Methods for increasing the fat content of a cell

Abstract

Described herein are novel methods for increasing the fat content of a cell or population of cells in culture. Also described are methods for producing cultured fat from a population of cells, processes for producing food products, and processes for transforming non fat storing cells into fat storing cells.

Description

[0001] METHODS FOR INCREASING THE FAT CONTENT OF A CELL

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods for increasing the fat content of a cell. The invention also relates to methods for producing cultured fat from a population of cells, and processes for producing food products.

[0004] BACKGROUND

[0005] Due to environmental and ethical reasons, meat free food products are growing in demand and production. In addition to vegetable based (e.g. soy, wheat gluten or other source) meat alternatives, lab grown (cultured) meat is expected to have a strong growth trajectory and become an important part of the food landscape. Globally, the cultured meat market was valued at ~$130M in 2021 and is expected to reach $320M by 2028 and $520M by 2030 according to market trend forecasts (KBV Research and Acumen Research & Consulting, respectively).

[0006] A current issue with cultured meat products is the low quality of the cultured meat, whereby a special concern is the addition of fat cells to improve the flavour, texture and nutrient quality of the final product. Because the production of fat cells is technologically challenging (Fish, K.D., Rubio, N.R., Stout, A.J., Yuen, J.S.K. & Kaplan, D.L. Prospects and challenges for cell-cultured fat as a novel food ingredient. Trends Food Sci Technol 98, 53-67 (2020)), the current cultured meat market is limited to processed end-products, such as burgers, meatballs, nuggets, or sausages, with especially the nuggets segment achieving a sizable market share in 2021 . Nevertheless, there is an increasing desire for high quality meat alternative products, evidenced by the emergency of luxury segment products such as lab grown foie gras (Gourmey) and plant-based filet mignon (Juicy Marbles).

[0007] Fat is an essential component of meat, with an average intramuscular content of 1 .6% (turkey) to 12.6% in some common cuts of beef, and >30% in Japanese Wagyu beef. Fat is nutritionally important, contributes significantly to a tender texture, and especially contributes to meat flavour through aroma and flavour molecules, especially through lipid oxidation. Lipid oxidation products also interact with products of the Maillard reaction that occur during cooking, creating aroma compounds that significantly influence the meat flavour after cooking, frying, roasting etc.

[0008] As a consequence of the increased desire for meat alternatives that have nutritional, textural and flavour profiles comparable with meat, fat cell production has become a focus of the cultured meat industry. However, a common bottleneck in fat production is the lengthy culturing process required for lipid storage to occur.

[0009] Thus, there is a need in the art for improved methods for the production of cultured fat. SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides a method for increasing the fat content of a cell, the method comprising modulation of a mechanosensitive signalling pathway in the cell.

[0011] In a second aspect, the invention provides a method for increasing the fat content of a cell, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and b. contacting the cell with an agent that activates a mechanosensitive ion channel; wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0012] In a third aspect, the invention provides the use of a Piezol agonist to increase the fat content of a cell.

[0013] In a fourth aspect, the invention provides the use of a Piezo2 agonist to increase the fat content of a cell.

[0014] In a fifth aspect, the invention provides a method for producing cultured fat from a population of cells, the method comprising culturing the cells under conditions suitable to increase the fat content of the cells, said conditions comprising: a. culturing the cells at a pressure from 10 to 200 kPa, and / or b. contacting the cells with a modulator of a mechanosensitive signalling pathway.

[0015] In a sixth aspect, the invention provides a cultured fat obtained or obtainable by the methods described herein.

[0016] In a seventh aspect, the invention provides a process for producing a food product, the process comprising: a. culturing a population of cells under conditions suitable to increase the fat content of the cells, said conditions comprising: i. culturing the cells at a pressure from 10 to 200 kPa, and / or ii. contacting the cells with a modulator of a mechanosensitive signalling pathway; and b. combining the population of cells with at least one other composition to produce the food product.

[0017] In an eighth aspect, the invention provides a process for producing a food product, the process comprising: a. providing a cultured fat produced according to the methods disclosed herein, or a cultured fat as disclosed herein, and b. combining the cultured fat with at least one other composition to produce the food product.

[0018] In a ninth aspect, the invention provides a method for differentiating a cell into an adipocyte or a preadipocyte, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with a modulator of a mechanosensitive signalling pathway.

[0019] In a tenth aspect, the invention provides a method for transforming a non-fat storing cell into a fat storing cell, comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with an agent that activates a mechanosensitive ion channel; wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Piezol localises to the vSMC nuclear lamina and controls calcium levels. Immunofluorescent micrographs show enrichment of Piezol at the nuclear membrane (A-C). A) Anti-piezo1 staining shows a dotted appearance around the nuclear membrane (zoom in inset). B) example line profile (line indicated in A) shows enrichment at the edges. C) quantification of enrichment against cytoplasm and nuclear intensity. *** P<0.001 from one-sample t-test, compared to a value of 1 , i.e. no enrichment. D) the nuclear localisation is further confirmed using a Piezol -GFP transfection (zoom in inset); here cotransfected with Tractin-Tomato. E-J) Yodal treatment leads to rapid Ca2+ transients in A7r5 (E,F), primary human (G,H) and primary rat (I, J) vSMCs. Data from three independent repeats with n=30-45 cells. Scale bar 100pm.

[0022] Figure 2: Yodal dependent calcium increase is blocked by Dookul . A) Yodal (Y1) treatment leads to rapid Ca2+ transients in A7r5 that are reduced by simultaneous Dookul treatment. B) Quantification of the Ca2+ transients, p-values from t-test: * P<0.05, * P<0.01 , *** P<0.001 .

[0023] Figure 3: Chronic Yodal treatment supports transition to a foam cell phenotype. A-B) Wound scratch assay shows reduced migration of Yodal treated cells. C-D) Click-EdU assay indicates higher cell proliferation following Yodal incubation. E) Nanoindentation indicates a lower Young's Modulus after Yodal treatment. F,G) qPCR testing indicate ncreased CD68 and lower ABCA1 transcription after 8- hour. H) Top 15 outcomes from causal reasoning testing to identify upstream regulators of the of quantitative proteomic data from 1 kPa atmospheric pressure vs HT pressure treated A7r5 cells(13). Yellow shading indicates molecules involved in lipid metabolism and foam cell formation; red shading indicates micro RNA hits; grey shading indicates molecules involved in histone modification, p-values from t-test: * P<0.05, * P<0.01 , *** P<0.001 . Figure 4: Hyperspectral stimulated Raman Scattering (hsSRS) indicates lipid droplet accumulation and lipid metabolic changes after pressure and Yoda stimulation. A,B) Acute Piezol activation and HT pressure stimulation leads to lipid accumulation in A7r5 cells. A) Lipid droplet segmentation from phasor blots (bottom row) overlaid in red over 2930cm-1 image for overall cell outlines (green); B) Quantification of area ratio of lipid droplets; C) lipid droplet raman spectrogram shows increase in total lipids after HT pressure and reduction in TAGs after Yodal treatment. D) Quantification of ratios of total lipids compared to proteins (D), TAGs compared to total lipids (E), and cholesterol esters (CEs) compared to total lipids (F).

[0024] Figure 5: Hyperspectral stimulated Raman Scattering (hsSRS) indicates lipid droplet accumulation and lipid metabolic changes during neointima formation. hsSRS was performed on tissue sections after carotid artery ligation (LGT) or in sham operated animals (Sham). Example images and segmentation of ECM, cellular regions and lipid droplets from phasor plots (right panels). Lipid droplet formation was increased in LGT arteries (B). C) Raman spectrograms show a reduction in TAGs in LGT vs Sham control. D) Spectrogram of segmented cytoplasm from A7r5 cells show increases in total lipids after hypertensive pressure stimulation and TAG decreases after Yodal treatment, comparable to the lipid droplets, thus confirming overall changes in the cellular lipid pools.

[0025] Figure 6: Lipidspot staining confirms changes in lipid droplets in A7r5 cells and pre-adipocytes. A) A7r5 cells have increased lipid droplet formation after Yodal treatment when assessed with the "lipidspot" lipid stain. B,C) Hypertensive pressure stimulation (B) and Yodal treatment (C) leads to increased lipid droplet formation in pre-adipocytes.

[0026] DETAILED DESCRIPTION

[0027] The invention described herein provides novel methods for increasing the fat content of a cell. The invention is based, at least in part, on the surprising discovery that stimulating a cell with low-level pressure causes increased lipid droplet formation within the cell, as well as increased lipid uptake. The inventors further discovered that this process could be recapitulated by contacting cells with an agent that activates mechanosensitive ion channels on the cell, such as with an agent that activates the mechanosensitive Ca2+ channel, Piezol . In addition, the inventors have discovered that the same processes can be used to enable non-fat cell types to store lipid droplets.

[0028] The methods described herein find particular utility in the food industry, where it is desirable to increase the fat content of cultured cells in order to produce cultured fat, which can be added to food products.

[0029] Another particular advantage of the methods described herein is that they can be used to increase the lipid content of cells without increasing the levels of cholesterol, providing benefits from a health perspective when those cells are used as a source of fat. Methods for increasing the fat content of a cell

[0030] Described herein are methods for increasing the fat content of a cell. Generally, the methods comprise modulation of a mechanosensitive signalling pathway in the cell. The mechanosensitive signalling pathway is typically a pressure-sensitive signalling pathway.

[0031] The inventors have discovered that modulation of the mechanosensitive signalling pathway can be achieved in several ways in order to increase the fat content of a cell. In general terms, one approach is to culture the cell under conditions of low pressure (with “low” pressure being defined herein as pressures from 10 to 200 kPa). Another approach is to contact the cell with a modulator of a mechanosensitive ion channel, such as with an agent that activates the mechanosensitive ion channel. It will be appreciated that these two approaches are not mutually exclusive and can both be used (simultaneously or sequentially) to increase the fat content of a cell.

[0032] Culturing the cell at low pressure

[0033] Methods for increasing the fat content of a cell may comprise culturing the cell at a pressure from 10 to 200 kPa, and more particularly at a pressure from 10 to 50 kPa. Such pressure ranges are referred to herein as “low” pressure. The pressures used in the methods of the invention are orders of magnitude lower than other methods used in the food industry, such as in high-pressure processing, which applies hundreds of MPa of pressure (compared to tens or hundreds of kPa in the present methods).

[0034] In standard cell culture, cells are generally cultured at atmospheric pressure. In the methods described herein, the preferred pressure(s) can be achieved using a dedicated tissue culture stimulator, such as the Mechano-Culture TR stimulator (CellScale). When cells are cultured in a bioreactor, the cells can be removed from the bioreactor and added to the culture stimulator for the duration of the pressure stimulation. The pressure-treated cells can then be returned to the bioreactor for further culturing, or used immediately as a source of cultured fat. Alternatively, the bioreactor itself may have pressure stimulating capabilities, so that the cells can be pressure-treated without needing to remove them from the bioreactor.

[0035] Generally, the methods described herein for increasing the fat content of a cell may comprise culturing the cell or cells at a pressure from 10 to 200 kPa. The cells may be cultured at a pressure from 10 to 175 kPa, 10 to 150 kPa, 10 to 125 kPa, 10 to 100 kPa, 10 to 90 kPa, 10 to 80 kPa, 10 to 70 kPa, 10 to 60 kPa, 10 to 50 kPa, 10 to 45 kPa, 10 to 40 kPa, 10 to 35 kPa, 10 to 30 kPa, 10 to 25 kPa, 10 to 20 kPa, 15 to 200 kPa, 15 to 175 kPa, 15 to 150 kPa, 15 to 125 kPa, 15 to 100 kPa, 15 to 90 kPa, 15 to 80 kPa, 15 to 70 kPa, 15 to 60 kPa, 15 to 50 kPa, 15 to 45 kPa, 15 to 40 kPa, 15 to 35 kPa, 15 to 30 kPa, 15 to 25 kPa, 20 to 200 kPa, 20 to 175 kPa, 20 to 150 kPa, 20 to 125 kPa, 20 to 100 kPa, 20 to 90 kPa, 20 to 80 kPa, 20 to 70 kPa, 20 to 60 kPa, 20 to 50 kPa, 20 to 45 kPa, 20 to 40 kPa, 20 to 35 kPa, 20 to 30 kPa, or 20 to 25 kPa. In some cases, the cells may be cultured at a pressure from 15 to 30 kPa. A method for increasing the fat content of a cell may comprise culturing the cell or a population of the cells at a pressure from 10 kPa. The cell or population of cells may be cultured at a pressure from 15 kPa, from 20 kPa, from 25 kPa, from 30 kPa, from 35 kPa, from 40 kPa, from 45 kPa, from 50 kPa, from 55 kPa, from 60 kPa, from 65 kPa, from 70 kPa, from 80 kPa, from 90 kPa, from 100 kPa, from 110 kPa, from 120 kPa, from 130 kPa, from 140 kPa, from 150 kPa, from 160 kPa, from 170 kPa, from 180 kPa, from 190 kPa or from 200 kPa.

[0036] A method for increasing the fat content of a cell may comprise culturing the cell or a population of the cells at a pressure up to 10 kPa. The cell or population of cells may be cultured at a pressure up to 15 kPa, up to 20 kPa, up to 25 kPa, up to 30 kPa, up to 35 kPa, up to 40 kPa, up to 45 kPa, up to 50 kPa, up to 55 kPa, up to 60 kPa, up to 65 kPa, up to 70 kPa, up to 80 kPa, up to 90 kPa, up to 100 kPa, up to 110 kPa, up to 120 kPa, up to 130 kPa, up to 140 kPa, up to 150 kPa, up to 160 kPa, up to 170 kPa, up to 180 kPa, up to 190 kPa or up to 200 kPa.

[0037] The cells may be cultured under conditions of low pressure (that is, a pressure from 10 to 200 kPa) for an amount of time from 30 minutes to 72 hours. For example, pressure may be applied for 30 minutes to 48 hours, 30 minutes to 24 hours, 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, 30 minutes to 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 5 hours, 2 hours to 4 hours, or 2 hours to 3 hours. In some cases, the pressure is applied for 2 to 8 hours.

[0038] In most cases, the pressure is applied for at least 30 minutes. For example, pressure may be applied to the cells for at least 45 minutes, at least 1 hour, at least 1 hour 30 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours.

[0039] The cells may be cultured under conditions of low pressure for up to 30 minutes, up to 45 minutes, up to 1 hour, up to 1 hour 30 minutes, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 8 hours, up to 10 hours, up to 12 hours, up to 24 hours, up to 48 hours, or up to 72 hours.

[0040] The methods described herein may comprise culturing the cells at a hydrostatic pressure, i.e., a pressure that remains relatively constant for the duration of the pressure stimulation. Thus, in some cases, the cell may be cultured at a constant pressure.

[0041] In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 30 minutes. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 1 hour. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 2 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 6 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 12 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 24 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 200 kPa for at least 48 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 30 minutes. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 1 hour. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 2 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 6 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 12 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 24 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 100 kPa for at least 48 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 30 minutes. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 1 hour. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 2 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 6 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 12 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 24 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 40 kPa for at least 48 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 30 minutes. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 1 hour. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 2 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 6 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 12 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 24 hours. In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a pressure from 10 to 30 kPa for at least 48 hours. Methods for increasing the fat content of a cell may also comprise culturing the cell at two different pressures, for example a first pressure and a second pressure. The first pressure may be lower than the second pressure. For example, the cells may be cultured at a first pressure from 10-20 kPa, and a second pressure from 21-30 kPa. Accordingly, a method for increasing the fat content of a cell may comprise culturing the cell at a first lower pressure and a second higher pressure. It will be understood that both “lower pressure” and “higher pressure” in this context still refer to pressures within the range of 10-200 kPa. The cells may be cultured at the lower pressure first, or the higher pressure first (in other words, the higher and lower pressures can be applied in any order).

[0042] In some cases, once the cell has been cultured at the first pressure and the second pressure, the first and second pressures may then be repeated. For example, the cell may be cultured at the first lower pressure, followed by the second higher pressure, followed by the first lower pressure, followed by the second higher pressure, and so on. Such repeating cycles of two different pressures (one lower and one higher) are described herein as culturing the cells at a “cyclic hydrodynamic” pressure. The number of cycles (wherein one cycle comprises the first lower pressure and the second higher pressure) can be varied in order to achieve the desired total pressure duration (such as a culturing the cell for up to 48 hours). Accordingly, culturing the cell may comprise alternating between the lower pressure and the higher pressure for a plurality of times or cycles. Culturing the cell may comprise alternating between the lower pressure and the higher pressure for at least 100 cycles, at least 200 cycles, at least 300 cycles, at least 400 cycles, at least 500 cycles, at least 600 cycles, at least 700 cycles, at least 800 cycles, at least 900 cycles, at least 1000 cycles, at least 1500 cycles, at least 1800 cycles, at least 2000 cycles, at least 2500 cycles, at least 3000 cycles, at least 4000 cycles, at least 5000 cycles, at least 6000 cycles, at least 7000 cycles, at least 8000 cycles, at least 9000 cycles, at least 10000 cycles, at least 10800 cycles, at least 1 1000 cycles, at least 12000 cycles, at least 13000 cycles, at least 14000 cycles, at least 15000 cycles, at least 16000 cycles, at least 17000 cycles, at least 18000 cycles, at least 19000 cycles, at least 20000 cycles, at least 21000 cycles, at least 25000 cycles, at least 30000 cycles, at least 35000 cycles, at least 40000 cycles, at least 45000 cycles or at least 50000 cycles.

[0043] The first pressure may be from 5-100 kPa, from 5-90 kPa, from 5-80 kPa, from 5-70 kPa, from 5-60 kPa, from 5-50 kPa, from 5-40 kPa, from 5-30 kPa, from 5-20 kPa, from 10-100 kPa, from 10-90 kPa, from 10-80 kPa, from 10-70 kPa, from 10-60 kPa, from 10-50 kPa, from 10-40 kPa, from 10-30 kPa, from I Q- 20 kPa, from 11-20 kPa, from 12-20 kPa, from 13-20 kPa, from 14-20 kPa, or from 15-20 kPa. The first pressure may be up to 100 kPa, up to 90 kPa, up to 80 kPa, up to 70 kPa, up to 60 kPa, up to 50 kPa, up to 40 kPa, up to 30 kPa, up to 20 kPa, up to 19 kPa, up to 18 kPa, up to 17 kPa, or up to 16 kPa. In some cases, the first pressure may be from 10-20 kPa, in particular 16 kPa.

[0044] The second pressure may be from 15-200 kPa, from 15-190 kPa, from 15-180 kPa, from 15-170 kPa, from 15-160 kPa, from 15-150 kPa, from 15-140 kPa, from 15-130 kPa, from 15-120 kPa, from 15-110 kPa, from 15-100 kPa, from 15-90 kPa, from 15-80 kPa, from 15-70 kPa, from 15-60 kPa, from 15-50 kPa, from 15-40 kPa, from 15-30 kPa, from 20-200 kPa, from 20-190 kPa, from 20-180 kPa, from 20- 170 kPa, from 20-160 kPa, from 20-150 kPa, from 20-140 kPa, from 20-130 kPa, from 20-120 kPa, from 20-110 kPa, from 20-100 kPa, from 20-90 kPa, from 20-80 kPa, from 20-70 kPa, from 20-60 kPa, from

[0045] 20-50 kPa, from 20-40 kPa, from 20-35 kPa, from 20-30 kPa, from 21-200 kPa, from 21-190 kPa, from

[0046] 21-180 kPa, from 21-170 kPa, from 21-160 kPa, from 21-150 kPa, from 21-140 kPa, from 21-130 kPa, from 21-120 kPa, from 21-110 kPa, from 21-100 kPa, from 21-90 kPa, from 21-80 kPa, from 21-70 kPa, from 21-60 kPa, from 21-50 kPa, from 21-40 kPa, from 21-35 kPa, or from 21-30 kPa. In some cases, the second pressure may be from 21-30 kPa, in particular 26 kPa.

[0047] In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa. In some cases, the first pressure may be about 16 kPa, and the second pressure may be about 26 kPa.

[0048] In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa, and culturing the cell may comprise alternating between the first (lower) pressure and the second (higher) pressure for at least 450 cycles. In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa, and culturing the cell may comprise alternating between the first (lower) pressure and the second (higher) pressure for at least 900 cycles. In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa, and culturing the cell may comprise alternating between the first (lower) pressure and the second (higher) pressure for at least 1 ,800 cycles. In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa, and culturing the cell may comprise alternating between the first (lower) pressure and the second (higher) pressure for at least 10,800 cycles. In some cases, the first pressure may be from 10-20 kPa, and the second pressure may be from 21-30 kPa, and culturing the cell may comprise alternating between the first (lower) pressure and the second (higher) pressure for at least 21 ,000 cycles.

[0049] The duration of each instance of first and second pressure can be adapted and optimised by the skilled person. In some cases, each application of the lower pressure may have a duration of up to 2 seconds, and each application of the higher pressure may have a duration of up to 2 seconds. For example, the cell may be cultured at the first pressure for up to 2 seconds, and the second pressure for up to 2 seconds, and the first and second pressures can then be repeated until the total culture duration is reached. The first and second pressures may each independently have a duration of up to 0.5 seconds, up to 1 second, up to 2 seconds, up to 3 seconds, up to 4 seconds, up to 5 seconds, up to 6 seconds, up to 7 seconds, up to 8 seconds, up to 9 seconds or up to 10 seconds. Each application of the lower pressure may have a duration of up to 1 second, and each application of the higher pressure may have a duration of up to 1 second. Each application of the lower pressure may have a duration of up to 3 seconds, and each application of the higher pressure may have a duration of up to 3 seconds. Each application of the lower pressure may have a duration of up to 4 seconds, and each application of the higher pressure may have a duration of up to 4 seconds. Each application of the lower pressure may have a duration of up to 5 seconds, and each application of the higher pressure may have a duration of up to 5 seconds. The first and second pressures may each independently have a duration of at least 0.5 seconds, at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds or at least 10 seconds. The first and second pressures need not have the same duration. For example, the first pressure may have a duration of up to 2 seconds, and the second pressure may have a duration of up to 3 seconds.

[0050] In some cases, a method for increasing the fat content of a cell comprises culturing the cell at a first pressure from 10-20 kPa and a second pressure from 21-30 kPa, wherein the first pressure has a duration of at least 1 second, the second pressure has a duration of at least 1 second, and wherein the first and second pressures are repeated such that the cell is cultured for at least 30 minutes. A method for increasing the fat content of a cell may comprise culturing the cell at a first pressure of about 16 kPa and a second pressure of about 26 kPa, wherein the first pressure has a duration of at least 1 second, the second pressure has a duration of at least 1 second, and wherein the first and second pressures are repeated such that the cell is cultured for at least 2 hours, preferably for about 12 hours.

[0051] Contacting the cell with a modulator of a mechanosensitive ion channel

[0052] Methods for increasing the fat content of a cell may comprise contacting the cell with a modulator of a mechanosensitive ion channel.

[0053] As used herein, the term “modulator” refers to an agent that can increase (stimulate / activate) or decrease (inhibit) the activity of the mechanosensitive ion channel. For example, the modulator may comprise an agent which activates the mechanosensitive ion channel, causing it to open and allow the passage of ions through the channel.

[0054] The ion channel may be a Ca2+ channel, such as the mechanosensitive Ca2+ channel, Piezol . Thus, the methods described herein may comprise contacting a cell with an agent that modulates Piezol . The methods described herein may comprise contacting a cell with an agent that activates Piezol . The methods described herein may comprise contacting a cell with a Piezol agonist.

[0055] The ion channel may be the mechanosensitive Ca2+ channel, Piezo2. Thus, the methods described herein may comprise contacting a cell with an agent that modulates Piezo2, in particular one that activates Piezo2. The methods described herein may comprise contacting a cell with a Piezo2 agonist. In some cases, the agent may modulate, activate, or be an agonist for both Piezol and Piezo2.

[0056] Suitable agents for modulating (i.e. activating or inhibiting) mechanosensitive ion channels include small molecules, such as the Piezol agonist Yodal , 2-(5-{[(2,6-Dichlorophenyl)methyl]sulfanyl}-1 ,3,4- thiadiazol-2-yl)pyrazine. The ability of Yodal to act as a Piezol agonist was first described in Syeda R. et al. (2015) eLife 4:e07369. The chemical structure of Yodal is shown below.

[0057] (Yodal)

[0058] Yodal analogues are also suitable agents for modulating (i.e. activating or inhibiting) mechanosensitive ion channels. A Yodal analogue (KC159, referred to as Yoda2) was described in Parsonage G et al., (2022) British Journal of Pharmacology 180:2039-2063 and shown to act as a Piezol agonist, as was its potassium salt (KC289).

[0059] Dookul is a further agent suitable for modulating (i.e. activating or inhibiting) mechanosensitive ion channels. Dookul has been described in Evans EL et al., (2018) British Journal of Pharmacology 175:1744-1759, as being capable of chemical antagonism of Yodal -evoked Piezol channel activity. The structure of Dookul is shown below. (Dookul)

[0060] Jedil is a further agent suitable for modulating (i.e. activating or inhibiting) mechanosensitive ion channels. Jedil has been described in Wang Y et al., (2018) Nature Communications 9:1300 as acting as an agonist for Piezol . The structure of Jedil is shown below. (Jedil) Jedi2 is a further agent suitable for modulating (i.e. activating or inhibiting) mechanosensitive ion channels. Jedi2 has been described in Wang Y et al., (2018) Nature Communications 9:1300 as acting as an agonist for Piezol . The structure of Jedi2 is shown below.

[0061] Accordingly, modulation of a mechanosensitive signalling pathway as described herein may comprise contacting the cell with Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof.

[0062] Other suitable agents for modulating mechanosensitive ion channels include peptides, proteins, antibodies, antibody fragments, antibody mimetics, fibronectin domains, oligonucleotides such as antisense oligonucleotides, or combinations thereof. Suitable agents can be identified by screening assays.

[0063] The modulator or agent may be added to the cells in culture for a duration of at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 20 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours. The modulator or agent may be added to the cells in culture for a duration of up to 5 minutes, up to 10 minutes, up to 20 minutes, up to 30 minutes, up to 45 minutes, up to 1 hour, up to 20 hours, up to 4 hours, up to 6 hours, up to 8 hours, up to 12 hours, up to 18 hours, up to 24 hours, up to 36 hours, or up to 48 hours. The modulator or agent may be added to the cells in culture for a duration of 5 minutes to 48 hours. Afterthe desired treatment time, the modulator can be washed out, for example by replacing the culture medium.

[0064] The cells may be contacted with the modulator at any suitable concentration that results in the modulation (e.g. activation) of the desired mechanosensitive ion channel. It will be appreciated that the appropriate concentration may be adapted depending on the culture conditions, for example the cell density within a bioreactor or culture chamber, and the size of the bioreactor or culture chamber. Suitable concentrations can be established by the skilled person using routine experimentation. For example, the modulator, agent, or Piezol agonist (such as Yodal or analogues thereof) may be contacted to the cell at a concentration of at least 1 pm, at least 5 pm, at least 10 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 35 pm, at least 40 pm, at least 45 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, or at least 100 pm. In some cases, methods for increasing the fat content of a cell may comprise a combination of the approaches described above. That is, cells may be treated with both pressure stimulation and an agent that modulates (for example activates) a mechanosensitive ion channel. The two treatments may be performed simultaneously, or they may be performed separately or sequentially, in any order.

[0065] Thus, described herein are methods for increasing the fat content of a cell, the method comprising: c. culturing the cell at a pressure from 10 to 200 kPa, and d. contacting the cell with an agent that modulates a mechanosensitive ion channel; wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0066] In particular, described herein are methods for increasing the fat content of a cell, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and b. contacting the cell with an agent that activates a mechanosensitive ion channel; wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0067] More specifically, described herein are methods for increasing the fat content of a cell, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and b. contacting the cell with an agent that activates Piezol ; wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0068] As will be understood, the invention also extends to cells that have been treated with the methods described herein. Accordingly, described herein is a population of cells, wherein the cells have increased fat content relative to cells which have not been subject to modulation of a mechanosensitive ion channel, for example cells which have been cultured according to standard protocols in the art. Relative fat content can be measured using the methods described herein, such as lipid staining, stimulated raman scattering (SRS) or hyperspectral stimulated raman scattering (hsSRS). In some cases, the cells have increased fat content relative to cells which have not been contacted with an agent that modulates a mechanosensitive ion channel, more specifically the cells described herein may have increased fat content relative to cells which have not been contacted with a Piezol agonist. Also described herein are cells which have increased fat content relative to cells that have not been cultured at a pressure from 10-200 kPa.

[0069] Also described herein is the use of an agent to increase the fat content of a cell, wherein the agent modulates a mechanosensitive ion channel. In particular, the agent may activate a mechanosensitive ion channel.

[0070] Also described herein is the use of a Piezol agonist to increase the fat content of a cell. Also described herein is the use of a Piezol agonist as a fat content increasing agent. The Piezol agonist may be a small molecule. The Piezol agonist may be Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, JedU , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof

[0071] Also described herein is the use of a Piezo2 agonist to increase the fat content of a cell. Also described herein is the use of a Piezo2 agonist as a fat content increasing agent. The Piezo2 agonist may be a small molecule. The Piezo2 agonist may be Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof.

[0072] Methods for producing cultured fat

[0073] As described above, the inventors have discovered that the fat content of cells can be increased by modulating mechanosensitive signalling pathways in those cells. By applying these techniques to a population of cells in culture, the inventors also provide new methods for producing cultured fat, such as fat which can be used in food products.

[0074] Accordingly, described herein are methods for producing cultured fat from a population of cells, the method comprising culturing the cells under conditions suitable to increase the fat content of the cells, said conditions comprising: a. culturing the cells at a pressure from 10 to 200 kPa, and / or b. contacting the cells with a modulator of a mechanosensitive signalling pathway.

[0075] Where the method comprises both steps (a) and (b), these steps may be performed simultaneously or sequentially in any order.

[0076] Such methods may optionally comprise a further step of harvesting fat from the cells in order to provide the cultured fat. Alternatively, the population of fat-containing cells may be used directly as the cultured fat. For example, the population of cells (e.g. a population of adipocytes) may be mixed directly with a source of protein in order to provide a food product.

[0077] Methods for producing cultured fat as described above may form part of a manufacturing process. In some cases, cells will be cultured in a bioreactor and the methods described above may be performed in the same bioreactor. The bioreactor may be adapted in order to provide variable pressures to the cells. In other cases, the cells may be temporarily removed from the bioreactor to perform the methods of the invention, for example by placing the cells in a separate pressure stimulator in order to apply low pressure as described above to increase their fat content, before returning the cells to the bioreactor for further culture. Also described herein are cultured fats obtained or obtainable by the methods described above. The cultured fat may comprise a population of cells, such as adipocytes or pre-adipocytes, with an increased fat content arising from the methods described herein. Generally, the fat content of the cells is increased relative to cells which have not been treated using a method as described herein (for example, cells that have not been treated with low pressure stimulation, or with an agent that modulates a mechanosensitive ion channel).

[0078] Also described herein is the use of a Piezol agonist to produce a cultured fat. The Piezol agonist may be a small molecule. The Piezol agonist may be Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof

[0079] Also described herein is the use of a Piezo2 agonist to produce a cultured fat. The Piezo2 agonist may be a small molecule. The Piezo2 agonist may be Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof.

[0080] Processes for producing a food product

[0081] The methods for increasing fat content of cells as described herein may also be usefully applied in processes for producing food products. In general, such processes may include culturing cells according to the methods described herein in order to increase the fat content of those cells, and subsequently combining the cells with at least one other composition in order to produce the food product.

[0082] Thus, described herein is a process for producing a food product, the process comprising: a. culturing a population of cells under conditions suitable to increase the fat content of the cells, said conditions comprising: i. culturing the cells at a pressure from 10 to 200 kPa, and / or ii. contacting the cells with a modulator of a mechanosensitive signalling pathway; and b. combining the population of cells with at least one other composition to produce the food product.

[0083] Where the method comprises both steps (i) and (ii), these steps may be performed simultaneously or sequentially in any order.

[0084] Also described herein is a process for producing a food product, the process comprising: a. providing a cultured fat produced according to the methods described herein, or a cultured fat obtained or obtainable by the methods described herein; and b. combining the cultured fat with at least one other composition to produce the food product.

[0085] The cells or cultured fat may be combined with other food compositions, such as protein sources, connective tissues, fats, carbohydrates, vitamins, minerals, preservatives, colourings, flavourings, other fat sources, or combinations thereof. Suitable protein sources may include animal protein, such as noncultivated or non-cultured animal protein, cultivated or cultured animal protein, plant-based protein sources, such as high moisture extrudate or textured vegetable protein (TVP) derived from any suitable plant source, fungal proteins, or yeast proteins. The at least one other composition may be selected from the group consisting of non-cultured protein, cultured protein, muscle cells, muscle tissue, connective tissue, fat, and combinations thereof.

[0086] Definitions

[0087] Below are provided certain definitions of terms, technical means, and embodiments used herein.

[0088] Fat

[0089] As used herein, “fat” or “fat content” may refer to saturated fat, unsaturated fat, monounsaturated fat, polyunsaturated fat, total fat, triacylglycerides, or a combination thereof.

[0090] The fat content of a cell can be increased via numerous different mechanisms. For example, the methods described herein may increase lipid droplet formation in the cell, may increase lipid retention by the cell, may increase lipid uptake by the cell, or may increase lipid phagocytosis by the cell, among others. The methods disclosed herein may increase the fat content in the cytoplasm of a cell, either as well as or in addition to increasing the lipid droplet content. By “increase”, it is meant that the fat content is increased relative to a cell cultured in substantially the same conditions, but without modulation of the mechanosensitive signalling pathway, for example without contacting the cell with an agent that modulates a mechanosensitive ion channel, or without culturing the cell at a pressure from 10-200 kPa, as described herein.

[0091] The fat content of a cell may be measured by any suitable technique known in the art. In particular, fat content may be measured by stimulated raman scattering (SRS) or hyperspectral stimulated raman scattering (hsSRS). These approaches offer label-free, non-destructive testing of cellular biomolecules. hsSRS also enables the chemical mapping of molecules with similar raman spectra, such as cholesterol esters (CEs) or triacylclycerides (TAGs), while the implementation of spectral phasor analysis of the hyperspectral datasets allows the differentiation of discrete cellular compartments, such as nuclei, membrane, cytoplasm or lipid droplets (W. J. Tipping et al., Chem Sci 13, 3468-3476 (2022); D. Fu et al., J Am Chem Soc 136, 8820-8828 (2014)). Fat content can also be measured using lipid stains such as LipidSpot™ (Biotium, US) or other fluorescent dyes that accumulate in lipid droplets. Such stains can be detected using conventional fluorescence microscopy techniques and analysed according to standard methods in the art. Metabolomics and lipidomics approaches can also be used to assess metabolic and lipid signalling pathways and profiles in cells.

[0092] As noted above, when using the methods described herein, the fat content of a cell may be increased relative to a control cell, wherein the control cell is cultured in substantially the same conditions, but without modulation of the mechanosensitive signalling pathway, for example without being cultured at low pressure (10-200 kPa) or without being contacted by an agent that modulates a mechanosensitive ion channel (such as a Piezol agonist). The fat content of a cell may be increased by at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to the control cell.

[0093] The lipid droplet content of a cell may be increased relative to a control cell, wherein the control cell is cultured in substantially the same conditions, but without being cultured at low pressure (10-200 kPa) and without being contacted by an agent that modulates a mechanosensitive ion channel (such as a Piezol agonist). The lipid droplet content of a cell may be increased by at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to the control cell.

[0094] Lipid uptake by a cell may be increased relative to a control cell, wherein the control cell is cultured in substantially the same conditions, but without being cultured at low pressure (10-200 kPa) and without being contacted by an agent that modulates a mechanosensitive ion channel (such as a Piezol agonist). Lipid uptake by a cell may be increased by at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to the control cell.

[0095] Cells and cell types

[0096] The methods for increasing fat content described herein can be applied to a wide range of cell types. Without limitation, the cell may be a prokaryotic cell, a eukaryotic cell, a non-mammalian cell, a mammalian cell, a fungal cell, or a yeast cell. In particular, cells suitable for use with the methods described herein include adipocytes, pre-adipocytes, dedifferentiated fat (DFAT) cells, stem cells, mesenchymal stem cells, satellite cells, vascular smooth muscle cells (VSMCs), induced pluripotent stem cells (iPSCs) and combinations thereof. In general, the cell is one that expresses Piezol . Generally, the cells are isolated cells. Generally, the cells are living cells.

[0097] While the methods described herein may refer to increasing the fat content of “a” cell, it will be appreciated that generally the methods will be applied to a population of cells, such as a number of cells in culture or in a bioreactor. The methods described herein are also useful for differentiating cells, such as stem cells, into adipocytes or pre-adipocytes. A method for differentiating a cell into an adipocyte or a pre-adipocyte may comprise:

[0098] (a) culturing the cell at a pressure from 10 to 200 kPa, and / or

[0099] (b) contacting the cell with a modulator of a mechanosensitive signalling pathway, such as a Piezol agonist as described herein.

[0100] A number of suitable cell types can be differentiated using the methods described herein. Stem cells are particularly suitable. The cell may be a dedifferentiated fat (DFAT) cell, a stem cell, a mesenchymal stem cell, a bone marrow derived stem cell, an induced pluripotent stem cell, or a satellite cell.

[0101] In addition, the methods described herein can transform or transdifferentiate a non-fat storing cell into a fat storing cell. In other words, applying the methods described herein to a typically non-fat storing differentiated cell enables or enhances the ability of the cell to store lipid droplets. A method for transforming a non-fat storing cell into a fat storing cell may therefore comprise a step of modulating a mechanosensitive signalling pathway in the cell. Specifically, a method fortransforming a non-fat storing cell into a fat storing cell may comprise (a) culturing the cell at a pressure from 10 to 200 kPa, and / or (b) contacting the cell with an agent that activates a mechanosensitive ion channel, wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

[0102] Where the methods are utilised as part of a process for producing food products, generally the cells may be animal in origin. For example, the cells may be derived from a pig, a cow, a bird (such as a turkey, a chicken, a duck, or a goose), a sheep, a goat, a horse, an insect, a reptile, an amphibian, or a fish. However, the cells may be derived from any other species. Cells used in the methods described herein can be from a single species type, or may comprise a combination of different species types. The cells may be mammalian cells, or may be non-mammalian cells, orthe cells used in the method may comprise a combination of mammalian and non-mammalian cells.

[0103] Described herein are cells or a population of cells obtained or obtainable by the methods disclosed herein.

[0104] Culturing

[0105] Cell culture as described above can be performed using standard methods in the art. In particular, the culture medium can be adapted according to the desired cell type and culture conditions, as long as the culture medium contains a source of fatty acids. Suitable culture media components include Dulbecco's Modified Eagle Medium (DMEM), buffers, hormones, growth factors, foetal bovine serum (FBS), calf and adult bovine serum, horse serum, pig serum, goat serum, and combinations thereof. Animal free media, i.e. media that does not contain any components derived from an animal source, can also be used. Fatty acid supplements can be used in conjunction with any suitable cell culture serum to provide a source of fat for uptake by the treated cells.

[0106] Optimal cell culture conditions such as temperature, pH, gas concentration (e.g. PO2) and agitation / mixing can all be determined by one of skill in the art, and tailored to suit the bioreactor or culture vessel and cell type.

[0107] The methods described herein may be performed in bioreactors, for example as part of a manufacturing process. Consequently, the methods may be described as in vitro methods or ex vivo methods.

[0108] Cholesterol

[0109] Advantageously, the methods described herein can be used to increase the fat content of a cell without increasing the amount of cholesterol in the cell. A high level of cholesterol in humans is associated with heart disease and other poor health outcomes, so avoiding an increase in cholesterol in fat-producing cells will be advantageous where the cells are used in food products. For example, adipocytes obtained by the methods described herein having higher fat content but with normal cholesterol levels can be incorporated directly into food products.

[0110] Cholesterol can be measured using any suitable technique known in the art, and in particular by stimulated raman scattering (SRS) or hyperspectral stimulated raman scattering (hsSRS) as described above. Using these techniques, cholesterol can be measured simultaneously with overall fat content.

[0111] Accordingly, described herein are methods for increasing the fat content of a cell, wherein the method does not increase the amount of cholesterol in the cell compared to a control cell cultured under substantially the same conditions but without being cultured at low pressure (10-200 kPa) and without being contacted by an agent that modulates a mechanosensitive ion channel (such as a Piezol agonist).

[0112] Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of’ or “consisting of’.

[0113] The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of and vice versa, wherever they occur herein.

[0114] It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise. The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0115] EXAMPLES

[0116] The experimental results below demonstrate that low-pressure stimulated vascular smooth muscle cells (VSMCs) (A7r5 and primary human and rat) display increased lipid droplet accumulation. The inventors further identified this increase in lipid accumulation was associated with calcium transients downstream of Piezol , and that Piezol activation can similarly induce lipid droplet accumulation in cultured VSMCs. The inventors further show that this mechanism is not limited to VSMCs, and that a similar response is observed when stimulating pre-adipocytes. Importantly, hyperspectral stimulated raman spectroscopy shows that Piezol alone can induce lipid droplet formation. In addition, a combination of pressure stimulation and Piezol activation leads to simultaneous lipid uptake / retention and lipid droplet formation. Thus, low-pressure stimulation and modulation of Piezol both lead to an increase in fat content in the treated cells.

[0117] Materials & Methods

[0118] Cell culture

[0119] A7r5 rat vascular smooth muscle cell line was obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1 % GlutaMAX, and 1 % penicillin / streptomycin. Primary vSMCs were obtained from explants of bovine aorta and from human aortic tissue from a healthy donor (04.35 Fll). Primary cells were used at passages 3-5 cultured in DMEM containing 20% FBS.

[0120] Antibodies, Plasmids and Reagents

[0121] Primary antibodies included Piezol (Novus Biologicals, NBP-78446), H3K9me3 (Cell Signaling, 13969S), cPLA (Santa Cruz Biotechnology, sc-454). Fluorescent secondary antibodies were from Jacksonlmmuno Research (Cy2-Goat Anti-Rabbit: 111-225-144 , Cy5-Goat Anti-Mouse: 115-175-146).

[0122] The following reagents were used at specified concentrations: Yodal (10pM, Bio-Techne, 5586 / 10), Dookul (50pM, Bio-Techne, 6568 / 10).

[0123] Human Piezo1-1591-EGFP was a kind gift from Charles Cox. ITPKA9-dTomato (Tractin-Tomato) was a gift from M. Schell. Immunostaining

[0124] Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 5 min and blocked with 5% bovine serum albumin (BSA) in PBS for 30 minutes. Cells were incubated overnight with primary antibodies diluted in 1 % BSA. The next day, cells were washed in PBS and incubated for 1 hour with secondary antibodies, again washed with PBS and mounted using Prolong Gold (P36930) from Invitrogen. Cells were imaged using Nikon Ti2 SoRa spinning disc microscope with 40x or 60x objectives, using a 1x or 2.8x SoRa disc.

[0125] For the LipidSpot™ assay, LipidSpot™ 488 Lipid Droplet Stain (Biotium, 1000x stock) was diluted in cell culture medium and cells were incubated with the diluted stain for 30 minutes at 37°C, protected from light. Subsequently cells were washed, fixed, stained with DAPI and imaged.

[0126] Real-Time PCR

[0127] A7R5 cells (3.5x105cells / well) were seeded in a six-well plate and exposed to DMSO or YODA1 10uM for 1 hour or 8 hours. Total RNA was extracted using the commercial kit RNeasy mini-Kit (Qiagen, Hilden, Germany). RNA purity and concentration were determined by spectrophotometry using Nanodrop ND-1000 (Thermo Fisher Scientific).

[0128] Total RNA (300 ng) was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) in a final volume of 25 pl. 5 pL of the cDNA solution were used for the RT-PCR experiments to measure the amount of CD68 and ABCA1 transcript. StepOnePlus Real Time PCR System (Applied Biosystems) was used to perform the Real-Time PCR reactions using Powerllp SYBR Green Master Mix (Applied Biosystems, A25742) according to the manufacturer’s protocol in a final volume of 12 pl. Primers were purchased from Integrated DNA Technologies (Coralville, USA) and used at 250 nM concentration. Differences in gene expression levels were determined by the 2-AACt formula, using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference gene. The following primers were used: CD68 forward 5’-CTTGGCTCTCTCATTCCCTTA C-3’, reverse 5’- TGTATTCCACTGCCATGTAGTT-3’; ABCA1 forward 5’-TTGGATTCGGCTGTGAGTATTT-3’, reverse 5’-GGACACTGAGGTGGTAAGATTG-3’; GAPDH forward 5’-GCAAGGATACTGAGAGCAAGAG-3’, reverse 5’-GGATGGAATTGTGAGGGAGATG-3’.

[0129] PDMS substrates

[0130] Polydimethylsiloxane (PDMS) was prepared as described before(36). In short, Sylgard 527 was mixed 1 :1 in order to obtain the 1 kPa stiffness. Placed on a roller for 45 min to thoroughly mix, then spin-coated with a 50i spin processor (SPS) onto coverslips and cured overnight at 60 C. Before cell plating, PDMS coverslips were plasma-treated and coated with fibronectin. Electroporation

[0131] Neon Transfection System (ThermoFisher Scientific) was used to electroporate the A7r5 cells. 3 x 105 cells were resuspended in a 10pl buffer and 2ug human Piezo1-1591-EGFP and 1 ug ITPKA9-dTomato were added. Two 20ms pulses at 1100 V were applied and then plated on fibronectin-coated coverslips.

[0132] Calcium Measurement

[0133] A7r5 cells were incubated 30min in 37C with Fluo4-AM. Next, cells were washed with a buffer and left to stabilize. Following the baseline recording, Yodal was added as a channel specific stimulator and recorded further for another 3-4 minutes. DMSO was used as control.

[0134] Scratch Assay

[0135] An equal number of A7r5 cells was plated on glass coverslips. The next day, a confluent cell monolayer was scratched with a sterile 10OOul tip. The cell debris were washed off and new media supplemented with Yodal or DMSO was added. After 24 hours at 37oC 5% CO2 the scratched area was assessed. Phase contrast images were taken using Leica DM IL Biological Inverted Microscope equipped with 10x objective lense. Images were quantified as percentage of wound closure using Imaged.

[0136] Cell proliferation assay

[0137] Cell proliferation was assessed using EdU-Click Assay and used according to the manufacturers protocol (Merck, BCK EDU-488). Briefly, cells were plated on fibronectin-coated glass coverslips. Treated accordingly and incubated with 5-Ethynyl-deoxyuridine (EdU), fixed, stained with DAPI and imaged using the SoRA Spinning Disc Confocal Microscope.

[0138] Pressure chamber

[0139] Pressure stimulation of A7r5 cells was performed in a Mechano-Culture TR stimulator, placed inside a tissue culture incubator. The stimulator was modified for low-pressure stimulation in the range of (human) normal (NBP) and hypertensive blood pressure (HT) and perfusion ports to supply presaturated cell culture medium. Cells were stimulated with a sinusoid profile (stretch: 0.5 s, duration: 1 s, hold: 0 s, recovery: 0.5 s) and alternating between set pressures of 16 kPa (peak load) and 8 kPa (pre-load) to reach a measured pressure profile of 120 / 60 mmHg for NBP stimulation and between 26 and 16 kPa for a measured pressure profile of 180 / 120 mmHg for hypertensive pressure stimulation. Cyclic pressure stimulations were performed for 12 hours, and static pressure stimulations (at peak pressure setting) were performed for 30 min. Control cells were placed inside the stimulator without applying pressure. Nanoindentation

[0140] Nanoindentation experiments were performed using an Opticsl 1 Chiaro Nanoindenter attached to a Leica DMI-8 microscope, as described previously. Cell measurements were performed above the nucleus with an R = 50 pm, k = 0.5 N / m probe (suitable for a stiffness range from approximately 0.5 and 80 kPa). The Hertzian contact model was used to fit the data.

[0141] Stimulated Raman Spectroscopy

[0142] An integrated laser system (picoEmerald™ S, Applied Physics & Electronics, Inc.) was used to produce two synchronised laser beams at 80 MHz repetition rate. A fundamental Stokes beam (1031 .4 nm, 2 ps pulse width) was intensity modulated by an electro-optic-modulator with >90% modulation depth, and a tunable pump beam (700-960 nm, 2 ps pulse width, <1 nm (10 cm'1) spectral bandwidth) was produced by a built-in optical parametric oscillator. The pump and Stokes beams were spatially and temporally overlapped using two dichroic mirrors and a delay stage inside the laser system and coupled into an inverted laser-scanning microscope (Leica TCS SP8, Leica Microsystems) with optimised near-IR throughput. SRS images were acquired using 40x objective (HC PL IRAPO 40x, N.A. 1.10 water immersion lens) with a 9.75-48 ps pixel dwell time over a 512 x 5i2 or a 1024 x 1024 frame. The Stokes beam was modulated with a 20 MHz EoM. Forward scattered light was collected by a S1 N. A. 1.4 condenser lens (Leica Microsystems). Images were acquired at 12-bit image depth. The laser powers measured after the objective lens were in the range 10-30 mW for the pump beam only, 10-50 mW for the Stokes beam only and 20-70 mW (pump and Stokes beams). The spatial resolution of the system is ~450 nm (pump wavelength = 792 nm).

[0143] Prior to imaging, the plates were aspirated and washed with PBS (2 x 2 mL), the cells were fixed with paraformaldehyde (4% in PBS, 15 min at rt), and washed with PBS (2 x 2 mL). The coverslips were then affixed to glass microscope slides with a PBS boundary between the glass layers prior to imaging. For spectral phasor analysis, a wavelength scan was conducted across the range 2800-3050 cm-1(0.4 nm re-tune, ~6 cm'1, 40 images) from a minimum of three replicate areas containing typically 15 cells per frame for each condition. The image stack was imported into Imaged and an average intensity projection was created. Segmentation of the phasor plot was performed manually using regions-of- interest to create images of discrete cellular locations including lipid droplets and cytoplasm regions. The corresponding average spectra for each ROI is plotted in Origin2018 and normalised between 0-1.

[0144] Piezol localization in vascular smooth muscle cells.

[0145] The inventors had previously studied vascular mechanosensing in response to pathophysiological arterial stiffness and hypertensive pressure. Thereby the inventors found that distinct mechanosensing pathways downstream of stiffness and pressure sensing converged to define the mechanoresponse (P. Swiatlowska et al., Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching. Sei Adv 8, eabm3471 (2022)). Especially, the inventors found elevated Ca2+ levels in vSMC following hypertensive pressure application (200 / 120 mmHg), with especially dramatic calcium increases in the nucleus, but the source of the calcium remained unclear. Piezol is a known Ca2+-permeable channel activated by mechanical stimuli that has been described in vSMC previously. The inventors hypothesized that Piezol was mediating the detected pressure response.

[0146] Super-resolution spinning disc confocal microscopy indicated the presence of Piezol in punctate localisation along the cell membrane, inside the cytoplasm and particularly pronounced in the nucleus and the nuclear membrane (Fig 1A-C). Quantification of the staining pattern indicated a ~7 fold enrichment in the nuclear lamina compared to the cytoplasm and a ~50% enrichment in the lamina compared to the nuclear staining. The inventors also verified the nuclear localisation and antibody specificity using transfection with a human Piezol -GFP plasmid (Fig 1 D).

[0147] To assess the physiological significance of Piezol activation in vSMC derived from different animal models, the inventors used Piezol specific activator, Yodal . A7r5, primary human, and primary bovine vSMC were incubated with Fluo-4 AM as a Ca2+ indicator. Following a baseline recording, cells were subjected to a 10pM Yodal . After treatment, an immediate response was observed with consistent Ca2+ increase in A7r5, primary human and bovine cells (Fig 1 E-J). The inventors again found a particular strong Ca2+ increase in the nucleus that was absent in the DMSO treated control groups. The response could be further suppressed, using the Piezol specific inhibitor Dookul , indicating that Piezol can mediate the mechanosensitive Ca2+ response in vSMCs (Fig 2).

[0148] Phenotypic changes following Piezol stimulation

[0149] The inventors tested the functional implications of Piezol stimulation on vSMC migration and proliferation, as hallmarks of phenotypic switching. Using an in vitro wound scratch assay, the inventors observed that following a 24-hour Yodal incubation, A7r5 cell motility was significantly decreased (Fig 3A,B). In contrast, using an EdU-Click Assay the inventors found significantly increased proliferation after Yodal treatment, when compared to the DMSO control (Fig 3C,D). Additionally, the inventors found a reduced cellular Young's modulus after Yodal treatment, consistent with the phenotypic transformation and previous observations after hypertensive pressure stimulation (Fig 3E) (Swiatlowska et al. (2022)). Moreover, gPCR testing after 8h Yodal treatment indicated increased CD68 and decreased ABCA1 levels, consistent with a macrophage-like or foam-cell phenotype (Fig 3F,G). Intriguingly, bioinformatic analysis for causal reasoning of the inventors previous proteomics data (Swiatlowska et al. (2022)) identified several molecules related to foam cell formation and lipid metabolism as potential regulators of the changes in the proteome between mechanically stimulated and control cells (Fig 3H, 6 out of top 12 hits; miRNA hits have been omitted from table for simplicity), further supporting the notion that mechanical stimulation would be sufficient to induce vSMC foam cell formation. Acute Piezol activation leads to rapid lipid droplet formation.

[0150] Since the inventors found expression of macrophage markers, the inventors next wanted to test for changes in the lipid content and lipid droplet formation. Stimulated raman scattering (SRS) offers label- free, non-destructive testing of cellular biomolecules. More recently, the development of hsSRS enabled the chemical mapping of molecules with similar raman spectra, such as cholesterol esters (CEs) or Triacylclycerides (TAGs), while the implementation of spectral phasor analysis of the hyperspectral datasets allows the differentiation of discrete cellular compartments, such as nuclei, membrane, cytoplasm or lipid droplets (W. J. Tipping et al., Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug-cell interactions. Chem Sci S, 3468-3476 (2022); D. Fu et al., In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J Am Chem Soc 136, 8820-8828 (2014)). The inventors therefore employed hsSRS for the analysis of lipid droplet content and chemical signatures after pressure treatment and / or Piezol activation.

[0151] The inventors found clear and rapid increases in lipid droplet formation after 30 min pressure stimulation or Piezol activation (Fig 4A,B). The lipid droplet formation was further enhanced through combined pressure stimulation and Yodal treatment. Yodal dependent lipid droplet formation was further confirmed using the lipid stain Lipidspot™ (Fig 6).

[0152] Closer inspection of the lipid composition suggested differences between pressure and Yodal treated samples. Namely, an overall increase in lipids (Fig 3C, total lipid peak at ~2850cm-1 (20); here shifted to 2868cm-1) in cytoplasm and lipid droplets in pressure treated cells, which was absent in Yodal treated cells, indicating that Piezol independent mechanisms were leading to additional lipid uptake in pressure stimulated cells. Moreover, the inventors detected a reduction of TAGs (peak at 3018cm-1) in Yodal treated cells. Because TAG synthesis is the preceding step for lipid droplet formation(21), this surprisingly suggested that the rapid lipid droplet formation, without simultaneous lipid uptake, was leading to a depletion in the bioavailable lipids. In agreement with this, the inventors found a dramatic increase in lipid droplet formation and overall increase in lipids (in cytoplasm and lipid droplets) after combined pressure and Yodal treatment and no significant reduction in TAGs. No changes were found for cholesterol esters (peak / shoulder at 2962cm-1).

[0153] The inventors further found similar changes in a mouse model for intima hyperplasia after carotid artery ligation, where hsSRS could identify the extracellular matrix, cytoplasm and lipid droplet signatures. Importantly, this is to the best of the inventor’s knowledge the first time this technique was applied to tissue samples, demonstrating the outstanding capabilities of this method. Lipid droplets were dramatically increased in intimal hyperplasia and especially in the phenotypically switched cells towards the luminal side (Swiatlowska et al. (2022)). Similar to Yodal treated cells and consistent with a lack of hypertensive pressure in this model, the inventors found reduced level of TAGs, but no change in overall lipids. The inventors tested if the effect of Yodal and hypertensive pressure was specific for VSMCs, or could be also detected in other lipid droplet forming cell types. Indeed, pre-adipocytes reacted to both pressure and Yodal treatment, with increased lipid droplet formation seen in both pressure treated and Yodal treated cells (Figure 6B, 6C).

[0154] Overall, these results demonstrate that Piezol activation alone can lead to a rapid stimulation of lipid droplet formation. Pressure stimulation also leads to rapid lipid droplet formation, accompanied by an increased uptake of extracellular lipids, thereby increasing the overall fat content of the cell.

[0155] The invention may further be understood by reference to the following numbered clauses:

[0156] 1 . A method for increasing the fat content of a cell, the method comprising modulation of a mechanosensitive signalling pathway in the cell.

[0157] 2. The method of clause 1 , wherein said modulation comprises culturing the cell at a pressure from 10 to 200 kPa.

[0158] 3. The method of clause 2, wherein the cell is cultured at a pressure from 10 to 40 kPa.

[0159] 4. The method of any one of clauses 2-3, wherein the cell is cultured at a pressure from 10 to 30 kPa.

[0160] 5. The method of any one of clauses 2-4, wherein the cell is cultured at a first lower pressure and a second higher pressure.

[0161] 6. The method of clause 5, wherein the lower pressure comprises a pressure from 10-20 kPa and the higher pressure comprises a pressure from 21-30 kPa.

[0162] 7. The method of clause 5 or 6, wherein culturing the cell comprises alternating between the lower pressure and the higher pressure for a plurality of cycles.

[0163] 8. The method of clause 7, wherein culturing the cell comprises alternating between the lower pressure and the higher pressure for at least 900 cycles.

[0164] 9. The method of clause 7 or 8, wherein culturing the cell comprises alternating between the lower pressure and the higher pressure for at least 1 ,800 cycles.

[0165] 10. The method of any one of clauses 5 to 9, wherein each application of the lower pressure and each application of the higher pressure independently has a duration of up to 5 seconds. The method of any one of clauses 2-4, wherein the cell is cultured at a hydrostatic pressure. The method of any one of clauses 2-11 , wherein the cell is cultured for 30 minutes to 72 hours. The method of any one of clauses 2-12, wherein the cell is cultured for 1 hour to 24 hours. The method of any one of clauses 2-13, wherein the cell is cultured for at least 2 hours. The method of any preceding clause, wherein said modulation comprises contacting the cell with a modulator of a mechanosensitive ion channel. The method of clause 15, wherein the modulator comprises an agent which activates the mechanosensitive ion channel. The method of clause 15 or 16, wherein the ion channel is a Ca2+ ion channel. The method of any one of clauses 15-17, wherein the ion channel is Piezol . The method of any one of clauses 15-17, wherein the ion channel is Piezo2. The method of any preceding clause, wherein said modulation comprises contacting the cell with a Piezol agonist. The method of any preceding clause, wherein said modulation comprises contacting the cell with a Piezo2 agonist. The method of any one of clauses 16-20, wherein the agent, Piezol agonist or Piezo2 agonist is a small molecule. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is Yodal . The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is a Yodal analogue. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is Yoda2. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is a Yoda2 analogue. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is Jedil . The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is a Jedil analogue. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is Jedi2. The method of any one of clauses 16-22, wherein the agent, Piezol agonist or Piezo2 agonist is a Jedi2 analogue. The method of any preceding clause, wherein the mechanosensitive signalling pathway is a pressure-sensitive signalling pathway. A method for increasing the fat content of a cell, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and b. contacting the cell with an agent that activates a mechanosensitive ion channel; wherein steps (a) and (b) are performed simultaneously or sequentially in any order. The method of any preceding clause, wherein the method increases lipid droplet formation in the cell. The method of any preceding clause, wherein the method increases lipid retention by the cell. The method of any preceding clause, wherein the method increases lipid uptake by the cell. The method of any preceding clause, wherein the method increases lipid phagocytosis by the cell. The method of any preceding clause, wherein the method increases fat content in the cytoplasm of the cell. The method of any preceding clause, wherein the fat content is measured by stimulated raman scattering (SRS) or hyperspectral stimulated raman scattering (hsSRS). 39. The method of any preceding clause, wherein the fat content comprises saturated fat, unsaturated fat, total fat, triacylglycerides, or a combination thereof.

[0166] 40. The method of any preceding clause, wherein the method does not increase the amount of cholesterol in the cell.

[0167] 41 . The method of any preceding clause, wherein the cell is selected from the group consisting of: a prokaryotic cell, a eukaryotic cell, a non-mammalian cell, a mammalian cell, a fungal cell, and a yeast cell.

[0168] 42. The method of any preceding clause, wherein the cell is selected from the group consisting of: an adipocyte, a pre-adipocyte, a dedifferentiated fat (DFAT) cell, a stem cell, a mesenchymal stem cell, a satellite cell, a vascular smooth muscle cell, and induced pluripotent stem cells (iPSCs).

[0169] 43. The method of any preceding clause, wherein the cell is derived from a pig, a cow, a bird, a turkey, a chicken, a duck, a goose, a sheep, a goat, a horse, an insect, a reptile, an amphibian, or a fish.

[0170] 44. Use of a Piezol agonist to increase the fat content of a cell.

[0171] 45. Use of clause 44, wherein the Piezol agonist is a small molecule.

[0172] 46. Use of clause 44 or 45, wherein the Piezol agonist is Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof.

[0173] 47. Use of a Piezo2 agonist to increase the fat content of a cell.

[0174] 48. Use of clause 47, wherein the Piezo2 agonist is a small molecule.

[0175] 49. Use of clause 47 or 48, wherein the Piezo2 agonist is Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Dookul , a Dookul analogue, Jedil , a Jedil analogue, Jedi2, a Jedi2 analogue, or any combination thereof.

[0176] 50. A method for producing cultured fat from a population of cells, the method comprising culturing the cells under conditions suitable to increase the fat content of the cells, said conditions comprising: a. culturing the cells at a pressure from 10 to 200 kPa, and / or b. contacting the cells with a modulator of a mechanosensitive signalling pathway. 51 . The method of clause 50, wherein the population of cells comprises cells selected from the group consisting of: adipocytes, pre-adipocytes, dedifferentiated fat (DFAT) cells, stem cells, mesenchymal stem cells, satellite cells, vascular smooth muscle cells (VSMCs), or a combination thereof.

[0177] 52. The method of any preceding clause, wherein the method is performed in a bioreactor.

[0178] 53. Cultured fat obtained or obtainable by the method of any one of clauses 50-52.

[0179] 54. A process for producing a food product, the process comprising: a. culturing a population of cells under conditions suitable to increase the fat content of the cells, said conditions comprising: i. culturing the cells at a pressure from 10 to 200 kPa, and / or ii. contacting the cells with a modulator of a mechanosensitive signalling pathway; and b. combining the population of cells with at least one other composition to produce the food product.

[0180] 55. A process for producing a food product, the process comprising: a. providing a cultured fat produced according to the method of any one of clauses SO- 52, or a cultured fat according to clause 53; and b. combining the cultured fat with at least one other composition to produce the food product.

[0181] 56. The method of any one of clauses 50-52, or the process of clause 54, wherein conditions (i) and / or (ii) cause differentiation of the population of cells into adipocytes or pre-adipocytes.

[0182] 57. The process of any one of clauses 54-56, wherein the at least one other composition is selected from the group consisting of: cultured protein, muscle cells, muscle tissue, connective tissue, fat, and combinations thereof.

[0183] 58. A method for differentiating a cell into an adipocyte or a pre-adipocyte, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with a modulator of a mechanosensitive signalling pathway.

[0184] 59. The method of clause 58, wherein the cell is selected from the group consisting of: a dedifferentiated fat (DFAT) cell, a stem cell, a mesenchymal stem cell, a bone marrow derived stem cell, an induced pluripotent stem cell, or a satellite cell. 60. A method for transforming a non-fat storing cell into a fat storing cell, comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with an agent that activates a mechanosensitive ion channel.

[0185] EQUIVALENTS AND SCOPE

[0186] Those skilled in the art will appreciate that the present invention is defined by the appended claims and not by the Examples or other description of certain embodiments included herein.

[0187] Similarly, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0188] Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art, or according to manufacturer’s specifications.

[0189] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

Claims

CLAIMS1 . A method for increasing the fat content of a cell, the method comprising modulation of a mechanosensitive signalling pathway in the cell.

2. The method of claim 1 , wherein said modulation comprises culturing the cell at a pressure from 10 to 200 kPa.

3. The method of claim 2, wherein the cell is cultured at a pressure from 10 to 40 kPa.

4. The method of claim 2 or 3, wherein the cell is cultured at a first lower pressure and a second higher pressure.

5. The method of claim 4, wherein the lower pressure comprises a pressure from 10-20 kPa and the higher pressure comprises a pressure from 21-30 kPa.

6. The method of claim 4 or 5, wherein culturing the cell comprises alternating between the lower pressure and the higher pressure for a plurality of cycles.

7. The method of any one of claims 2-6, wherein the cell is cultured for 30 minutes to 72 hours.

8. The method of any preceding claim, wherein said modulation comprises contacting the cell with a modulator of a mechanosensitive ion channel.

9. The method of claim 8, wherein the ion channel is a Ca2+ ion channel.

10. The method of claim 8 or 9, wherein the ion channel is Piezol or Piezo2.

11. The method of any preceding claim, wherein said modulation comprises contacting the cell with a Piezol agonist and / or a Piezo2 agonist.

12. The method of any one of claims 8-11 , wherein the modulator, Piezol agonist or Piezo2 agonist is a small molecule.

13. The method of any one of claims 8-12, wherein the modulator, Piezol agonist or Piezo2 agonist is Yodal , a Yodal analogue, Yoda2, a Yoda2 analogue, Jedil , a Jedi 1 analogue, Jedi2, or a Jedi2 analogue.

14. A method for increasing the fat content of a cell, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and b. contacting the cell with an agent that activates a mechanosensitive ion channel;wherein steps (a) and (b) are performed simultaneously or sequentially in any order.

15. The method of any preceding claim, wherein the method increases lipid droplet formation in the cell, increases lipid retention by the cell, increases lipid uptake by the cell, increases lipid phagocytosis by the cell, and / or increases fat content in the cytoplasm of the cell.

16. The method of any preceding claim, wherein the method does not increase the amount of cholesterol in the cell.

17. The method of any preceding claim, wherein the cell is selected from the group consisting of: a prokaryotic cell, a eukaryotic cell, a non-mammalian cell, a mammalian cell, a fungal cell, and a yeast cell.

18. The method of any preceding claim, wherein the cell is selected from the group consisting of: an adipocyte, a pre-adipocyte, a dedifferentiated fat (DFAT) cell, a stem cell, a mesenchymal stem cell, a satellite cell, a vascular smooth muscle cell, and induced pluripotent stem cells (iPSCs).

19. Use of a Piezol agonist to increase the fat content of a cell.

20. Use of a Piezo2 agonist to increase the fat content of a cell.21 . A method for producing cultured fat from a population of cells, the method comprising culturing the cells under conditions suitable to increase the fat content of the cells, said conditions comprising: a. culturing the cells at a pressure from 10 to 200 kPa, and / or b. contacting the cells with a modulator of a mechanosensitive signalling pathway.

22. Cultured fat obtained or obtainable by the method of claim 21 .

23. A process for producing a food product, the process comprising: a. culturing a population of cells under conditions suitable to increase the fat content of the cells, said conditions comprising: i. culturing the cells at a pressure from 10 to 200 kPa, and / or ii. contacting the cells with a modulator of a mechanosensitive signalling pathway; and b. combining the population of cells with at least one other composition to produce the food product.

24. The process of claim 23, wherein the at least one other composition is selected from the group consisting of: cultured protein, muscle cells, muscle tissue, connective tissue, and combinations thereof.

25. A method for differentiating a cell into an adipocyte or a pre-adipocyte, the method comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with a modulator of a mechanosensitive signalling pathway.

26. A method for transforming a non-fat storing cell into a fat storing cell, comprising: a. culturing the cell at a pressure from 10 to 200 kPa, and / or b. contacting the cell with an agent that activates a mechanosensitive ion channel.

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