Methods for producing a dairy product or a dairy-like component and compositions thereof

Abstract

The present disclosure provides methods of producing a dairy product or a dairy-like component or a mixture thereof, comprising: growing cells in a structured apparatus, which are procured and optimized for relevant functionality; inducing growing cells to obtain secretion of the dairy product or dairy like component, into a designated compartment of the structured apparatus; and extracting the dairy product or dairy like component or the mixture using an extraction process, from the designated compartment of the structured apparatus. The present disclosure also provides a milk composition comprising: a) milk or milk components obtained from cell culture and b) milk or milk components obtained from a natural source.

Description

[0001] METHODS FOR PRODUCING A DAIRY PRODUCT OR A DAIRY-LIKE COMPONENT AND COMPOSITIONS THEREOF

[0002] TECHNICAL FIELD

[0003] 5 The present disclosure relates to methods for producing dairy products or dairy-like components or mixtures thereof from a cell culture. The present disclosure also relates to a dairy composition comprising the dairy product or dairy-like components or mixtures thereof, wherein the dairy product and / or dairy-like components are obtained from a cell culture and / or a natural source like human and animals.

[0004] 10

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Milk and dairy products have long been recognized as fundamental nutritional sources, providing essential proteins, lipids, carbohydrates, and micronutrients necessary for human health. Traditionally, milk production has relied on dairy farming, which, despite its widespread use, 15 presents numerous challenges. Conventional dairy farming contributes significantly to environmental concerns, including greenhouse gas emissions, deforestation, and excessive water consumption. Additionally, ethical concerns related to animal welfare and the use of antibiotics in livestock further complicate the sustainability of dairy farming. Moreover, fluctuations in milk production due to seasonal variations, disease outbreaks, and the genetic limitations of livestock 20 result in inconsistent supply chains, affecting the availability and affordability of dairy products worldwide.

[0007] To address these concerns, researchers have explored the possibility of producing milk components using cell culture techniques. Researchers have attempted to develop in vitro milk production 25 methods, leveraging bioreactors and genetically engineered cells. These efforts aim to replicate the natural lactation process within a controlled environment.

[0008] Current methods disclosed in the art of producing milk using cell culture techniques often result in milk that is intermixed with culture media components, leading to contamination and affecting 30 the purity and composition of the final product. This poses a significant challenge in obtaining milk that retains its natural nutritional and bioactive properties without unwanted residues from

[0009] 1 the culture medium. The present disclosure attempts to address this problem by providing a method allows efficient extraction of dairy products or dairy-like components, ensuring minimal / no contamination / mixing of dairy products or dairy-like components with culture media and preserving the integrity of the final product.

[0010] 5

[0011] SUMMARY OF THE DISCLSOURE

[0012] The present disclosure provides a method of producing a mammalian dairy product or a dairy-like component or a mixture thereof, comprising:

[0013] (a) growing cells in a structured apparatus, which are obtained and optimized for specific 10 functionality;

[0014] (b) inducing growing cells to obtain secretion of the dairy product or dairy like component, into a designated compartment of the structured apparatus; and

[0015] (c) extracting the dairy product or dairy like component or the mixture using an extraction process, from the designated compartment of the structured apparatus.

[0016] 15

[0017] The present disclosure also provides a device comprising the structured apparatus (e.g., Figure 1) for any biotechnology, food, medical, or pharmaceutical application.

[0018] The present disclosure also provides a process of growing cells involving application of external 20 voltage gradient across the cells for any biotechnology, food, medical, or pharmaceutical application.

[0019] The present disclosure also provides a method comprising an extraction process for separation of any mixture or extraction application; wherein the wherein the extraction process comprises: 25 a) collecting all outputs or secretions or media from the designated space;

[0020] b) loading the collected outputs or secretions or media on an extraction setup comprising of components like centrifugal filter and spinning the loaded centrifugal filter one or more times at 500rcf or higher;

[0021] c) repeating step b) with the extraction setup comprising of components like multiple 30 membranes or filters or same filters multiple times;

[0022] 2 d) collecting a retentate and subjecting it to a freezing mechanism or flash freezing or lyophilizing or several of those in succession to obtain a refined output; and e) mixing the refined output with water as needed to obtain the desired dairy product.

[0023] 5 The present disclosure also provides a milk composition comprising: a) milk or milk components obtained from cell culture and b) milk or milk components obtained from a natural source.

[0024] The present disclosure also provides a combination milk product comprising a human origin milk, milk component, or milk product and a non-human origin milk, milk component, or milk product.

[0025] 10

[0026] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0027] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures.

[0028] 15 The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

[0029] Figure 1 shows exemplary structured apparatuses (100, 200, 300, 400) according to one 20 embodiment of the present disclosure.

[0030] FIGs. 2A-2F shows exemplary schematics of the extraction process used for extracting the dairy product or dairy-like component or the mixture from the designated compartment of the structured apparatus.

[0031] 25

[0032] Figure 3: Panel A shows an exemplary schematic of the centrifugal filtration process used for extracting the dairy product or dairy-like component or the mixture from the designated compartment of the structured apparatus. Panel B shows an exemplary schematic of the dialysis process used for extracting the dairy product or dairy-like component or the mixture from the 30 designated compartment of the structured apparatus.

[0033] 3 Figure 4 illustrates an exemplary procedure for the propagation of cells for use in the generation of the milk components and milk products of the disclosure.

[0034] Figure 5 illustrates an exemplary strategies for the conversion of isolated cells into lactogenic 5 mammary epithelial cells.

[0035] FIGS. 6A-6C illustrate exemplary gene editing strategies for the genetic engineering of the cell lines provided herein, for use in the generation of the milk components and milk products of the disclosure.

[0036] 10

[0037] Figure 7 illustrates an exemplary lab-scale culturing system, for use in the generation of the milk components and milk products of the disclosure.

[0038] FIGS. 8A-8B illustrate seeding an exemplary bioreactor of the disclosure.

[0039] 15

[0040] FIGS. 9A-9E illustrate various combinatorial milk products of the disclosure.

[0041] Figure 10 illustrates an exemplary lab-scale culturing system where cells are grown by applying an external voltage gradient (V1-V2) across the cells for use in the generation of the milk 20 components and milk products of the disclosure.

[0042] Figure 11 schematically illustrates a lumen and interstitial space formed by mammary epithelial cells in vivo and the major fluxes of macronutrients during lactation.

[0043] 25 DETAILED DESCRIPTION

[0044] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” 30 suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout

[0045] 4 this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having”, or “including but not limited to” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers 5 or steps.

[0046] Reference throughout this specification to “one embodiment”, “an embodiment”, or “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, 10 the appearances of the phrases “in one embodiment”, “in an embodiment”, or “in some embodiments” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in 15 the context of a single embodiment, may also be provided separately or in any suitable sub- combination.

[0047] The term “about” as used herein encompasses variations of + / -5% and more preferably + / -2.5%, as such variations are appropriate for practicing the present invention.

[0048] 20

[0049] The present disclosure provides a method for producing a mammalian dairy product or a dairy- like component or a mixture thereof, comprising:

[0050] a) growing cells in a structured apparatus, which are obtained and optimized for specific functionality;

[0051] 25 b) inducing growing cells to obtain secretion of the dairy product or dairy like component, into a designated compartment of the structured apparatus; and

[0052] c) extracting the dairy product or dairy like component or the mixture using an extraction process, from the designated compartment of the structured apparatus.

[0053] 30 The term “dairy product” as used herein refers to a product containing any mammalian milk as well as products obtained / derived from mammalian milk, such as yoghurt, curd, cheese, butter,

[0054] 5 cream, ice cream, condensed milk, powdered milk, infant formula, infant milk, kefir, buttermilk, skyr, lassi, ayran, clarified butter, kulfi, oil, ghee and the like.

[0055] The term “dairy-like component” as used herein refers to any component present in mammalian 5 milk such as lactose, casein, whey protein, fats, including modified or engineered versions of the same.

[0056] The term “structured apparatus” refers to any apparatus that can be used for growing cells. For example, in some embodiments, the structured apparatus can be cell culture plates, cell culture 10 flasks, cell culture inserts, a bioreactor comprising a compartment where cells can be grown, or any other structure that can support the growth of cells. Figure 1 shows a few exemplary structured apparatuses that may be employed in the present methods. Accordingly, in some embodiments, the structured apparatus is 100, 200, 300, or 400 as shown in Figure 1.

[0057] 15 In Figure 1:

[0058] • numeral 1 indicates growth / differentiation media;

[0059] • numeral 2 indicates a designated space for the secretion of the dairy product or dairy component that may be filled with media, an isotonic solution (PBS, saline, etc) or similar or combination thereof or that may be empty, i.e., devoid of any media or any other 20 solution;

[0060] • numeral 3 indicates an extracellular matrix such as collagen, laminin, Matrigel, Geltrex, etc., that provides support for growing cells;

[0061] • numeral 4 indicates cells capable of producing the dairy product or a dairy like component such as mammary epithelial cells;

[0062] 25 • numeral 5 indicates a cell culture vessel such as cell culture plates, petri dishes, etc.; and • numeral 6 indicates a cell culture insert.

[0063] • numeral 7 indicates an optional cell insert support structure

[0064] In some embodiments, the structured apparatus comprises a cell culture insert that is placed on a 30 cell culture vessel or inside a cell culture vessel and supports the growth of cells. Exemplary designs of the cell culture inserts are shown in panels A-D of Figure 1 where numeral 6 indicates

[0065] 6 the cell culture inserts. In some embodiments, the cell culture inserts are coated with an extracellular matrix such as collagen, laminin, Matrigel, Geltrex, etc. that provide support to growing cells.

[0066] 5 In some embodiments, a cell culture insert with a base of a permeable membrane hangs on the walls of a cell culture plate or petri dish (see, for example, Type 1 structured apparatus shown in panel A of Figure 1).

[0067] In some embodiments, a cell culture insert fits inside another concentric ring holding a permeable 10 membrane in between and hanging on the walls of a cell culture plate or petri dish (see, for example, Type 2 structured apparatus shown in panel B of Figure 1).

[0068] In some embodiments, a cell culture insert fits inside another concentric ring holding a permeable membrane in between and kept on the base of a cell culture plate or petri dish (see, for example, 15 Type 3 structured apparatus shown in panel C of Figure 1).

[0069] In some embodiments, the structured apparatus is a bioreactor which has tubular scaffolds with permeable membranes (see, for example, Type 4 apparatus shown in panel D of Figure 1).

[0070] 20 In some embodiments, the term “cells” as used herein refers to cells capable of producing a dairy product or a dairy-like component. In some embodiments, the cells are primary cells or a cell line.

[0071] In some embodiments, cells are mammary cells or lactogenic cells, i.e., capable of producing milk or a milk component. In some embodiments, mammary cells are primary mammary epithelial cells, 25 immortalized mammary epithelial cells, mammary organoids, or mammary stem cells and the like.

[0072] In some embodiments, mammary cells are mammary epithelial cells. In some embodiments, mammary epithelial cells are selected from human mammary epithelial cells (HMECs), bovine mammary epithelial cells (BMECs), goat mammary epithelial cells, sheep mammary epithelial 30 cells, camel mammary epithelial cells, or any other mammalian mammary epithelial cells as well as cell lines derived from them.

[0073] 7 The term “specific functionality” as used herein refers to an ability to produce a mammalian dairy product or a dairy-like component or a mixture thereof.

[0074] In some embodiments, the method for producing a mammalian dairy product or a dairy-like 5 component or a mixture thereof, comprises a) growing cells in a structured apparatus, which are obtained and optimized for specific functionality; b) inducing growing cells to obtain secretion of the dairy product or dairy-like component, into a designated compartment of the structured apparatus; and c) extracting the dairy product or dairy like component or the mixture using an extraction process, from the designated compartment of the structured apparatus.

[0075] 10

[0076] As described above, the cells such as mammary cells are grown in a structured apparatus (e.g., 100, 200, 300, or 400 of Figure 1). The growing cells are induced to secrete the dairy product or dairy-like component into a designated compartment (e.g., indicated by numeral 2 in Figure 1) of the structured apparatus. In some embodiments, the cells are grown to about 60% to 100% 15 confluency, including values and ranges thereof, such as about 70%-100%, 70%-90%, 80%-100%, or 80%-90% confluency and then induced to secrete the dairy product or dairy-like component. The secreted dairy product or dairy-like component or the mixture thereof is then extracted from the designated compartment of the structured apparatus.

[0077] 20 In some embodiments, the cells are grown in a culture medium comprising Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) and one or more components selected from, HEPES, phenol red, bovine serum albumin (BSA), transferrin, glutamine, thiamine, epidermal growth factor (EGF), phosphatidylethanolamine (PE), or any combination thereof. BSA and the growth factors may also be recombinant in nature.

[0078] 25

[0079] In some embodiments, the step of growing cells in a structured apparatus is performed in the absence of applying any external voltage gradient across the cells. In some other embodiments, the step of growing cells in a structured apparatus is performed by applying an external voltage gradient across the cells. For example, V1 and V2 shown in Figures 1 and 10 indicate a voltage 30 gradient applied externally across the cells. In some embodiments, in the step of growing cells, the external voltage gradient applied across the cells ranges from 0-90 mV, including values and ranges

[0080] 8 thereof, such as 0-85 mV, 0-80 mV, 0-75 mV, 0-70 mV, 0-60 mV, 0-55 mV, 0-50 mV, 0-45 mV, 0- 40 mV, 0-35 mV, 0-30 mV, 0-25 mV, 0-20 mV, 0-15 mV, 0-10 mV, 0-5 mV, 5-90 mV, 5-85 mV, 5- 80 mV, 5-75 mV, 5-70 mV, 5-60 mV, 5-55 mV, 5-50 mV, 5-45 mV, 5-40 mV, 5-35 mV, 5-30 mV, 5-25 mV, 5-20 mV, 5-15 mV, 5-10 mV, 10-90 mV, 10-85 mV, 10-80 mV, 10-75 mV, 10-70 mV, 10- 5 60 mV, 10-55 mV, 10-50 mV, 10-45 mV, 10-40 mV, 10-35 mV, 10-30 mV, 10-25 mV, 10-20 mV, 10-15 mV, 20-90 mV, 20-80 mV, 20-70 mV, 20-60 mV, 20-50 mV, 20-40 mV, 20-30 mV, 30-90 mV, 30-80 mV, 30-70 mV, 30-60 mV, 30-50 mV, 30-40 mV, 40-90 mV, 40-80 mV, 40-70 mV, 40- 60 mV, 40-50 mV, 50-90 mV, 50-80 mV, 50-70 mV, 50-60 mV, 60-90 mV, 60-80 mV, 60-70 mV, 70-90 mV, or 80-90 mV. The external voltage gradient can be applied by connecting a power source 10 across the top and the bottom of the cells, wherein the power source supplied he voltage gradient in the ranges described above.

[0081] Figure 10 shows an exemplary lab-scale set-up for culturing cells by applying an external voltage gradient across the cells. The range for the voltage gradient is described above. The external 15 voltage gradient can be applied to cells growing in any of the structured apparatuses described here, such as Type 1 (100), 2 (200), 3 (300), or 4 (400) or any combination of them.

[0082] In some embodiments, the growing cells are induced to produce the dairy product or the dairy-like component or the mixture by contacting the cells with an induction media.

[0083] 20

[0084] In some embodiments, the induction media comprise one or more components along with the growth media described above for growing of cells.

[0085] In some embodiments, the induction media is serum-free. In some embodiments, the serum free 25 media is selected from but not limited to M199 Medium, RPMI 1640, DMEM / F12 and Ham’s F- 12, preferably DMEM / F12. In some embodiments, the serum free media is DMEM / F12.

[0086] In some embodiments, the induction media comprises DMEM / F12, HEPES, phenol red, BSA, transferrin, EGF, thiamine, glutamine, PE, oleic acid, palmitic acid, prolactin, hydrocortisone or 30 some combination thereof

[0087] 9 In some embodiments, the growing cells are contacted with the induction media for at least about 24 hours, for about 24-48 hours, for about 24-72 hours, for about 1-3 days, about 1-5 days, about 2-5 days, about 3-5 days, or about 3-4 days to produce the dairy product or the dairy-like component or the mixture thereof. After the step of induction, all outputs or secretions or media 5 from the designated space is collected for the extraction of the dairy product or the dairy-like component or the mixture thereof.

[0088] In some embodiments, the dairy product or the dairy-like component or the mixture thereof secreted by the cells grown in the structured apparatus are extracted by an extraction process. In 10 some embodiments, the extraction process comprises:

[0089] a) collecting all outputs or secretions or media from the designated space;

[0090] b) loading the collected outputs or secretions or media on an extraction setup comprising of components like centrifugal filter and spinning the loaded centrifugal filter one or more times at 500rcf or higher;

[0091] 15 c) repeating step b with extraction setup comprising of components like multiple membranes or filters or same filters multiple times;

[0092] d) collecting a retentate and subjecting it to a freezing mechanism or flash freezing or lyophilizing or several of those in succession to obtain a refined output;

[0093] e) mixing the refined output with water as needed to obtain the desired dairy product.

[0094] 20

[0095] In some embodiments, the extraction process comprises:

[0096] a) collecting all outputs or secretions or media from the designated space;

[0097] b) loading the collected outputs or secretions or media on a 1-200kDa centrifugal filter and spinning the loaded centrifugal filter one or more times at 900-2000rcf;

[0098] 25 c) repeating step b) with multiple filters or same filters multiple times;

[0099] d) collecting a retentate and subjecting it to a freezing mechanism or flash freezing or lyophilizing or any combination of those in succession to obtain a refined output; and e) mixing the refined output with water as needed to obtain the desired dairy product

[0100] 30 An exemplary schematic for the extraction process is shown in Figure 2.

[0101] 10 In some embodiments, the extraction setup comprises a dialysis tube or chromatography or beads or electrophoresis or similar extraction mechanisms or a combination thereof. In some embodiments, the extraction setup comprises centrifugal filters or membrane filters. In some embodiments, the extraction setup comprises multiple membranes or filters or same filters multiple 5 times.

[0102] The media can be collected over a period time ranging from 1 day to 6 weeks or more as suitable. In some embodiments, the centrifugal filter used to filter the collected media has a pore size range of 1-200kDa, including values and ranges thereof, such as 1-3kDa, 3-5kDa, 3-10 kDa, 3-15 kDa, 10 5-15 kDa, 5-12 kDa, 5-10 kDa, 10-15 kDa, 20-30kDa, 30-50kDa, 50-100kDa, 100-150kDa, or 150-200kDa and the like.

[0103] In some embodiments, the centrifugal filtration is performed for about 5-105 minutes, including values and ranges thereof, such as about 10-100 minutes, 10-90 minutes, 20-100 minutes, 20-50 15 minutes, 25-30 minutes, 5 minutes, 15 minutes, 25 minutes, 40 minutes, 65 minutes, 90 minutes, or 105 minutes and the like.

[0104] In some embodiments, the centrifugal filtration is performed at a speed in the range of 500-2000rcf, including values of 500 rcf, 750 rcf, 1000 rcf, 1250rcf, 1500 rcf, 1800 rcf, or 2000 rcf and the like.

[0105] 20 The above steps can be repeated multiple times like 2, 3, 6, 8 or similar. Post this more specific extraction and quantification techniques like chromatograraphy, electrophoresis, mass spectrometry, nano-filteration, ligand affinity based extraction, bead based extraction or similar or a combination thereof can be applied to obtain the final product.

[0106] 25 In some embodiments, the centrifugal filter can be a dialysis tube having a size between 1-200kDa.

[0107] In some embodiments, the centrifugal filter can be a series of filters in succession.

[0108] Figure 3 shows an exemplary schematic of the filtration process. In Figure 3, numeral 8 indicates the retentate; numeral 9 indicates a filter; and numeral 10 indicates the filtrate.

[0109] 30

[0110] 11 In some embodiments, the structured apparatus employed for growing cells is a cell culture device that comprises an upper chamber and a lower chamber. Accordingly, in some embodiments, provided herein is a method for producing a mammalian dairy product or a dairy-like component or a mixture thereof, comprising:

[0111] 5 a) growing cells in a cell culture device comprising an upper chamber and a lower chamber;

[0112] wherein the cells are grown in the upper chamber; and a culture medium is added to the upper chamber and the lower chamber;

[0113] b) inducing growing cells to obtain secretion of the dairy product or dairy-like component or the mixture thereof, into the upper chamber; and

[0114] 10 c) collecting the culture media containing the secreted dairy product or dairy-like component or the mixture from the upper chamber; and

[0115] d) extracting the dairy product or dairy-like component or the mixture from the culture media using an extraction process.

[0116] 15 In some embodiments, provided herein is a method for producing a dairy product or a dairy-like component or a mixture thereof, comprising:

[0117] a) growing cells in a cell culture device comprising an upper chamber and a lower chamber; wherein the cells are grown in the upper chamber that is devoid of media or any other solution; and a culture medium is added to the lower chamber;

[0118] 20 b) inducing growing cells to obtain secretion of the dairy product or dairy-like component or the mixture thereof, into the upper chamber; and

[0119] c) collecting the secreted dairy product or dairy-like component or the mixture from the upper chamber.

[0120] 25 The culture media used for growing cells and the induction media used for inducing secretion of the dairy product or the dairy-like component from the growing cells is described above. In these embodiments, where the cells are grown in the cell culture device comprising the upper chamber and the lower chamber, the induction medium is added to the lower chamber. The extraction process used to extract the dairy product or the dairy-like component or the mixture thereof from 30 the culture media collected from the upper chamber includes filtration, dialysis, lyophilization, or

[0121] 12 a combination thereof. In some embodiments, the dairy product or dairy-like component or the mixture thereof is extracted from the culture medium by filtration followed by lyophilization.

[0122] In some embodiments, the dairy product or dairy-like component or the mixture thereof is 5 extracted from the culture medium collected from the upper chamber by the extraction process described above.

[0123] In some embodiments, the dairy product or dairy-like component or the mixture thereof is extracted from the culture medium collected from the upper chamber by the process shown in 10 Figure 2.

[0124] The present disclosure also provides a device comprising the structured apparatus (100, 200, 300, 400) for any biotechnology application.

[0125] 15 The present disclosure also provides a process of growing cells involving application of external voltage gradient across the cells for any biotechnology application. In some embodiments, cells are grown by applying an external voltage gradient across the cells. In some embodiments, the external voltage gradient applied across the cells ranges from 0-90 mV, including values and ranges thereof, such as 0-85 mV, 0-80 mV, 0-75 mV, 0-70 mV, 0-60 mV, 0-55 mV, 0-50 mV, 0-45 mV, 0-20 40 mV, 0-35 mV, 0-30 mV, 0-25 mV, 0-20 mV, 0-15 mV, 0-10 mV, 0-5 mV, 5-90 mV, 5-85 mV, 5- 80 mV, 5-75 mV, 5-70 mV, 5-60 mV, 5-55 mV, 5-50 mV, 5-45 mV, 5-40 mV, 5-35 mV, 5-30 mV, 5-25 mV, 5-20 mV, 5-15 mV, 5-10 mV, 10-90 mV, 10-85 mV, 10-80 mV, 10-75 mV, 10-70 mV, 10- 60 mV, 10-55 mV, 10-50 mV, 10-45 mV, 10-40 mV, 10-35 mV, 10-30 mV, 10-25 mV, 10-20 mV, 10-15 mV, 20-90 mV, 20-80 mV, 20-70 mV, 20-60 mV, 20-50 mV, 20-40 mV, 20-30 mV, 30-90 25 mV, 30-80 mV, 30-70 mV, 30-60 mV, 30-50 mV, 30-40 mV, 40-90 mV, 40-80 mV, 40-70 mV, 40- 60 mV, 40-50 mV, 50-90 mV, 50-80 mV, 50-70 mV, 50-60 mV, 60-90 mV, 60-80 mV, 60-70 mV, 70-90 mV, or 80-90 mV. The external voltage gradient can be applied by connecting a power source across the top and the bottom of the cells, wherein the power source supplied he voltage gradient in the ranges described above.

[0126] 30

[0127] 13 The present disclosure also provides a method comprising an extraction process for separation of any mixture or extraction application as shown in Figure 2.

[0128] The present disclosure also provides a milk composition comprising: a) milk or milk components 5 obtained from the methods described herein and b) milk or milk components obtained from a natural source. In some embodiments, the natural source is a human or a non-human mammal. In some embodiments, the non-human mammal is a cow, a buffalo, a doe, a sheep, a goat, or a camel.

[0129] Lactation

[0130] 10 Reference is made to Figure 11, which schematically shows a lumen and interstitial space formed by MECs in vivo and the major fluxes of macronutrients during lactation. STAT5 and ligand-bound GR contribute to the expression of adhesion proteins that during lactation form a strong tight junction, which effectively functions as a blood-milk barrier and prevents the paracellular transport of both small and large molecules. Bovine tight junctions are formed by 15 proteins including but not limited to Claudin-1, -3, -4 and -7 and Occludin. The strength of the tight junction is determined by measurement of transepithelial resistance upon application of a voltage difference across the epithelial membrane. Treatment with Dexamethasone and Prolactin-containing bovine pituitary extract caused a four-fold increase in transepithelial resistance, when compared to control.

[0131] 20 Milk-producing mammary epithelial cells have a cobblestone appearance (as shown in Figure 11) under light microscopy and have a signature expression (including but not limited to) of CK18, β-casein and α-lactalbumin, which may be detected using appropriate antibodies in immunofluorescence experiments.

[0132] Biochemical Components of Milk

[0133] 25 Table 1 summarizes basic biochemical components of milk produced by different animals, including humans, in accordance with some embodiments described herein. Table 2 summarizes fatty acid composition of milk from the different animals, in accordance with some embodiments

[0134] 14 described herein. Table 3 summarizes protein fractions separated from milk produced by the different animals, in accordance with some embodiments described herein.

[0135] Table 1

[0136]

[0137] 5 Table 2

[0138]

[0139] Table 3

[0140]

[0141] 10 Table 4 summarizes contribution to daily dietary reference intakes and health benefits for a selection of milk components, in accordance with some embodiments described herein.

[0142] 15 Table 4

[0143]

[0144] Table 5 summarizes vitamin and mineral composition of milk produced by human, cow, buffalo, goat, and sheep, in accordance with some embodiments described herein.

[0145] 16 Table 5

[0146]

[0147] In some embodiments, provided herein is a method for generating a milk-based product from a culture of mammalian cells. Figure 4 shows a flow chart showing aspects of the method. For

[0148] 17 convenience of presentation, the method may be referred to herein as a “cellular milk production method”.

[0149] In some embodiments, a cellular milk production method comprises obtaining a sample of cells comprising: obtaining a sample of lactogenic cells (block 101) and generating a cell line from the 5 lactogenic cells (block 103). In some embodiments, the cellular milk production method may further comprise creation of a cell bank of the cell line (block 105). In some embodiments, the cellular milk production method may further comprise differentiation and / or proliferation of the lactogenic cell line prior to seeding a milk production bioreactor (block 107). In some embodiments, the cellular milk production method may further comprise proliferation of the cell 10 line in a milk production bioreactor and triggering lactation by the proliferated cell line (block 109).

[0150] Isolation of BMECs

[0151] In certain embodiments, obtaining a sample of lactogenic cells (block 101) may comprise isolating MECs, by way of example Bovine mammary epithelial cells (BMECs) by biopsy.

[0152] 15 Bovine mammary epithelial cells (BMECs) are obtained by using the following methodology:

[0153] • Isolate bovine mammary gland from a cow.

[0154] • Mince into small pieces and incubate the minced tissue with collagenase.

[0155] • Centrifuge and resuspend the cell pellet in DMEM with TrypLE and leave at room temperature such that mammary epithelial fragments are free of contaminating cells, like 20 fibroblasts and myoepithelial cells.

[0156] Towards a goal of creating cruelty-free and sustainable products, obtaining a sample of lactogenic cells (block 101) may comprise alternative strategies that comprise differentiating non-invasively obtained cells including but not limited to stem cells and satellite cells into having MEC features.

[0157] 25 In some embodiments, obtaining a sample of lactogenic cells (block 101) comprises isolating mammary epithelial stem cells (MESCs) from milk, of which an example procedure is provided herein-below:

[0158] 18 a) Collect milk from a lactating cow. Wash and disinfect bovine teats 70% ethanol, discard the first milk jets and collect milk samples in sterile bottles added with 1% penicillin / streptomycin and 1% amphotericin B.

[0159] b) Dilute milk with sterile PBS and centrifuge at 1,400 rpm for 15 min at RT. Discard 5 the supernatant and fat layer and wash the cellular pellet twice in wash solution composed of sterile sodium chloride with 1% penicillin / streptomycin and 1% amphotericin B. Suspend the cells in a DMEM supplemented with, 1% penicillin / streptomycin, and 1% L-glutamine, and grow in 5% CO2 and 37 degrees Celsius incubator.

[0160] 10 c) Change the media every 3 days and passage the cells after 7–10 days, when the cultures reach 70%–80% confluence. Use cells between passages 3–6.

[0161] d) Stain for surface antigens by incubating 5 x 105cells with primary antibody againstCD73, CD90, CD105 for 30 min on ice, followed by staining with the appropriate Alexa488-labeled secondary antibody for 30 min on ice.

[0162] 15 e) Sort cells positive for these 3 markers and separate them.

[0163] f) Plate the cells on attachment plates and suspend them in a DMEM supplemented with, 1% penicillin / streptomycin, and 1% L-glutamine, and grow in 5% CO2 and 37C incubator

[0164] g) Culture and passage as appropriate.

[0165] 20

[0166] For differentiation of these stem cells, cells may be incubated in Roswell Park Memorial Institute medium (RPMI) 1640 with L-glutamine (Invitrogen) supplemented with, 4 μg / ml insulin (Invitrogen), 20 ng / ml epidermal growth factor (EGF) (Invitrogen), 0.5 μg / ml hydrocortisone (Sigma-Aldrich), 5% antibiotic-antimycotic, and 2 μL / ml fungizone. The above protocol for 25 differentiation may be used for any mesenchymal stem cell isolated from another tissue including but not limited to bone marrow, dental, adipose tissue, uterus, umbilical cord, placental and fetal fluid, etc. In some embodiments, obtaining a sample of lactogenic cells (block 101) comprises isolation of bovine embryonic stem cells from pre-implantation embryo obtained by in vitro fertilization of bovine sperm and egg.

[0167] 19 In some embodiments, obtaining a sample of lactogenic cells (block 101) comprises generating induced pluripotent stem cells (iPSCs) from bovine fibroblasts. A example methodology for generating iPSCs may be as follows:

[0168] 1. Passage primary culture of bovine fibroblasts two to three times in fibroblast 5 medium (DMEM high glucose with, 1% nonessential amino acids supplement, and 1% penicillin–streptomycin) at 37°C with 5% CO2.

[0169] 2. Generate retrovirus for reprogramming:

[0170] i) Clone pMX retroviral vector with cDNAs coding for the bovine OCT4 (also called POU5F1), SOX2, KLF4, MYC, LIN28, and NANOG genes.

[0171] 10 ii) Transfect GP2 cells with the cloned polycistronic vector using polyethyleneimine transfection reagent.

[0172] iii) Culture the infected cells in HEK293 media and change media at 30 hours post infection.

[0173] iv) Collect the virus-containing supernatants at 48 and 72 hours post infection.

[0174] 15 v) Pool the supernatants and pass through a 0.45 µm filter.

[0175] 3. Infect the cultured bovine fibroblasts with retrovirus in medium supplemented with 6 g / ml Polybrene on Day 0 and Day 2.

[0176] 4. Seven days after infection, plate cells on irradiated mouse embryonic fibroblast feeders, and change culture medium to reprogramming medium (DMEM / F12 20 containing 15% Knockout Serum Replacement, 1% nonessential amino acids supplement, 1% penicillin–streptomycin, 0.1 mM -mercaptoethanol, and growth factors—20 ng / ml of human basic fibroblast growth factor and 10ng / ml of human leukemia inhibitory factor).

[0177] 5. Maintain cells in 37°C and 5% CO2 for 25 days. Colonies are isolated mechanically 25 from day 21-35 using a 200 μl pipette and transferred to feeder-coated tissue culture dishes and maintained as a biPSC line.

[0178] There are multiple strategies of converting these isolated cells into lactogenic mammary epithelial cells (Figure 5) including but not limited to treatment in any combination of hormones including but not limited to glucocorticoid, insulin, transferrin, apo transferrin, progesterone, 30 prolactin, ethanolamine, estrogen, oxytocin, fibroblast growth factor (FGF), FGF receptor, T-box

[0179] 20 3 (TBX3), NRG3 / ERBB4, Wnt / LEF1, insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), nuclear factor KB (NF-KB), pTHrP, non- phospho β-catenin, p-p65, RELA, CTNNB1, SMAD3, relaxin, HCG, thyroxin or growth hormone. The glucocorticoid may include but not be limited to one or a combination of

[0180] 5 beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone. The cells may be grown over any period of time under variable environmental conditions of temperature, humidity, oxygen concentration, CO2 concentration, pH, basal surface tension (e.g., growing on a stretched silicone surface and then releasing the stretch, etc.

[0181] 10 Modification of BMEC or progenitor / stem cells

[0182] In certain embodiments, once lactogenic cells are obtained (block 101), the cells may be used to generate a cell line (block 103).

[0183] In certain embodiments, the obtained cells may be immortalized by any one or a combination of the following methods including but not limited to:

[0184] 15 1) Spontaneous immortalization with mutations

[0185] 2) Transfection of primary BMECs with the simianvirus 40 Large T (SV40LT) gene

[0186] 3) Transfection of primary BMECS with telomerase reverse transcriptase (TERT) gene

[0187] 4) shRNA against p16lnk4a

[0188] 5) Overexpression of c-Myc4

[0189] 20

[0190] Reference is made to Figs.6A-6C. In certain embodiments, the obtained cell line may be genetically engineered using any of the following, including but not limited to transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs) with Cas nucleases such as C2C1, 25 C2C2, C2C3, Cas1, Cas 1B, Cas2, Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas9d, Cas10, Cas10d, Cas11, Cas12, Cas13, Cas14, CasF, CasG, CasH, CasX, CasY, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csxl6, CsaX, Csx3, Csx1, Csxl5, Csf1, Csf2, Csf3, Csf4, to delete

[0191] 21 (Fig.6A), increase (with VP16, VP64, p65, etc.) or decrease expression of (with KRAB, EnR,SID, etc.) (Fig.6B) or alter (Fig.6C) a single or combination of gene(s) of choice.

[0192] Transgene expression, or shRNA / siRNA use may also be used.

[0193] In certain embodiment, the response to the obtained cell line to a hormone or a signaling factor 5 may be modified by modulating the activity of genes, including but not limited to those encoding USF, GATA-1, GATA-2, GATA-3 c-Myc:Max, ATF, USF, CREB, TATA, SREBP-1, Amt, USF, Tal- 1beta, NRF-2, FOXD3, HNF-3beta, v-Myb, FOXJ2, FOXO1, Evi-1, F0X04, ARP-1, Staf, NF-kappaB, myogenin, AML- la, Elk-1 Oct-1, Tax / CREB, HNF-1, AP-1, Ahr, Bachl, RP58, AREB6, NKX3A, XFD- 1, deltaEF1, poly A downstream element, Pax-4, Sox-5, Sox-9, 10 Zic3, E47, LPL, AGPAT6, CD36, SCD, GPAM, BTN1A1, ACACA, ACSL1, LPIN1, FASN, FABP3, AGPAT6, SREBP1, INSR, PRLR, EGFR.

[0194] Propagation / maintenance of mammary epithelial cells:

[0195] With reference to block 105 shown in Figure 4, in certain embodiments, the obtained cell line may be maintained or propagated in culture prior to induction of lactation.

[0196] 15 Milk production from mammary epithelial cells

[0197] In certain embodiments, lactogenic cells obtained in block 101 and / or a cell line obtained in block 103 may be induced to lactation in a lab-scale culturing system. A lab-scale culturing system may be useful in testing culturing environments, treatments, and cell-lines for production efficiency of a desired milk product prior to scaling up to large-volume bioreactors. An example 20 lab-scale culturing system is shown in Fig.7, and an exemplary method for culturing a cell line for production of milk product using the culturing system shown in Fig.7 is provided herein below:

[0198] Culture BMECs in DMEM / F12 media containing cell culture elements such as HEPES, BSA, EGFF, glutamine, thiamine, PE, as suitable. Change media in both chambers every 25 1-3 days.2. Milk will be produced in the upper chamber.

[0199] Fig.7 schematically shows BMECs growing on a cell-culture insert coated with collagen containing milk-inducing medium in the bottom chamber (outside the cell-culture insert) and

[0200] 22 milk being secreted into the upper chamber (inside the cell-culture insert). The cell culture insert is made of a porous material which allows free flow of materials below a certain size, which is determined by the size of the average pore (e.g., 0.4 μm in the case of BMECs).

[0201] In certain embodiments, culture media used in the system shown in Fig.7 may include

[0202] 5 components as follows:

[0203] DMEM / F12 media:

[0204] ComponentsMolecularConcentration

[0205] Weight (mg / L)mMAmino Acids

[0206] Glycine 75.0 18.75 0.25

[0207] L-Alanine 89.0 4.45 0.049999997 L-Arginine hydrochloride 211.0 147.5 0.69905216 L-Asparagine-H2O 150.0 7.5 0.05

[0208] L-Aspartic acid 133.0 6.65 0.05

[0209] L-Cysteine hydrochloride- H2O 176.0 17.56 0.09977272 L-Cystine 2HCl 313.0 31.29 0.09996805 L-Glutamic Acid 147.0 7.35 0.05

[0210] L-Glutamine 146.0 365.0 2.5

[0211] L-Histidine hydrochloride- H2O 210.0 31.48 0.14990476 L-Isoleucine 131.0 54.47 0.41580153 L-Leucine 131.0 59.05 0.45076334 L-Lysine hydrochloride 183.0 91.25 0.4986339 L-Methionine 149.0 17.24 0.11570469 L-Phenylalanine 165.0 35.48 0.2150303 L-Proline 115.0 17.25 0.15

[0212] 23ComponentsMolecularConcentration

[0213] Weight (mg / L)mML-Serine 105.0 26.25 0.25

[0214] L-Threonine 119.0 53.45 0.44915968 L-Tryptophan 204.0 9.02 0.04421569 L-Tyrosine disodium salt dihydrate 261.0 55.79 0.21375479 L-Valine 117.0 52.85 0.4517094 Vitamins

[0215] Biotin 244.0 0.0035 1.4344263E-5 Choline chloride 140.0 8.98 0.06414285 D-Calcium pantothenate 477.0 2.24 0.0046960167 Folic Acid 441.0 2.65 0.0060090707 Niacinamide 122.0 2.02 0.016557377 Pyridoxine hydrochloride 206.0 2.0 0.009708738 Riboflavin 376.0 0.219 5.824468E-4 Thiamine hydrochloride 337.0 2.17 0.0064391694 Vitamin B12 1355.0 0.68 5.0184503E-4 i-Inositol 180.0 12.6 0.07 Inorganic Salts

[0216] Calcium Chloride (CaCl2) (anhyd.) 111.0 116.6 1.0504504 Cupric sulfate (CuSO4-5H2O) 250.0 0.0013 5.2E-6 Ferric Nitrate (Fe(NO3)3”9H2O) 404.0 0.05 1.2376238E-4 Ferric sulfate (FeSO4-7H2O) 278.0 0.417 0.0015 Magnesium Chloride (anhydrous) 95.0 28.64 0.30147368 Magnesium Sulfate (MgSO4) 120.0 48.84 0.407 (anhyd.)

[0217] 24ComponentsMolecularConcentration

[0218] Weight (mg / L)mMPotassium Chloride (KCl) 75.0 311.8 4.1573334 Sodium Bicarbonate (NaHCO3) 84.0 1200.0 14.285714 Sodium Chloride (NaCl) 58.0 6995.5 120.61207 Sodium Phosphate dibasic 142.0 71.02 0.50014085 (Na2HPO4) anhydrous

[0219] Sodium Phosphate monobasic 138.0 62.5 0.45289856 (NaH2PO4-H2O)

[0220] Zinc sulfate (ZnSO4-7H2O) 288.0 0.432 0.0015 Other Components

[0221] D-Glucose (Dextrose) 180.0 3151.0 17.505556 HEPES 238.0 3574.5 15.018908 Hypoxanthine Na 159.0 2.39 0.015031448 Linoleic Acid 280.0 0.042 1.4999999E-4 Lipoic Acid 206.0 0.105 5.097087E-4 Putrescine 2HCl 161.0 0.081 5.031056E-4 Sodium Pyruvate 110.0 55.0 0.5 Thymidine 242.0 0.365 0.0015082645

[0222] Seeding the Bioreactor

[0223] Reference is made to block 107 of the flowchart shown in Figure 4. In certain embodiments, following generation of a cell line from biopsied and / or differentiated lactogenic cells, the cell 5 line may be expanded to prepare for seeding in a bioreactor. In an exemplary procedure, the cells may be cultured in collagen-coated flasks (Fig.8A).

[0224] Reference is made to block 109 of the flowchart shown in Figure 4. In certain embodiments, the proliferated cell line produced in block 105 may be used to see a milk production bioreactor. In

[0225] 25 an exemplary procedure, once approximately 80% confluence is reached in the collagen-coated flasks, the cells may be detached from the flask and transfer to bioreactor (Fig.8B).

[0226] In certain embodiments, to prepare the bioreactor for cell culture, the growth surface comprised in the bioreactor on which the cells are grown may be prepared as follows: the surface is initially 5 immersed in PBS for 24 hours, then 24 hours in PBS containing an appropriate mixture of one or a combination of extra-cellular matrix (ECM) proteins, including but not limited to collagen, gelatin, fibrin, alginate, agar, cassava, maize, chitosan, gellan gum, com-starch, chitin, cellulose, chia, recombinant silk, decellularized tissue (plant or animal), hyaluronic acid, fibronectin, laminin, hemicellulose, glucomannan, textured vegetable protein, heparan sulfate, chondroitin 10 sulfate, tempeh, keratan sulfate, or synthetic materials including but not limited to hydroxyapatite, polyethylene terephthalate, acrylates, polyethylene glycol, polyglycolic acid, polycaprolactone, polylactic acid, and respective copolymers. Subsequently, the ECM protein- containing PBS may be flushed out with any one or a combination of cell growth media, including but not limited to DMEM / F12, DMEM, F12, RPMI, MCDB 170 (see below), WIT-P 15 or M87A media (see below).

[0227] Composition of MCDB 170

[0228] 26

[0229]

[0230] p p g e- ionized water)

[0231] Glycine 13.1285

[0232] L-Alanine 6.6795

[0233] L-Arginine hydrochloride 105.355

[0234] L-Asparagine-H2O 78.8

[0235] L-Aspartic acid 9.98

[0236] L-Cysteine hydrochloride-H2O 14.925

[0237] L-Cystine 2HCl 15.645

[0238] L-Glutamic Acid 11.03

[0239] L-Glutamine 328.6

[0240] L-Histidine hydrochloride-H2O 26.225

[0241] L-Isoleucine 33.795

[0242] L-Leucine 49.205

[0243] L-Lysine hydrochloride 63.895

[0244] L-Methionine 10.858

[0245] L-Phenylalanine 20.218

[0246] L-Proline 11.5025

[0247] L-Serine 28.89

[0248] L-Threonine 44.59

[0249] L-Tryptophan 7.573

[0250] L-Tyrosine disodium salt dihydrate 32.425

[0251] L-Valine 44.005

[0252] Biotin 0.0054145

[0253] Choline chloride 11.47

[0254] 27 Composition of M87A media: Component Concentration (in mg / L of distilled de- ionized water)

[0255] D-Calcium pantothenate 1.23915

[0256] Folic Acid 1.328008

[0257] Niacinamide 4.0625

[0258] Pyridoxine hydrochloride 1.03084

[0259] Riboflavin 0.16595

[0260] Thiamine hydrochloride 1.25365

[0261] Vitamin B12 0.40775

[0262] Lipoic Acid 0.0535315

[0263] myo-Inositol 15.31

[0264] Calcium Chloride (CaCl2) (anhyd.) 205.3

[0265] Cupric sulfate (CuSO4—5H2O) 0.00077485

[0266] Ferric Nitrate (Fe(NO3)3ʺ9H2O) 0.025

[0267] Ferrous sulfate (FeSO4—7H2O) 0.9035

[0268] Magnesium Chloride (anhydrous) 14.32

[0269] Magnesium Sulfate (MgSO4) (anhyd.) 222.72

[0270] Potassium Chloride (KCl) 249.1

[0271] Sodium Bicarbonate (NaHCO3) 600

[0272] Sodium Chloride (NaCl) 7004.75

[0273] Sodium Phosphate dibasic (Na2HPO4) 35.51

[0274] anhydrous

[0275] Sodium Phosphate monobasic 31.25

[0276] (NaH2PO4—H2O) 0.2879

[0277] Zinc sulfate (ZnSO4—7H2O)

[0278] H2SeO30.0019345

[0279] MnSO45H2O 0.00006025

[0280] Na2SiO39H2O 0.07105

[0281] (NH4)6Mo7O244H2O 0.000618

[0282] NH4VO30.0002925

[0283] NiCl26H2O 5.945E−07

[0284] SnCl22H2O 0.000000564

[0285] KH2PO434.025

[0286] D-Glucose (Dextrose) 2296.15

[0287] Hypoxanthine Na 1.26255

[0288] Linoleic Acid 0.021

[0289] Putrescine 2HCl 0.04058055

[0290] Sodium Pyruvate 82.5

[0291] Thymidine 0.21883

[0292] HEPES 5361.75

[0293] Using a bioreactor with the above-noted cells, any kind of milk, milk product or milk components including but not limited to lipids (mono-, di- and tri-glycerides, phospholipids, free

[0294] 28 fatty acids, cholesterol, cholesteryl esters, etc.), proteins (αS1-, αS2-, β- and κ- Casein, α- lactalbumin, β-lactoglobulin, lactotransferrin, etc.), carbohydrates (lactose, glucose, galactose), vitamins (vitamin A, B1, B2, B5, B6, B12, C, D, E and K), minerals (Calcium, Magnesium, Selenium, Sodium, Potassium, Phosphorus, Iron, Copper, Zinc, Manganese, Iodine, etc.), 5 immunogenic and antimicrobial components (see combinatorial milk product formulations below), may be produced.

[0295] Moreover, the milk or milk-like composition provided by the cultured cells may be used to create any dairy, dairy-related or other edible / inedible product including but not limited to butter, cheese, ice cream, whipped cream, oil, ghee, yogurt, buttermilk, whey, fermented milk, milk 10 powder, caseinate, milk concentrate, etc.

[0296] Combinatorial Milk Products

[0297] In some embodiments, the present disclosure provides a combinatorial milk products (also interchangeably referred to herein as a combination milk product) comprising milk, milk products (e.g. cream, cheese, yoghurt and butter), or milk components derived from separate 15 mammalian sources, e.g. human and cow. The combination milk products of the disclosure may comprise any combination of biomolecules from two or more mammalian species along with any natural or artificial components for creating a desirable / viable / stable product. In some variations, the combination milk products of the disclosure may further comprise microbial cells corresponding to the generally found flora / fauna in milk, food, animal gut or body.

[0298] 20 Reference is made to Figs.9A-9F. In certain embodiments, the combinatorial milk product may combine human milk or milk product and non-human milk or milk product in any proportion ranging from >0% milk or milk product of human origin combined with <100% milk or milk product of non-human origin to <100% milk or milk product of human origin combined with >0% milk or milk product of non-human origin. The human origin milk or milk product may be 25 produced by a human or by a cultured cell line of lactogenic cells derived from a human source as described herein. The non-human milk or milk product may be produced by a non-human mammal or by a cultured cell line of lactogenic cells derived from the non-human mammal. The non-human mammal may be a bovid. The bovid may be one of a bison, water buffalo, a sheep, a goat, or a domestic cow.

[0299] 29 Figs.9A-9E illustrate various combinatorial milk products of the disclosure. In some variations, human lactogenic cells and non-human lactogenic cells may be cultured separately (Fig.9A), cultured together (Fig.9B) or cultured sequentially (Fig.9C). In some variations, non-human milk or milk product may be mixed with human milk or milk product produced by cultured 5 human lactogenic cells (Fig.9D). In some variations, human milk or milk product derived from humans are mixed with non-human milk or milk product produced by cultured non-human lactogenic cells (Fig.9E). In some variations, non-human milk or milk product derived from cows are mixed with human milk or milk product derived from humans (Fig.9F).

[0300] Certain formulations of the combinatorial milk product of the disclosure may use bovine milk as 10 a base and include the useful components of human milk that make it nutritious for a human newborn. Bovine milk (as opposed to human milk) is a better base for further addition of nutrients to make such a product because: (i) its taste is popular, (ii) it contains more protein, which is one of the main reasons for its consumption, and (iii) it is convertible to cheese, ice cream, butter, etc., which are one of milk’s greatest utility. Optional biochemical components of 15 human milk for addition to bovine milk in order to produce the combinatorial milk product of the disclosure are listed herein below:

[0301] 1. PUFAs – omega-3 (e.g., docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA)) and omega-6 (e.g., arachidonic acid(AA)) have been shown to be efficacious outside infancy also including in diseases such as heart disease, diabetes, alzheimers, etc.

[0302] 20 2. Glycerol monolaurate – GML possesses general antibacterial activity

[0303] 3. Proteins including but not limited to: Lysozyme, Lactoferrin, SIgA, A1BG, ACE, ACTB, ACTG1, ACTN1, ACTN4, ADAM11, AFM, ALB2, AGT, AHSG, ALB, AMBP, AMY1, ANG, ANPEP, APOA1, APOA4, APOC1, APOD1, APOE, APOH, ASM3B, ATRN, AZGP1, B2M, B4GALT1, BDNF, BLC / CXCL13, BRMS1, BTD, BTN1A1, BTN2A1, 25 C1RL, C2, C3, C4A, C4BPA, C5, C6, C7, C8A, C9, CA6, CALCA, CALR, CAMP, CCL1,

[0304] CCL11, CCL13 / MCP4, CCL15 / MIP1d, CCL17 / TARC, CCL18 / PARC, CCL2 / MCP1, CCL20 / MIP3, CCL22 / MDC, CCL24, CCL26, CCL4 / MIP1b, CCL5, CCL7 / MCP3, CCL8 / MCP2, CD14, CD36, CD59, CD9, CEACAM1, CEL / BAL, CF1, CFB, CHI3L1, CHI3L2, CHRDL2, CLEC11A, CLU, CP, CRABP2, CRISP3, CSF1, CSF3, CSN1S1,

[0305] 30 CSN2, CSN3, CST3, CTBS, CTSC, CTSD, CTSS, CX3CL1, CXCL12 / SDF, CXCL2, CXCL5, CXCL9, DAG1, DDR1, DEFA1, DEFA3, DEFA5, DEFA6, DEFB4, EFEMP1, EGF, ENO1, EPO, ERAP1, EZR, F2, FABP3, FGB, FGF4, FGF6, FGF7, FGF9, FGG, FOLR1, FSTL1, GC, GDNF, GGT1, GHI, GHRH, GM-CSF, GNB1, GNB2, GOLM1, 5 GPR158, GPRC5B, GPX4, GRO / CXCL1, HAPLN3, HEBP1, HGF, HLA-DRB5, HNP-1, HP, HPX, HSP90B1, HSPA5, HSPA8, HSPG2, ICAM1, IFNG, IGF1, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFPB4, IGHA1, IGHA2, IGHG1, IGHM, IGJ, IGKC, IGLC, IL10, IL12, IL13, IL15, IL16, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8 / CXCL8, INS, IP10 / CXCL10, KLK11, KLK12, KLK6, KNG1, KRT5, KRT7, LALBA, LCN2, LCP1, LEP, 10 LGALS3BP, LHRH, LIF, LPO, LRG1, LTA, LTF, LYPD3, LYZ, MFGE8, MIF, MIPF, MPO, MRC1, MUC1, MUC4, MUC5B, N-APP, NRP-1, NUCB1, NUCB2, OLFM4, OPG, ORM1, ORM2, OSM, OXT, P4HB, PCDHA10, PCGF6, PDGFB, PDIA3, PDIA6, PDZD4, PIGF, PIGR, PIP, PLA2G7, PLG, PLIN2, PLIN3, PLTP, PODXL, PPIA, PPP2R3A, PRL, PROM1, PRSS8, PRTN3, PSAP, PTHR, QSOX1, RHOA, RNASET2, S100A11, S100A8, 15 S100A9, SAR1B, SCF, SCGB1D2, SCGB3A1, SDCBP, SELENBP1, SERPINA1,

[0306] SERPINA3, SERPINA6, SERPINB1, SERPINC1, SERPING1, SIAE, SIL1, SORT1 / NT3, SPARCL1, SPINT1, SPP1, SST, STC2, STOM, TCN1, TF, TGFA, TGFB1, TGFB2, TGFB3, TGFBR3, THBS1, THPO, TIMP1, TIMP2, TNC, TNF, TPI1, TSH, TTR, UBB, VCAM1, VEGF, VTN, VWA1, WNT2B, XDH, YWHAZ, ZG16B

[0307] 20 4. Human or other milk-specific carbohydrates and sugars including but not limited to D- Glucose, D-Galactose, N-Acetylglucosamine, L-Fucose, N-acetylneuraminic acid, Lactose, Lacto-N-biose, N-acetyllactosamine, Galactosyllactose, Lacto-N-tetraose, Lacto-N- neotetraose, lacto-N-fucopentaose (I, II, III), Lacto-N-hexaose, Lacto-N- neohexaose, 2’Fucosyllactose, fucosyllacto-N-hexaose, 3-Fucosyllactose, Difucosyllactose,

[0308] 25 difucosyllacto-N-tetraose, difucosyllacto-N-hexaose, Lacto-N- fucopentaose, 3’Sialyllactose,

[0309] 6’Sialyllactose, Sialyllacto-N-tetraose (b and c), Disalyllacto-N-tetraose, disialyllacto-N- hexaose, etc.

[0310] 5. Beneficial micro-organisms, pro-biotics.

[0311] 6. Immunological factors, hormones and other entities – 11S sIgA, 7S IgA, IgG, IgM, IgE, IgD,

[0312] 31 secretory component, complement proteins, properdin (factor P), interferon, α- fetoprotein, Bifidus factor, Antisaphylococcal factor(s), Antiadherence substances, epidermal growth factor, folate uptake enhancer, antiviral factor(s), migration inhibition factor, gangliosides, nucleotides, antisecretory factor, spermine, soluble CD14, lactoferrin, transferrin, Vitamin 5 B12-binding protein, corticoid-binding protein, lysozyme, lipoprotein lipase, leukocyte enzymes, T-lymphocytes, B-lymphocytes, neutrophils, macrophages, epithelial cells, stem cells, epidermal growth factor, prostaglandins, relaxin, neurotensin, somatostatin, bombesin, gonadotropins, ovarian steroids, thyroid-releasing hormone, thyroid-stimulating hormone, thyroxine, triiodothyronine, adrenocorticotropin, corticosteroids, prolactin, erythropoietin, 10 insulin, cytokines, interleukins, globotriaosylceramide, GM3, chondroitin sulfate, sulfatide, mucin, glycosaminoglycans, lactadherin, MUC1, IL-1β, IL-1RA, IL-2, sIL-2R IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-15, IFN-α, TNF-α, G- CSF, M-CSF, Interferon-γ, EGF, TGF-α, TGF-β1, TGF-β2, SCF, TNF-α, MIP, RANTES, GRO-α, MCP-1, LIF, nucleotides, nucleosides and nucleic acids, lactoferrin, lactoperoxidase, α2-macroglobulin 15 protein, α1-antitrypsin, bile salt-stimulated lipase, catalase, alpha-tocopherol, cysteine, ascorbic acid, histaminase, arylsulfatase, α1-antichymotrypsin, etc.

[0313] In certain embodiments, milk product produced by cultured lactogenic cells derived from biopsied MECs or from stem cells as noted above may be modified through addition of an amount of a milk protein including but not limited to αS1-, αS2-, β-, κ-casein, α-lactalbumin, β-20 lactoglobulin, lactoferrin, lactoperoxidase, lysozyme, haptocorrin, osteopontin, complements C3 and C4, etc. Alternatively or in combination, the expression of one or more of the above-noted proteins may be achieved through modulating expression of the corresponding gene in the cultured lactogenic cell.

[0314] In certain embodiments, milk product produced by cultured lactogenic cells derived from 25 biopsied MECs or from stem cells as noted above may be modified through addition of an amount of one or a combination of milk carbohydrates, including but not limited to 2’- fucosyllactose, 3-fucosyllactose, difucosyllactose, difucosyllacto-N-tetrose (DFLNT), difucosyllacto-N-hexaose, fucosyllacto-N-hexaose (FLNH), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, 3’-sialyllactose, 6’- sialyllactose, disialyllacto-N-hexaose (DSLNH), 30 disialyllacto-N-tetraose (DSLNT), fucodisialyllacto-N-hexaose (FDSLNH), sialyl-lacto-N- 32 tetraose b (LSTb), and sialyl-lacto-N- tetraose c (LSTc), lacto-N-hexaose, lacto-N-neotetraose (LNnT), lacto-N-tetrose (LNT), and lactose. Alternatively or in addition, modification of milk carbohydrates in the milk product may be achieved through modulating expression of a corresponding gene in the cultured lactogenic cell, including but not limited to a gene encoding 5 LALBA, galactosyltransferase B4GALT1, galactosyltransferase B4GALT2, LGALS1, LGALS2, LGALS3, LGALS8, LGALS9, LGALS12, LGALS13, LGALS14, LGALS16, LCT, GLUT1, FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, sialyltransferase, GALM, GLTP, LGALSL, N-acetylglucosaminyltransferase, or glycosyltransferase.

[0315] In certain embodiments, Milk product produced by cultured lactogenic cell derived from

[0316] 10 biopsied MECs or from stem cells as noted above may be modified through addition of an amount of one or a combination of fats / lipids, including but not limited to palmitic acid, myristic acid, stearic acid, butyric acid, caproic acid, oleic acid, linoleic acid, α-linoleic acid, vaccenic acid, eicosapentaenoic acid, docosahexaenoic acid, calendic acid, γ-linoleic acid, eicosadienoic acid, dihomo-γ- linoleic acid, arachidonic acid, docosadienoic acid, adrenic acid, asbond acid, 15 tetracosatetraenoic acid, tetracospentaenoic acid, hexadecatrienoic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, rumenic acid, caprylic acid, capric acid. Alternatively or in addition, modification of milk lipid in the milk product may be achieved through modulating expression of a corresponding gene in the cultured lactogenic 20 cell, including but not limited to those encoding CIDEA, XDH, C / ΕΒΡβ, HDAC, DGAT1, DGAT2, acyl- CoA:cholesterol acyltransferase, AKT1, S14, LPL, BTN / BTN1A1, ADFP, ACACA / B, FASN, ACLY, S- acyl fatty acid synthase thioesterase, medium chain / OLAH, ACOT, SCD, ELOVL, FADS, ACSS, ACSL, GDP1L, GK5, DGKA, GPAM, AGPAT1, LPIN1, LPIN2, SLC27A, FABP, ABCG2, FDFT1, SC4MOL, SC5DL, IDI1, MVD, CH25H, DHCR7, 25 PLIN2, SREBP1, MUC1, or MFGE8.

[0317] Additional Exemplary Aspects

[0318] Below are additional exemplary aspects of the disclosure as contemplated herein:

[0319] 33 1. Fusing gametes (egg and sperm) to create a starting cell line including

[0320] but not limited to embryonic stem cells for eventual lactation.

[0321] 2. Extraction of stem cells / epithelial cells / mammary epithelial cells from mouth / teeth of an organism or milk or any other body part or from a secretion

[0322] 5 or excretion.

[0323] 3. Differentiation of stem cells into lactating mammary epithelial cells for improved resource utilization and maximizing efficiency.

[0324] 4. Cultivation of derived population of differentiated cells on a bench-scale cell insert setup for in vitro lactation.

[0325] 10 5. Induction of in vitro lactation using a combination of hormones and other molecules.

[0326] 6. Transfer to scale-up bioreactor for optimized lactation to create our

[0327] cultured milk product, which may be referred to as “UnReal Milk”.

[0328] 7. A combinatorial milk product - A multi-species milk / milk-component

[0329] 15 mixture, by way of example a mixture of human and bovine milk and or

[0330] milk components. Advantageously, the combinatorial milk product can offer health and other benefits when compared to milk or milk products produced from a single species.

[0331] 8. Conversion of milk or milk-like product obtained from cultured cells into 20 any dairy or dairy-product or any other product.

[0332] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-25 known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0333] The foregoing descriptive matter is illustrative of the disclosure and not a limitation. While the present disclosure is susceptible to various modifications and alternative forms, specific aspects 30 thereof have been shown by way of examples and drawings and are described in detail below.

[0334] However, it should be understood that it is not intended to limit the invention to the particular

[0335] 34 forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the embodiments of the disclosure.

[0336] 5 EXAMPLES

[0337] The following examples are provided to illustrate certain embodiments of the invention. These examples are not intended to limit the scope of the claims and should be construed as exemplary only. Unless otherwise specified, all percentages are expressed by weight.

[0338] Example 1: Isolation of Primary BMECs

[0339] 10 Bovine mammary gland was isolated from a cow and minced into small pieces. The minced tissue was incubated with collagenase. The collagenase-treated tissue was centrifuged and the cell pellet was resuspended in DMEM (Sigma-Aldrich) with TrypLE and left at room temperature such that mammary epithelial fragments are free of contaminating cells, like fibroblasts and myoepithelial cells.

[0340] 15 Example 2: Growth and Induction of Lactation in Primary BMECs

[0341] The cell pellet obtained Example 1 was resuspended in growth media containing a combination of DMEM and recombinant growth factors, like epidermal growth factor, etc. phosphatidylethanolamine and penstrep. The cells were seeded in the upper chamber of a cell culture insert and the growth media was added to both chambers. The media was changed every 20 1-3 days until cells reached confluence. After confluence, the media in the lower chamber was changed to an induction media, which contained a combination of growth media and additional growth factors and fatty acids, like prolactin, oleic acid, palmitic acid, etc. The media in the upper chamber can be changed to DMEM. The media in both the chambers was changed every day and the media from the upper chamber was collected for analysis.

[0342] 25

[0343] Example 3: Analysis of Secreted Proteins and Sample Processing

[0344] Media collected from the upper chamber was filtered using centrifugal filters, one or more times at 500rcf or higher. This step can also be repeated with multiple filters or same filters multiple times. The retentate was subjected to a freezing process by immersion in a slurry of dry ice and

[0345] 35 isopropyl alcohol. The frozen sample was placed into a freeze dryer. After lyophilization, the sample was resuspended in water to a consistency close to milk.

[0346] Milk proteins in the sample can be measured using western blotting or mass spectrometry and have 5 been verified using one or more of the same. For western blotting, this sample was denatured and reduced and run on SDS-polyacryamide gels. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane utilizing an iBlot 2 machine. The membrane was blocked using PBST containing 2% BSA for 1.5 hours at room temperature. Following blocking, the membrane was incubated with appropriate dilutions of primary antibody overnight at 4°C. After washing, the 10 membrane was incubated with suitable dilutions of secondary antibody for 1 hour at room temperature and washed again with PBST. Imaging of the membrane was performed using the Odyssey Imaging System. Induction media was used as a negative control, while whole milk was used as a positive control for comparison.

[0347] 15 Carbohydrates can be measured using lactose assay, HPLC or biosensor tubes and have been verified using one or more of the same. Fats can be quantified using triglyceride enzymatic assays or mass spectrometry and have been verified using one or more of the same.

[0348] 36

Claims

We claim:

1. A method of producing a mammalian dairy product or a dairy-like component or a mixture thereof, comprising:a) growing cells in a structured apparatus, which are obtained and optimized for 5 specific functionality;b) inducing growing cells to obtain secretion of the dairy product or dairy like component or a mixture, into a designated compartment of the structured apparatus; andc) extracting the dairy product or dairy like component or the mixture using an 10 extraction process, from the designated compartment of the structured apparatus.

2. A device comprising the structured apparatus for any biotechnology, food, medical, or pharmaceutical application.

3. A process of growing cells involving application of external voltage gradient across the cells for any biotechnology, food, medical or pharmaceutical application.15 4. A method comprising the extraction process for separation of any mixture or extraction application.

5. The method of claim 1 or 4, wherein the extraction process comprises:a) collecting all outputs or secretions or media from the designated space;b) loading the collected outputs or secretions or media on an extraction setup 20 comprising of components like centrifugal filter and spinning the loaded centrifugal filter one or more times at 500rcf or higher;c) repeating step b) with extraction setup comprising of components like multiple membranes or filters or same filters multiple timesd) collecting a retentate and subjecting it to a freezing mechanism or flash freezing or 25 lyophilizing or several of those in succession to obtain a refined output; and e) mixing the refined output with water as needed to obtain the desired dairy product.

6. The method of claim 5, wherein the extraction setups comprises a dialysis tube or chromatography or beads or electrophoresis or similar extraction mechanisms or a combination 30 thereof.

377. The method of claim any one of claims 1 and 3-6, wherein the growing cells are mammary epithelial cells isolated from a biopsy.

8. The method of any one of claims 1 and 3-6, wherein the growing cells are generated by: a) isolating mammary epithelial stem cells (MESCs) isolated from milk; and5 b) differentiating the MESCs into the lactogenic cells.

9. The method of any one of claims 1 and 3-6, wherein the growing cells are generated by: a) isolating stem cells from a pre-implantation embryo; andb) differentiating the stem cells into the lactogenic cells.

10. The method of any one of claims 1 and 3-9, wherein the step of growing comprises growing 10 cells to confluence in a cell culture device in the presence of a culture medium.

11. The method of any one of claims 1 and 3-10, wherein the step of inducing production of the dairy product or the dairy like component or the mixture thereof comprises contacting the cells with an induction medium.

12. The method of claim 11, wherein the cells are contacted with the induction medium for at least 15 24 hours.

13. The method of any one of claims 1 and 3-12, wherein the cell culture device or the structured apparatus comprises an upper chamber and a lower chamber; the cells are grown in the upper chamber; and the culture medium or specific solution is added to the upper chamber and the lower chamber.20 14. The method of any one of claims 1 and 3-12, wherein the cell culture device or the structured apparatus comprises an upper chamber and a lower chamber; the cells are grown in the upper chamber; and the upper chamber is empty.

15. The method of claim 13 or 14, wherein, in the step of inducing milk production, the induction medium is added to the lower chamber.25 16. The method of any one of claims 1 and 3-15, wherein the dairy product or dairy-like component or the mixture thereof is separated from the culture medium by a method selected from filtration, dialysis, lyophilization, vibratory membrane filtration or a combination thereof.3817. The method of any one of claims 1 and 3-16, wherein the dairy product or dairy-like component or the mixture thereof is separated from the culture medium by filtration followed by lyophilization.

18. A milk composition comprising: a) milk or milk components obtained from cell culture and b) 5 milk or milk components obtained from a natural source.

19. The milk composition of claim 18, wherein the natural source is a human or a non-human mammal.

20. The milk composition of claim 18 or 19, wherein the non-human mammal is a cow, a buffalo, a doe, a sheep, a goat, or a camel.10 21. A combination milk product comprising a human origin milk, milk component, or milk product and a non-human origin milk, milk component, or milk product.

22. The combination milk product of claim 21, wherein:a) the human origin milk or milk product is produced by a cultured cell of lactogenic cells derived from a human source; and15 b) the non-human origin milk, milk component, or milk product is produced by a cultured cell of lactogenic cells derived from a non-human source.

23. The combination milk product of claim 21, wherein:a) the human origin milk, milk component, or milk product is produced by a cultured cell of lactogenic cells derived from a human source; and20 b) the non-human milk, milk component, or milk product is a milk produced by a non- human mammal.

24. The combination milk product of claim 21, wherein:a) the human milk, milk component, or milk product is a milk produced by a human;and25 b) the non-human origin milk, milk component, or milk product is produced by a cultured cell of lactogenic cells derived from a non-human source.

25. The combination milk product of claim 21, wherein:a) the human milk, milk component, or milk product is a milk produced by a human;and39b) the non-human milk, milk component, or milk product is a milk produced by a non- human mammal.40

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