In vitro insect fat culture for cell agricultural applications

Abstract

Described herein are methods of making edible fat products using insect cells. The method comprises isolating a population of insect cells, which may be precursor cells; inoculating the population of insect cells onto a scaffold; and contacting the population of insect cells with a free fatty acid composition or a lipid composition. The cells inoculated on the scaffold accumulate lipids, thereby forming an edible adipose tissue. Further disclosed are compositions of matter prepared by the method of preparing edible adipose cells, food products comprising the compositions of matter, and in vitro edible adipose tissues comprising a population of insect precursor cells inoculated on the scaffold.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 366,532, filed on June 16, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the production of edible cultured fats. In particular, the production and / or accumulation of edible lipids in insect cells. Background Art

[0004] Cultured meat technology has the potential to address some of the public health, environmental degradation, and animal welfare issues associated with factory-farmed meat. However, technical challenges related to cell sources, scale-up, and high production costs have hampered commercialization. For example, cultured mammalian cells need to grow at physiological temperatures, accumulate toxic byproducts (e.g., lactic acid) at high rates, are often sensitive to fluctuations in temperature and pH, and require supplementation with recombinant proteins and growth factors during serum-free culture. In addition to muscle tissue production in cultured meat, fat is another key tissue type essential in cultured meat production. In traditional meat (e.g., beef, pork, poultry), adipose tissue is a key factor affecting texture, flavor, and nutrient distribution.

[0005] In the field of tissue engineering, lipid accumulation (e.g., adipogenesis) in mammalian adipogenic precursor cells is usually achieved by supplementing the culture medium with a hormone cocktail containing insulin, dexamethasone, and isobutylmethylxanthine. However, for cell agriculture applications, the use of lower-cost food-grade culture medium supplements is preferred.

[0006] In cultured meat production, cells are typically grown on biomaterial scaffolds to increase the surface area for attachment, resulting in structured and ordered tissue. Useful scaffold systems need to be abundantly available, edible, and inexpensive, while supporting the in vitro culture of target cell types. Some plant-based biomaterials meet these criteria and have been proposed as potential scaffolds for cultured meat. For example, plants with a high cellulose content are decellularized to remove the plant genetic material, leaving behind the natural structure of cellulose.

[0007] Decellularized spinach, celery, and grass support the attachment and differentiation of myogenic cells. When compared to aligned muscle tissue, adipose tissue does not require a strict architecture, but it should benefit from a 3D environment to achieve robust differentiation and mimic the sensory properties of natural fat. These efforts illustrate the need for cell-cultured meat tissues (such as adipose) that avoid the shortcomings currently associated with cultured mammalian cells and whose structure is organized in a way that allows cultured adipose tissue to mimic the sensory properties of natural meat fat. Summary of the invention

[0008] The present disclosure provides a method for producing an edible cultured fat product, which can simulate the organoleptic properties of natural meat fat. The cultured fat product may include cells that can produce and / or accumulate lipids, such as insect cells or insect precursor cells. The cultured fat product can be produced in vitro by: an insect cell colony or an insect precursor cell colony is seeded on a scaffold, wherein the scaffold is two-dimensional or three-dimensional, and the insect cell colony or the insect precursor cell colony seeded on the scaffold is contacted with a cell culture medium containing a free fatty acid composition or a lipid composition, thereby accumulating lipids in the insect cell colony or the insect precursor cell colony, and producing edible adipose tissue therefrom. The cultured fat product can be produced in vitro by the following method, the method comprising (1) isolating an insect cell population or an insect precursor cell population; (2) inoculating the insect cell population or the insect precursor cell population onto a three-dimensional or two-dimensional scaffold or into a two-dimensional tissue culture plastic, such as a well of a culture dish or container; (3) optionally freezing and thawing the insect cell population or the insect precursor cell population inoculated on the scaffold; and (4) feeding the insect cell population or the insect precursor cell population inoculated on the scaffold with a free fatty acid composition or a lipid composition, thereby accumulating lipids in the insect cell population or the insect precursor cell population and producing edible adipose tissue therefrom. A material composition prepared by the above method is further disclosed. A food comprising the material composition prepared by the above method is also disclosed. An in vitro edible adipose tissue is also disclosed, the tissue comprising an insect cell population or an insect precursor cell population inoculated on a scaffold. The insect cell population or the insect precursor cell population can be enriched in lipid content by exposure to a free fatty acid composition or a lipid composition.

[0009] The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Methods for the isolation of embryonic cell lines, fabrication of scaffolds, and in vitro lipid accumulation are presented.

[0011] Figure 2 A-2C shows the culture and isolation of M. sexta embryonic precursor cells (Ms PCs). Figure 2 A: Phase contrast image of Ms PC culture 14 days after isolation. Some attached and elongated cells are visible underneath the spherical cell aggregates. Figure 2 B: Cell proliferation (P7) was quantified over 5 days at increasing cell seeding densities by fluorescence-based DNA quantification assay. Error bars are standard deviations, statistical significance is indicated by p-values ​​(n=3, p-value<0.05). Figure 2 C: Phase contrast images of Ms PC before and after freezing (scale bar is 100 μm). Cells were passaged on day 5 after cryopreservation.

[0012] Figure 3 Fluorescence microscopy images of striated muscle cells derived from Manduca sexta embryonic cell cultures P0 (top row) and P2 (bottom row) are shown. Cells were isolated, cultured for 2 months (P0) or 10 days (P2), and immunostained for myosin heavy chain (red) and counterstained for nuclei (blue). Scale bar is 100 μm.

[0013] Figure 4 A-4D shows fatty acid-induced lipid accumulation of Ms PC. Panel A: Fluorescence microscopy images of Ms PC stained with BODIPY (green) and DAPI (blue) after 7 days of culture with growth medium (control) or supplemented with 0.01, 0.1 or 1 mM Intralipid or oleic acid (scale bar is 100 μm). Figure 4 B: Fluorescence microscopy images of Ms PCs stained with BODIPY (green) and DAPI (blue) on day 1, 4, 7, or 14 after culture with growth medium (control) or medium supplemented with 0.1 mM Intralipid (scale bar is 100 μm). Figure 4 C: Bright field images of MsPCs stained with Oil Red O (red) after 7 days of culture with growth medium (control) or supplemented with 0.01, 0.1 or 1 mM intralipid or oleic acid (scale bar is 100 μm). Figure 4 D: Lipid quantification by Oil Red O dye elution of Ms PC cultures at day 7. Error bars are standard deviations, statistical significance is indicated by p-values ​​(n=3, p-value<0.05).

[0014] Figure 5 A-5B shows fatty acid-induced lipid accumulation in Dm PC. Figure 5A: Fluorescence microscopy images of Dm PCs (green) stained with BODIPY (red) and DAPI (blue) after 7 days of culture with oleic acid (scale bar is 100 μm). Figure 5 B: Lipid quantification by Oil Red O dye elution of Dm PC cultures at day 7. Error bars are standard deviations, statistical significance is indicated by p-values ​​(n=3, p-value<0.05).

[0015] Figure 6 A-6C shows the internal and external fat of Manduca sexta. Figure 6 A: Fluorescence microscopy images of frozen sections of in vivo fat body tissue of 5th instar larvae stained with BODIPY (green) or Phalloidin (pink) and DAPI (blue) (scale bar is 100 μm). Figure 6 B: Fluorescence microscopy images of in vivo fat body tissue of cryosectioned 5th instar larvae stained with phalloidin (pink) and DAPI (blue) (scale bar = 100 μm). Figure 6 C: Confocal microscopy images of Ms PC aggregates after culturing in 0.1 mM Intralipid for 14 days and staining with BODIPY (green) and DAPI (blue) (scale bar is 100 μm). Figure 6 D: Quantification of neutral lipids in Ms PC samples cultured with or without 0.1 mM Intralipid for 7 days. Error bars are standard deviations, statistical significance is indicated by p-values ​​(n=3, p-value<0.05).

[0016] Figure 7 In vivo and in vitro lipidomics are shown. The ratios of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PFA) in wandering-stage tobacco worms (M. sexta) compared to Ms PCs that were untreated or treated with 0.1 mM Intralipid for 7 days, and reported tissue fat distribution in livestock animals.

[0017] Figure 8 A-8G shows a mycelium scaffold. Figure 8 A: Mycelial scaffolds were sterilized by 70% ethanol, 10% bleach, autoclaving, or ethylene oxide gas. Scale bar is 1 mm. Figure 8 B: Scanning electron microscopy image of mycelium. Scale bar is 2 μm. Figure 8 C: Dm PC (green) grown in 2D on mycelial fibers dissolved with 4% bleach. Figure 8 D: Dm PC (white arrow) grown in 3D manner on living mycelial fibers (red arrow). Figure 8E: Cryosections (15 μm thick) of untreated and decellularized mycelia stained with DAPI. Scale bar is 100 μm. Inset: Photographs of untreated and decellularized scaffolds. Figure 8 F: Cryosections of decellularized mycelia inoculated with Ms PC and cultured in control medium or medium supplemented with 0.1 mM Intralipid for 7 days, then fixed and stained with BODIPY (green) and DAPI (blue). Scale bar is 100 μm. Figure 8 G: Comparison of the elastic modulus of decellularized scaffolds without cells, with cells, or with cells and treated with 0.1 mM Intralipid (all cultured for two weeks). DETAILED DESCRIPTION

[0018] It should be understood that, as will be appreciated by those skilled in the art, the present invention is not limited to the specific methods, protocols and reagents, etc. described herein, and may therefore vary. The terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present disclosure. The present invention described herein is limited only by the claims.

[0019] As used herein and in the claims, unless the context clearly indicates otherwise, singular forms include plural references, and vice versa. Except in the operating examples or where otherwise indicated, all numbers used herein expressing quantities of ingredients or reaction conditions should be understood as modified by the term "about" in all cases.

[0020] For the purpose of describing and disclosing the methods described in such disclosures, for example, which may be used in conjunction with the present invention, all identified patents and other disclosures are expressly incorporated herein by reference. Only the disclosures disclosed in these disclosures prior to the filing date of the present application are provided. Nothing in this regard should be construed as admitting that the inventor has the right to precede such disclosures by virtue of prior inventions or for any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any known methods, devices and materials can be used for the practice or testing taught herein, the methods, devices and materials of this aspect are described herein.

[0022] Cultured meat technology has the potential to address some of the public health, environmental degradation and animal welfare issues associated with factory-farmed meat 1 However, technical challenges related to cell sources, scalable production, and high production costs have hampered commercialization. 2Insect cell culture has been introduced as a favorable platform for cultured meat production based on insect-specific bioprocess benefits related to increased scalability and reduced costs. 3 Specifically, compared to most vertebrate cells, insect cells grow at near-ambient temperatures, accumulate toxic byproducts (e.g., lactate) at lower rates, are generally more tolerant to fluctuations in temperature and pH, and do not require supplementation with recombinant proteins and growth factors during serum-free culture. 4,5 Insect progenitor cells can be expanded in vitro and induced to differentiate into mature tissues to produce meat analogs 6 The administration of key hormones (e.g., juvenile hormone, ecdysone) and scaffolds (e.g., silicone molds, chitosan membranes, and sponges) has been demonstrated. 7-9 Muscles were grown from Drosophila melanogaster (common fruit fly) and Manduca sexta (tobacco hornworm) embryonic cells using a combination of proteins to regulate proliferation and differentiation. Fat is another key tissue type necessary for cultured meat production. 10 In conventional meats (e.g., beef, pork, poultry), adipose tissue is a key contributor to texture, flavor, and nutrient profile. 10 The analogue of adipose tissue in insects is the fat body organ, which is an important site for lipid storage, metabolism, endocrine secretion, and systemic immunity. 11 The major cell type of adipose tissue is the trophoblast, which stores lipids in the form of triglycerides. 11 Insect fat body cells have been cultured and characterized for decades but have not been relevant for food production applications. 6,12,13 .

[0023] Lipid accumulation (e.g., adipogenesis) in mammalian adipogenic precursor cells is typically achieved by supplementing the culture medium with a hormone cocktail containing insulin, dexamethasone, and isobutylmethylxanthine. 10 For cell agriculture applications, the use of lower-cost food-grade media supplements is preferred. To this end, free fatty acids (from plant oils, e.g., erucic acid, oleic acid) have been reported to induce in vitro lipid accumulation in livestock cells at concentrations of 50-300 μM. 14,15 Interestingly, free fatty acids (e.g., oleic acid at 400-1000 μM) have also been reported to induce lipid accumulation in insect cell cultures (Drosophila melanogaster embryonic cell line) 16,17 For example, treatment of S2 cells with 1 mM oleic acid for 24 h increased triglyceride content by 5-fold 17 .

[0024] In cultured meat production, cells are often grown on biomaterial scaffolds to increase the surface area for attachment, thereby forming a structured and ordered tissue. 18 Useful scaffold systems need to be abundantly available, edible, and inexpensive, while supporting the in vitro culture of target cell types. Many plant-based biomaterials meet these criteria and have been proposed as potential scaffolds for cultured meat. 19 For example, plants with a high cellulose content are decellularized to remove the plant genetic material, leaving behind the natural structure of cellulose. 18 Decellularized spinach, celery, and grass support myogenic cell attachment and differentiation 19-21 In addition, the natural alignment structures of celery and grass can induce myotube alignment 19,21 When compared to aligned muscle tissue, adipose tissue does not require a strict architecture, but it should benefit from a 3D environment to achieve robust differentiation and mimic the sensory properties of native adipose tissue. 10 To achieve this goal, mycelium (e.g., filamentous fungi) is a promising candidate scaffold. Mycelium is abundant, already consumed as a meat analog, and is inexpensive to produce. 22 Mycelium is composed of interconnected hyphal fibers that can encapsulate cells for three-dimensional culture 23 Decellularization may facilitate the removal of fungal genetic material, thereby enhancing analysis of cell-scaffold constructs (e.g., imaging, DNA quantification) 20 .

[0025] The present disclosure provides a kind of edible cell cultured adipose tissue (such as tissue formed by cell collection) and method for preparing adipose tissue.Adipose tissue is generated by insect cells (such as cultured precursor / embryo insect cells) that produce and / or accumulate lipids.These cells can be induced to produce and / or store lipids.In some embodiments, insect cells are seeded on a scaffold to provide structure for fat products.The scaffold can be a natural product containing mycelium.The lipid-rich cells and scaffold can jointly produce a fat product that imitates animal fat in both taste and appearance.

[0026] In some embodiments, the insect cell may include any cell or cell type of the class Insecta. For example, an insect cell line may include any cell or cell type of the order Insecta, Lepidoptera, Hymenoptera, Coleoptera, Orthoptera, Odonata, Hemiptera, and Diptera. For example, an insect cell or cell type may include any species of insects (e.g., moths and butterflies) of the order Lepidoptera, including but not limited to Manduca sexta (e.g., Manduca sexta larvae).

[0027] In certain embodiments, the insect cell can be any cell type capable of accumulating lipids, including but not limited to germ cells, oocysts, adipocytes, precursor cells, and fat body cells. Precursor cells refer to cells that are not fully differentiated. Insect precursor cell populations are easily found in eggs and other non-adult stages of insects.

[0028] In certain embodiments, the cells are grown in any cell culture medium (e.g., growth medium) that can maintain cell growth or longevity. These cell culture media may include, but are not limited to, M3+BPYE growth medium, Shields and Sang M3 medium, Grace insect medium, Schneider insect medium, and SF9-based medium. The cell culture medium may include any supplements, including, but not limited to, serum (e.g., bovine, calf, fetal bovine, goat) and antibiotics. For example, the cell culture medium may include 10% fetal bovine serum.

[0029] In certain embodiments, cells are contacted with lipid compositions. The lipid compositions can include any lipid material or lipid-containing material, including but not limited to oleic acid, erucic acid and intralipid. Intralipid is a sterile, pyrogen-free fat emulsion prepared for intravenous administration as a source of calories and essential fatty acids. In one form, intralipid includes 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerol and water. Lipid can be derived from plant sources, including but not limited to soybean oil, corn oil or other oils produced by vegetables, cereals, seeds or beans. In some embodiments, the lipid composition is an oil emulsion, such as a seed oil emulsion. Lipid can also be derived from animals. In some embodiments, the lipid composition includes a cell culture medium (e.g., a growth medium).

[0030] Cell culture medium can include or be supplemented with lipid or lipid composition between 0.001mM and 100mM, lipid or lipid composition between 0.01mM and 10mM, or lipid and lipid composition between 0.1mM and 2mM.For example, cell culture medium can include or be supplemented with lipid or lipid composition of about 0.1mM or about 1mM.Substratum can include or be supplemented with lipid or lipid composition between 0.000032% (V / V) and 3.2% (V / V), lipid or lipid composition between 0.00032% (V / V) and 0.32% (V / V), or lipid or lipid composition between 0.0032% (V / V) and 0.064% (V / V).For example, cell culture medium can include or be supplemented with lipid or lipid composition of about 0.0032% (v / v) or about 0.032% (V / V).

[0031] In certain embodiments, the lipid profile of the lipid accumulated is controlled by exposing lipid or lipid mixture / composition to cells under different conditions (such as lipid concentration, lipid type, exposure time, cell culture medium selection, culture temperature or other cell growth and cell culture characteristics). For example, specific lipid types can be added to cells based on known palatable (e.g., simulating the organoleptic properties of natural meat fat), healthy or the predicted lipid profile with other desired properties. In another example, the time when lipid composition is exposed to cells can be limited, because extended exposure may produce unpalatable lipids.

[0032] In some embodiments, adding lipid or lipid composition to cell culture medium causes increase in intracellular lipid content. Compared with lipid-free control, the increase in intracellular lipid content can be increased by 10% to 1000%, 20% to 500%, 30% to 250%, 40% to 200% or 50% to 100% (e.g., by mass, concentration or v / v). For example, compared with control, adding lipid to cell culture medium may increase intracellular lipid content by about 50% or increase by about 1.5 times (e.g., by mass, concentration or v / v). Adding lipid or lipid concentration to cell culture medium may also increase the extracted cellular lipid, which is comparable to the above ranges and approximate values.

[0033] As used herein, the term "enrichment" is defined as increasing the concentration or amount of a material from a control or basal state. For example, adding a lipid or lipid composition to an insect cell can effectively increase or enrich the cell with lipids compared to a lipid-free control. For example, adding a lipid or lipid composition to the cell can enrich the cell with lipids in the above ranges and approximate values.

[0034] In some embodiments, the insect cells contacted with lipid or lipid composition can be enriched in specific lipid species. These enriched lipid species can include but are not limited to polyunsaturated fatty acids (PUFA), octadecenoic acid (18:1), eicosenoic acid (20:1), octadecadienoic acid (18:2) and alpha linoleic acid (18:3). The lipid of cell accumulation can include any lipid listed in Table 2.

[0035] As used herein, "adipose tissue" is defined as a structured product containing insect cells of the present disclosure. The term "adipose product" is defined as any product that includes adipose tissue in some form. Adipose products can include adipose tissue as a structured product, or adipose tissue as a diffuse or processed product. For example, adipose products can include adipose tissue that has been processed in a blender.

[0036] In certain embodiments, adipose tissue can be included in a composition of matter (including but not limited to food). Food can include any edible product that generally includes lipids, particularly edible foods that traditionally have the appearance and / or taste of animal fat. For example, adipose tissue can be added to steak (e.g., prepared beef products). In another example, adipose tissue can be added to steak to make its appearance and / or taste similar to animal products, but does not include animal products (such as steaks), where the muscle part is made of plants or other materials. Foods containing adipose tissue can also simulate processed lipid-containing animal products, such as lard, tallow and butter. Food can also be used for cooking and processing other foods. For example, food can include adipose tissue that has been used for cooking / frying vegetables.

[0037] Methods for insect cell isolation and scaffold preparation are described below and as demonstrated herein, see e.g. Figure 1 In certain embodiments, embryonic precursor cells are isolated from Manduca sexta eggs using a protocol adapted from previous work (Baryshyan, AL et al., Isolation and Maintenance-Free Culture of Contractile Myotubes from Manduca sexta Embryos. PLoS ONE 7, e31598 (2012), which is incorporated by reference in its entirety. A few days after isolation, subpopulations of cells form a network of contractile muscles. After two weeks, the cultures are characterized by the presence of three major populations: (1) proliferating, loosely adherent, aggregated, or suspended spherical cells ( Figure 2, A); (2) adherent elongated cells forming a monolayer on the surface of the culture vessel; and (3) a network of contractile muscle cells stained positive for myosin heavy chain ( Figure 3 ). The first generation colony is passaged by shedding and transferred to a fresh culture container, in which the cells are proliferated and passaged approximately once a week. After passage, a subpopulation of cells adheres to the wall and elongates, while other cells remain aggregated and spherical. A stable growth rate is observed until approximately the 15th generation, when the cells continue to look healthy, but grow significantly slower. The adherent colony is passaged by trypsinization or cell scraping and transferred to a fresh culture container, where growth is significantly slower than that of the first generation colony. Robust and sustained (e.g., weeks) cell network contraction is observed in the initial separation, while the frequency of contracted networks observed in the passage culture colony is lower. The method may include thawing cryopreserved insect precursor cells onto a scaffold, and collecting and / or cryopreserving cells from the scaffold.

[0038] As used herein, "contacting" is defined in the context of a cell as bringing two entities (such as a cell and a lipid composition) together so that the entities can interact (e.g., a cell can accumulate lipids from a lipid composition). Contacting in this context may mean "touching" or "almost touching." In some cases, contact of a lipid composition with a cell may also be referred to as "feeding" (e.g., feeding a cell with a lipid composition).

[0039] The proliferating spheroid cell population used for subsequent experiments was termed Ms PC (Manduca sexta precursor cells). These cells were selected based on their growth rate, ease of passaging, homogeneous morphology, and loose adherence - suggesting potential for future adaptation to suspension culture 7 In the example experiment, a growth curve of Ms PC was plotted to determine the optimal inoculum density for propagation ( Figure 2 , B). Adherent mammalian cells are usually grown at 5,000 cells / cm 2 Insect cells are relatively small (e.g., the volume of Ms PC is about 1 / 5 of that of C2C12) and are usually plated at higher densities (e.g., 50,000 cells / cm 2 ) 7,24 Over 5 days, 25,000, 50,000, and 100,000 cells / cm 2 The average specific growth rates of the inoculated Ms PC were 0.0017, 0.0037, and 0.006 h -1 Based on this result, 100,000 cells / cm 2After Ms PCs have been expanded to passage 4, they are cryopreserved at high density (i.e., 9-10E6 cells / mL) in growth medium supplemented with fetal bovine serum (10-30%) and dimethyl sulfoxide (10%). Figure 2 , C). After thawing, the cryopreserved cells initially had low viability (75% compared to 86% before cryopreservation), but recovered rapidly after 5 days (91%) (Table 1). After 10 days, the recovered cells were morphologically similar to the initial cell population and continued to grow at a nearly equivalent rate (reaching approximately 90% confluence once a week). Ms PC was also tested for mycoplasma contamination. In certain embodiments, insect cells are cultured at or about 20,000 cells / cm 2 Up to or about 250,000 or more cells / cm 2 The 2D seeding density can be at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in the range between the above values. Insect cells can be inoculated at a range including: one or about one cell / cm 2 , 20,000 or about 20,000 cells / cm 2 , 25,000 or about 25,000 cells / cm 2 , 30,000 or about 30,000 cells / cm 2 , 35,000 or about 35,000 cells / cm 2 40,000 or about 40,000 cells / cm 2 45,000 or about 45,000 cells / cm 2 , 50,000 or about 50,000 cells / cm 2 , 55,000 or about 55,000 cells / cm 2 , 60,000 or about 60,000 cells / cm 2 , 65,000 or about 65,000 cells / cm 2 , 70,000 or about 70,000 cells / cm 2, 75,000 or about 75,000 cells / cm 2 , 80,000 or about 80,000 cells / cm 2 , 85,000 or about 85,000 cells / cm 2 , 90,000 or about 90,000 cells / cm 2 , 95,000 or about 95,000 cells / cm 2 , 100,000 or about 100,000 cells / cm 2 , 125,000 or about 125,000 cells / cm 2 , 150,000 or about 150,000 cells / cm 2 , 175,000 or about 175,000 cells / cm 2 , 200,000 or about 200,000 cells / cm 2 or about 250,000 or more cells / cm 2 .

[0040] In certain embodiments, where the scaffold is three-dimensional, seeding can be performed at a three-dimensional density that is three-dimensionally equivalent to a two-dimensional equivalent density, such as a plate seeding density per square centimeter as described above. For example, a three-dimensional seeding density can be equivalent to about at least 15,000 cells / cm 3 , at least 20,000 cells / cm 3 or at least 25,000 cells / cm 3 , and up to 1,000,000 cells / cm 3 , 500,000 cells / cm 3 , up to 250,000 cells / cm 3 or up to 200,000 cells / cm 3 , or within the range of the above values ​​or in the range between the above values. The seeded three-dimensional scaffold can also be seeded at a density of at least 100,000 cells / cm 3 , at least 500,000 cells / cm 3 or at least 2 million cells / cm 3 , and up to 1e9 cells / cm 2 , up to 1e8 cells / cm 3 or up to 1e7 cells / cm 3 The seeding may be performed at a density of at least one cell / cm 3 , at least 15,000 cells / cm 3, at least 20,000 cells / cm 3 or at least 25,000 cells / cm 3 , and up to 1,000,000 cells / cm 3 , up to 500,000 cells / cm 3 , up to 250,000 cells / cm 3 or up to 200,000 cells / cm 3 , or within the range of the above values ​​or in the range between the above values. The three-dimensional seeding density can also be a three-dimensional density equivalent to the following two-dimensional seeding density: at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in the range between the above values.

[0041] Table 1

[0042] Pre-cryopreservation 24 hours after cryopreservation 5 days after cryopreservation 10 days after cryopreservation

[0043]

[0044] In certain embodiments, the precursor cells are treated with a lipid composition to stimulate lipid production and / or storage. Vertebrate and invertebrate cells accumulate intracellular lipid droplets in response to fatty acid medium supplementation 14,17 In an exemplary experiment, Ms PCs were treated with different concentrations of water-soluble oleic acid or intralipid in the form of fatty acids (e.g., soybean oil emulsion or other lipid compositions). After 7 days in culture, although oleic acid-treated cells showed unhealthy morphology, minimal growth, and were considered non-viable, intralipid conditions at 0.1 and 1 mM and oleic acid conditions at 1 mM, BODIPY ( Figure 4 , A) and Oil Red O ( Figure 4 , C) staining to visualize the formation of lipid droplets. Lipid content was also quantified by eluting Oil Red O dye and comparing absorbance levels ( Figure 4, D). The assay confirmed that Intralipid induced more lipid accumulation in Ms PCs than oleic acid, and 0.1 and 1 mM were the optimal concentrations tested. A similar trend was observed in Drosophila melanogaster cells ( Figure 5 , A, B). Lipid accumulation in Ms PC by 0.1 mM Intralipid was observed within two weeks ( Figure 4 , B), during which lipid accumulation steadily increased relative to that of control cells. Neutral lipid content (e.g., triglycerides) was also quantified; treatment with 0.1 mM intralipid for 14 days resulted in a significant increase in neutral lipid content in Ms PCs (p = 0.0044) ( Figure 6 , D). In certain embodiments, the lipid composition added to the insect cells and / or scaffold comprises any lipid-containing substance, including but not limited to lipid emulsions, oil emulsions, seed oil emulsions, and solutions containing fatty acids.

[0045] In certain embodiments, lipidomics is used to characterize the fatty acids produced by the precursor cells. For example, lipidomics was used to characterize the fatty acid profiles in untreated and treated Ms PCs (Table 2). Here, lipidomics data was separated into the proportions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) for both phospholipids (important for cell membranes and taste) and triglycerides (the storage form of fat that makes up the majority of lipids found in animals). 25-27 In triglycerides, intralipid treatment resulted in a shift from MUFA and SFA to a higher proportion of PUFA content ( Figure 7 ). It is important to note that lipidomics data represent relative amounts of fatty acids, not total or absolute amounts, and it was estimated that the triglyceride content of cells treated with Intralipid was approximately 30% higher than that of untreated cells ( Figure 6 , D). The decrease in SFA content was mainly due to the decrease in palmitic acid (16:0) (the most common SFA in animals, plants and microorganisms) and a slight decrease in stearic acid (18:0) (Table 2) 28 The shift in MUFA was due to a large decrease in hexadecenoic acid (16:1) and a slight increase in octadecenoic acid (18:1) and eicosenoic acid (20:1). The change in PUFA was mainly due to an increase in octadecadienoic acid (18:2) (Table 2) 29 There was also a significant increase in alpha linoleic acid (18:3), an omega-3 fatty acid found in soybean oil (the main component of Intralipid). 30Among the phospholipids, MUFAs were relatively reduced, PUFAs were increased, and SFAs were slightly increased under introlipid treatment relative to controls. Again, octadecadienoic acid (18:2) was the main cause of the increase in PUFAs (Table 2). When the fatty acid content of the fat produced by Ms PC was compared to published data on traditional fat sources from livestock, the SFA content of the in vitro Ms PC fat was similar to that of chicken fat and was lower than that of beef and pork fat. 31 Likewise, the PUFA content of the fat produced by Ms PC was slightly higher than that of chicken and much higher than that of beef and pork. These fatty acid ratios were also similar to values ​​previously published in whole tobacco worm caterpillars. 32 .

[0046] Table 2

[0047] Lipidomic results of Ms PC cells, separated by phospholipids and triglycerides, of untreated and 7-day treated Ms PC cells with 0.1 mM Intralipid.

[0048]

[0049]

[0050] p-value * <0.0332; ** <0.0021; *** <0.0002; **** <0.0001

[0051] In certain embodiments, decellularized fungi or fungal mycelium is used as a scaffold for inoculating precursor cells (e.g., a decellularized mycelium scaffold). Decellularized mycelium was selected as a scaffold material based on the benefits of scalability, cost, and established use in meat substitutes (e.g., Quorn). 22 .like Figure 8 As indicated, decellularization can be performed to eliminate fungal DNA by gentle rinsing in a detergent solution (Table 3). The zeta potential, pore density, and pore size between untreated and decellularized scaffolds were also compared, and the results showed that decellularization had no significant effect on the surface charge or pore size of the scaffolds (Table 3). The mechanical properties were also studied, and the elastic modulus of the decellularized scaffolds was significantly reduced compared to the untreated scaffolds (Table 3). Before inoculating cells into the scaffolds, an appropriate sterilization method was established by comparing four methods: treatment with 70% ethanol for 1 hour, treatment with 10% bleach for 1 hour, autoclaving, or treatment with ethylene oxide. The scaffolds deformed during autoclaving and bleaching treatments ( Figure 8A). The autoclaved samples appeared charred and had volume shrinkage, and the bleached samples were brittle and had lost their original mass and shape. Ethylene oxide treatment was chosen because scaffolds treated in this way maintain their structure and it is also an established sterilization method in the food industry 33 Prior to working with primary Ms PCs, initial experiments were performed with Drosophila melanogaster progenitor cells (Dm PCs) due to their fast growth rate (e.g., doubling time of 24–48 h) and ease of imaging of GFP expression. 7 Dm PCs were initially cultured in 2D with dissociated mycelial fibers ( Figure 8 , C), where cells grew on top of and between hyphae and remained viable based on GFP expression. Dm PCs were also cultured on live (pre-sterilized) mycelium. Mycelium was viable and began to grow in insect cell culture medium, where Dm PCs were loosely associated with hyphal fibers in 3D for several days ( Figure 8 , D). Although initially an interesting result, the mycelium quickly outcompeted the insect cells and outgrew the culture. Once the decellularization, sterilization, and inoculation methods for the mycelium scaffolds had been optimized, Ms PCs were inoculated at 5 to 6 million cells per scaffold. When inoculated directly onto the mycelium (i.e., passive inoculation), Ms PCs infiltrated the scaffold and dispersed throughout the construct ( Figure 8 , F). After 14 days in lipid accumulation medium (0.1 mM Intralipid as defined in 2D experiments), Ms PCs grown in 3D accumulated lipid droplets based on staining with BODIPY ( Figure 8 , F). After 14 days, the mechanical properties of the 3D decellularized scaffold cell system were also studied by comparing the elastic modulus of the cells without cells, the control (no Intralipid treatment) and the 0.1 mM Intralipid treatment ( Figure 8 , G). Adding cells to the scaffolds had no significant effect on the mechanical properties.

[0052] Table 3 Characterization of untreated and decellularized mycelium scaffolds

[0053]

[0054]

[0055] a p-value 0.0005, b p-value 0.0050, c p-value 0.0317, d p-value 0.0103

[0056] In certain embodiments, the scaffold is derived from fungal mycelium. The scaffold may include other fungi, including but not limited to yeast (i.e., Saccharomyces), and edible members of the Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, and Glomeromycota. For example, the scaffold may be derived from any fungus capable of producing mycelium.

[0057] In summary, Manduca sexta embryonic primary cells (Ms PCs) were isolated, and a proliferating cell population was identified and expanded. Ms PCs were stimulated to produce lipids by incubation with free fatty acids, and subsequent experiments showed that: (1) incubation with 0.1 mM intralipid (e.g., soybean oil emulsion) resulted in robust lipid accumulation over 7 days, (2) the appearance and fatty acid composition of cultured fat reflected in vivo Manduca sexta fat, (3) 0.1 mM intralipid treatment resulted in a shift from relative SFA to PUFA content and an increase in linoleic acid, and (4) decellularized mycelial scaffolds supported 3D lipid accumulation with mechanics appropriate for a fat-based diet.

[0058] Previously established primary cell cultures were generated from the contractile muscles of Manduca sexta, but isolated cells in culture did not proliferate after multiple passages and could not withstand cryopreservation. 8 The addition of the broad-spectrum antibiotic primocin during isolation and the focus on the proliferative, loosely adherent, spherical cell population rather than the adherent, contractile muscle cell population appear to overcome these limitations. Culture methods have also been developed for the midgut epithelial cells of Manduca sexta, and cell lines from embryonic tissue have been previously characterized. 34-36 Previous work has not investigated Manduca sexta cells for food production or cultured meat. The present work demonstrates an isolation method for producing a relatively homogeneous population of cells that consistently produce lipids when stimulated with free fatty acids.

[0059] “Foonomics” describes an emerging group of analytical approaches that aim to assess the quality, nutritional value, and safety of food through high-throughput methods such as metabolomics, proteomics, transcriptomics, and lipidomics. 37 Lipidomics was used here to compare the fatty acid profiles of intralipid-treated and untreated Ms PC. Fatty acids are generally classified into four groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and trans fats. 38SFA (e.g., butter, coconut oil) contain a carbon atom bonded to two hydrogen atoms. This composition makes SFA very stable, but high levels of consumption can lead to an increased risk of coronary heart disease and other negative health effects, so limiting SFA intake is recommended. 39-41 MUFAs are fatty acids that have a pair of carbon atoms whose double bond replaces a hydrogen-carbon bond. PUFAs have two or more carbon-carbon pairs and are generally considered healthier than SFAs. Increasing the intake of MUFAs and PUFAs is thought to reduce the risk of coronary heart disease and may also be important in the prevention and management of a variety of diseases. 42,43 Trans fats are made by adding hydrogen atoms to unsaturated oils and do not appear in the MsPC lipid data. In the triglycerides treated with Intralipid, a decrease in SFA content was observed, and an increase in PUFA content, suggesting a possible shift toward a “healthier” fat content. In Ms PC fat, the ratios of SFA, MUFA, and PUFA were found to be similar to previously published reports on chicken fat. 31 Poultry is often praised for its higher nutritional value compared to red meat, a distinction that comes primarily from differences in SFA and PUFA content. 44 Another notable finding was an increase in linoleic acid (PUFA) in triglycerides after 14 days of intralipid treatment. Preliminary studies have shown that replacing SFA with linoleic acid has multiple benefits, including reducing brain microglial inflammation to prevent coronary heart disease; however, the literature on the benefits of linoleic acid is controversial. 45-48 .

[0060] While lipidomics was performed using Intralipid due to the observation that it accumulates lipids more efficiently compared to oleic acid, it is anticipated that exploring other fatty acid treatments or concentrations may result in altered ratios of SFAs, PUFAs, and MUFAs. Thus, multiple products could be prepared from the same cell type using different fatty acid treatments to induce a desired nutritional and flavor profile.

[0061] In some embodiments, the scaffold can be configured as a two-dimensional (2D) or approximately 2D structure. The 2D scaffold is relatively flat and is used to generate a thin layer of tissue constructs. The 2D scaffold can include living tissue or previously living tissue, such as mycelium fibers or other fibers as described herein. Cells can also be plated or cultured as 2D cultures with or without a scaffold. For example, cells can be cultured in a 2D tissue culture dish (e.g., a plastic-based tissue culture dish). Mycelium-based scaffolds can be decellularized and / or otherwise processed. For example, mycelium-based scaffolds can be processed to remove DNA. In some embodiments, the scaffold is edible. For example, the scaffold can include mycelium that can be safely ingested by a subject.

[0062] In certain embodiments, the scaffold can be configured as a three-dimensional (3D) structure. While 2D cell culture is useful for experimental research, large tissue constructs produced for consumption would benefit from 3D scale-up. Scaffold materials for 3D applications can include materials such as mushroom chitosan, alginate, or cellulose. 3 In the present disclosure, Ms PCs were successfully cultured in decellularized mycelial scaffolds, demonstrating the presence of nuclei throughout the construct. Notably, the elastic modulus of the decellularized scaffolds was lower when compared to untreated scaffolds. Although the decellularized scaffolds were still within the modulus range of in vivo fat products, 49 , but if a “tougher” product is desired, different mycelium strains, fermentation methods, or post-processing methods could be explored to reinforce the scaffold. The main bottleneck in producing larger cultured meat constructs is the difficulty in engineering a functional vasculature that delivers oxygen and nutrients throughout the larger construct. The natural mycelium matrix is ​​able to encapsulate cells in 3D without the need for tissue-engineered vasculature or microfabricated scaffolds, which is a significant benefit for future scaling. Insect cells may also be more amenable to large-scale 3D culture because insects are grown in vivo without a closed circulatory system.

[0063] While the current disclosure shows proof-of-concept results that Ms PCs can be used as a fat source for culture, future considerations include the adaptability of Ms PCs to serum-free media and the optimization of Ms PCs for scale-up growth in suspension culture. Drosophila melanogaster primary cells (Dm PCs) can be adapted to commercial serum-free media, and similar formulations may support the growth of Ms PCs. 5,7 . Suspension culture is a useful method for the large-scale production of cultured meat because growth is not limited by surface area. Ms PC survive and proliferate in single cell suspensions (data not shown), and cells grown in suspension are used to seed 3D scaffolds; however, suspension cultures must be optimized to achieve growth rates that support large-scale production. Although the purpose of the studies here was to produce cultured fat, manipulation through treatment with other naturally occurring insect hormones (e.g., 20-hydroxyecdysone or juvenile hormone) may direct these cells to different fates. It is envisioned that Ms PC can be maintained as a "starter cell" population that can be used to produce a variety of meat-related cell types in addition to lipid production. The fat produced by Ms PC can be used for a variety of purposes: (1) combined with Ms PC muscle constructs to produce combined muscle and fat products, (2) incorporated as a fat source into other cultured meat products (e.g., cattle, poultry), or (3) used to provide animal-derived nutrition and fat flavor in combined plant-based meat products and cultured meat products.

[0064] Although conventional livestock species have also been shown to accumulate lipids after treatment with fatty acids, Ms PCs are more amenable to this approach due to their ease of growth, broader tolerance to environmental perturbations, and potential for scale-up. This is reflected in their ability to grow at room temperature in the absence of CO2, antibiotics, and adaptability to both suspension and adherent cultures with minimal culture medium changes.

[0065] In certain embodiments, the present disclosure relates to any of the following numbered paragraphs:

[0066] 1. An in vitro method for preparing edible adipose tissue, the method comprising:

[0067] seeding a population of insect cells onto a scaffold, wherein the scaffold is two-dimensional or three-dimensional; and

[0068] The insect cell population is contacted with a cell culture medium containing a lipid composition, thereby accumulating lipid in the insect cell population and producing edible adipose tissue therefrom.

[0069] 2. The method according to paragraph 1, wherein the insect cell population is a precursor cell population.

[0070] 3. The method according to paragraph 1 or paragraph 2, wherein the insect precursor cell population is a Lepidoptera cell population or a Manduca sexta precursor cell population.

[0071] 4. The method according to any of the preceding paragraphs, wherein the scaffold is a fungal scaffold.

[0072] 5. The method according to paragraph 4, wherein the fungal scaffold is a decellularized mycelium scaffold.

[0073] 6. The method according to any one of paragraphs 1 to 5, wherein the scaffold is two-dimensional and the seeding is performed at the following two-dimensional seeding density: at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in the range between the above values.

[0074] 7. The method according to any one of paragraphs 1 to 5, wherein the scaffold is three-dimensional and the seeding is performed at a three-dimensional seeding density, which is three-dimensional and equivalent to the following two-dimensional seeding density: at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in a range between the above values, or the following three-dimensional seeding density: at least one cell / cm 3 , at least 15,000 cells / cm 3 , at least 20,000 cells / cm 3 or at least 25,000 cells / cm 3 , and up to 1,000,000 cells / cm 3 , up to 500,000 cells / cm 3 , up to 250,000 cells / cm 3 or up to 200,000 cells / cm 3 , or within the range of the above values ​​or in the range between the above values.

[0075] 8. The method according to any one of the preceding paragraphs, further comprising the steps of: cryopreserving and thawing the insect precursor cell population seeded on the scaffold.

[0076] 9. according to the method described in any one of the preceding paragraphs, the lipid concentration from the lipid composition in the cell culture medium is in the range of 0.1mM to 10mM, wherein the lipid composition is an emulsion.

[0077] 10. The method according to paragraph 9, wherein the lipid composition is a seed oil emulsion, preferably a soybean oil emulsion.

[0078] 11. The method of any of the preceding paragraphs, wherein the lipid composition and exposure time are selected to produce a desired lipid profile in the edible adipose tissue.

[0079] 12. The method according to any one of the preceding paragraphs, further comprising the step of isolating a population of insect precursor cells.

[0080] 13. A composition of matter prepared according to the method of any of the preceding paragraphs.

[0081] 14. A food product comprising the composition of matter of paragraph 13.

[0082] 15. An in vitro edible adipose tissue comprising an insect precursor cell population seeded on a scaffold, wherein the insect precursor cell precursor population is enriched in lipid content by exposure to a lipid composition.

[0083] 16. The method of paragraph 15, wherein the insect precursor cell population is a Lepidopteran cell population.

[0084] 17. The in vitro edible adipose tissue according to paragraph 16, wherein the insect precursor cell population is a Manduca sexta precursor cell population.

[0085] 18. The in vitro edible adipose tissue according to any one of paragraphs 15 to 17, wherein the scaffold is a fungal scaffold.

[0086] 19. The in vitro edible adipose tissue according to paragraph 18, wherein the fungal scaffold is a decellularized mycelium scaffold.

[0087] 20. The in vitro edible adipose tissue according to any one of paragraphs 15 to 19, wherein the lipid composition is an oil emulsion.

[0088] 21. The in vitro edible adipose tissue of paragraph 19, wherein the lipid composition is a seed oil emulsion.

[0089] 22. An in vitro method for preparing edible adipose tissue, the method comprising:

[0090] inoculating a population of insect precursor cells into a container; and

[0091] The insect precursor cell population is contacted with a cell culture medium containing a lipid composition, thereby accumulating lipid in the insect precursor cell population and producing edible adipose tissue therefrom.

[0092] Example

[0093] Example 1. Cell isolation and culture

[0094] Manduca sexta embryonic cells (i.e., Ms PCs) were isolated as previously described. 8Briefly, Manduca sexta eggs were staged at 19-22 hours, sterilized with 50% sodium hypochlorite solution for 10 minutes, rinsed with M3+BPYE growth medium (Shields and Sang M3 medium (S8398, Sigma-Aldrich, St. Louis, MO) supplemented with 0.5 g / L potassium bicarbonate, 2.5 g / L bacto-peptone, 1 g / L yeast extract, and 10 (vol. / vol.)% heat-inactivated fetal bovine serum), 500 ng / mL methoprene (33375, Sigma-Aldrich), 1% antibiotic-antimycotic (15062, ThermoFisher), and 100 mg / mL primocin (NC9141851, Fisher Scientific), and homogenized. 50 The homogenate was filtered, centrifuged (380 x g, 10 minutes) twice to rinse, and the cell pellet was resuspended in plating medium (growth medium supplemented with 1.14 mg / mL ethylene glycol-bis(2-aminoethyl)-N,N,N9,N9-tetraacetic acid). The cells were cultured at 220,000 / cm 2 Plates were inoculated and incubated for two hours at 27°C in a non-humidified incubator without CO2 gas exchange. The culture vessels were placed on a shaking plate for 10 minutes, non-adherent cells were aspirated, and the cells were refreshed with growth medium before the vessels were returned to the incubator. The cells were grown undisturbed for two weeks before passaging and characterization. Cells were routinely subcultured every 7 days at 90% confluence in medium without antibiotics and plated at 100,000 cells / cm 2 Drosophila melanogaster embryonic cells (i.e., Dm PCs) (EF4005, Kerafast, Boston, MA) were also cultured in M3+BPYE growth medium. Dm PCs were routinely plated at 50,000 cells / cm 2 Plated, passaged at 90% confluence, replenished with fresh medium once a week, and incubated at 25°C in a non-humidified incubator without carbon dioxide gas exchange.

[0095] Example 2. Cell proliferation assay

[0096] Cell growth was quantified by CyQuant cell proliferation assay (C7026, Thermo Fisher Scientific). MsPCs were plated at 25,000, 50,000, or 100,000 cells / cm 2Triplicate was seeded in 96-well plates. On day 1, day 3, and day 5, the culture medium was aspirated and the plates were frozen at -80 degrees Celsius. At the time of the assay, the plates were thawed and the cells were incubated in the dark for 5 minutes with CyQuant working solution. Fluorescence was quantified at 480 nm excitation and 520 nm emission on a microplate reader.

[0097] Example 3. Cryopreservation of cells

[0098] Ms PC was expanded in T25 and T75 cell culture flasks. When the cells reached 90% confluence, the cells were collected by trypsinization at 27°C for 5 minutes. The cells were centrifuged at 380 x g for 5 minutes and resuspended in freezing medium (i.e. 60% growth medium, 30% heat-inactivated fetal bovine serum and 10% dimethyl sulfoxide) at 9E6 cells / mL. The cells were transferred to a freezing vial and frozen overnight in a freezing bowl at -80°C, and then re-placed in long-term liquid nitrogen storage. The cells were thawed by incubation in a room temperature water bath for two minutes, the cells were diluted in growth medium and centrifuged, then counted, resuspended and counted at 100,000 viable cells / cm 2 After cryopreservation, cells were monitored for 10 days and passaged after 5 days. TM Cell viability and aggregation were determined by ChemoMetec (Chemometec, Denmark). The cell aggregation rate was defined as the percentage of cells in aggregates of five or more cells.

[0099] Example 4. Mycoplasma testing

[0100] The presence of mycoplasma was investigated by MycoAlert assay (LT07, Lonza, Basel, Switzerland). Ms PC cell suspensions were centrifuged at 380 x g for 5 minutes and the supernatant was transferred to a 96-well plate. Samples, negative controls and positive controls were incubated with reagent solution for 5 minutes and luminescence was measured on a microplate reader. The samples were then incubated with substrate solution for 10 minutes and luminescence was measured again. The second reading was divided by the first reading to generate a luminescence ratio. Samples, negative controls and positive controls were measured in duplicate.

[0101] Example 5. Measurement of lipid accumulation

[0102] To screen for lipid accumulation in Ms PC, cells were plated at 100,000 cells / cm 2Inoculated in 48-well plates. After culturing for 24 hours, the culture medium was renewed with the growth medium supplemented with water-soluble oleic acid (01257, Sigma-Aldrich) or Intralipid (I141, Sigma-Aldrich) of different concentrations (0, 0.01, 0.1, 1mM). After culturing for seven days, cells were dyed with BODIPY (D3922, Thermo Fisher Scientific) and NucBlue (R37605, Thermo Fisher Scientific) at 1:2,000 dilution, or fixed and dyed with Oil Red O (00625, Sigma-Aldrich). Cells were imaged on a fluorescence microscope (BZ-X800, Keyence). In order to quantify lipid accumulation, the Oil Red O dye under each condition was eluted with isopropanol, and absorbance (500nm) was measured on a microplate reader. To study lipid accumulation over time, cells were treated with 0.1 mM Intralipid and stained with BODIPY and NucBlue on days 1, 4, 7, and 14.

[0103] Example 6. Extraction of lipids from cells

[0104] Ms PC (p7) was prepared at 100,000 / cm 2 Inoculate to control or treated conditions. After 48 hours, 0.1 mM of Intralipid was added to treated cells. The culture medium of all cells was renewed after 7 days. After 14 days, cells were harvested and lipids were extracted by methyl tert-butyl ether (MTBE) as previously described. 51 . Briefly, cells were detached, counted, and each sample was split into 2E6 cells. The samples were resuspended and vortexed with methanol, then MTBU was added and incubated for 1 hour. Water was added, the samples were vortexed, and centrifuged at 10,000xg for 5 minutes. The upper organic phase was removed and the MTBE extraction was repeated. The double-extracted MTBE phase was dried under liquid nitrogen and stored at -20°C.

[0105] Example 7. Lipidomic analysis

[0106] Lipids were extracted as described above. Non-targeted lipidomic analysis (n=5) was performed by high-resolution liquid chromatography combined with mass spectrometry (QExactive Plus / HFOrbitrap HR-LC-MS / MS, Thermo Fisher Scientific) at the Mass Spectrometry Core of Beth Israel Deaconess Medical Center in Boston, Massachusetts. Data processing was performed in Python (v3.8.5). Python's pandas (v 1.2.3) was used to organize data for manipulation. Lipids were binned according to their classification (e.g., phospholipids, triglycerides). In each classification, the lipid peak areas of the relevant fatty acids were added to determine the total fatty acid area. Fatty acids were sorted as saturated, monounsaturated, or polyunsaturated.

[0107] Example 8. Neutral lipid determination

[0108] Lipids were extracted as described herein. Neutral lipids were quantified using a neutral lipid assay kit (ab242307, Abcam) according to the manufacturer's instructions. In short, the sample was diluted in a 2:1 mixture of methanol and chloroform. The sample was incubated at 55°C for 30 minutes until dry, briefly cooled at 4°C, rehydrated in isopropanol, and incubated with a fluorescent reagent for 15 minutes. Fluorescence was quantified at 490nm excitation and 585nm emission on a microplate reader. Standard curves were prepared using lipid standards to estimate the lipid values ​​for each sample.

[0109] Example 9. Mycelium decellularization and scaffold preparation

[0110] Ecovative Design (Green Island, NY) generously donated Excell TM Mycelium scaffolds. Mycelium was sectioned with a 6-8 mm biopsy punch to produce cylindrical samples of 6-8 mm in diameter and 3-6-8 mm in height. For decellularization, the samples were immersed in 70% ethanol on an orbital shaker at 100 rpm for 1 hour. The samples were then immersed in deionized water (24 hours), 1% sodium dodecyl sulfate (24 hours), 10% sodium hypochlorite bleach containing 0.1% Tween 20, deionized water (24 hours), and finally 10 mM Tris buffer (pH 9.0) (24 hours). The samples were removed from the solution, frozen at -20°C for at least 12 hours, and lyophilized for 48 hours. Prior to cell culture, the samples were sterilized with ethylene oxide.

[0111] Example 10. Sterilization screening

[0112] To compare sterilization methods, mycelial samples were either untreated, soaked overnight in 70% ethanol, soaked overnight in 20% bleach, autoclaved, or exposed to ethylene oxide gas. Sterility was screened by incubating scaffolds in basal medium at 37°C for two weeks and observing under a microscope for signs of visible contamination.

[0113] Example 11. DNA quantification

[0114] DNA content in control and decellularized mycelia was quantified by CyQuant assay (C7026, Thermo Fisher Scientific, Waltham, MA). Mycelial samples were frozen in liquid nitrogen and ground into powder using a mortar and pestle. Samples were suspended in cell lysis buffer and CyQuant reagent, centrifuged at 380 x g for 5 minutes, and the supernatant was analyzed on a microplate reader at 480 nm excitation and 520 nm emission.

[0115] Example 12. 3D lipid accumulation

[0116] As described above, the mycelium scaffolds were decellularized and sterilized. Ms PC was inoculated with 6E6 cells per scaffold and allowed to adhere for 24 hours. For mechanical testing, Ms PC was inoculated with approximately 15M cells per scaffold (the exact inoculation density is unknown because not all cells adhere to the scaffold). Non-adherent cells were removed and the culture medium was renewed with growth medium (control) or growth medium supplemented with 0.1mM Intralipid (treated). 50% of the culture medium was replaced after 7 days, and the scaffolds were fixed for BODIPY / DAPI staining or for mechanical testing after 14 days. For BODIPY / DAPI staining, the cell-loaded scaffolds were stained with BODIPY (D3922, Thermo Fisher Scientific) at a dilution of 1:2,000 for 30 minutes. Next, it was fixed overnight in 4% paraformaldehyde, immersed in 15% sucrose (24 hours), 30% sucrose (24 hours) and optimal cutting temperature (OCT) compound (24 hours). After fixation, the bracket was embedded in the OCT in the freezing mold and sliced ​​with 15mm with a cryostat (CM1950, Leica, Wetzlar, Germany). The bracket of the frozen section was mounted on a slide with a sealing agent (ab104139, Abcam, Cambridge, United Kingdom) with DAPI, and imaged on a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan).

[0117] Example 13. Fat Body Histology

[0118] First, the 5th instar larvae of the second day of tobacco moth were anesthetized with ice and carbon dioxide, and the fat body tissue was dissected out. The fat body tissue was placed in 10% neutral buffered formalin for 24 hours and then rinsed 3 times with phosphate buffered saline. In order to prepare frozen sections, the tissue was immersed in 15% sucrose (24 hours), 30% sucrose (24 hours) and optimal cutting temperature (OCT) compound (24 hours). The OCT-embedded tissue was transferred to a freezing mold and frozen with dry ice. The tissue was sliced ​​into 15mm slices with a cryostat (CM1950, Leica) and transferred to a microscope slide. The tissue was sliced ​​with BODIPY in phosphate buffered saline at a dilution of 1:2,500. TM (D3835, Thermo Fisher Scientific) or phalloidin (A12381, Thermo Fisher Scientific) diluted 1:100 in phosphate-buffered saline and mounted with mounting medium containing DAPI (ab104139, Abcam). Slides were imaged on a fluorescence microscope (BZ-X800, Keyence). Images were edited using Fiji software.

[0119] Example 14. Texture Analysis

[0120] Texture analysis (e.g., double compression test) was performed with a dynamic mechanical analyzer. Cylindrical scaffolds and cell scaffold constructs were compressed to 10% of their original height (in the range of about 1.5-3 mm) over a 30 second time span for a total of two cycles. The elastic modulus was calculated by measuring the slope between 5% and 15% strain of the secondary compression.

[0121] Example 15. Confocal Microscopy

[0122] The cell-laden scaffolds were stained with BODIPY 488 (D3922, Thermo Fisher Scientific) at 1:1,000 for 30 minutes, fixed with 4% paraformaldehyde for 2 hours at room temperature, rinsed with PBS, and stained with DAPI (D1306, Thermo Fisher Scientific) in PBS at 1:1,000 for 15 minutes. The scaffolds were imaged on a glass plate on a confocal microscope (TCS SP8, Leica).

[0123] Example 16. Statistical analysis

[0124] Unless otherwise stated, all experiments were performed in triplicate. Statistical analysis was performed using GraphPad Prism version 9 software. Statistical significance was determined when p values ​​were less than 0.05. Analysis of variance (ANOVA) was performed using post hoc Tukey unless otherwise stated.

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Claims

1. An in vitro method for preparing edible adipose tissue, the method comprising: seeding a population of insect cells onto a scaffold, wherein the scaffold is two-dimensional or three-dimensional; as well as The insect cell population is contacted with a cell culture medium containing a free fatty acid composition or a lipid composition, thereby accumulating lipids in the insect cell population and producing edible adipose tissue therefrom.

2. The method of claim 1, wherein the insect cell population is a precursor cell population.

3. The method of claim 1, wherein the insect cell population is a Lepidoptera cell population or a Manduca sexta precursor cell population. The method of claim 1 , wherein the scaffold is a fungal scaffold. The method of claim 4 , wherein the fungal scaffold is a decellularized mycelium scaffold.

6. The method according to any one of claims 1 to 5, wherein the scaffold is two-dimensional and the seeding is performed at the following two-dimensional seeding density: at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in the range between the above values.

7. The method according to any one of claims 1 to 5, wherein the scaffold is three-dimensional and the seeding is performed at a three-dimensional seeding density, which is three-dimensional and equivalent to the following two-dimensional seeding density: at least one cell / cm 2 , at least 15,000 cells / cm 2 , at least 20,000 cells / cm 2 or at least 25,000 cells / cm 2 , and up to 1,000,000 cells / cm 2 , up to 500,000 cells / cm 2 , up to 250,000 cells / cm 2 or up to 200,000 cells / cm 2 , or within the range of the above values ​​or in a range between the above values, or the following three-dimensional seeding density: at least one cell / cm 3 , at least 15,000 cells / cm 3 , at least 20,000 cells / cm 3 or at least 25,000 cells / cm 3 , and up to 1,000,000 cells / cm 3 , up to 500,000 cells / cm 3 , up to 250,000 cells / cm 3 or up to 200,000 cells / cm 3 , or within the range of the above values ​​or in the range between the above values.

8. The method according to any one of claims 1 to 5, further comprising the following steps: The insect cell population seeded on the scaffold is cryopreserved and thawed.

9. The method according to any one of claims 1 to 5, wherein the concentration of lipids from the lipid composition in the cell culture medium is in the range of 0.1 mM to 10 mM, wherein the lipid composition is an emulsion.

10. The method of claim 9, wherein the lipid composition is a seed oil emulsion.

11. The method of any one of claims 1 to 5, wherein the lipid composition and exposure time are selected to produce a desired lipid profile in the edible adipose tissue.

12. The method according to any one of claims 1 to 5, further comprising the step of isolating a population of insect cells.

13. A composition of matter prepared according to the method of any one of claims 1 to 5.

14. A food product comprising the composition of matter according to claim 13.

15. An in vitro edible adipose tissue comprising an insect cell population seeded on a scaffold, wherein the insect cell population is enriched in lipid content by exposure to a free fatty acid composition or a lipid composition.

16. The method of claim 15, wherein the insect cell population is a precursor cell population.

17. The in vitro edible adipose tissue according to claim 16, wherein the insect cell population is a Lepidoptera cell population or a Manduca sexta precursor cell population.

18. The in vitro edible adipose tissue of claim 15, wherein the scaffold is a fungal scaffold.

19. The in vitro edible adipose tissue according to claim 18, wherein the fungal scaffold is a decellularized mycelium scaffold.

20. The in vitro edible adipose tissue according to any one of claims 15 to 19, wherein the lipid composition is an oil emulsion.

21. The in vitro edible adipose tissue of claim 19, wherein the lipid composition is a seed oil emulsion.

22. An in vitro method for preparing edible adipose tissue, the method comprising: inoculating a colony of insect cells into a container; as well as The insect cell population is contacted with a cell culture medium containing a free fatty acid composition or a lipid composition, thereby accumulating lipids in the insect cell population and producing edible adipose tissue therefrom.

23. The in vitro method according to claim 22, wherein the insect cell is a precursor cell.

24. The method of claim 10, wherein the lipid composition is a soybean oil emulsion.

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