2 (c) of European Council Directive 91 / 321 / EEC 2006 / 141 / EC on December 22, 2006, about infant formula milk powder and larger infant formula milk powder). Infant formula food includes starting infant formula food and follow-up or follow-on (follow-up or follow-on) formula food. Usually, the starting formula milk powder is applicable to newborn babies, as a breast milk substitute, and the follow-up formula milk powder is applicable to infants more than 6 months old.

[0288] Other expected products include, but are not limited to, milk, butter, cream, cheese, ice cream, yogurt. These products can be products obtained in culture, such as cultured milk; or natural milk, wherein at least part of the components are obtained by culture, such as lipid or FG.

[0289] In some embodiments, lipid droplets (e.g., FG) can be added to the nutritional composition by replacing an equal amount of the remainder of the total fat mixture normally present in the nutritional composition. In some embodiments, a certain amount of oil used as a fat source (not comprising milk fat globules as described herein) can be replaced with lipid droplets (e.g., FG). In another embodiment, the nutritional composition can be supplemented with lipid droplets, such as GF. In some embodiments, lipid droplets, such as FG, can be the only fat source added to the nutritional composition.

[0290] In some embodiments, the nutritional compositions described herein include a fat source. The lipid droplets described herein, such as FG, can be the only fat source, or can be used in combination with any other suitable fat or lipid source of the nutritional compositions known in the art. Suitable fat sources include, but are not limited to, animal sources, such as milk fat, butter, butter fat, egg yolk lipids; marine sources, such as fish oil, marine fish oil (marine oil), single cell oil; vegetable and plant oils, such as corn oil, rapeseed oil, sunflower oil, soybean oil, palm oil, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, flaxseed (flaxseed or linseed) oil, cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and fatty acid esters; and any combination thereof.

[0291] In some embodiments, the disclosed nutritional compositions described herein may also include a prebiotic source. The term "prebiotic" as used herein refers to an indigestible food ingredient that produces a health benefit to the host. This health benefit may include, but is not limited to, selectively stimulating the growth and / or activity of one or a limited number of beneficial intestinal bacteria, stimulating the growth and / or activity of ingested probiotic microorganisms, selectively reducing intestinal pathogens, and the favorable effects on the distribution of intestinal short-chain fatty acids. This prebiotic can be naturally occurring, synthetic, or developed by genetically manipulating organisms and / or plants, whether this new source is now known or developed later. Prebiotics useful in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics containing fructose, xylose, soy, galactose, glucose, and mannose.

[0292] More specifically, the prebiotics useful in the present disclosure may include polydextrose, polydextrose powder, lactulose, lactofructo-oligosaccharide, raffinose, gluco-oligosaccharide, inulin, oligofructose, isomaltooligosaccharide, soybean oligosaccharide, lactofructo-oligosaccharide, xylo-oligosaccharide, chito-oligosaccharide, manno-oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, galacto-oligosaccharide and gentio-oligosaccharide. In a preferred embodiment, the prebiotics include galacto-oligosaccharide, polydextrose, or a mixture thereof.

[0293] In some embodiments, the disclosed compositions described herein may also include a probiotic source. The term "probiotic" refers to a microorganism that has a beneficial effect on host health. Any probiotic known in the art is acceptable in this embodiment. In a specific embodiment, the probiotic may be selected from any Lactobacillus, Lactobacillus rhamnosus (ATCC No. 53103), Bifidobacterium, Bifidobacterium longum BB536 (BL999, ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382), Bifidobacterium breve AH1205 (NCIMB: 41387), Bifidobacterium infantis 35624 (NCIMB: 41003) and Bifidobacterium animalis subspecies lactis BB-12 (DSM No. 10140), or any combination thereof.

[0294] The nutritional composition of the present invention may optionally include one or more of the following flavoring agents, including but not limited to: flavoring extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavorings, cookie crumbs, vanilla or any commercially available flavoring agents. Examples of useful flavoring agents include but are not limited to pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure mint extract, honey, imitation pineapple extract, imitation rum extract, imitation strawberry extract or vanilla extract; or volatile oils, such as balm oil, laurel oil, bergamot oil (bergamotoil), cedarwood oil, cherry oil, cinnamon oil, clove oil or mint oil; peanut butter, chocolate flavoring, vanilla cookie crumbs, butterscotch (butterscotch), toffee, and mixtures thereof. The amount of flavoring agent can vary greatly depending on the flavoring agent used. The type and amount of flavoring agent can be selected as known in the art.

[0295] The nutritional composition of the present disclosure can provide minimal, partial or complete nutritional support. The composition can be a nutritional supplement or a dietary substitute. The composition can (but not necessarily) be nutritionally comprehensive. In one embodiment, the nutritional composition of the present disclosure is nutritionally comprehensive and contains lipids, carbohydrates, proteins, vitamins and minerals of suitable types and amounts.

[0296] As used herein, the term "about" refers to ± 10%.

[0297] The terms "comprises," "comprising," "includes," "including," "having" and their conjugates mean "including but not limited to."

[0298] The term "consisting of" means "including and limited to."

[0299] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0300] As used herein, the singular forms "a", "an" and "the" include plural forms unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0301] Throughout the application, various embodiments of the present invention may be presented in the form of a range. It should be understood that the description of the range form is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, it is applicable.

[0302] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The expressions "a range between a first indicated number and a second indicated number" and "a range from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first indicated number and the second indicated number and all numbers and integers therebetween.

[0303] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developed from known manners, means, techniques and procedures.

[0304] It should be understood that, for the sake of clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the invention described in the context of a single embodiment may also be provided individually, or in any suitable sub-combination, or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment does not function without those elements.

[0305] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0306] Example

[0307] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0308] Materials and methods

[0309] Primary culture

[0310] Primary cultures of MECs were isolated from mammary biopsies. Briefly, mammary tissue was collected from lactating cows at a commercial slaughterhouse and immediately immersed in ice-cold growth medium DMEM-F12 containing 1000 U / ml penicillin, 1 mg / ml streptomycin, 2.5 μg / ml amphotericin B cocktail, and 0.02 mg / ml heparin to prevent cell clumping after digestion. The study protocol followed the regulations of the Israeli Department of Health.

[0311] After being transferred to the laboratory, the tissue is minced and vibrated at 37 ℃ for 3 hours with digestion at a speed of 100 rpm in a growth medium supplemented with collagenase (1 mg / ml), hyaluronidase (1 mg / ml) and 0.02 mg / ml heparin. After incubation, the suspension is filtered through a metal mesh (250 μm), and the filtrate is centrifuged for 5 minutes at 350 g. The precipitate is treated with trypsin-EDTA and 0.04% (w / v) DNase. The cells are then washed with a growth medium supplemented with heparin, and treated with a single DNase, filtered through a 100 μm cell strainer (BD Falcon, Bedford, MA), and washed with a growth medium.

[0312] Cells were grown in plastic culture dishes containing DMEM / F12 supplemented with 10% (w / v) FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B supplemented with insulin (1 μg / ml), hydrocortisone (0.5 μg / ml) and prolactin (1 μg / ml).

[0313] Induction medium:

[0314] DMEM / F12 was supplemented with 10% (w / v) FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B supplemented with insulin (1 μg / ml), hydrocortisone (0.5 μg / ml) and prolactin (1 μg / ml).

[0315] deal with

[0316] 1. Oleic acid treatment: Oleic acid was added to the induction medium at a final concentration of 360-1500 mM. The treatment was carried out for 1 to 7 days.

[0317] 2. LXRa Treatment: Liver X receptor agonist (LXRa), a known mouse LXR activator, is proposed to act similarly on the human and bovine orthologs (NR1H3). LXRa was added to the medium with or without oleic acid. LXRa was added to the medium with or without oleic acid. Two LXR agonists were tested: GW3965 (LXR1, SIGMA) and T0901317 (LXR2, SIGMA), each at a concentration of 1 μM / ml, incubated at least overnight (ON). OA was added after the cells reached at least 80% confluence.

[0318] Lipid extraction and analysis

[0319] After treatment, cells were harvested with trypsin (0.05%), washed with 0.9% (w / v) NaCl, and stored at -20°C until lipid extraction.

[0320] Total lipids were extracted from intact cells by NP-40 and from the culture medium by a cold extraction procedure developed by Folch et al., but with slight modifications. In brief, 5 ml of aliquot of methanol: chloroform solution (2:1, v / v) was added to each sample. After incubation at room temperature, 1 ml of double distilled water was added. After incubation overnight at 4 ° C, the upper phase was discarded and the lower phase was filtered with a Pasteur pipette with glass wool. The sample was then dried under a stream of nitrogen at 65 ° C and diluted with 100 μl of 0.5% NP-40. It is worth noting that lipid metabolites are measured by Cobas measurement, which is a parameter of lipid activity, but glycerol is also measured. On the other hand, the plate assay specifically measures TG while eliminating glycerol background.

[0321] After treatment, the culture medium was collected and centrifuged at 500g for 10 minutes. The supernatant was transferred to a fresh test tube and kept at 4°C until analysis. The triglyceride content of the culture medium was quantified using a triglyceride quantification kit (lipase assay Abcam, Cambridge, UK). Quantification was performed against an external standard curve and expressed as 1mL / 10μL viable cells. The number of viable cells was determined using a hemocytometer after trypan blue staining.

[0322] Lipid droplet staining

[0323] Cells grown on glass coverslips were rinsed three times with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The coverslips were then rinsed four times with PBS and stained with Nile red for 15 min. The coverslips were then rinsed three times with PBS and stained with DAPI for 5 min. Finally, the coverslips were rinsed four times with PBS and mounted with fluorescent mounting medium. Fluorescence microscopy and lipid droplet size measurement Slides were observed using an Echo fluorescence microscope equipped with a digital camera. Lipid droplet analysis was performed using ImageJ software.

[0324] Gene expression

[0325] RT-PCR protocol

[0326] Using TYRPLE TM The cells were harvested using Nzytech TM Total RNA Isolation Kit (MB13402) was used from 1×10 6 RNA was extracted from each cell using Rapid TM cDNA was prepared using a cDNA kit (0.5 μg rRNA / 20 μl reaction mixture). Rapid PCR with 1.25-20 ng cDNA / well was used. TM RT-PCR was performed using an RT-PCR kit and the following primers.

[0327] Table 1

[0328]

[0329]

[0330] Example 1

[0331] LXR agonists affect gene expression of lipid synthesis and metabolism genes

[0332] The activation of lipid metabolism of LXR downstream genes SREBP1, FASN, SLC25A1, and SQLE was tested by RT-PCT. A 5-fold overexpression of SREBP1 and a 2-fold overexpression of FASN were observed, while there was no change in gene expression of SLC25A1 and SQLE. These results suggest that LXR agonists activate the LXR receptor NR1H3. The reduction of signaling along this pathway implies a signaling switch from gene expression regulation to a metabolic feedback loop. Although lipid droplets were observed after OA incubation, OA had no effect on LXR-dependent gene expression of lipid metabolism, while LXR pathway activation by LXR agonists was OA-independent.

[0333] Example 2

[0334] LXR agonists enhance lipid metabolism and ameliorate the negative effects of oleic acid in tissue culture

[0335] Oleic acid treatment had a negative impact on cell viability when used at high concentrations above 300 μM or for extended periods of time exceeding one day. This may be due to the accumulation of lipid bodies in the cytoplasm to abnormally high concentrations, leading to loss of cell function, cytotoxic effects, and ultimately cell death ( Figure 2 Indeed, for cells treated with oleic acid, intracellular accumulation of lipid bodies was observed (white-yellowish bodies in bright field). The presence of 360 μM OA or 1100 μM OA with or without LXRa (TO901317) was analyzed. Another observation was the massive cell death, suggesting a correlation between lipid accumulation, cytotoxic effects and cell death.

[0336] A major cause of lipid accumulation is the lack of lipid body release from cells into the culture medium.

[0337] Addition of 1 μM / ml LXR increased lipid metabolism and also resulted in lipid release into the culture medium and improved cell survival. Interestingly, despite the release of lipids into the culture medium by cells in response to LXRα, total TG measured at 1 M cells was similar or higher in cells treated with LXR-OA compared to OA alone ( Figure 3 ). This suggests that the lipids are more evenly distributed in the cells and therefore less cytotoxic, and that lipid production is higher in LXRa-treated cells.

[0338] When oleic acid was used at concentrations of 360 μM or lower, additive effects of LXR agonists on lipids measured intracellularly were mainly observed. However, for higher oleic acid concentrations, there was less or no effect on cellular TG detected by plate assays; lipid metabolite levels in the culture medium were higher for LXR-treated samples.

[0339] More specifically, LXRa treatment (1 μl / ml) did not reduce total TG observed in the cells for any concentration of OA treatment. For OA concentrations above 360 ​​μM, no effect of the LXR agonist was observed within the cells, but only on the media metabolites. For 360 μM OA treatment, the LXRa treated group showed 60% higher intracellular TG levels. IM control: Induction media control. GM control: Growth media control. OA control: Oleic acid was loaded on BSA without the addition of NaOH.

[0340] Example 3

[0341] Preparation of Milk Fat Globule-like (MFGL) Structure

[0342] MEC cultures—as described above.

[0343] Lipid synthesis induction - with LXRa and OA was performed as described above.

[0344] Lipid extraction and analysis—as described above.

[0345] Membrane and lipid separation

[0346] Nuclear and mitochondrial membranes and lipid fractions were cleared by collecting the upper fraction of the lysed cell pellet after prolonged centrifugation or by using column-based separation to collect the lipophilic fraction by gently grinding the cells, incubating on ice for 10 min, and then centrifuging vigorously at 16,000 × g. The supernatant was collected and further subjected to gradient centrifugation as described above (Eski et al., 2020).

[0347] Lipid and lipid droplet staining

[0348] For lipid staining, the collected pellets were mixed with 10 μl of Nile red (0.1% w / v in ethanol). After incubation for 1 hour, the samples were thoroughly mixed with an equal volume of 0.5% low melting point agarose in 10 mM sodium dihydrogen phosphate buffer (pH 6.8), and then 20 μl of the stained samples were placed on a glass chamber slide fluorescence microscope and the lipid droplet size measurement slide was observed through the RFP channel using an Echo fluorescence microscope equipped with a digital camera. Lipid droplet analysis was performed using ImageJ software.

[0349] Formation of lipid membrane layer

[0350] To produce lipid micelles, a step using a combination of membrane and lipid droplet fractions is included to produce a lipid membrane lamella. This method, described by H. Zhang in 2017 and W. Zhang et al. in 2018, produces a heterogeneous product containing membrane and lipid droplet elements in the form of liposome-like structures. This step is the initial stage in the production of MFGL structures. Another method for producing MFGLs is through 30 heating-cooling cycles of 2 minutes each, with 3 minutes of vortexing while cooling, which also provides heterogeneous MFGLs. The size ranges from 200nM to 10μM.

[0351] MFGL was homogenized by sonication (Mendez & Banerjee, 2017) or extrusion (Gven et al., 2009). Both methods homogenized MFGL to a size of <20 μM. The purpose of homogenizing the final product was to increase its stability and increase the membrane fraction by increasing the (membrane / lipid droplet size) ratio.

[0352] Example 4

[0353] Effects of transgenic LXR and PRLR on TAG metabolism in human mammary epithelial cells

[0354] The human LXR gene (hNR1H3 [NM_001251935.2]) and the human PRLR gene (hPRLR [NM_000949.7]) were inserted into a plasmid that confers puromycin resistance. Mammary epithelial cells were transfected with the plasmid using PolyJet transfection reagent at a concentration of 1 μg DNA / 1×10 6 Cells were then seeded into 6-well plates, incubated at room temperature for 10 minutes, and then 2 ml of growth medium was added to each well. The plates were then moved to a 37°C incubator with 5% CO2. The next day, the growth medium was replaced with fresh medium containing 1 μg / ml puromycin, and the cells were cultured for 10 days. Lipid profiles were obtained using HPLC-CAD, and triglycerides were quantified using Abnova's ELISA kit.

[0355] Table 2 below shows the synergistic effects of LXR and PRLR on TAG metabolism in human mammary epithelial cells from the mammary gland. Compared with WT and PRLR, LXR provided higher TAG content, but the combination of LXR and PRLR provided synergistic effect in TAG production.

[0356] Table 2

[0357] ID genetically modified TAGμg / 1M cell 1 WT 7 2 PRLR 34 3 LX 44 4 LXR+PRLR 60.3

[0358] Example 5

[0359] Mammary epithelial cells grown as spheroids in suspension

[0360] Spheroid formation in Aggrewell

[0361] Mammary epithelial cell spheroids were generated in large quantities using the Aggrewell plate platform (Stemcell technologies). Each well in these plates contained a standardized microwell array of 400 μm or 800 μm in size. Plates were pre-coated with an anti-adhesion solution (Stemcell technologies). After coating, 50, 100, 150, 200, 500 or 900 cells / microwell were inoculated according to the Stemcell technologies standard protocol. Stable spheroids were observed after 1-3 days after inoculation. Spheroids were collected from Aggerwell and transferred to suspension cultures.

[0362] Example 6

[0363] Growth of mammary epithelial cells on suspension beads

[0364] First, in the absence of Ca 2+ and Mg 2+ The Cytodex I microcarrier beads were hydrated and activated in PBS at a density of 225 mg microbeads per 50 to 100 mL / g PBS and then incubated overnight at room temperature. Prior to inoculation, the carriers were washed twice according to the manufacturer's preparation protocol; and centrifuged at 1200 rcf for 10 minutes. MEC (as described above) were inoculated on Cytodex I carriers in a 500 ml spinner bottle for 2 hours. The suspension was then transferred to a 1.6 L bioreactor; the stirring speed was 50 rpm. The final inoculation density in the bioreactor was 0.1-0.5 mg microbeads / ml.

[0365] Although the present invention has been described in conjunction with the specific embodiments of the present invention, it is obvious that many substitutions, modifications and changes will be apparent to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and changes that fall within the spirit and broad scope of the appended claims.

[0366] It is the intention of the applicant that all publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent or patent application were specifically and individually incorporated into this specification by reference. In addition, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. As far as the section headings used are concerned, they should not be construed as necessary limitations. In addition, the entire contents of any one or more priority documents of the present invention are incorporated herein by reference in their entirety.

[0367] References

[0368] (Other references cited in the document)

[0369] ■Eski,SE,Dubois,C.,&Singh,SP(2020).Nuclei isolation from wholetissue using a detergent and enzyme-free method.Journal of VisualizedExperiments,2020(160),1–10.www(.)doi(.)org / 10.3791 / 61471

[0370] ■Gven,A.,Ortiz,M.,Constanti,M.,&O’Sullivan,C.K.(2009).Rapid andefficient method for the size separation of homogeneous fluorescein-encapsulating liposomes.Journal of Liposome Research,19(2),148–154.www(.)doi(.)org / 10.1080 / 08982100802674419

[0371] ■Hernell,O.,Timby,N., M.,& B.(2016).ClinicalBenefits of Milk Fat Globule Membranes for Infants and Children.Journal ofPediatrics,173,S60–S65.www(.)doi(.)org / 10.1016 / j.jpeds.2016.02.077

[0372] ■Mendez,R.,&Banerjee,S.(2017).Sonication-based basic protocol forliposome synthesis.In Methods in Molecular Biology(Vol.1609,pp.255–260).Humana Press Inc.www(.)doi(.)org / 10.1007 / 978-1-4939-6996-8_21

[0373] ■Timby,N., M., B.,&Hernell,O.(2017).Supplementationof infant formula with bovine milk fat globule membranes.In Advances inNutrition(Vol.8,Issue 2,pp.351–355).American Society for Nutrition.www(.)doi(.)org / 10.3945 / an.116.014142

[0374] ■Zhang,H.(2017).Thin-film hydration followed by extrusion method forliposome preparation.In Methods in Molecular Biology(Vol.1522,pp.17–22).Humana Press Inc.www(.)doi(.)org / 10.1007 / 978-1-4939-6591-5_2

[0375] ■Zhang,W.,Li,C.,Jin,Y.,Liu,X.,Wang,Z.,Shaw,J.P.,Baguley,B.C.,Wu,Z.,&Liu,J.(2018).Multiseed liposomal drug delivery system using micelle gradientas driving force to improve amphiphilic drug retention and its anti-tumorefficacy.Drug Delivery,25(1),611–622.www(.)doi(.)org / 10.1080 / 10717544.2018.1440669。

Claims

1. A method of increasing lipid synthesis or accumulation and optionally secretion from a culture of mammary epithelial cells (MECs), the method comprising contacting the MECs with an LXR agonist in the presence of fatty acids, thereby increasing lipid synthesis or accumulation and optionally increasing secretion of lipids.

2. The method according to claim 1, wherein the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.

3. The method of claim 1, wherein the fatty acid is selected from the group consisting of oleic acid and palmitic acid.

4. The method of claim 1, wherein the fatty acid is oleic acid.

5. The method according to any one of claims 1 to 4, wherein the contacting is performed in the presence of at least one of a phenolic composition, insulin, hydrocortisone, prolactin, and beta-hydroxybutyric acid (BHBA).

6. The method of any one of claims 1 to 5, further comprising harvesting lipids from the MECs.

7. The method of any one of claims 1 to 5, further comprising harvesting lipids from the culture medium of the MEC.

8. The method of any one of claims 1 to 7, wherein the LXR agonist is GW3965 or T0901317.

9. The method of any one of claims 1 to 7, wherein the LXR agonist is 9-cis-13,14-dihydroretinoic acid.

10. The method of any one of claims 1 to 7, wherein the LXR agonist is at least one nucleic acid agent that upregulates LXR, its activators and / or effectors.

11. The method of claim 10, wherein the nucleic acid agent encodes LXR.

12. The method of claim 10, wherein the at least one nucleic acid agent encodes LXR and PRLR.

13. The method of claim 10, wherein the at least one nucleic acid agent introduces a nucleic acid alteration in the genomic sequence of the LXR, activator thereof and / or effector.

14. The method of claim 13, wherein the genomic sequence is a coding sequence.

15. The method of claim 13, wherein the genomic sequence is a non-coding sequence.

16. The method of claim 15, wherein the nucleic acid alteration renders the LXR, the activator and / or effector thereof constitutively active.

17. The method of claim 15, wherein the nucleic acid alteration causes regulation of the LXR, the activator and / or effector thereof in an inducible manner.

18. The method of any one of claims 15 to 18, wherein the nucleic acid alteration renders the LXR response element (LXRE) constitutively active.

19. The method of any one of claims 15 to 18, wherein the nucleic acid alteration renders the LXR response element (LXRE) inducible.

20. The method of any one of claims 5 to 19, wherein the culture comprises the LXR agonist, fatty acids, and the phenolic composition.

21. The method according to any one of claims 5 to 20, wherein the phenolic composition is selected from the group consisting of flavonols, flavanols, flavones, flavanones and anthocyanidins.

22. The method according to any one of claims 5 to 20, wherein the phenolic composition is selected from the group consisting of gallic acid or myricetin or derivatives thereof.

23. The method according to any one of claims 1 to 22, performed in the presence of albumin.

24. The method of claim 23, wherein the albumin is bovine serum albumin (BSA).

25. The method of claim 23, wherein the albumin is BSA or plant albumin.

26. The method of any one of claims 1 to 23, wherein the MEC is human or bovine MEC.

27. A composition comprising a cell or a secretome or a fraction of the cell or the secretome obtainable according to the method of any one of claims 1 to 26.

28. A method for producing synthetic fat globules, the method comprising: (a) Isolation of lipid droplets from cultured cells; (b) Encapsulating the droplets with a lipid bilayer, thereby producing synthetic fat globules.

29. The method of claim 28, wherein the cells are mammary epithelial cells (MECs).

30. The method of any one of claims 28 to 29, wherein the lipid bilayer is a cell membrane.

31. The method of claim 30, wherein the cell membrane is of a mammary epithelial cell (MEC).

32. The method of any one of claims 28 to 29, wherein the lipid bilayer is a synthetic membrane.

33. The method of any one of claims 28 to 32, wherein the lipid bilayer comprises a peptide.

34. The method of any one of claims 28 to 33, wherein the membrane and the lipid droplet contents are from different sources.

35. The method of claim 34, wherein one of the different sources comprises MEC.

36. The method of any one of claims 1 to 33, wherein the membrane and the lipid droplet contents are from the same source.

37. The method of claim 36, wherein the source is mammary epithelial cells (MEC).

38. The method of any one of claims 28 to 37, further comprising subjecting the cells to a lipid synthesis induction regimen prior to step (a).

39. The method of claim 38, wherein the lipid induction protocol is according to any one of claims 1-23.

40. A composition comprising synthetic fat globules obtainable according to any one of claims 28-39.

41. A composition comprising synthetic fat globules comprising a lipid droplet core coated with a lipid bilayer.

42. The composition according to any one of claims 40 to 41, wherein the synthetic fat globules have a size range of 100 nm - 15 μm.

43. A nutritional product comprising the composition of any one of claims 40 to 42.

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