Methods of manufacturing myocyte-laden scaffolds

Abstract

The present disclosure provides methods of manufacturing myocyte-laden scaffolds. Particularly, the methods as disclosed herein comprise forming a fibre scaffold from myocyte precursor cells such that the cells are confined in the fibre under a static tension, and culturing the myocyte precursor cells confined in the fibre from under the static tension for differentiating the myocyte precursor cells into myocytes to obtain the myocyte-laden scaffold.

Description

METHODS OF MANUFACTURING MYOCYTE-LADEN SCAFFOLDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the Singapore provisional application no.10202500214X filed on 23 January 2025, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cellular agriculture. Particularly, it relates to methods of manufacturing myocyte-laden scaffolds for cultivating meat in vitro.BACKGROUND

[0003] Cellular agriculture is an emerging field that aims to manufacture agricultural products derived from cell culture technology rather than traditional farming and harvesting of livestock or plants. Cellular agriculture has promising potential in large scale production of food products, with less environmental burden. In addition, cellular agriculture offers benefits such as no slaughtering of animals, easier pathogen control, and potentially antibiotic-free and pollution-free production in the production process.

[0004] Cell-based meat, also referred to as cultured meat, clean meat, lab-grown meat or cultivated meat, utilizes cellular agriculture techniques and biomanufacturing technology of animal cell lines in order to create edible food structures similar to animal meats.

[0005] Scalability remains a problem in the production of cultivated meat. Known methods for cultivating meat typically involve fabricating scaffolds separately and then seeding cells onto the fabricated scaffolds in order to guide cell attachment, proliferation, and differentiation of cells into edible meat products. However, these methods often require multiple steps, including scaffold fabrication, sterilization, and subsequent cell seeding, which can complicate scalability and increase production cost of producing cultivated meat.

[0006] There is therefore an unmet need for alternative methods for cultivated meat applications.SUMMARY OF INVENTION

[0007] In one aspect, the present disclosure relates to a method of manufacturing a myocyteladen scaffold, wherein the method comprises: (a) preparing a mixture comprising myocyte pre-cursor cells and a food-grade polymeric solution; (b) forming a fibre from the mixture from(a) such that the myocyte-precursor cells are confined in the fibre under a static tension; (c) culturing the myocyte-precursor cells confined in the fibre from (b) under the static tension for differentiating the myocyte pre-cursor cells into myocytes to obtain the myocyte-laden scaffold.

[0008] In another aspect, the present disclosure relates to a myocyte-laden scaffold obtained or obtainable from the methods as disclosed herein.

[0009] In another aspect, the present disclosure relates to a cultivated meat product obtained or obtainable from the method as disclosed herein.

[0010] In another aspect, the present disclosure relates to a kit for obtaining a myocyte-laden scaffold comprising: (a) one or more food-grade scaffold materials; (b) one or more populations of myocyte precursor cells; (c) a food-grade culture medium; and (d) a fibre-forming system.BRIEF DESCRIPTION OF THE DRAWINGS[OH] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0012] Figure 1 provides an illustration to show the one-step process of fabricating adipose-derived stem cell (ADSC)-laden scaffold in an example as disclosed herein. In this example, the ADSC-laden microfiber scaffolds which were spun and collected on the drum sleeves, were submerged in basal growth culture medium for culture without being removed from the drum sleeves. Without removing the spun cell-laden microfiber from the drum sleeves, the micro fiber is held under a constant static tension.

[0013] Figure 2 provides data showing the storage (G’) and loss (G”) modulus of hydrogel discs obtained from a polymeric solution comprising: 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate -4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4). The hydrogel discs were subjected to an amplitude sweep of 0.1-100% strain rate, at an angular frequency of 10 rad / s and at 37 °C.

[0014] Figure 3 provides data showing the storage (G’j and loss (G”) modulus of hydrogel discs obtained from a polymeric solution comprising: 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4). The hydrogel discs were subjected to a frequency sweep of 0.1-100 rad / s, at a strain of 1%, within the linear viscoelastic region (LVR) and at 37°C.

[0015] Figure 4 provides data showing the gel strength or stiffness of hydrogel discs obtained from a polymeric solution comprising: 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / valginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) as measured by storage modulus. The storage modulus of the hydrogels taken at 1% strain and at angular frequency of 1 Hz. Statistical analysis: *samples are significantly different from each other with a p-value of < 0.05.

[0016] Figure 5 provides data showing that adipose-derived stem cells (ADSCs) can be differentiated into myocytes in vitro. The photomicrographs show ADSCs cultured on treated tissue culture plates for about 21 days in a myogenic differentiation media comprising DMEM, 2% horse serum and 1% penicillin / streptomycin. The cells were stained with DAPI, f-actin and the late-stage myogenic marker, myosin heavy chain (mhc). The percentage indicate the average myogenic yield of cells expressing mhc. Scale bar = 100 pm.

[0017] Figure 6 provides data to show the diameter of wet-spun microfiber scaffolds obtained from polymeric solution comprising alginate and gelatin of different concentrations. The top panel shows representative images of micro fibers from a polymeric solution comprising 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) wet-spun at flow rate of 0.1 ml / min, and collected at a drum speed of 80 round per minute (rpm). The lines across the micro fibers on the images indicate the diameter of the micro fibers. The bottom panel shows a bar chart presenting the average and standard errors of the diameters of the microfiber scaffolds. Statistical analysis: samples labelled * are significantly different from each other with a p-value < 0.05; samples labelled # are significantly different from each other with p-value < 0.05; sample size, n=100. Scale bar = 100 pm.

[0018] Figure 7 provides photomicrographs to show that adipose-derived stem cells (ADSCs) encapsulated in 3% w / v alginate-3% w / v gelatin (A3G3) and 3% w / v alginate-4% w / v gelatin (A3G4) microfiber scaffolds are viable. The encapsulated cells were cultured in a growth medium comprising DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Representative images of ADSC-laden microfiber scaffold stained with Calcein-AM (LIVE) and Ethidium homodimer- 1 (DEAD), to indicate live and dead cells respectively, after about 21 days in culture. Scale bar = 200 pm.

[0019] Figure 8 provides data to show that culturing of adipose-derived stem cells (ADSCs) confined within a micro fiber scaffold improves myogenesis compared to two-dimensional (2D) culturing of the cells on a tissue culture plate in the presence of a myogenic differentiation medium. The figure shows qPCR analysis of early stage myogenic marker, myf5, and late stage myogenic marker, mhc in (i) cells that are cultured on tissue culture plate under 2D conditionswith basal growth media (Untreated); (ii) cells cultured on tissue culture plate under 2D conditions with myogenic differentiation medium comprising DMEM, 2% horse serum and 1% penicillin / streptomycin (Differentiation Media); and (iii) cells confined within a scaffold comprising 3% w / v alginate-4% w / v gelatin cultured in a basal growth medium (A3G4). Statistical analysis: * p-value < 0.05; # p-value < 0.1; sample size: n=3.

[0020] Figure 9 provides data showing the effect of stiffness of the microfiber scaffold on myogenesis of the adipose-derived stem cells (ADSCs) encapsulated in it. The encapsulated cells were cultured in a growth medium comprising DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin for 21 days. ADSC-laden micro fiber scaffolds comprising different alginate-gelatin concentration, each representing different stiffness or gel strength, were stained with the late-stage myogenic marker, myosin heavy chain (mhc), f-actin and DAPI, after about 21 days in culture. In accordance with Figure 4, the micro fiber scaffold from 3% w / v alginate-3% w / v gelatin (A3G3) and 3% w / v alginate-4% w / v gelatin (A3G4) has a gel strength of 9.84 ± 0.238 kPa and 19.2 ± 0.252 kPa, respectively; while the microfiber scaffold formed from 4% w / v alginate-4% w / v gelatin (A4G4) is the strongest with a gel strength or stiffness of 32.8 ± 0.259 kPa. The percentages in the figure indicate the myogenic yield, which is determined by the percentage of cells expressing mhc within the respective microfiber scaffold. The A4G4 microfiber scaffold, which represents the highest gel strength, has the highest percentage of cells stained with mhc, indicating that higher stiffness of the scaffold promotes myogenesis of ADSCs. Scale bar = 100 pm.

[0021] Figure 10 provides data showing the myogenic yield of adipose-derived stem cells (ADSCs) encapsulated in 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) micro fiber scaffolds at day 7 (D7), day 14 (DI 4) and day 21 (D21) in culture. The encapsulated cells were cultured in a growth medium comprising DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Myogenic yield was determined by the percentage of cells expressing the late-stage myogenic marker, myosin heavy chain (mhc). As can be seen from the graph, higher percentages of cells confined in A3G4 and A4G4 microfibers express mhc, indicating that higher gel strength or stiffness promotes myogenesis of ADSCs. Statistical analysis: #fO samples with the same symbol were significantly different from each other with p-value < 0.05; sample size, n=3

[0022] Figure 11 provides data to show that the myogenic yield of adipose-derived stem cells (ADSCs) encapsulated in 3% w / v alginate-4% w / v gelatin is reduced when the cell-laden microfibers are cultured with reduced static tension. In the ADSC-laden microfibers cultured in static tension (A3G4 group), the cell-laden micro fibers were wet-spun and collected on drumsleeves, and submerged in growth medium comprising DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin for culture without being removed from the drum sleeves. Thus, the cell-laden microfibers were held under a constant tension as they remained wound on the drum sleeve. In the ADSC-laden microfibers cultured under reduced static tension (A3G4TLR group), the cell-laden micro fibers were removed from the drum sleeves after wet spinning, and were cultured in suspension with the same culture medium. The cell-laden microfibers were released from static tension upon removal from the drum sleeve and thus experienced reduced static tension. The cell-laden micro fibers of A3G4 and A3G4TLR groups were stained with late-stage myogenic marker, myosin heavy chain (mhc), f-actin and DAPI on day 21 of culture. Myogenic yield is determined by the percentage of cells expressing the late-stage myogenic marker, myosin heavy chain (mhc), and are indicated in the figure. The A3G4 group had higher myogenic yield at 76% compared to the A3G4TLR, which had lower myogenic yield at 53%. The data indicates that reduced static tension in the cell-laden scaffold reduces myogenic yield. Scale bar = 100 pm.

[0023] Figure 12 provides data showing the myogenic yield of adipose-derived stem cells (ADSCs) encapsulated in 3% w / v alginate-4% w / v gelatin cultured under static tension (A3G4 group) or reduced static tension (A3G4TLR group). The percentages of cells stained positively with early-stage myf5, mid-stage myg and late-stage mhc myogenic markers encapsulated in the cell-laden micro fibers of A3G4 and A3G4TLR groups on day 21 of culture are shown in the bar chart. Culturing of cell-laden microfibers under reduced static tension reduces myogenic yield as seen by the reduction in the percentage of mhc-positive cells. Statistical analysis: ‘n.s.’, not significantly different; *p-value < 0.05; #p-value < 0.01. Sample size, n=3.

[0024] Figure 13 provides an analysis of the stress relaxation profiles of cell-laden microfiber comprising 3% w / v alginate-4% w / v gelatin, which are held in tension (A3G3) or released from tension (A3G4TLR). In the process of being wet-spun, the microfibers experience mechanical stress as they are being drawn from the needle to the drum sleeves. However, the alginate-gelatin composite microfibers being viscoelastic will experience stress relaxation over time. To understand the stress relaxation profile of the micro fiber, the characterization of the stress relaxation of a gel disc, made from the same material and composition, using a rheometer was conducted. To mimic cell-laden microfibers held in static tension, a constant strain of 0.851 was applied to hydrogel discs made from 3% w / v alginate-4% w / v gelatin with a rheometer for 30 minutes (1800s; A3G4 group). To mimic the condition with reduced static tension (i.e., the wet-spun micro fibers were removed from drum sleeves 15 minutes after wet-spinning), a strain of 0.851 was first applied to separate hydrogel discs for 15 minutes (900s) before it was reducedto a strain of 0.1, for the next 15 minutes (900s; A3G4TLR group). As can be seen from the graph, while the strain (tension) applied to the microfibers remain constant, the microfibers undergo relaxation over time, which is consistent with the behavior of a viscoelastic material. Even though both A3G4 and A3G4TLR hydrogels undergo relaxation, the stress experienced by the A3G4 hydrogels remained higher due to the applied strain (tension), at about 700 Pa after 15 minutes, compared to A3G4TLR hydrogels, which is lower than 100 Pa.

[0025] Figure 14 provides a comparison between the stress relaxation profiles of hydrogel discs mimicking cell-laden microfibers held in static tension obtained from rheological measurement (A3G4 raw data), and modeling using Equation 1 (A3G4 fitted curve). Equation 1 represents the constitutive equation of the SLS (standard linear solid) model with a finite loading element, where kGR and 0<k<l. Time is represented by t, ^ relaxation (t) refers to stress as a function of time during the stress relaxation process, and E l represents the parameter in the standard linear model relevant to viscoelastic properties and is to be determined in the analysis. The strain rate is represented by r, and r_c and r_R are the creep time constant and relaxation time constant respectively. The constant strain during the stress relaxation process is represented by £_0. The coefficient of determination (R2) for this curve fitting is 0.9981, which is indicative of high correlation between the experimental data and the modelled data.

[0026] Figure 15 provides a comparison between the stress relaxation profiles of hydrogel discs mimicking cell-laden microfibers with reduced static tension obtained from rheological measurement (TLR Raw data), and modeling using Equation 1 as shown in Figure 14 (TLR Fitted curve). This curve fitting was performed after reducing the strain from 0.851 to 0.1 at 900 seconds, which simulates the release of tension as the microfibers were removed from the drum sleeves. The R2value for this curve fitting measures at 0.9991, which indicates high correlation between the experimental data and the modelled data.

[0027] Figure 16 provides the modeling of stress relaxation profiles for the microfibers cultured under tension (A3G4 Fitted 21 days) or reduced tension (TLR Fitted 21 days) over 21 days in culture using SLS model with finite loading element according to Equation 1 shown in Figure 14. The modeled data shows that stress experienced by the microfibers of the A3G4 group consistently exceeded the stress experienced by A3G4TLR group throughout the entire 21 -day culture period. A3G4TLR group experienced a rapid and substantial reduction, reaching a plateau in less than a day; while A3G4 group exhibited a significant initial drop in stress and reached a stress of about 700 Pa, the stress experienced by the A3G4 micro fibers did not reach the same low levels as observed for A3G4TLR group.

[0028] Figure 17 provides data showing the difference in morphology of adipose-derived stem cells (ADSCs) confined in the microfibers held in tension (A3G4) or reduced tension (A3G4TLR) over different days in culture. The cell-laden microfibers were stained with f-actin and DAPI on day 1 (DI), day 3 (D3) and day 7 (day 7) in culture. The cell aspect ratio is larger in cells of A3G4 group compared to cells in A3G4TLR group, which indicates that the cells confined in microfibers held in tension are more elongated in morphology compared to the cells confined in micro fibers under reduced static tension.

[0029] Figure 18 provides data showing the difference in morphology of the nuclei of cells confined in micro fibers held in tension (A3G4) or reduced tension (A3G4TLR). Photomicrographs of immunofluorescence staining of f-actin, DAPI, and merged images of the cell-laden micro fibers A3G4 group and A3G4TLTR group are shown alongside their respective phase-contrast images 21 days (D21) in culture. Scale bar = 50pm. The adjacent bar chart compares the nucleus aspect ratio of the two groups, and shows that nuclei in the A3G4 group had a higher aspect ratio than the nuclei in the A3G4TLR group, which is indicative that the nuclei of cells confined in microfibers held in tension have a more elongated morphology compared to the nuclei confined in microfibers with reduced tension. Statistical analysis: *p-value < 0.1; sample size, n=3.

[0030] Figure 19 shows photomicrographs of f-actin-stained adipose-derived stem cells (ADSCs)-laden microfibers held in tension (A3G4-D21) or reduced tension (A3G4TLR-D21) and cultured for about 21 days, and their respective fast fourier transformation (FFT) frequency distribution images. The top panel shows Z-stacked confocal image of cell-laden microfibers stained with f-actin. Scale bar = 50 pm. The middle panel shows cropped and rotated images of z-stacked confocal image of the cell-laden microfibers for analysis of f-actin orientation. The image is rotated such that the longitudinal axis of the microfiber aligns with the east direction and is set as the 0° reference. Scale bar = 20 pm. The bottom panel shows the FFT frequency distribution of the middle panel after it was rotated by 90° to correct for mathematical transformation. The pixels arrangement in the A3G4-D21 group showed a non-random, elliptical distribution, whereas the pixels distribution in the A3G4TLR-D21 group are in a more circular pattern as the pixel intensities are randomly distributed without specific orientation.

[0031] Figure 20 provides line plots illustrating the difference in directionality or alignment of cells confined in microfibers held in static tension during culture (A3G4, top panel) and cells within confined in micro fibers with reduced static tension during culture (A3G4TLR, bottom panel) by plotting the angle of orientation of actin filaments against it normalized frequency. The longitudinal direction of the micro fibers was established as 0°. The results showed that theactin filaments of cells within microfibers held in static tension exhibited a clear and singular peak at 0°, indicating that the actin filaments were predominantly oriented along the longitudinal axis of the microfibers, unlike the actin filaments of cells within microfibers with reduced static tension, which lack a specific preference for alignment along any particular axis.

[0032] Figure 21 provides an example of a cultivated meat prototype made by stacking myocyte-laden micro fibers obtained from about 21 days in culture on drum sleeves in a growth media containing DMEM + 10% FBS + 1% penicillin / streptomycin. Alginate was used as a food binder to glue and bind multiple cell-laden microfibers in place. Scale bar = 1 cm.

[0033] Figure 22 provides a bar chart for showing the water content of pork loin and an example of the cultivated meat prototype, PA3G4, fabricated with wet-spun cell-laden microfibers and assembled by binding a plurality of the differentiated myocyte-laden micro fibers together after about 21 days in culture. Statistical analysis: * groups are significantly different from each other with p-value < 0.01; sample size, n=3.

[0034] Figure 23 provides images showing pork loin and examples of the cultivated meat prototype, PA3G4, before (raw) and after cooking with 10 minutes of boiling or 20 seconds of frying.

[0035] Figure 24 provides data showing the loss of mass detected in pork loin and PA3G4 cultivated meat prototype after boiling and frying. Statistical analysis: * significantly different from each other with a p value of < 0.01; sample size, n=3.

[0036] Figure 25 provides data illustrating the total amount of soluble protein content, with reference to their respective dry weights, in raw pork loin samples (Pork), cultivated meat prototype (PA3G4) and 3% w / v alginate-4% w / v gelatin hydrogel (A3G4) samples. Statistical analysis: * significantly different from each other with a p-value < 0.005; sample size, n=3.

[0037] Figure 26 shows the typical force profile of a sample when it is subjected to a two-compression test with a texture profile analyser.

[0038] Figure 27 shows the texture profile analysis of pork loin (PORK) and the cultivated meat prototype (PA3G4) when subjected to the two-compression test.

[0039] Figure 28 provides data illustrating the hardness and chewiness of pork loin (PORK vol) and the prototype cultivated meat (PA3G4_vol) samples, normalized to their respective volumes. The cultivated meat prototype was found to be softer, and less chewy than pork loin. Statistical analysis: groups labelled with * and #, respectively, are significantly different from each other, with a p-value < 0.01; sample size: n=3.DEFINITION OF TERMS

[0040] As used herein, the term “scaffold” refers to a three-dimensional structural framework comprising a material or a composition of materials that provides a surface suitable for adherence and proliferation of cells. A scaffold can provide mechanical stability and support. A scaffold can be in a particular shape or form so as to influence or delimit a three-dimensional shape or form assumed by a population of cells. Such shapes or forms can include, but are not limited to, fdms, ribbons, cords, sheets, flat discs, cylinders, spheres, fibres, or any three-dimensional amorphous shapes. Amorphous means that the surface shape is not uniform, for example, the shape of a scaffold material having irregularities. Many cells, for example stem cells, require a three-dimensional surrounding to grow and, in case of stem cells, differentiate into a desired cell type.

[0041] As used herein, a “cell-laden scaffold” refers to a biomaterial construct that incorporates living cells into a three-dimensional structural support network formed from scaffold materials. In other words, the cells are encapsulated by materials that formed the scaffold and held in a three-dimensional structure. The cells are encapsulated in the scaffold while the cell-laded scaffold is formed using methods described herein, such as wet spinning or other forms of fiber extrusion. In the present disclosure the cell-laden scaffold comprises edible (or food-grade), cell-supporting three-dimensional structures that provide framework for cell growth. In some examples, the cell-laden scaffold can be a hydrogel. In some examples, the cell-laden scaffold as disclosed herein can be a myocyte precursor cell-laden scaffold. In some examples, the cellladen scaffold as disclosed herein can be a myocyte-laden scaffold, wherein the myocytes precursor cells encapsulated in the scaffold have differentiated into mature myocytes.

[0042] As used herein, the term “static tension” refers to a persistent, constant force applied to a material / structure or created in a material / structure without any movement of dynamic variation. When static tension is applied externally, the material experiences an internal stress. The internal stress experienced by the material evolves over time due to creep and relaxation. Thus, a stress-relaxation response describes how the internal stress in a viscoelastic material, such as a fiber or polymer, decreases over time when it is held at constant tension or strain. As described herein, the changes in internal stress of a fiber or the stress-relaxation response of the fiber can be measured using a rheometer over a period of time, or can be calculated using a viscoelastic model. In the context of a fiber, static tension can be the force applied to the fiber during its formation or processing that keeps the fiber stretched and aligned. As described in the present disclosure, static tension is applied during fiber processing, for example, through the use of drum sleeves, the fibers can be maintained in an extended state and thus reducing the relaxation of the fibers during collection.

[0043] As disclosed herein, the term “stiffness” refers to a mechanical characteristic of a material or structure to resist deformation under an applied force. As disclosed herein, storage modulus represents a readout of mechanical stiffness. The storage modulus reflects the elastic response of the material structure under dynamic oscillatory strain. As described herein, storage modulus is used for the determining stiffness of fiber structures, such as hydrogel fibers exemplified in the present application because their final geometry is amenable to oscillatory shear rheology, which reports the elastic response under shear deformation. Storage modulus can be measured using a rheometer, which will provide information of the material or structure’s ability to resist deformation under an applied force.

[0044] As disclosed herein, the term “hydrogel” refers to is a three-dimensional network of hydrophilic polymers that can absorb and retain large amount of water without dissolving. Hydrogels are prepared using a variety of polymeric materials, which can be broadly divided into natural or synthetic material. In the present disclosure, for example, the materials used for forming the hydrogel are food-safe, edible polymeric material, which can include, but are not limited to alginate, pectin, agarose, carrageenan, gellan gum, konjac glucomannan, xanthan gum, guar gum, locust bean gum, gum arabic, starches and derivatives thereof, pullulan; (3-glucans, cellulose and derivatives thereof, gelatin and derivatives thereof, collagen, whey protein, casein, caseinates, soy protein, pea protein, gluten, glutenin, zein and combinations thereof. In some examples, the carrageenan can be kappa, iota, or lambda carrageenan, characterized by different gelling properties. In some examples, the cellulose derivatives can include, but are not limited to carboxymethyl cellulose (CMC), methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC), or bacterial nanocellulose. In some examples, the gelatin can be recombinant gelatin obtained from nonanimal sources or microbial systems. In some examples, the gelatin can be hydrolyzed gelatin, also known as gelatin hydrolysates, wherein the gelatin is broken down into smaller peptides by hydrolysis. In some examples, the gelatin derivatives can include, but are not limited to succinylated gelatin, acetylated gelatin or gelatin-polysaccharide blends, which are gelatin combined with a polysaccharide group such as, but are not limited to carrageenan, starch, or pectin.

[0045] As used herein, the term “food-grade” is used to describe materials, substances and equipment safe enough for direct contact with food or for human and animal consumption being non-toxic and unlikely to leach harmful substances or foreign particles into the food.

[0046] As used herein, the term “polymeric solution” refers to a mixture where polymer molecules are evenly dispersed and dissolved in a liquid solvent, such as water or an organicliquid. The term “polymeric solution” is used interchangeably with “polymer solution”. An example of a polymeric solution is a pre-gel solution. Polymer molecules in a polymeric solution can be crosslinked to form a three-dimensional structure. In the context of this disclosure, a polymeric solution comprises material suitable for forming a scaffold wherein cells can be confined or encapsulated for growth and differentiation.

[0047] As used herein, the term “mechano -transduction” refers to a process by which cells sense mechanical properties of their surrounding microenvironment and convert these cues or stimuli into intracellular biochemical signals that regulate cell fate (i.e. differentiation), cell function and cellular response.

[0048] As disclosed herein, the term “myocyte” refers to a muscle cell, which is a building block of a muscle. Muscle is a soft tissue that contacts to produce movement. There are three types of muscles in meat animals: skeletal muscle, smooth muscle, and cardiac muscle. Skeletal muscles constitute the bulk (35-65%) of the carcass weight of meat animals and are organs of the muscular system that are attached directly or indirectly to bones. Smooth muscle is the muscle type found in the walls of internal organs, such as the stomach, intestines, bladder, and blood vessels. Cardiac muscle is the muscle tissue that forms the heart. Accordingly, skeletal muscles are made up of skeletal muscle cells or myocytes, smooth muscles are made up of smooth muscle cells or myocytes, and the heart is made up of cardiac muscle cells or myocytes. Thus, the term “myocyte” or “muscle cell” as used in the present disclosure refers to all types of muscle cells. Myocytes in smooth muscles are referred to as smooth myocytes or smooth muscle cells; myocytes in the heart are referred to as cardiomyocytes or cardiac muscle cells; while myocytes in the skeletal muscles are referred to as skeletal myocytes, skeletal muscle cells or muscle fibers. As can be appreciated by a person skilled in the art, while all three types of muscles discussed above can be consumed in the diet, the skeletal muscles of animals are the most consumed muscles and are synonymous with meat.

[0049] As used herein, the term “skeletal myocytes” or “skeletal muscle cells” refers to the building block of skeletal muscles. Skeletal muscle cells are elongated and tubular with a striated appearance. Therefore, skeletal myocytes or muscle cells are also known as muscle fibers or skeletal muscle fibers. Skeletal muscle cells are multi-nucleated and have the high number of mitochondria per cell. Each skeletal muscle fiber can contain hundreds of myofibrils. A myofibril is a long, cylindrical organelle in skeletal muscle cells, made of protein filament. Myofibrils are composed of repeating segments called sarcomeres, linked from end-to-end, which form the basic contractile unit of a skeletal muscle fiber.

[0050] As used herein, the term “myocyte precursor cells” refers to cells that have the capability or the ability to differentiate into myocytes. Examples of myocyte precursor cells include but are not limited to pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells, mesenchymal stem cells such as adipose-derived stem cells or myosatellite cells, or myoblasts.

[0051] As used herein, the term “cell differentiation” or “differentiation” means the development of immature or less specialized cell into specific cell types with specific functions. It is the process by which unspecialized cells, such as stem cells, become specialized to perform distinct functions. For example, in the present disclosure, myocyte precursor cells develop or differentiate into mature myocytes.

[0052] As used herein, the term “differentiation medium” or “cell differentiation medium” refers to a cell culture medium that promotes cells to develop from one type to another, typically from a less specialized type to a more specialized type. A differentiation medium for promoting differentiation of stem cells into neurons is referred to as a neurogenic differentiation medium, while a differentiation medium that promotes differentiation of stem cells into a myocyte or muscle cell is referred to as a myogenic differentiation medium. A differentiation medium can comprise factors that can direct a cell to a particular cell fate, for example, from a myocyte precursor cell into the fate of a myocyte. Such factors can include, but are not limited to growth factors, morphogenetic factors, and / or small molecules. These factors can activate cellular pathways in the less specialized cell to induce lineage commitment. Cell differentiation media for the differentiation of myocytes as described herein are known in the art. For example, a basic myogenic differentiation medium, also referred to as a basic myogenic induction medium, can comprise a basal growth medium and horse serum. Other biochemical factors can be added to the basic myogenic differentiation medium to improve the efficacy of myogenesis. In some examples, such a myogenic differentiation medium comprising such biochemical factors are referred to as a specialized myogenic differentiation medium. The biochemical factors that can be added to the specialized myogenic differentiation medium can include, but are not limited to growth factors, such as: insulin-like growth factors (IGF), hepatocyte growth factors (HGF), epidermal growth factors (EGF), fibroblast growth factors (FGF) and platelet-derived growth factors (PDGF); small molecules, such as: CHIR99021, a small molecule inhibitor for glycogen synthase kinase 3 (GSK-3), SB-431542 , a small molecule inhibitor of transforming growth factor-beta (TGF-(3) signaling, or N-[N-(3, 5-difLuorophenacetyl-L-alanyl)]-S-phenylglycine tert-butyl ester (DAPT), a small molecule inhibitor of Notch signaling; hormones and steroids, such as: insulin, dexamethasone,hydrocortisone, or triiodothyronine (T3); or epigenetic modifiers, such as: 5-azacytidine or histone deacetylase (HD AC) inhibitors.

[0053] As used herein, the term “myogenic yield” refers to the percentage of cells encapsulated in the cell-laden scaffold which have differentiated into mature myocytes. Cells which express the late-stage myogenic marker, myosin heavy chain (mhc) are taken to be cells that have differentiated into mature myocytes.DETAILED DESCRIPTION

[0054] Cultivated meat, a form of cellular agriculture, is one of the alternatives to livestock production. In general, cultivated meat is produced by first taking a sample of animal cells, which include, but are not limited to, embryonic stem cells, multipotent stem cells, or tissuespecific stem cells, or induced pluripotent stem cells. The cell samples are then proliferated and differentiated into myocytes or skeletal muscle fiber lineage, which makes up the most of a meat product. These myocytes or skeletal muscle fibers are subsequently harvested and assembled to form the final cultivated meat product.

[0055] To produce cultivated meat, scaffolds can be utilized so that higher order of muscle tissue structure can be achieved. Scaffolds can be used in conjunction with the differentiation of stem cells to bridge the difference in structural and textural properties between cultivated meat products and meat. The problems associated with fabrication of scaffolds for production of cultivated meat include achieving the correct mechanical properties of the scaffold, ensuring scalability and affordability, and making scaffolds edible and safe for consumption.

[0056] The present disclosure describes methods of manufacturing cell-laden scaffolds through a scalable process suitable for used in manufacture of cultivated meat. Specifically, the present disclosure describes methods of manufacturing a myocyte-laden scaffold. The methods described herein involve confining myocyte precursor cells within a three-dimensional environment that is formed from one or more scaffold materials, which enables the differentiation of the myocyte precursor cells into myocytes through mechano-transduction cues. By confining myocyte precursor cells within a mechanically compliant microfiber of a specified stiffness, the present disclosure shows that the myocyte precursor cells confined in the scaffold are viable (Figure 7) and can be differentiated into mature myocytes without the use of specialized myogenic differentiation medium (Figures 8-10), thereby eliminating the need for soluble factors in the culture medium, which can reduce the overall cost in cultivated meat production. Moreover, during the fabrication process, the cell-laden scaffold, which is in the form of a fiber, is held under a static tension, which provides mechanical cues to promotedifferentiation of myocyte precursor cells into mature myocytes, such as skeletal muscle fibers, which resemble the structure of meat obtained from livestock.

[0057] Therefore, in one aspect, there is provided a method of manufacturing a myocyte-laden scaffold, wherein the method comprises: (a) preparing a mixture comprising myocyte precursor cells and a food-grade polymeric solution; (b) forming a fibre from the mixture from (a) such that the myocyte-precursor cells are confined in the fibre under a static tension; (c) culturing the myocyte-precursor cells confined in the fibre from (b) under the static tension for differentiating the myocyte pre-cursor cells into myocytes to obtain the myocyte-laden scaffold. In some examples, the myocytes in the myocyte-laden scaffold can be smooth myocytes, cardiomyocytes or skeletal myocytes.

[0058] Known methods in the art for manufacturing cultivated meat requires multiple steps during the manufacturing process, which include a step of fabricating or producing a scaffold, followed by seeding the precursor cells onto the fabricated scaffold. The methods known in the art comprise multiple steps which require more time and additional cost for quality control to ensure that each manufacturing step meets food safety standards. In one of many aspects, the methods as disclosed herein are different as they combine the fabrication of scaffold and seeding of cells in one step, that is, a three-dimensional myocyte precursor cell-laden scaffold is formed directly from a mixture comprising myocyte precursor cells and a polymeric solution comprising scaffold material, which could be directly cultured and grown in a food-grade cell culture medium, thus obviating the need for the multiple steps known in the art, which will reduce the risk of contamination.

[0059] In the methods of manufacturing a myocyte-laden scaffold as described herein, the myocytes confined within the scaffold can be differentiated from myocyte precursor cells. As described herein, myocyte precursor cells are cells that can divide and differentiate into myocytes or muscle cells. In some examples, the myocyte precursor cells can include, but are not limited to myoblasts, mesenchymal stem cells such as but not limited to adipose-derived stem cells or myosatellite cells, pluripotent stem cells such as but not limited to embryonic stem cells or induced-pluripotent stem cells.

[0060] As known in the art, satellite cells or myosatellite cells are a common source of myocyte precursor cells for obtaining mature skeletal muscle cells or muscle fibers for cultivated meat applications. Satellite cells are primary precursor cells for skeletal myocytes, or skeletal muscle cells, acting as stem cells for skeletal muscle repair and growth. Satellite cells are characterized by the expression of the paired type homebox transcription factor, Pax7. Under normal conditions, satellite cells are dormant or in a quiescent state. Satellite cells can be activated bysignals from muscle damage, exercise or stress, which trigger them to exit their quiescent state and begin dividing. The signals to activate satellite cells can include, but are not limited to, factors released from damage myofibers, mechanical strain, and signaling molecules such as nitric oxide, hepatocyte growth factor, fibroblast growth factors, and certain sphingolipids. Once activated, satellite cells can proliferate to give rise to myoblasts. Myoblasts are singlenucleus stem cells that can further proliferate to give rise to more myoblasts, or can differentiate into committed skeletal muscle cells, expressing specific transcription factors such as MyoD and myogenin. The differentiated myoblasts can fuse together to form multinucleated myotubes, which can further mature into myofibril, and can integrate into existing skeletal muscle fibers. Satellite cells also have properties of self-renewal to maintain a stem cell pool for cell replenishment. The process of formation of skeletal muscle fiber starting from proliferation of satellite cells, differentiation of myoblasts and fusion of differentiated myoblast to form myo tubes is known as myogenesis.

[0061] Myogenesis can be divided into an early stage, a mid-stage and a late stage, marked or characterized by expression of different cellular markers. For example, the early stage of myogenesis is marked by expression of myf5 and myoD, which are markers of terminal specification of skeletal muscle linage. The mid stage of myogenesis is marked by expression of myogenin, which is a transcription factor that promotes differentiation of myoblasts into myotubes; while the late stage is marked by expression of myosin heavy chains (mhc), which are structural proteins that are incorporated into mature muscle fiber. The expression of mhc is indicative of a mature myocyte or a mature skeletal muscle cell.

[0062] While many known methods in the art use satellite cells as a source of skeletal myocyte precursor cells for differentiating into skeletal muscle cells in cultivated meat application, these cells are limited in availability in vivo and comprise about 2.5% to 6% of muscle nuclei of adult skeletal muscles. Repeated biopsies are also required from live animals to obtain these satellite cells for cultivated meat application. Therefore, in some examples as disclosed herein, other sources of myocyte precursor cells can be used in the method disclosed herein. In some examples, induced pluripotent stem cells can be used as the myocyte precursor cells for differentiating into mature myocytes. In some examples, the myocyte precursor cells for differentiating into mature myocytes are embryonic stem cells or mesenchymal stem cells. In some examples, adipose-derived stem cells (ADSCs) can be used as the myocyte precursor cells for differentiating into mature myocytes.

[0063] Adipose-derived stem cells (ADSCs) are mesenchymal stem cells found in fat or adipose tissue, and have the ability to self-renew and differentiate into multiple cell types.Adipose-derived stem cells (ADSCs) are more abundant in animals and are easier to isolate, making them a more practical and scalable option for use in cultivated meat applications. Thus, in some examples, in the method of manufacturing a myocyte-laden scaffold as disclosed herein, the myocyte precursor cells can be adipose-derived stem cells (ADSCs). Therefore, in some examples, the method as disclosed herein comprises the step of preparing a mixture comprising myocyte precursor cells, and a food-grade polymeric solution, wherein the myocyte precursor cells are adipose-derived stem cells (ADSCs). In some examples, the adipose-derived stem cells (ADSCs) have multipotent capacity, which means that they can differentiate into more than one cell type, for example, into chondrocytes, osteocytes or myocytes. In one example, ADSCs can differentiate into skeletal myocytes as shown in Figure 5.

[0064] In other examples, sources of myocyte precursor cells can include, but are not limited to pluripotent stem cells, such as induced-pluripotent stem cells or embryonic stem cells. As described herein, the myocyte precursor cells are of animal origin. In some examples, the myocyte precursor cells can be of avian, amphibian, bovine, fish, ovine, porcine or reptilian origin. In some examples, the myocyte precursor cells is of porcine origin.

[0065] In the method of manufacturing a myocyte-laded scaffold as disclosed herein, the method comprises the step of preparing a mixture comprising myocyte precursor cells and a food-grade polymeric or polymer solution. In some examples, the food-grade polymeric solution can comprise one or more types of polymeric material suitable for constructing the cell-laden scaffold. In some examples, the one or more types of material can include, but are not limited to alginate, gelatin and derivatives thereof, pectin, agarose, carrageenan, gellan gum, konjac glucomannan, xanthan gum, guar gum, locust bean gum, gum arabic, starches and derivatives thereof, pullulan; (3-glucans, cellulose and derivatives thereof, collagen, whey protein, casein, caseinates, soy protein, pea protein, gluten, glutenin, zein and combinations thereof. In some examples, the carrageenan can be kappa, iota, or lambda carrageenan, characterized by different gelling properties. In some examples, the cellulose derivatives can include, but are not limited to carboxymethyl cellulose (CMC), methylcellulose (MC), hydroxy ethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC) or bacterial nanocellulose. In some examples, the gelatin can be recombinant gelatin obtained from nonanimal sources such as microbial systems. In some examples, the gelatin can be hydrolyzed gelatin, also known as gelatin hydrolysates, wherein the gelatin is broken down into smaller peptides by hydrolysis. In some examples, the gelatin derivatives can include, but not limited to succinylated gelatin, acetylated gelatin or gelatin-polysaccharide blends, which are gelatin combined with a polysaccharide group such as, but not limited to carrageenan, starch, or pectin.

[0066] In some examples, the polymeric solution can comprise one type of material. In some examples, the polymeric solution can comprise more than one type of material. Thus, in these examples, the polymeric solution can comprise a combination or a composition of scaffold materials. In some examples, the polymeric solution can comprise two, three, four, five, six, seven, eight, nine or ten types of material. In some examples, the polymeric solution can comprise two types of material. In some examples, the polymeric solution comprises alginate. In some examples, the polymeric solution comprises gelatin or derivatives thereof. In some examples, the polymeric solution can comprise alginate and gelatin.

[0067] In some examples, the one or more material in the polymeric solution is present at an amount of about 0.1% to about 6.0% weight / volume (w / v) for each material. In other examples, the one or more material in the polymeric solution is present at an amount of about 0.5% to 5.0%, about 1.0% to 4.0%, about 1.5% to 3.0%, about 1.0% to 2.0% w / v or about 3.0% to 4.0% w / v for each material. In some examples, the one or more material in the polymeric solution is present at an amount of about 0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5% or 6% w / v for each material.

[0068] In some examples, the polymeric solution comprises alginate. In some examples, the polymeric solution comprises about 0.1% to about 6.0% weight / volume (w / v) of alginate. In some examples, the polymeric solution comprises about 0.5% to 5.0%, about 1.0% to 4.0%, about 1.5% to 3.0%, about 1.0% to 2.0% w / v or about 3.0% to 4.0% w / v of alginate. In some examples, the polymeric solution comprises about 0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5% or 6.0% w / v of alginate.

[0069] In some examples, the polymeric solution comprises gelatin. In some examples, the polymeric solution comprises about 0.1% to about 6.0% weight / volume (w / v) of gelatin. In some examples, the polymeric solution comprises about 0.5% to 5.0%, about 1.0% to 4.0%, about 1.5% to 3.0%, about 1.0% to 2.0% w / v or about 3.0% to 4.0% w / v of gelatin. In some examples, the polymeric solution comprises about 0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5% or 6.0% w / v of gelatin.

[0070] In some examples, the polymeric solution comprises alginate and gelatin. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 0.1 %-6.0% w / v and wherein gelatin is present at an amount of about 0.1 %-6.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 0.5% to 5.0%, about 1.0% to 4.0%, about 1.5% to 3.0%, about 1.0% to 2.0% w / v or about 3.0% to 4.0% w / v, and wherein gelatin is present at an amount of about 0.5% to 5.0%, about 1.0% to 4.0%, about 1.5% to 3.0%, about 1.0% to 2.0%or about 3.0% to 4.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5% or 6.0% w / v and wherein gelatin is present at an amount of about 0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5% or 6.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 3.0% to 4.0% w / v and wherein gelatin is present at an amount of about 3.0% to 4.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 3.0% w / v and wherein gelatin is present at an amount of about 3.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 3% w / v and wherein gelatin is present at an amount of about 4.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 4.0% w / v and wherein gelatin is present at an amount of about 3.0% w / v. In some examples, the polymeric solution comprises alginate and gelatin, wherein alginate is present at an amount of about 4.0% w / v and wherein gelatin is present at an amount of about 4.0% w / v.

[0071] As described in the methods disclosed herein, the method of manufacturing a myocyteladen scaffold comprises the step of preparing a mixture comprising myocyte precursor cells and a food-grade polymeric solution to form a polymer-cell mixture. One or more types of polymer material can be mixed together with one population of myocyte precursor cells or more than one population of myocyte precursor cells to prepare the mixture as disclosed here.

[0072] In some examples, the mixture comprises about 5 million to 20 million myocyte precursor cells / mL. In some examples, the mixture comprises about 8 million to 15 million cells / mL, about 8 million to 12 million cells / mL, about 10 million to 20 million cells / mL, about 10 million to 20 million cells / mL or about 10 million to 15 million cells / mL. In some examples, the mixture comprises about 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 17 million, 18 million, 19 million or 20 million cells / mL. In some examples, the mixture comprises about 10 million cells / mL mix with one or more types of polymer material as disclosed herein.

[0073] In some examples, the mixture can comprise alginate, gelatin, and adipose-derived stem cells. In some examples, the mixture can comprise 0.1% to 6.0% w / v alginate, 0.1% to 6.0% w / v gelatin, and 5 million to 20 million adipose-derived stem cells. In some examples, the mixture can comprise 3.0% to 4.0% w / v alginate, 3.0% to 4.0% w / v gelatin, and 5 million to 20 million adipose-derived stem cells. In some examples, the mixture can comprise 0.1%-6.0% w / v alginate, 0.1%-6.0% w / v gelatin, and 8 million to 15 million adipose-derived stem cells.In some examples, the mixture can comprise 3.0% to 4.0% w / v alginate, 3.0% to 4.0% w / v gelatin, and 8 million to 15 million adipose-derived stem cells. In some examples, the mixture can comprise 3.0% to 4.0% w / v alginate, 3.0% to 4.0% w / v gelatin, and 10 million adipose-derived stem cells. In some examples, the mixture can comprise 3.0% w / v alginate, 3.0% w / v gelatin, and 10 million adipose-derived stem cells. In some examples, the mixture can comprise 3.0% w / v alginate, 4.0% w / v gelatin, and 10 million adipose-derived stem cells. In some examples, the mixture can comprise 4.0% w / v alginate, 3.0% w / v gelatin, and 10 million adipose-derived stem cells. In some examples, the mixture can comprise 4.0% w / v alginate, 4.0% w / v gelatin, and 10 million adipose-derived stem cells.

[0074] In some examples, the mixture comprising myocyte precursor cells and one or more types of scaffold material can further comprise a crosslinking agent. A crosslinking agent is a substance that forms bonds between polymers or polymer chains to create a three-dimensional network structure. As can be appreciated by a person skilled in the art, the crosslinking agent to be added to the mixture can be selected based on the polymer present in the mixture. For example, if the polymer is gelatin, a suitable crosslinking agent can include, but are not limited to transglutaminase, tyrosinases, horseradish peroxidases, genipin, glyceraldehyde or citric acid. In another example, if the polymer is alginate, if the material is alginate, the crosslinking agent can be a multivalent cation. Examples of such multivalent cations can include but are not limited to calcium ions, barium ions, strontium ions or iron (III) ions. In some examples, the crosslinking agent for alginate is calcium ions (Ca2+) from calcium chloride.

[0075] In the method as disclosed herein, the method comprises the step of forming a three-dimensional (3D) environment from the mixture comprising a polymeric solution and myocyte precursor cells, such that the myocyte precursor cells are confined in the 3D environment. As described herein, the myocyte precursor cells can be confined into different three-dimensional structural forms for culturing and differentiation into mature myocytes such as skeletal myocytes. Thus, the resulting structure of the myocyte-laden scaffold is dependent on the shape of the 3D environment which the cells are confined or encapsulated in. In some examples, the cells can be confined into a sphere, fibre, sheet, film, ribbon, cord, flat disc, cylinder or any three-dimensional amorphous shapes. Therefore, the resultant manufactured myocyte-laden scaffold is in the structural form of a sphere, fibre, sheet, film, ribbon, cord, flat disc, cylinder or any three-dimensional amorphous shapes. In one example shown in Figure 6, the myocyte precursor cells can be confined in the form of a fiber as fabricated by a wet spinning method as shown in Figure 1. Therefore, in one example, the method as disclosed herein comprisesthe step of forming a fiber scaffold from the mixture comprising a polymeric solution and myocyte precursor cells.

[0076] In some examples, the myocyte precursor cells can be confined in a scaffold in the form of a fiber. In some examples, the fiber scaffold can have a diameter of about 50 pm to about 600 pm. In some examples, the fiber can have a diameter of about 80 pm to about 500 pm, about 100 pm to about 400 pm, about 150 pm to about 300 pm, about 80 pm to about 150 pm, about 90 pm to about 120 pm. In some examples, the fiber scaffold can have a diameter of about 50 pm, 60 pm, 70 pm 80 pm, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm or 600 pm. In some examples, the cell-laden fiber scaffold can have a diameter about 100 pm. Without being bound by theory, encapsulating myocyte precursor cells within micro fibers of about 90 pm to 120 pm in diameter is preferred, as fibers of these diameters facilitate nutrients and waste exchange resulting in less occurrence of necrotic centers. In examples as disclosed herein, myocytes precursor cells encapsulated within microfibers of about 90 pm to 120 pm (Figure 6) showed high degree of cell alignment (Figures 19 and 20) and differentiation into mature skeletal myocytes (Figure 9), making the myocyte-laden scaffold obtained from the method disclosed herein suitable for cultivated meat applications.

[0077] In the method for manufacturing a myocyte-laden scaffold as disclosed herein, the myocyte precursor cells confined in the fiber are cultured for about 5 days to 23 days to obtain a myocyte-laden scaffold comprising mature myocytes or skeletal muscle cells. In some examples, the myocyte precursor cells are cultured for about 7 days to 21 days, about 10 days to 20 days, about 12 days to 18 days or about 14 days to 21 days. In some examples the myocyte precursor cells are cultured for about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days or 23 days. In one example, the myocyte precursor cells encapsulated in the fiber scaffold are cultured for about 14 days. In another example, the myocyte precursor cells encapsulated in the fiber scaffold are cultured for 21 days. As shown in Figure 9, culturing of myocyte precursor cells, such as adipose-derived stem cells (ADSCs), using the method as disclosed herein can achieve a myogenic yield of up to 78%, as measured by the percentage of cells expressing the late-stage myogenic marker, myosin heavy chain (mhc).

[0078] In another aspect, the present disclosure provides a myocyte precursor cell-laden scaffold or myocyte-laden scaffold comprising mature myocytes, obtained from the methods as disclosed herein. In another aspect, the present disclosure provides a myocyte-laden scaffold comprising one or more food-grade scaffold materials and one or more types of cells, whereinthe myocyte-laden scaffold is characterized by a stiffness of about 8 kPa to 35 kPa. In some examples, the myocyte-laden scaffold is characterized by a stiffness of about 12 kPa to 32 kPa, about 15 kPa to 30 kPa, about 18 kPa to 28 kPa, or about 20 kPa to 25 kPa. In some examples, the myocyte-laden scaffold is characterized by a stiffness of about 8 kPa, 9 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, 24 kPa, 25 kPa, 26 kPa, 27 kPa, 28 kPa, 29 kPa, 30 kPa, 31 kPa, 32 kPa, 33 kPa, 34 kPa, or 35 kPa. In some examples, the stiffness of the myocyte-laden scaffold is about 10 kPa. In some examples, the stiffness of the cell-laden scaffold is about 19 kPa. In some examples, the stiffness of the myocyte-laden scaffold is about 20 kPa. In some examples, the stiffness of the myocyte-laden scaffold is about 30 kPa. In some examples, the stiffness of the myocyteladen scaffold is about 32 kPa. Without being bound by theory, the stiffness of the cell-laden scaffold can change over time due to cell-mediated remodeling, or enzymatic degradation of the scaffold. For example, by day 21 of culture, cell-mediated remodeling, or enzymatic degradation may cause measurable softening or stiffening of the scaffold. Nonetheless, as described herein, the stiffness of the fiber or myocyte-laden scaffold in the range of about 8 kPa to about 35 kPa supports the differentiation of myocyte precursor cells into mature myocyte when cultured under static tension as disclosed herein. The stiffness of the myocyte precursor cell-laden scaffold or myocyte-laden scaffold is contributed by the composition of the scaffold material and amount of material used to form the scaffold. As exemplified in Example 1, the stiffness of a scaffold comprising of 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) is 9.84 ± 0.238 kPa, 19.2 ± 0.252 kPa and 32.8 ± 0.259 kPa, respectively.

[0079] In some examples, the cell-laden scaffold or myocyte-laden scaffold can be a hydrogel. As is known in the art, a hydrogel is a three-dimensional network of hydrophilic polymers that can absorb and retain large amounts of water without dissolving. Therefore, the food-grade polymers or scaffold materials used for manufacturing the cell-laden or myocyte-laden scaffold as disclosed herein can form a three-dimensional structure while holding large amount of water. In some examples, the cell-laden or myocyte-laden scaffold comprises one or more food-grade scaffold material. In some examples, the one or more types of material can include, but are not limited to alginate, gelatin and derivatives thereof, pectin, agarose, carrageenan, gellan gum, konjac glucomannan, xanthan gum, guar gum, locust bean gum, gum arabic, starches and derivatives thereof, pullulan; P-glucans, cellulose and derivatives thereof, collagen, whey protein, casein, caseinates, soy protein, pea protein, gluten, glutenin, zein and combinations thereof. In some examples, the carrageenan can be kappa, iota, or lambda carrageenan,characterized by different gelling properties. In some examples, the cellulose derivatives can include but are not limited to carboxymethyl cellulose (CMC), methylcellulose (MC), hydroxy ethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC) or bacterial nanocellulose. In some examples, the gelatin can be recombinant gelatin obtained from nonanimal sources such as microbial systems. In some examples, the gelatin can be hydrolyzed gelatin, also known as gelatin hydrolysates, wherein the gelatin is broken down into smaller peptides by hydrolysis. In some examples, the gelatin derivatives can include, but not limited to succinylated gelatin, acetylated gelatin or gelatin-polysaccharide blends, which are gelatin combined with a polysaccharide group such as, but not limited to carrageenan, starch, or pectin.

[0080] In some examples, the food grade polymeric material used for fabricating cell-laden hydrogel micro fiber as disclosed herein serve as model materials to demonstrate the feasibility of the method as disclosed herein. For food-grade commercialization, the method as disclosed herein can be adapted by substituting any of the material disclosed herein with edible or regulatory-approved biopolymers, such as plant-derived proteins or polysaccharides, while maintaining equivalent rheological and mechanical behaviour necessary for successful fiber formation and cell encapsulation.

[0081] In some examples, myocyte precursor cells encapsulated within microfiber scaffold showed improved myogenic differentiation potential, even in the absence of myogenic-inducing soluble factors commonly used in the art to promote myogenesis. In these examples, the physiological environment of the microfiber scaffold, such as stiffness of the scaffold, provides suitable mechanical cues to promote differentiation of myocyte precursor cells into mature myocytes such as skeletal muscle cells. For example, as shown in Example 4, adipose-derived stem cells (ADSCs) encapsulated in 3% w / v alginate-4% w / v gelatin (A3G4) microfiber scaffolds show a larger increase in expression of myogenic markers, myf5 and mhc as compared to ADSCs cultured in a myogenic differentiation medium without encapsulation in a scaffold (Figure 8).

[0082] In a further example as shown in Figure 9, higher stiffness or gel strength of the scaffold can improve myogenicity of the encapsulated or confined ADSCs as compared to lower stiffness or gel strength of the scaffold. In this example, ADSCs encapsulated in a scaffold comprising 3% w / v alginate-4% w / v gelatin (A3G4) or 4% w / v alginate-4% w / v gelatin (A4G4), which have a stiffness of about 19 kPa and 33 kPa, respectively, can achieve more than 75% myogenic yield after about 21 days in culture, while at a lower stiffness of about 10 kPa, ADSCs encapsulated in 3% w / v alginate-3% w / v gelatin (A3G3) showed a lower myogenic yield of about 40% after about 21 days in culture, wherein myogenic yield ismeasured by the percentage of cells expressing the late-stage myogenic marker, myosin heavy chain (mhc). As described in the disclosure, the stiffness of the scaffold is dependent on the composition of materials and the amount of material present in the scaffold. Thus, stiffness of the myocyte-laden scaffold can be modified by changing the scaffold material or the amount of material used to manufacture the myocyte-laden scaffold.

[0083] In the methods as disclosed herein, the myocyte precursor cells are confined or encapsulated in the fiber scaffold under a static tension. In some examples, the myocyte precursor cells are cultured under static tension, which facilitated differentiation of the myocyte precursor cells into myocytes to obtain a myocyte-laden scaffold. In some examples, the myocytes can be skeletal myocytes or skeletal muscle cells.

[0084] Thus, in the method as disclosed herein, there is provided a method comprising the step of forming a fiber held under a static tension from the mixture as disclosed herein, such that the myocyte-precursor cells are confined in the fiber. In some examples, the fiber is formed by a fiber forming or fiber generating method or process that includes but is not limited to wet spinning, microfluidic spinning, electrospinning, microextrusion bioprinting, interfacial polyelectrolyte complexation, template-assisted fibre fabrication, co-axial spinning or 3D bioprinting. In some examples, the fiber held under a static tension is formed by wet spinning.

[0085] As known in the art, wet spinning is a fiber forming method used for manufacturing fibers from polymers that can be dissolved in a suitable solvent. As described herein, wet spinning methods can be used to manufacture cell-laden scaffolds comprising myocytes, by extruding the mixture comprising myocyte precursor cells and polymeric solution through tiny holes in a device called spinneret to form continuous filaments. The extruded fibers then enter a coagulation bath wherein the extruded fibers, which contain the precursor cells and scaffold material, become solidified and are pulled by rollers to be collected onto drum sleeves at a controlled speed.

[0086] During the wet spinning process, static tension can applied to the fiber such that the fiber experiences an internal stress. In some examples, static tension can be applied to the extruded fibers by the drag force in the coagulation bath, wherein as the extruded fibers move through the coagulation bath, the liquid exerts resistance on the fiber, which creates tension along the fiber length. In other examples, differential solidification of the extruded fiber, wherein the outer layer of the fiber solidifies before the inner core, can also cause internal stresses. In some examples, the coagulation bath can comprise one or more crosslinking agents for crosslinking the materials contained in the polymeric solution to solidify the extruded fibers. As can be appreciated by a person skilled in the art, the crosslinking agents present inthe coagulation bath can be selected based on the polymer present in the mixture. For example, if the polymer is gelatin, a suitable crosslinking agent can include, but are not limited to transglutaminase, tyrosinases, horseradish peroxidases, genipin, glyceraldehyde or citric acid. In another example, if the material is alginate, the crosslinking agent can be a multivalent cation. Examples of such multivalent cations can include but are not limited to calcium ions, barium ions, strontium ions or iron (III) ions. In some examples, the crosslinking agent for alginate is calcium ions (Ca2+) from calcium chloride.

[0087] In some examples, static tension on the extruded fiber can also be created when the take up speed of the roller pulls and collects the fibers on the rotating drum sleeve faster than they are being extruded, which causes the fibers to be stretched, adding to static tension. Thus, in some examples, the mixture is extruded at a flow rate of about 0.05 ml / min to 0.2 ml / min. In some examples, the mixture is extruded at a flow rate of about 0.07 ml / min to 0.14 ml / min. In some examples, the mixture is extruded at a flow rate of about 0.09 ml / min to 0.12 ml / min. In some examples, the mixture is extruded at a flow rate of about 0.05 ml / min, 0.06 ml / min, 0.07 ml / min, 0.08 mFmin, 0.09 ml / min, 0.10 ml / min, 0.11 ml / min, 0.12 ml / min, 0.13 ml / min, 0.14 ml / min, 0.15 ml / min, 0.16 ml / min, 0.17 ml / min, 0.18 ml / min, 0.19 ml / min, or 0.2 ml / min. In some examples, the extruded fiber is collected onto the rotating drum sleeve rotating at a speed of about 60 to about 90 rounds per minute (rpm), about 65 rpm to about 85 rpm, about 70 to about 80 rpm. In some examples, the extruded fiber is collected onto the rotating drum sleeve rotating at a speed of about 60 rpm, 65 rpm, 70 rpm, 75 rpm, 80 rpm, 85 or 90 rpm. In some examples, the extruded fiber is collected onto the rotating drum sleeve rotating at a speed of about 80 rpm. In a preferred example, the flow rate is about 0.1 ml / min and the rotating speed is about 80 rpm. In some examples, the diameter of the drum sleeve is about 1 cm to about 5 cm, about 2 cm to about 4 cm, or about 2.5 cm to about 3.5 cm. In some examples, the diameter of the drum sleeve is about 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4.5 cm or 5 cm.

[0088] In some examples, upon extrusion, the cell-laden fibers experience strain as they are taken up by roller and collected on the drum sleeves in the coagulation bath. In this example, the change in cross-sectional area of extruded fiber can be used to quantify strain as it directly reflects the degree of deformation the fiber undergoes perpendicular to its length. Thus, in this example, taking strain as the change in cross-sectional area of microfiber during wet spinning, the strain experienced by wet spun fibers comprising 3% w / v alginate-4% w / v gelatin (A3G4) is 85.1%. When strain or static tension is applied to the cell-laden fibers as discussed above, the cell-laden fibers experience an internal stress. In some examples, the fiber under static tension has an internal stress of about 500 Pa to 900 Pa. In some examples, the fiber understatic tension has an internal stress of about 600 Pa to 800 Pa. In some examples, the fiber under static tension has an internal stress of about 650 Pa to 750 Pa. In some examples, the fiber under static tension has an internal stress of about 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, 800 Pa, 850 Pa or 900 Pa. In some examples, the fiber under static tension has an internal stress of about 700 Pa. In some examples, the internal stress of the fiber is about 500 Pa to 900 Pa. In some examples, the internal stress of the fiber is about 600 Pa to 800 Pa. In some examples, the internal stress of the fiber is about 650 Pa to 750 Pa. In some examples, the internal stress of the fiber experienced by the myocyte precursor cells is about 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, 800 Pa, 850 Pa or 900 Pa. In some examples, the internal stress of the fiber is about 700 Pa.

[0089] In the method as disclosed herein, the step of culturing the myocyte precursor cells confined in the fiber is performed by contacting the fiber spun unto the drum sleeve in a foodgrade culture medium without removing the fiber from the drum sleeve. Thus, in the method as disclosed herein, keeping the cell-laden fiber in the drum sleeve maintains the static tension applied on the fibers, that is created during the wet spinning process; thus, the encapsulated myocyte precursor cells experience the mechanical cues to promote their differentiation into mature myocytes. In some examples, the static tension applied on the fiber can be reduced by removing the cell-laden fiber from the drum sleeve. Thus, in these examples, the myocyte precursor cells experience reduced static tension, which results in reduced efficacy of differentiation of myocyte precursor cells into mature myocytes. In one example, the myogenic yield of adipose derived stem cells (ADSCs) encapsulated in 3% w / v alginate-4% w / v gelatin (A3G4) is reduced from 76% to 53% when the cell-laden micro fibers are cultured with reduced static tension (A3G4TLR; Figure 11 and Figure 12).

[0090] As described herein, there is a difference in the morphology and alignment of the cells between myocytes obtained from myocyte precursor cells cultured under static tension, and myocytes obtained from myocyte precursor cells cultured under reduced tension. In one example, the actin filaments of myocytes within microfibers held under static tension appeared more elongated and stretched, as compared to myocytes within microfibers with reduced tension, which showed a more rounded morphology (Figure 17). In other examples, it is shown that the structural organization and alignment of cellular components can be influenced by the static tension experienced by the encapsulated cells. In these examples, myocytes encapsulated within microfibers held under static tension showed an elliptical distribution, which is indicative of a degree of alignment or orientation, while in myocytes within microfibers held in reduced static tension showed a circular distribution, which is indicative of a lack ofalignment (Figures 18, 19 and 20). In the above examples, it is shown that static tension can affect cell morphology and alignment, indicating that static tension in the microfiber experienced by the myocyte precursor cells play a role in guiding myogenesis and enhancing myogenic maturation.

[0091] The methods of manufacturing a myocyte-laden scaffold comprising mature skeletal myocytes as disclosed herein rely on mechanotransduction mechanisms to enhance myogenesis of myocyte precursor cells. For example, using mechanical cues such as stiffness of the scaffold, which is influenced by the composition of the scaffold material used, and static tension, which is created in the scaffold during the fiber fabrication process and maintained during the culturing of the myocyte-laden fiber, the methods disclosed herein can direct myogenesis of myocyte precursor cells such as adipose-derived stem cells (ADSCs), without the need for expensive biochemical differentiation factors. For example, methods known in the art have shown that addition of such biochemical factors can improve efficacy of myogenic differentiation. These biochemical factors include, but are not limited to growth factors, such as: insulin-like growth factor (IGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF); small molecules, such as: CHIR99021, a small molecule inhibitor for glycogen synthase kinase 3 (GSK-3), SB-431542 , a small molecule inhibitor of transforming growth factor-beta (TGF-P) signaling, or N-[N-(3, 5-difhiorophenacetyl-L-alanyl)]-S-phenylglycine tert-butyl ester (DAPT), a small molecule inhibitor of Notch signaling; hormones, such as: insulin, dexamethasone, hydrocortisone, or triiodothyronine (T3); or epigenetic modifiers, such as: 5-azacytidine or histone deacetylase (HD AC) inhibitors. Accordingly, the method as disclosed herein can achieve more than 75% myogenic yield (Figure 10) when cultured in a food-grade culture medium, which is an improvement over methods known in the art.

[0092] Thus, in the examples as disclosed herein, the myocyte precursor cells encapsulated in the scaffold can be cultured using a food-grade culture medium which does not contain any of the abovementioned biochemical factors. In these examples, the food-grade culture medium can comprise a basal cell growth culture medium. As can be appreciated by a person skilled in the art, a basal cell growth medium is a fundamental nutrient medium that can be used to support the growth and proliferation of cells cultured in vitro. A basal cell growth medium provides essential nutrients like amino acids, vitamins, glucose, salts, L-glutamine and sodium pyruvate. The basal cell growth medium can be supplemented with serum such as fetal bovine serum or horse serum, or other factors to support specific cell types. In some examples, the basal growth medium can be any suitable basal growth medium commonly used formammalian cell culture. In some examples, the basal growth medium can include, but are not limited to Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute medium (RPMI-1640), Minimum Essential Medium (MEM), a-MEM or Ham’s F-12. In some examples disclosed herein, the basal growth medium is Dulbecco's Modified Eagle Medium (DMEM). In some examples, the food-grade medium can optionally include low amount of antibiotics, such as penicillin and / or streptomycin. As known in the art, these antibiotics are commonly used in mammalian cell culture to prevent bacterial contamination and also used as an injectable to treat bacterial infection in livestock.

[0093] In another aspect, there is provided a myocyte-laden scaffold obtained or obtainable from the methods as disclosed herein. In some examples, a plurality of myocyte-laden scaffolds obtained from the method as disclosed herein can be assembled using food binders to obtain a cultivated meat product. In some examples, a cultivated meat prototype can be assembled by stacking multiple myocyte-laden scaffolds after about 14 days to 21 days in culture. Thus, in another aspect, there is provided a cultivated meat product obtained or obtainable from the methods as disclosed herein. An example of a cultivated meat product or prototype is shown in Figure 21. Some examples providing the characteristics of the cultivated meat obtained from the method as disclosed herein are shown in Figures 22 to 28 of Example 8.

[0094] In another aspect, there is provided a kit for obtaining a myocyte-laden scaffold comprising: (a) one or more food-grade scaffold materials; (b) one or more populations of myocyte precursor cells; (c) a food-grade culture medium; and a fibre-forming or generating system, such as a fiber-spinning system.

[0095] In the present disclosure, the methods as disclosed rely on mechanotransduction mechanisms to induce the differentiation of myocyte precursor cells. When confined in a hydrogel scaffold in the form of a fiber, mechanical cues such as, but are not limited to, stiffness of the hydrogel and static tension applied to the scaffold material promote differentiation of myocyte precursor cells into mature myocytes without the reliance of biochemical factors, such as growth factors, small chemical molecules and hormones. Eliminating the use of such biochemical factors in culture medium can be cost saving for cultivated meat applications in commercial scale, wherein large volume of culture medium is required for producing cultivated meat. Further, the scaffold materials as used in the method as disclosed herein are widely available, inexpensive, biocompatible, and food-safe, making them suitable for use in cultivated meat applications.

[0096] The method as disclosed herein can be translated into industrial-scale production of cell-laden hydrogel microfibers for cultivated meat applications. In some examples, at thelaboratory level, the process involves the extrusion of a polymer-cell mixture through a syringe or single nozzle into a coagulation bath, forming individual hydrogel microfibers that encapsulate viable cells. This process can be adapted to large-scale production through integration with continuous manufacturing systems. For example, in a commercial setting, scalability can be achieved by employing multi-nozzle or spinneret arrays capable of simultaneous fiber extrusion, allowing continuous and parallel production of hydrogel micro fibers. In some examples, these systems can be integrated with automated flow control, real-time viscosity monitoring, and synchronized crosslinking modules to maintain uniform fiber geometry and cell distribution. In other examples, the coagulation and crosslinking baths can be configured as a continuous belt or tubular reactor system with controlled flow of crosslinking.

[0097] In other examples, the scalability of the method as disclosed herein is dependent on several parameters that influence fiber integrity and cell performance, including polymer concentration, viscosity, flow rate, crosslinking kinetics, and cell encapsulation density. In these examples, the balance of these parameters has to be maintained to ensure that the resulting fabricated fibers retain adequate mechanical strength, shape fidelity, and cell viability. As described in the present disclosure, the microfiber properties, particularly stiffness and static tension of the cell-laden microfiber, are important to induce stem cells toward the myogenic lineage. In other examples, industrial-scale adaptation would also require closed-loop feedback systems and environmental controls to prevent contamination and maintain sterility throughout the spinning and culture process.

[0098] Overall, the method of manufacturing myocyte-laden scaffolds as disclosed herein is a technologically scalable and modular platform for cultivated meat production that integrates scaffold fabrication, cell encapsulation, and mechanotransduction-driven differentiation into a single continuous process. The scalability potential of the method as disclosed herein can have industrial relevance while preserving flexibility for adaptation to diverse cell types, hydrogel systems, and end-use applications.

[0099] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Further, as used herein the term “consisting essentially of’ describes an invention that includes the listed elements but also allows for other components, provided those additional components do not materially affect the invention's basic and novel characteristics. Additionally, the terms and expressions employed herein have been used as terms ofdescription and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0100] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0101] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0102] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0103] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0104] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0105] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0106] It should further be appreciated that the exemplary examples are only examples, and are not intended to limit the scope, applicability, dimensions, or configuration of the disclosure in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the disclosure, it being understood that various changes may be made in the function and arrangement of elements and method of fabrication described in an exemplary embodiment without departing from the scope of the disclosure as set forth in the appended claims.EXAMPLESExample 1: Gel Strength of Alginate-Gelatin Composites

[0107] The mechanical properties of the hydrogels comprising different amounts of alginate and gelatin were characterized using the rheometer. Hydrogel discs of 25 mm diameter were prepared and subjected to oscillatory tests with a plate-plate rheometer to determine the gel strength of the alginate-gelatin composites.

[0108] When hydrogels were subjected to an amplitude sweep of 0.1-100% strain rate, at an angular frequency of 10 rad / s and at 37°C, the storage and loss modulus of hydrogels were recorded by a rheometer. At low strain rates, the mechanical response of the hydrogel is linear and this is known as the linear viscoelastic region (LVR), where the modulus of the hydrogels is independent of the applied deformation. This region can be determined by subjecting hydrogels to an amplitude sweep as shown in Figure 2, with the LVR indicated with the double headed arrow. Having identified the linear viscoelastic region, the hydrogels were subjected to a frequency sweep of 0.1-100 rad / s, at a strain of 1%, which lies within the LVR and at 37°C. The storage and loss modulus recorded during the experiment is presented in Figure 3 below. With these data, the gel strength, or stiffness, can be determined.

[0109] Figure 4 provides the gel strength data for the respective alginate-gelatin composite hydrogels, comprising of 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4). Gel strength or stiffness was taken as the storage modulus of the hydrogels at 1% strain, and at the angular frequency of 1Hz. The angular frequency is taken at 1 Hz as most research papers use this value as a reference, furthermore, most physiological activity and responses occur approximately at this frequency. Based on data in Figure 4, A3G3 hydrogels possess a gel strength of 9.84 ± 0.238 kPa (n=3), while A3G4 hydrogels possess a gel strength of 19.2 ± 0.252 kPa (n=3) and A4G4 hydrogels possess a gel strength of 32.8 ± 0.259 kPa (n=3).Example 2: Myogenic Capability of adipose-derived stem cells (ADSCs)

[0110] Cells were cultured in on treated culture plates, which are suitable for culturing adherent cells, in myogenic differentiation medium, comprising DMEM, supplemented with 2% horse serum and 1% penicillin-streptomycinAfter about 21 days in culture, the cells were stained for mhc, representing the late-stage marker of myogenesis (Figure 5), confirming myogenicity of the extracted ADSCs.

[0111] A separate set of samples were prepared and cultured under the same conditions, but the cell layer was removed with 0.25% Trypsin-EDTA before they were collected and pelleted. The pelleted cells were stained in suspension with mhc and the cells were counted with ARTHUIR™ Fluorescent Cell Counter. This method provides an estimation of the myogenic yield when ADSCs that were seeded on treated cell culture plate and induced towards myogenesis with myogenic induction media containing DMEM, supplemented with 2% horse serum and 1% penicillin-streptomycin, for 21 days. After about 21 days of culture in myogenic induction media, 25 ± 1.3% of cells expressed mhc. The data in this example indicated that ADSCs possess myogenic capabilities.Example 3: Wet-spun Microflbers

[0112] To produce cell-laden microfibers that are as thin as possible, the pre -gel solutions containing 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) were spun at low flow rate and high rotating drum speed. With the set-up in the lab, the lowest flow rate setting of the syringe pump used was 0.1 ml / min and the highest rotating speed of the drum collector was 80 rpm. The drum sleeves used to collect the microfibers have an outer diameter of 3 cm.

[0113] Representative images of wet-spun micro fibers obtained from each pre-gel solution are shown in the top panel of Figure 6; while the average diameters of each alginate -gelatin micro fiber composites obtained from the respective pre -gel solutions are plotted in the bar chart in Figure 6. A3G3 micro fibers have an average diameter of 99.44 ± 3.76pm, while the diameters of A3G4 micro fibers are at an average about 100.23 ± 3.65 pm. The diameters of A3G3 and A3G4 micro fibers are very similar while the diameters of A4G4 micro fibers arelarger with an average of 113.86 ± 3.52pm and is significantly higher than other two compositions.Example 4: Myogenesis within microflbers

[0114] To show the viability of the adipose-derived stem cells (ADSCs) encapsulated with alginate-gelatin microfibers, ADSCs encapsulated in 3% w / v alginate-3% w / v gelatin (A3G3) and 3% w / v alginate -4% w / v gelatin (A3G4) microfiber were stained with Calcein-AM and Ethidium homodimer- 1, to label live and dead cells respectively. As shown in Figure 7, the cells are shown to be viable with up to 98% of the cells shown to be stained with Calcein-AM.

[0115] Figure 8 provides data to show that culturing of ADSCs confined within an alginate-gelatin microfiber improves myogenesis compared to culturing in the presence of a myogenic differentiation medium. For this experiment, qPCR analysis of early stage myogenic marker, myf5, and late stage myogenic marker, mhc were performed in (i) cells that are cultured with basal growth media (Untreated); (ii) cells cultured in myogenic differentiation medium comprising DMEM, 2% horse serum and 1% penicillin / streptomycin (Differentiation Media); and (iii) cells confined within a micro fiber comprising 3% w / v alginate-4% w / v gelatin cultured in a basal growth medium (A3G4). The highest expression of myf5 and mhc were seen in the A4G4 group, indicating that ADSCs confined within microfibers and cultured in basal growth medium improves myogenesis as compared to culturing in the myogenic differentiation medium alone.

[0116] To show late-stage myogenic differentiation of ADSCs when they were cultured within the alginate-gelatin composite microfibers, the cell-laden microfibers were stained with the late-stage myogenic marker, mhc, after about 21 days of culture. The cells that have differentiated and are at the late stage of myogenesis are stained with mhc. All cells are counterstained with f-actin and DAPI. The actin filaments and positions of the nuclei within the cell-laden microfiber can be seen in Figure 9. This data shows that ADSCs are able to undergo late-stage myogenic differentiation when confined in micro fibers obtained from 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) pre-gel solution in the absence of a specialized myogenic differentiation medium.

[0117] At day 7 (D7), day 14 (D14) and day 21 (D21) in culture, the cells confined within each composite microfibers were stained and quantified with Arthur™ Fluorescent Cell Counter to determine the myogenic yield of ADSCs encapsulated in 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% w / v gelatin (A4G4) micro fiber. The bar chart in Figure 10 below provides the percentage of cells stained positivewith late-stage marker, mhc, at D7, D14 and D21. It was observed that for all alginate-gelatin composites, there was an upward trend in percentages of cells expressing late-stage myogenic marker, mhc, over time. The rate of increase also showed an upward trend within the cells confined in A3A4 and A4A4 micro fibers. The result indicates that the rate of differentiation or maturation of cells encapsulated within A3G4 and A4G4 micro fibers were faster than those in A3G3 micro fibers.

[0118] For cells encapsulated within A3G3 microfibers, 12 ± 1.0% of cells stained positive with mhc on D7, while 19 ± 3.1% of cells stained positive with mhc on DI 4, and 40 ± 1.4% of cells stained positive with mhc on D21. For cells encapsulated within A3G4 micro fibers, 20 ± 0.7% of cells stained positive with mhc on D7, while 34 ± 1.1% of cells stained positive with mhc on D14, and 76 ± 2.1% of cells stained positive with mhc on D21. For cells encapsulated within A4G4 microfibers, 23 ± 1.5% of cells stained positive with mhc on D7, while 49 ± 1.2% of cells stained positive with mhc on D14, and 78 ± 2.8% of cells stained positive with mhc on D21. While the cells encapsulated within A4G4 micro fibers matured at a faster rate than those in A3G4 micro fibers, on D21, the population of cells expressing mhc were comparable and were not statistically different.

[0119] Table 1 below presents the myogenic yield of ADSCs induced towards myogenesis with myogenic induction media, or by mechanotransduction when confined in alginate-gelatin composite micro fibers, on day 21. It was observed that the method of mechanotransduction was more effective in inducing ADSCs towards myogenesis since cells induced by biochemical cues resulted in the lowest myogenic yield.

[0120] Table 1: Myogenic yield (average ± standard error margin) on day 21 with myogenic induction media, or mechanotransduction with alginate-gelatin composite microfibers.| % cells expressing mhcMyogenic Induction Media | 25 ± 1.3A3G3 40 ± 1.4A3G4 76 ± 2.1A4G4 78 ± 2.8Example 5: Effect of static tension during culture on myogenesis

[0121] In order to understand the effect of static tension on myogenesis and maturation of ADSCs into myocytes confined in alginate-gelatin microfibers, the study compared cell-laden microfibers cultured in static tension and reduced static tension (also referred to as cultured in suspension). Samples were prepared using A3G4 hydrogel composite comprising 3% w / v of alginate and 4 % w / v of gelatin. For the group cultured in static tension (A3G4 group), cell-laden fibers were wet-spun and collected on metal drum sleeves, and cultured on metal drum sleeves throughout the 21 days of culture. For the group cultured in reduced static tension (i.e., in suspension, A3G4TLR group), the cell-laden microfibers were removed from the drum sleeves, and were transferred to empty wells for culture, 15 minutes after they were wet-spun onto the drum sleeves. Upon 21 days in culture, immunocytochemical staining was performed on the cell-laden microfibers cultured in static tension and reduced static tension, to evaluate the effect of static tension on myogenic differentiation of ADSCs. The late-stage marker mhc, in conjunction with f-actin, and DAPI were stained to provide a comprehensive view of cell morphology and characteristics. The resulting representative confocal images, including merged images, are shown in Figure 11. Both culture conditions showed marked expression of the late-stage myogenic marker, mhc, indicating myogenic differentiation of ADSCs into mature myocytes. Based on the f-actin staining data, it could be seen that the morphology of the cells differed in both conditions. The actin filaments of cells encapsulated within the A3G4 group were more elongated and stretched, while the actin filaments of cells encapsulated within A3G4TLR group were more rounded.

[0122] The myogenic yield of mature myocytes from ADSCs is measured by the expression of three myogenic markers, the early-stage marker, myf5, the mid-stage marker, myogenin (myg) and the late-stage marker, myosin heavy chain (mhc) at day 21 of culture. The data is presented in the bar chart in Figure 12. For myf5, the percentage of cells positively stained for the early-stage marker in the A3G4 and A3G4TLR groups was 17 ± 1.7% and 17 ± 2.5% respectively, which was not statistically different. As for myg, 14 ± 1.2% of cells in the A3G4 group and 20 ± 1.5% of cells in the A3G4TLR group were stained positive with the mid-stage marker. In the context of mhc, 76 ± 2.1% of cells in the A3G4 group and 53 ± 1.7% of cells in the A3G4TLR group were stained positive with the late-stage marker. The data indicates that between the two conditions, the ADSCs that were cultured under static tension had higher myogenic yield than those cultured under reduced static tension. Specifically, at late-stage myogenesis, which is marked by the expression of mhc, the A3G4 group had more than about 42% more cells that were mhc-positive compared to the A3G4TLR group, thus showing that static tension during culture promotes late-stage myogenesis.Example 6: Stress relaxation profile of wet-spun microfibers

[0123] In the process of being wet spun, the micro fibers experience mechanical stress as they are being extruded to the drum sleeves. Since the alginate-gelatin composite microfibers are viscoelastic, they will experience stress relaxation over time. A stress relaxation experiment was performed on alginate-hydrogel discs with a rheometer to excess the stress relaxation ofthe alginate-gelatin composite micro fiber over time. Upon extrusion from the dispensing needle, the cell-laden microfibers experience strain as they were wet-spun onto the drum sleeves in the coagulation bath. The change in cross-sectional area of microfiber can be used to quantify strain as it directly reflects the degree of deformation the fiber undergoes perpendicular to its length. Taking strain as the change in cross-sectional area of micro fiber during wet-spinning, the strain experienced by wet-spun micro fibers from a polymeric solution containing 3% w / v alginate and 4% w / v gelatin (referred to as A3G4) is 85.1%.

[0124] A constant strain of 0.851 was thus applied to A3G4 hydrogel discs with a rheometer for 30 minutes (1800s) to study the stress relaxation profile of micro fibers held in static tension. To mimic the stress relaxation profile of micro fibers with reduced static tension (A3G4TLR micro fibers, i.e., the wet-spun micro fibers were removed from drum sleeves 15 minutes after wet-spinning), a strain of 0.851 was first applied to another set of hydrogel disc samples for 15 min (900s) before it was reduced to a strain of 0.1, for the next 15 min (900s). The stress relaxation profiles of A3G4 hydrogels representing micro fibers held in static tension and A3G4TLR hydrogels with reduced static tension over a duration 30 minutes was plotted in Figure 13.

[0125] A viscoelastic model was employed to supplement the rheological data to further predict the evolution of stress over 21 days. The viscoelastic model used herein is an alternative model derived from the standard linear solid (SLS) model with a finite loading element, the constitutive equation of which is shown as Equation 1 below, to model the stress relaxation profile of A3G4 and A3G4TLR.°relaxation E) ~ Eir (Equation 1)

[0126] The raw data for A3G4 hydrogel obtained from rheological measurement is shown in Figure 14, alongside with the fitted curve generated using Equation 1, which represents the constitutive equation of the SLS model with a finite loading element, where kGR and 0<k<l. Time is represented by t, ^ relaxation (t) refers to stress as a function of time during the stress relaxation process, and E l represents the parameter in the standard linear model relevant to viscoelastic properties and is to be determined in the analysis. The strain rate is represented by r, and r_c and r_R are the creep time constant and relaxation time constant respectively. The constant strain during the stress relaxation process is represented by £_0. The coefficient of determination (R2) for this curve fitting is exceptionally high, measuring at 0.9981.

[0127] In Figure 15, the raw data for A3G4TLR hydrogel obtained from rheological measurement is presented alongside with the fitted curve produced using Equation 1. This curve fitting was performed after reducing the strain from 0.851 to 0.1 at 900 seconds, simulating the release of tension as the microfibers were removed from the drum sleeves. The R2value for this curve fitting measures at 0.9991. Since the R2values for both fitted curves for A3G4 hydrogel and A3G4TLR hydrogel and their respective raw data are very close to 1, this shows that the model can be used to predict stress relaxation of the material accurately over time.

[0128] The stress relaxation modeling profiles for A3G4 hydrogel (representing micro fibers held in tension in culture for 21 days) and A3G4TLR hydrogel (TLR in the graph; representing micro fibers held in reduced tension in culture for 21 days) using the SLS model with finite loading element was extrapolated and plotted in Figure 16. The modeling data shows that the stress experienced by cells encapsulated in A3G4 hydrogel is consistently higher than the stress experienced by cells encapsulated in A3G4TLR hydrogel throughout the entire 21 -day culture period. Furthermore, the modeling data showed that A3G4TLR hydrogel experienced a rapid and substantial reduction in stress, reaching a plateau of less than 500 Pa in less than a day. In contrast, A3G4 exhibited a significant initial drop in stress but did not reach the same low level as seen in A3G4TLR hydrogel. This difference in stress relaxation between A3G4 and A3G4TLR hydrogels highlights the role of static tension in influencing the stress relaxation response over time.Example 7: Characterization of differentiated myocytes encapsulated within microfibers cultured under static tension or microfibers cultured under reduced static tension

[0129] There is a difference in the cell morphology between myocytes obtained from ADSCs encapsulated in microfibers under tension, and myocytes obtained from ADSCs encapsulated in micro fibers that were under reduced tension. For example, the actin filaments of cells within micro fibers held under static tension appeared more elongated and stretched. In contrast, those in micro fibers with reduced tension displayed a more rounded morphology (see Figure 17).The changes in cell aspect ratio of cells encapsulated within the micro fibers were examined on days 1, 3, 7 of culture as these timepoints correlate with the micro fibers experiencing the most drastic changes in stress based on the stress relaxation modeling data. The cell-laden microfibers were stained with f-actin and DAPI at each pre-determined time point. Subsequently, the cell aspect ratio was established, calculated as the ratio of A (representing the longest end-to-end span of the f-actin stain) to B (representing the perpendicular dimensionof A), depicted in Figure 17. A higher cell aspect ratio signifies a more elongated cell shape, while an aspect ratio close to 1 indicates a cell with a more rounded morphology.

[0130] The cell morphology of the myocytes obtained from ADSCs encapsulated in micro fibers under tension or under reduced tension was also examined on day 21 of culture. The phase-contrast images of the cell-laden microfibers, along with their stained counterparts for f-actin, DAPI, and the merged images, are shown in Figure 18. The accompanying bar chart in Figure 18 shows the average nucleus aspect ratio based on measurements obtained from 50 nuclei. As can be seen in the photomicrograph in Figure 18, the ends of the actin filaments within a single cell are no longer distinguishable. Hence, quantitative analysis was performed on the nucleus aspect ratio instead. Based on the measurements taken, cells encapsulated within A3G4 microfibers (i.e., microfibers held under static tension) had an average nucleus aspect ratio of 3.13 ± 0.16, while cells within A3G4TLR micro fibers (microfibers held under reduced static tension) had an average cell aspect ratio of 2.73 ± 0.16 on day 21, indicating a significant difference in nuclear morphology between the two groups at the end of the culture duration.

[0131] The cells within A3G4 micro fiber also appeared to align and orientate in the longitudinal direction of the micro fibers, as compared to the cells within A3G4TLR micro fiber. Hence, cell morphology in terms of the alignment of the cells within the microfibers were examined. Cell-laden microfibers stained with f-actin, its cropped images for Fast Fourier Transform (FFT) analysis and their corresponding FFT frequency distribution images are shown in Figure 19. The FFT frequency distribution images were rotated 90° to correct for the mathematical transformation.

[0132] The resulting FFT frequency distribution image of A3G4-D21, representing f-actin stained, cell-laden micro fibers held in static tension and cultured for 21 days, shows the pixels arranged in a non-random, elliptical distribution, as pixel intensities are preferentially distributed with specific orientation. In contrast, the resulting FFT frequency distribution image of A3G4TLR-D21, representing f-actin stained, cell-laden micro fibers released from static tension (hence reduced tension) and cultured for 21 days, shows the pixels distributed in a more circular pattern as the pixel intensities are randomly distributed without specific orientation.

[0133] The Directionality Plugin in ImageJ software was used to quantitatively assess the orientation of the cropped figures in Figure 19. The angle of orientation, represented against the normalized frequency, is depicted in the line graphs in Figure 20. To provide a representative view of f-actin orientation distribution within the micro fibers for each condition, three samples were analyzed and plotted. In these analyses, the longitudinal direction of themicro fibers was established as 0°. The results showed that the actin filaments of cells within micro fibers held under static tension exhibited a clear and singular peak at 0°, indicating that the actin filaments were predominantly oriented along the longitudinal axis of the micro fibers. The actin filaments within microfibers cultured in condition wherein the microfibers had reduced static tension showed multiple peaks in the orientation distribution, indicating that the actin filaments within these cells had a more randomized orientation, lacking a specific preference for alignment along any particular axis.Example 8: Characterization of cultivated meat prototype obtained from the culture method disclosed herein

[0134] After about 21 days of culture, food binders can be added to the myocyte-laden microfiber, which resembles muscle fiber, to form a cultured meat product. Figure 21 shows a prototype fabricated by removing myocyte-laden microfiber from the drum sleeves after about 21 days of culture, before they were stacked and assembled using alginate as a binder.

[0135] To investigate the water content of the cultivated meat prototype, which is obtained by culturing the ADSCs confined in 3% w / v alginate-4% w / v gelatin (PA3G4) for 21 days, the assembled prototype samples were weighed before and after drying. The percentage of weight loss after drying the samples with a freeze dryer was taken to be the water content in the cultivated meat prototype. Store-bought pork loin samples were prepared in the same manner as a comparison. The water content in pork loin samples was found to be 72 ± 1.6%, which was similar to that reported in the FoodData of U.S. Department of Agriculture. The water content of PA3G4 cultivated meat prototype was found to be 95 ± 0.1% (Figure 22). Since the hydrogel was used as a scaffold, the water content in the prototype was found to be much higher than that of raw pork loin.

[0136] The loss of mass after cooking provides information of the water holding capacity of the prototype after cooking. The loss of mass during cooking results from protein structural changes. At elevated temperatures, the myofibrillar proteins shrinks starting from 40°C. This protein coagulation reduces volume and ability to retain water as it strengthens the structure of muscle fiber. Figure 23 provides images of pork loin and prototype samples (PA3G4), showing the appearance of the samples before and after cooking. Two cooking methods were used in this experiment: (1) boiling for 10 minutes in water and (2) frying for 20 seconds in cooking oil. As can be seen in Figure 23, the physical appearance of the pork loin samples and PA3G4 prototypes looks similar both before and after cooking. The prototypes were found to be able to withstand cooking conditions as they remained intact after cooking. The colour of the cultivated meat prototypes before cooking (i.e., Raw) appears yellowish, instead of reddishseen in pork loin due to the lack of myoglobin. The colour of the cooked prototype meat is visually similar to the colour of the cooked pork loin.

[0137] Cooking loss can be used as an indication of the moisture content left in meat or cultivated meat samples after cooking. Thus, a high cooking loss is indicative of poor water retention, which reflects the loss of juiciness of the meat. The cooking loss of the pork loin and prototype samples (PA3G4) is shown in Figure 24. After boiling for 10 minutes in an 80°C-water bath, the cooking loss of pork loin was found to be 22.1 ± 0.80%, while its cooking loss after being fried in oil for 20 s at 160°C was found to be 39 ± 2.7%. After boiling, the mass loss of the prototype cultivated meat was 25 ± 4.9%, while after frying, the mass loss of the prototype cultivated meat was 67 ± 3.3%. The cooking loss of pork loin and PA3G4 prototype samples are similar when boiled in water, indicating that the cultivated meat prototype can replicate the water holding capacity of meat and thus, the cultivated meat prototype can be as juicy as pork loin after boiling. The cooking loss of the cultivated meat prototype was much higher than that of pork loin when pan-fried, indicating that the cultivated meat prototype was unable to retain as much moisture as compared to pork loin when cooked at a temperature that exceeds the boiling point of water at 100°C. The higher cooking loss could also be attributed to the higher water content in the cultivated meat prototype PA3G4 as compared to pork loin.

[0138] The protein content of cultivated meat prototype PA3G4 is also compared vis-a-vis pork loin and the scaffold material (Figure 25). It was found that there were approximately 16 ± 0.15 g of soluble protein in 100 g of dried pork loin samples, approximately 11 ± 0.28g of soluble protein in 100 g of PA3G4 prototype samples, and 3.0 ± 0.02g of soluble protein in 100 g of scaffold material comprising 3% w / v alginate-3% w / v gelatin (A3G4), in terms of dry weight. This result shows that the soluble protein content in PA3G4 prototype is comparable to that of pork loin. Although the scaffold material comprises gelatin, which also contributes to protein content, it only takes up about 28% of the total protein content present in the cultivated meat prototype. This shows that a significant portion of the protein content came from the mature myocytes present in the cultivated meat prototype.

[0139] The texture profile analysis of raw pork loin and PA3G4 cultivated meat samples provides information on the hardness and chewiness of meat. To evaluate the textural properties of the meat and cultivated meat samples, a two-compression test, which mimics the biting of food, is conducted on samples, with compression speed of 2 mm / s. Figure 26 provides an illustration of a typical force profile of a meat sample. The hardness and chewiness of a sample can be calculated from their respective force profiles with the equations below.Hardness (A) = FlTime 2 Area 2Chewiness (A) — Fl x - Time 1 Area 1

[0140] The highest peak force measured during the first compression is determined as the hardness of the samples. Chewiness refers to the energy required to chew solid food and is determined by the ratio of the first compression positive force area to the second compression positive force area, multiplied by the hardness of the sample and the springiness of the sample, which is the height at which food can recover between end of first compression and the beginning of second. The force profiles experienced by a raw pork loin sample and a prototype sample during the two-compression test are shown in Figure 27 for comparison.

[0141] The hardness and chewiness data were normalized against the volume of the samples and are shown in Figure 28. The hardness of raw pork loin samples was approximately 4.94 ± 0.38mN / mm3and the hardness of the PA3G4 cultivated meat prototype samples was approximately 1.23 ± 0.56mN / mm3. The chewiness of the pork loin samples was approximately 1.17 ± 0.05mN / mm3and the chewiness of the PA3G4 samples were approximately 0.38 ± 0.11mN / mm3. Based on the normalized hardness and chewiness data above, the cultivated meat prototype PA3G4 was found to be softer, and less chewy than the pork loin sample analyzed in this study.Cell culture

[0142] Prior to encapsulation, adipose-derived stem cells (ADSCs) from porcine source are seeded into T175 flasks at a density of 6,000 cells / cm3and fed with culture media, comprising Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. The culture medium was replaced every 2 to 3 days till 80-90% confluency is reached. The cells were then dissociated with 0.25% Trypsin-EDTA at 37°C for 5 min, before equal amount of culture media was added to neutralize the dissociation media. Cell suspension solution was collected into a 15 mL centrifuge tube, and cells were pelleted via centrifugation at 300 G for 5 min prior to cell encapsulation with the pre -gel solution. Preparation of Pre-Gel and Crosslinking Solutions

[0143] Alginate and gelatin solutions are prepared separately by dissolving powered form of alginic acid sodium salt from brown algae (A0682; Sigma) and gelatin from porcine skin (G1890; Sigma), in deionized water and phosphate buffer solution (PBS) respectively. The powders were subjected to Ethylene Oxide Gas (EtO) sterilization and the buffers used were autoclaved in advance to ensure sterility. Alginate powder was weighed and dissolved indeionized water using a magnetic stirrer at 350 rpm and undergoes heat treatment at 80°C for 2 hours; where gelatin powder was weighed and dissolved in PBS. The gelatin solution was left to homogenize in a 37°C shaking incubator at 120 rpm for an hour. Once the powders were fully dissolved, the alginate and gelatin solutions were mixed to obtain the desired amount of each material and homogenized in the 37°C shaking incubator at 120 rpm for at least 30 minutes. The pre-gel solution containing the desired amount of alginate and gelatin were prepared in sterile conditions, in a biosafety cabinet.

[0144] The crosslinking solutions, calcium chloride and microbial transglutaminasem, were prepared by dissolving the respective powders in deionized water. 150 mM of calcium chloride was prepared in advance and autoclaved to make the coagulation bath, while stock solution of microbial transglutaminase was prepared at a concentration of 50 U / ml and stored at 4°C after it was sterile filtered.Fabrication of cell-laden microflbers using wet-spinning method

[0145] The pre-gel solution was left in a water bath at 37°C before use. Adipose-derived stem cells (ADSCs) from porcine source were first dissociated from the culture flasks and subsequently resuspended with alginate-gelatin pre-gel solution consisting of a range from 3% w / v alginate-3% w / v gelatin (A3G3), 3% w / v alginate-4% w / v gelatin (A3G4) and 4% w / v alginate-4% gelatin w / v (A4G4). The encapsulation cell density used was 10 million cells / ml. Microbial transglutaminase (mTgase), an enzymatic crosslinker for gelatin was added to the pre-gel cell solution and homogenized using a vortex for 5 s. The pre -gel cell solution was transferred to a syringe and tube system before injected into a 150 mM calcium chloride coagulation bath using a syringe pump at the rate of 0.1 ml / min, via a 25 gauge blunt tip dispensing needle. The cell-laden microfibers were then collected with rotating drum sleeves of 30 mm outer diameter, at 80 rounds per minute (rpm). After wet-spinning, the drum sleeves with cell-laden micro fibers were then removed from the drum and placed into a 50 U / ml mTgase bath and incubated at 37°C for 15 min before they were transferred into petri dish with comprising a cell culture medium for culture at 37°C in air with 5% CO2. The drum sleeves holding the cell-laden micro fibers were then placed in petri dishes and submerged in the culture medium for culturing. The cell culture medium was changed every 2-3 days. The cell culture media used in this context comprises DMEM,10% FBS and 1% Penicillin-Streptomycin. Storage Modulus of alginate-gelatin composites

[0146] Alginate-gelatin composites are prepared in petri dish and cut into 25-mm discs. A rheometer (Anton Paar Rheometer MCR 501) was used to measure the storage modulus of the hydrogel samples. The hydrogels were first subjected to an amplitude sweep of 0-100% strain,at an angular frequency of 10 rad / s. The linear viscoelastic region (LVR) was identified, and at the strain of 0.1%, within the LVR, a frequency sweep of 0.1-100 rad / s. The tests were conducted at 37°C, with a 25 mm diameter parallel plate.Immunofluorescence staining

[0147] At specified timepoints, cell-laden microfibers or cell suspension samples obtained from the cells released from the cell-laden microfibers were characterized by immunocytochemical staining with various markers of myogenesis. Firstly, the samples were fixed with 4% paraformaldehyde and left to stand at room temperature for 15 minutes. The fixation buffer was aspirated, and samples were washed thrice with PBS and left to stand at room temperature, for 5 minutes for each wash. 0.1% Triton X-100 (Sigma) in PBS was added to permeabilize the cell-laden microfibers and cell suspension, and the samples were left to stand at room temperature for 15 minutes, before the permeabilization solution was aspirated, and samples were washed thrice again. Following which, the samples were blocked with 2% bovine serum albumin (BSA; Sigma) and left to stand at room temperature for 1 hour, before the blocking buffer solution was replaced with the primary antibody and incubated overnight at 4°C. Samples were washed thrice with PBS the next day and subsequently stained with the corresponding secondary antibody, and samples were left to stand at room temperature in the dark for 1 hr. The samples were also counterstained with 4',6-diamidino-2-phenylindole (DAPI; Chemicon) and CFO488A conjugated-Phalloidin (Biotium), which stains for the nucleus and the filamentous actin (f-actin), respectively. The antibodies and fluorescent probes used are listed in Table 2 below.

[0148] Table 2: List of all antibodies and fluorescent probes usedPrimary antibodiesOther fluorescent probesMyogenesis of cells cultured within Microfibers

[0149] The myogenesis of cells cultured within microfibers were characterized via immunocytochemical staining. At specific timepoints of culture, immunocytochemical staining as described previously in sub-chapter 3.3.4, “Immunocytochemical Staining”, was performed on cell-laden microfiber samples. Cell-laden microfibers, if cultured on drum sleeves, were removed prior to staining. The stained cell-laden microfibers go through a last washing step with PBS before they were mounted onto glass slides for imaging under Zeiss LSM 710 Confocal Microscope.

[0150] The myogenic yield of cells within the microfibers was determined using cell suspension samples. To collect cells from cell-laden microfibers, the microfibers were treated with 0.25% Trypsin-EDTA (Gibco) for 8-10 minutes at 37°C. After treatment with Trypsin, the microfibers were digested, and cells were released. Equal volume of culture media was added to stop the reaction. The released cells were collected and subsequently collected and pelleted in a 1.5-ml microtube via centrifugation at 15 G for 5 minutes, while the supernatant was aspirated. The cells were subjected to immunocytochemical staining (see previous section) prior to cell count to determine the number of cells that were positively stained with the late phase myogenic marker, myosin heavy chain (mhc), which is indicative of the myogenic yield.Cell viability staining

[0151] The cell viability test of ADSCs were performed using the LIVE / DEAD cell viability assay (Life Technologies). At specific time points, cell laden micro fibers were removed from drum sleeves and incubated in 2 p Calcein-AM, which stains the cytoplasm of live cells, and 4 pM Ethidium homodimer (EthD-1), which stains the nucleus of dead cells, for 30 min at 37 °C and 5% CO2. The cell laden micro fiber samples were imaged via confocal microscopy (Carl Zeiss LSM 700, AG, Oberkochen, Germany).qPCR of myf5 and mhc

[0152] Total RNA was extracted using the PureLink RNA Mini Kit. cDNA was synthesized using iScript (Bio-Rad). qPCR was performed using SYBR Fast qPCR Master Mix (KAPA) on a CFX96 system (Bio-Rad). Relative gene expression of myogenic factor 5 (myf5) and myosin heavy chain (mhc) was normalized to GAPDH as the housekeeping gene using the AACt method. The primer sequences used were as follows: for myf5, forward 5'-GCCTGTCCGCAGAAGATGG-3' and reverse 5'-CGTGGCTCAAACTCGTCCC-3'; and for mhc, forward 5'-TCCCAAGGAGAGACCACTGT-3' and reverse 5'-CCGTCAGCTTGTACATGGAGT-3'. All qPCR reactions were performed in technical and biological triplicates.Two compression test for texture profile analysis

[0153] Texture profile analysis was performed with a texture analyzer (TA.XT plus C, Stable Micro Systems) and a cylindrical, 75 mm diameter, probe. A two-compression test was conducted on samples with the length x width x height of approximately 10 mm x 10 mm x 5 mm. The exact dimensions of the samples were measured with a vernier calliper right before sample loading. The fiber axis of the samples was placed perpendicular to the force axis during analysis and the tests were conducted at ambient temperature. Samples were compressed twice at a crosshead speed of 3 mm / s to 50% of its original height, with a time interval of 1 s in between the two compression cycles. The force-time plot experienced by the samples were presented and the hardness and chewiness values were calculated. For comparison across samples, the hardness and chewiness of the samples were normalized to their volume. Triplicate samples were employed for analysis.Statistical Analysis

[0154] The studies were performed with triplicates (n=3) and expressed as mean ± standard deviation unless otherwise specified. The statistical significance was analyzed using Kruskal-Wallis non-parametric one-way analysis of variance and Mann- Whitney U test, where the data was considered statistically significant with a p value of < 0.05.

Claims

CLAIMSWhat is claimed are:

1. A method of manufacturing a myocyte-laden scaffold, wherein the method comprises:(a) preparing a mixture comprising myocyte pre-cursor cells and a food-grade polymeric solution;(b) forming a fibre from the mixture from (a) such that the myocyte-precursor cells are confined in the fibre under a static tension;(c) culturing the myocyte precursor cells confined in the fibre from (b) under the static tension for differentiating the myocyte pre-cursor cells into myocytes to obtain the myocyte-laden scaffold.

2. The method of claim 1, wherein the static tension is created in the fibre when the mixture is extruded and collected onto a rotating drum sleeve in a coagulation bath.

3. The method of claim 2, wherein step (c) is performed by contacting the fibre spun unto the drum sleeve in a food-grade culture medium without removing the fibre from the drum sleeve.

4. The method of any one of claims 1-3, wherein the fibre of step (b) is formed by a fiberforming method comprising any one of wet spinning, microfluidic spinning, electrospinning, microextrusion bioprinting, interfacial polyelectrolyte complexation, template-assisted fibre fabrication, co-axial spinning or 3D bioprinting.

5. The method of claim 4, wherein the fibre of step (b) is formed by wet spinning, and wherein the mixture is extruded at a flow rate of about 0.05 to about 0.2 ml / min and the extruded fiber is collected onto the rotating drum sleeve rotating at a speed of about 60 to about 90 rpm.

6. The method of claim 5, wherein the flow rate is about 0.1 ml / min and the rotating speed is about 80 rpm.

7. The method of any one of claims 1-6, wherein step (c) is performed for at least about 14 days.

8. The method of any one of claims 1-7, wherein the cell density in the mixture is between about 8 million cells / mL to 15 million cells / mL.

9. The method of any one of claims 1-8, wherein the food grade polymeric solution comprises one or more materials selected from the group consisting of: alginate, pectin, agarose, carrageenan, gellan gum, konjac glucomannan, xanthan gum, guar gum, locust bean gum, gum arabic, starches, modified starches, pullulan, P-glucans including curdlan, cellulose, cellulose derivatives including carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, bacterialnanocellulose, gelatin, collagen, whey proteins, casein or caseinates, soy proteins, pea proteins, gluten, glutenin, gliadin, zein and combinations thereof.

10. The method of any one of claims 1 -9, wherein the one or more material in the polymeric solution is present at an amount of about 3.0-4.0% weight / volume (w / v) for each material.

11. The method of claim 9, wherein the food grade polymeric solution comprises a composition comprising alginate and gelatin.

12. The method of claim 11, wherein the amount of alginate or gelatin is about 3.0-4.0% weight / volume (w / v).

13. The method of any one of claims 1-12, wherein the myocyte precursor cells are selected from the group consisting of myoblast, mesenchymal stem cells, adipose- derived stem cells, myostatellite cells, embryonic stem cells, induced-pluripotent stem cells and a combination thereof.

14. The method of any one of claims 1-13, wherein the fibre and the myocyte-laden scaffold are characterized by a stiffness of 10 kPa -35 kPa.

15. The method of any one of claims 1-14, wherein the diameter of the fibre and the myocyte-laden scaffold is about 50 pm to about 600 pm.

16. The method of any one of claims 1-15, wherein the myocytes are characterized by expression of the late-stage myogenic marker, myosin heavy chains (mhc).

17. The method of any one of claims 1-16, wherein the fiber under the static tension has an internal stress about 500 Pa to about 900 Pa.

18. The method of any one of claims 1-17, wherein the method is used for cultivating meat.

19. The method of any one of claims 1-18, wherein the method further comprises adhering the myocyte-laden scaffolds together using a food binder to obtain a meat product.

20. The method of claim 19, wherein the food binder is selected from the group consisting of: alginate, pectin, agarose, carrageenan, gellan gum, konjac glucomannan, xanthan gum, guar gum, locust bean gum, gum arabic, starches, modified starches, pullulan, P- glucans including curdlan, cellulose, cellulose derivatives including carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, bacterial nanocellulose, gelatin, collagen, whey proteins, casein or caseinates, soy proteins, pea proteins, gluten, glutenin, gliadin, zein and combinations thereof.

21. A myocyte-laden scaffold obtained or obtainable from the method of any one of claims 1-17.

22. A cultivated meat product obtained or obtainable from the method of claim 19 or 20.

23. A kit for obtaining a myocyte-laden scaffold comprising:(a) one or more food-grade scaffold materials;(b) one or more populations of myocyte precursor cells;(c) a food-grade culture medium; and(d) a fibre-forming system.

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