Single-phase edible microcarrier for the three-dimensional culture of stem cells for the preparation of a cultured meat product

Abstract

The present invention relates to a single-phase, spherical edible microcarrier for the three-dimensional culture of stem cells intended for the preparation of cultured meat. The microcarrier comprises a homogeneous mixture of edible ionic polysaccharide (0.375-1.5%), edible protein (1-2.5%), edible polymer (0.025-0.1%) and water (95.9-98.6%). The invention also relates to a method for preparing these microcarriers by mixing three solutions: ionic polysaccharide in saline solution, edible protein in saline solution and edible polymer in water. The mixture is subjected to a process involving microspraying or electrospraying with calcium chloride in order to form spherical microcarriers of 50-300 micrometres. The design of the single-phase, spherical edible microcarrier simplifies the production of cultured meat by dispensing with the need for core-shell structures, thereby improving cell anchorage efficiency and facilitating cell recovery.

Description

[0001] SINGLE-PHASE EDIBLE MICRO-SUPPORT FOR THREE-DIMENSIONAL CULTURE OF STEM CELLS FOR THE PREPARATION OF A CULTURED MEAT PRODUCT

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to edible spherical micro-supports for the three-dimensional cultivation of stem cells in order to prepare a cultured meat product, as well as to the method of producing said micro-supports, the use thereof, and cultured meat products based on these micro-supports.

[0004] BACKGROUND OF THE INVENTION

[0005] Meat is an essential source of protein in the human diet. Due to growing concerns about animal welfare and the environmental impacts associated with the traditional meat industry, as well as increasing global demand for meat products, developing sustainable production alternatives is crucial. Tissue engineering technology offers an opportunity to produce edible sources of animal protein without the environmental problems linked to livestock farming. However, growing meat from cells presents significant challenges, including scaling up the process.

[0006] Micro-supports are promising tools for scaling up anchorage-dependent cell culture in bioreactors, as they provide a large surface area to volume ratio that facilitates cell anchorage. The current trend in micro-support development is to produce them with a core and a layer, as described in US patent application US-20220395537A1 and international PCT patent applications WO-2022239810A1 and WO-2023104768A1. US-20220395537A1 describes micro-supports consisting of a sodium alginate core covered with a gelatin layer. WO-2022239810A1 describes dry, spherical cell culture micro-supports composed of an alginate core with a gelatin layer, or vice versa.WO2023104768A1 describes an edible micro-support for culturing cells to prepare cultured meat products; the micro-support consists of a core made of hydrogel and a layer of crosslinked biopolymer with a crosslinking agent.

[0007] However, these core-coated micro-scaffolds have several disadvantages. First, the presence of a coating can affect the cells' ability to anchor efficiently, resulting in less effective cell growth. Furthermore, the production of these micro-scaffolds is more complex and expensive, as the coating process involves additional steps and the use of chemical agents that are not always compatible with food safety standards.

[0008] Another significant disadvantage is the difficulty in separating the cells from the micro-support at the end of the culture process. If the coating is biodegradable, it must be ensured that it does not affect the quality of the final product; if it is not biodegradable, additional filtration processes are required. Furthermore, ensuring that the core and layer materials comply with food safety standards can limit material options and increase costs. Maintaining the mechanical stability of these micro-supports under culture conditions is also challenging, as they must be robust enough to avoid premature degradation without compromising their biocompatibility. Finally, these micro-supports may exhibit poor cell release performance, as cells can become trapped between the core and the layer, hindering their retrieval.

[0009] Due to these disadvantages, it is necessary to develop single-phase micro-supports that simplify production, improve cell anchoring efficiency, ensure food compatibility, maintain mechanical stability, and facilitate cell recovery at the end of the culture process.

[0010] SUMMARY OF THE INVENTION

[0011] The object of the present invention is to provide an edible spherical micro-support for three-dimensional culture of stem cells for the preparation of a cultured meat product. The micro-support is formed from a homogeneous mixture of 0.375 to 1.5% by weight of an edible ionic polysaccharide, 1 to 2.5% by weight of an edible protein, 0.025% to 0.1% by weight of an edible polymer, and 95.9 to 98.60% by weight of water; such that the micro-support is single-phase.

[0012] The present invention also provides a method for preparing edible spherical micro-supports for stem cell culture. The method comprises the steps of: preparing a first solution of at least one edible ionic polysaccharide in a saline solution; preparing a second solution of at least one edible protein in a saline solution; preparing a third solution of at least one edible polymer in distilled water; homogeneously mixing the first, second, and third solutions; subjecting the homogeneous mixture to a microspraying or electrospraying process with calcium chloride, allowing the formation of single-phase spherical micro-supports; and collecting the spherical micro-supports.It is also an object of the present invention to provide a method for cultivating stem cells on the micro-supports of the present invention, by the steps of: preparing a crosslinking solution with a crosslinking agent; immersing the micro-supports in the crosslinking solution; conditioning the crosslinked micro-supports with culture medium; seeding stem cells at a density of 4,500 to 5,500 cells / cm. 2 ; and cultivate the cells under controlled temperature and CO2 conditions.

[0013] Another object of the present invention is to provide a method for manufacturing a cultured meat product by the steps of: cultivating stem cells on the micro-carriers of the present invention; collecting the micro-carriers with the cultured cells; and processing the micro-carriers with cultured cells by 3D bioprinting to form a cultured meat product.

[0014] The present invention also aims to provide the use of micro-supports in a 3D bioprinting process for the preparation of cultured meat products.

[0015] Finally, the object of the present invention is to provide a cultured meat product formed by micro-supports of the present invention; and cultured stem cells selected from the group consisting of mesenchymal stem cells, cells of embryonic origin, induced pluripotent stem cells, and combinations thereof, derived from animal tissues selected from the group consisting of pig, bovine, poultry, fish and combinations thereof.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] The features of the present invention will become evident from the following detailed description considered in conjunction with the accompanying drawings. It should be understood, however, that the drawings are provided solely for illustrative purposes and not as a limiting definition of the invention, in which:

[0018] Figure 1 illustrates a flow diagram of a method for preparing single-phase edible spherical micro-supports for stem cell culture for the preparation of a cultured meat product according to the present invention.

[0019] Figure 2 illustrates a flow diagram of a method for growing stem cells on single-stage edible spherical micro-supports according to the present invention.

[0020] Figures 3A and 3B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of alginate-gelatin-cellulose micro-supports according to the present invention. Figures 4A, 4B, 4C, and 4D illustrate 10x phase-contrast microscope micrographs of alginate-gelatin-cellulose micro-supports seeded with stem cells derived from avian muscle tissue according to the present invention, Figure 4A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 4B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 4C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 4D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0021] Figures 5A, 5B and 5C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on alginate-gelatin-cellulose micro-supports for 7 days in accordance with the present invention, Figure 5A being a fluorescence micrograph with DAPI staining (nuclei), Figure 5B a fluorescence micrograph with phalloidin staining (actin), and Figure 5C a fluorescence micrograph with combination staining.

[0022] Figures 6A and 6B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of chitosan-gelatin-cellulose micro-supports in accordance with the present invention.

[0023] Figures 7A, 7B, 7C and 7D illustrate 10x phase-contrast microscope micrographs of chitosan-gelatin-cellulose micro-supports seeded with stem cells derived from avian muscle tissue in accordance with the present invention, Figure 7A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 7B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 7C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 7D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0024] Figures 8A, 8B and 8C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on chitosan-gelatin-cellulose micro-supports for 7 days in accordance with the present invention, Figure 8A being a fluorescence micrograph with DAPI staining (nuclei), Figure 8B a fluorescence micrograph with phalloidin staining (actin), and Figure 8C a fluorescence micrograph with combination staining.

[0025] Figures 9A and 9B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of alginate-gelatin-cellulose micro-supports in accordance with the present invention.

[0026] Figures 10A, 10B, 10C and 10D illustrate 10x phase-contrast microscope micrographs of alginate-gelatin-cellulose micro-supports seeded with stem cells derived from avian muscle tissue in accordance with the present invention, Figure 10A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 10B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 10C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 10D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0027] Figures 11A, 11B and 11C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on alginate-gelatin-cellulose micro-supports for 7 days in accordance with the present invention, Figure 11A being a fluorescence micrograph with DAPI staining (nuclei), Figure 11B a fluorescence micrograph with phalloidin staining (actin), and Figure 11C a fluorescence micrograph with combination staining.

[0028] Figures 12A and 12B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of alginate-gelatin-xanthan gum micro-supports in accordance with the present invention.

[0029] Figures 13A, 13B, 13C and 13D illustrate 10x phase-contrast microscope micrographs of alginate-gelatin-xanthan gum micro-supports seeded with stem cells derived from avian muscle tissue according to the present invention, Figure 13A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 13B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 13C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 13D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0030] Figures 14A, 14B and 14C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on alginate-gelatin-xanthan gum micro-supports for 7 days in accordance with the present invention, Figure 14A being a fluorescence micrograph with DAPI staining (nuclei), Figure 14B a fluorescence micrograph with phalloidin staining (actin), and Figure 14C a fluorescence micrograph with combination staining.

[0031] Figures 15A and 15B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of alginate-pea protein-cellulose micro-supports in accordance with the present invention.

[0032] Figures 16A, 16B, 16C and 16D illustrate 10x phase-contrast microscope micrographs of alginate-pea protein-cellulose micro-supports seeded with stem cells derived from avian muscle tissue according to the present invention, Figure 16A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 16B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 16C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 16D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0033] Figures 17A, 17B and 17C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on alginate-pea protein-cellulose micro-supports for 7 days in accordance with the present invention, Figure 17A being a fluorescence micrograph with DAPI staining (nuclei), Figure 17B a fluorescence micrograph with phalloidin staining (actin), and Figure 17C a fluorescence micrograph with combination staining.

[0034] Figures 18A and 18B illustrate 4x and 10x phase-contrast microscope micrographs, respectively, of pectin-gelatin-cellulose micro-supports in accordance with the present invention.

[0035] Figures 19A, 19B, 19C and 19D illustrate 10x phase-contrast microscope micrographs of pectin-gelatin-cellulose micro-supports seeded with stem cells derived from avian muscle tissue in accordance with the present invention, Figure 19A showing the follow-up of cell adhesion and growth on the micro-supports at 0 hours, Figure 19B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 19C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, and Figure 19D showing the follow-up of cell adhesion and growth on the micro-supports at 7 days.

[0036] Figures 20A, 20B and 20C illustrate 10x fluorescence microscope micrographs of stem cells derived from avian muscle tissue seeded on pectin-gelatin-cellulose micro-supports for 7 days in accordance with the present invention, Figure 20A being a fluorescence micrograph with DAPI staining (nuclei), Figure 20B a fluorescence micrograph with phalloidin staining (actin), and Figure 20C a fluorescence micrograph with combination staining.

[0037] Figures 21A, 21B, 21C, 21D, 21E, 21F, 21G, and 21H illustrate 10x phase-contrast microscope micrographs of alginate-gelatin-cellulose micro-supports seeded with stem cells derived from avian muscle tissue according to the present invention, Figure 21A showing the follow-up of cell adhesion and growth on the micro-supports at 4 hours, Figure 21B showing the follow-up of cell adhesion and growth on the micro-supports at 7 hours, Figure 21C showing the follow-up of cell adhesion and growth on the micro-supports at 3 days, Figure 21D showing the follow-up of cell adhesion and growth on the micro-supports at 4 days, Figure 21E showing the follow-up of cell adhesion and growth on the micro-supports at 7 days, and Figure 21F showing the follow-up of cell adhesion and growth on the micro-supports. Figure 21G shows the monitoring of cell adhesion and growth on micro-supports at 10 days, and at 12 days,and Figure 21H shows the monitoring of cell adhesion and growth on the micro-supports at 14 days.

[0038] Figure 22 illustrates 10x fluorescence microscope micrographs using calcein-AM staining (live cells) and ethidium homodimer staining (dead cells) to evaluate the cell viability of a dynamic culture of alginate-gelatin-cellulose micro-supports seeded with porcine muscle tissue-derived stem cells monitored for 14 days, in accordance with the present invention.

[0039] Figure 23 illustrates 4x and 10x fluorescence microscope micrographs of dynamic culture of alginate-gelatin-cellulose micro-supports seeded with porcine muscle tissue-derived stem cells for nuclei and actin staining monitored for 14 days, in accordance with the present invention.

[0040] Figures 24A, 24B, 24C, 24D, 24E, 24F, 24G, and 24H illustrate 10x phase-contrast microscope micrographs of alginate-gelatin-microcrystalline cellulose micro-supports seeded with porcine muscle tissue-derived stem cells according to the present invention, Figure 24A showing the follow-up of cell adhesion and growth on the micro-supports at 3 hours, Figure 24B showing the follow-up of cell adhesion and growth on the micro-supports at 24 hours, Figure 24C showing the follow-up of cell adhesion and growth on the micro-supports at 4 days, Figure 24D showing the follow-up of cell adhesion and growth on the micro-supports at 6 days, Figure 24E showing the follow-up of cell adhesion and growth on the micro-supports at 7 days, and Figure 24F showing the follow-up of cell adhesion and growth on the micro-supports at 8 days. micro-supports after 10 days,Figure 24G shows the monitoring of cell adhesion and growth on the micro-supports at 12 days, and Figure 24H shows the monitoring of cell adhesion and growth on the micro-supports at 14 days.

[0041] Figure 25 illustrates 10x fluorescence microscope micrographs using calcein-AM staining (live cells) and ethidium homodimer staining (dead cells) to evaluate the cell viability of a dynamic culture of alginate-gelatin-microcrystalline cellulose micro-supports seeded with porcine muscle tissue-derived stem cells monitored for 14 days, in accordance with the present invention.

[0042] Figure 26 illustrates 4x and 10x fluorescence microscope micrographs of dynamic culture of alginate-gelatin-microcrystalline cellulose micro-supports seeded with porcine muscle tissue-derived stem cells for nuclei and actin staining monitored for 14 days, in accordance with the present invention.

[0043] Figure 27 illustrates 4x, 10x and 20x phase contrast microscopy and fluorescence micrographs of oil red and lipid tox staining respectively of the adipogenic differentiation process of adipose tissue-derived stem cells of porcine origin in edible single-phase micro-supports in dynamic culture according to the present invention, monitored for 21 days.

[0044] Figure 28 illustrates 10x fluorescence microscope micrographs of surface marker stains of porcine muscle tissue-derived stem cells in accordance with the present invention: ac) PAX 7, df) CD44.

[0045] Figure 29 illustrates 20x fluorescence microscope micrographs of surface marker stains of mesenchymal stem cells derived from porcine adipose tissue in accordance with the present invention: ac) CD44, df) CD90.

[0046] Figure 30 illustrates 20x phase-contrast microscope micrographs of follow-up adipogenic differentiation of stem cells derived from porcine adipose tissue with oil red staining to identify lipid formation in accordance with the present invention: a) 3 days, b) 7 days, c) 14, d) 21 days.

[0047] Figure 31 illustrates a schematic 3D bioprinting process with micro-supports cultured with pre-differentiated stem cells in a fillet model according to the present invention.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] The characteristic details of this invention are described in the following paragraphs, which have the objective of defining the invention, but without limiting its scope.

[0050] The present invention relates to a method for preparing edible spherical micro-supports designed for stem cell culture, for the purpose of producing cultured meat products. This method is based on the preparation of single-phase micro-supports, overcoming the disadvantages of traditional micro-supports that have a core and shell structure.

[0051] In the context of the present invention, the term "single-phase" refers to a homogeneous and uniform structure throughout the micro-support, without the distinction of a core and a separate layer. In this context, it means that the components of the micro-support are completely integrated and mixed so that there is no visible difference or separation between an inner core and an outer layer. This homogeneity ensures that the micro-support has consistent properties throughout its structure, facilitating better cell anchorage and simplifying both its production and use in cell culture processes for cultured meat.

[0052] The term "cultured meat" refers to food products comprising animal cells produced by in vitro culture. Figure 1 illustrates a flow diagram of a method for preparing single-stage edible spherical micro-supports for stem cell culture, intended for the preparation of a cultured meat product according to the present invention. In step 100, a first solution of at least one edible ionic polysaccharide from 0.75% to 3% w / v in a first solvent from 0.8% to 1% is prepared, and left under magnetic stirring until completely dissolved.

[0053] The edible ionic polysaccharide, due to its ability to form hydrogels through bonding with divalent cations such as calcium or anions such as tripolyphosphate, is necessary for this invention. The edible ionic polysaccharides may be selected from alginate, pectin, chitosan, and combinations thereof.

[0054] Alginate, based on its degree of polymerization and, consequently, its molecular weight, is classified as low molecular weight alginate (10,000 to 80,000 g / mol), medium molecular weight alginate (85,000 to 250,000 g / mol), and high molecular weight alginate (300,000 to 600,000 g / mol). The higher the molecular weight of the alginate, the greater the viscosity of the resulting solution, which means that the gel formed will have greater rigidity. Furthermore, the greater the rigidity of the gel, the greater its tendency to syneresis (water loss through gel exudation). For the present invention, alginate from the medium molecular weight range is used, seeking solutions that can flow easily during the fabrication of the micro-supports by electrospraying or air microspraying.

[0055] Low-methoxyl pectin can also be used to manufacture micro-supports, as it behaves similarly to alginates. In a system saturated with calcium ions, pectin can form gels stabilized by these divalent ions. Chitosan is another base material for micro-support production because it can form hydrogels in the presence of anions such as tripolyphosphate. Chitosan is available in different molecular weight ranges: high molecular weight (310,000 to 400,000 Da), medium molecular weight (190,000 to 310,000 Da), and low molecular weight (50,000 to 190,000 Da). In this case, low- to medium-molecular-weight chitosan is preferred.

[0056] The first solvent can be selected from saline solution, distilled water, solutions with divalent cations, dilute acetic acid, organic acids, and combinations thereof. Solutions with divalent cations include calcium chloride, calcium sulfate, magnesium chloride, magnesium sulfate, calcium lactate, and combinations thereof. Generally, the choice of the first solvent depends on the specific properties of the edible ionic polysaccharide and the objective of the first solution. In step 200, a second solution of at least one edible protein at a concentration of 2 to 6% w / v in a 0.8 to 1% w / v saline solution is prepared by magnetic stirring and heating to a temperature of 45 to 50°C until the edible protein is completely dissolved. The edible protein used in the present invention contains amine or amide groups, such as peptones or proteins of animal or vegetable origin.Examples of such edible proteins include gelatin, collagen, elastin, alpha-lactalbumin, peptides, soy proteins, and combinations thereof. These edible proteins have the ability to cross-link via covalent bonds using enzymes such as transglutaminase or compounds such as glutaraldehyde and genipin, among others. This cross-linking provides resistance to dissolution of the micro-support in the culture medium and creates anchoring zones for cell adhesion.

[0057] Mammalian gelatin has a molecular weight of 95,000 to 100,000 Da, and its Bloom number (an indicator of gel strength proportional to molecular weight) ranges from 90 to 300 g. Its molecular weight determines its properties, such as viscosity and gel strength. Being of polypeptide origin, gelatin can exhibit various molecular conformations, from globular-like structures to random structures. The unique properties of gelatin include the presence of ionizable functional groups from the amino acids that make up its polypeptide chains, such that acidic or basic groups can be found within the same molecule. Another unique characteristic is its ability to form triple-stranded helical structures, usually produced through collagen denaturation.For these reasons, different types of gelatin exhibiting varying gel strengths are found in the food industry, characterized by their Bloom number, a penetration strength measured with a texture analyzer, as well as by their melting points. For the present invention, gelatin with a molecular weight of 30,000 to 45,000 Da and a Bloom number of 90 to 120 g is used.

[0058] In step 300, a third solution of at least one edible polymer is prepared at a concentration of 0.2 to 0.3% w / v in filtered water, stirring until completely dissolved. The present invention utilizes edible polymers due to their ability to act as soluble modifiers and water retainers, and because they can also have chemical interactions with the other components of the micro-support. Among the edible polymers used are cellulose derivatives (such as microcrystalline cellulose, methylcellulose, and ethylcellulose). Hydrocolloids such as gums, including xanthan gum, guar gum, carrageenan, locust bean gum, and starches, such as tapioca starch and combinations thereof, can also be employed.

[0059] Steps 100, 200, and 300 can be performed sequentially or simultaneously. Once the first, second, and third solutions are prepared, they are subjected, in an alternative step 400, to a sterilization process, which can be carried out in an autoclave at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored under refrigeration at a temperature of around 4°C.

[0060] Alternatively, in step 500, the first, second, and third solutions are tempered in a water bath at 35–40°C for at least 15–20 minutes. Then, in step 600, the first, second, and third solutions are homogenously mixed in a 40–60 / 30–50 / 10–20 volume / volume ratio. That is, to prepare 10 mL of the homogenous mixture, 5 mL of the first solution (0.75–3%), 4 mL of the second solution (2–4%), and 1 mL of the third solution (0.2–0.3%) are added. This homogenization can be carried out in a biosafety cabinet to avoid compromising sterilization.

[0061] In stage 700, the homogeneous mixture is subjected to an electro-spraying or air micro-spraying process with 0.5 to 1.5% w / v calcium chloride or 5 to 10% w / v sodium tripolyphosphate, allowing the formation of single-phase spherical micro-supports. During the electro-spraying or air micro-spraying process, the formation of surface aggregates must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes.

[0062] Finally, in stage 800, the edible single-phase spherical micro-supports are collected, left to stand for 15 to 30 minutes in a 0.5 to 1.5% w / v calcium chloride solution or 5 to 10% w / v sodium tripolyphosphate, and then subjected to washing with sterile water.

[0063] As a result of the described method, single-phase edible spherical micro-supports are obtained for use in the three-dimensional culture of stem cells. The micro-support comprises a homogeneous mixture of 0.375 to 0.67 wt% of an edible ionic polysaccharide, 1 to 1.5 wt% of an edible protein, 0.025 to 0.1 wt% of an edible polymer, and 97.73 to 98.48 wt% water.

[0064] Figure 2 illustrates a flow diagram of a method for culturing cells on single-phase edible spherical micro-supports according to the present invention. In step 900, a crosslinking solution of at least one crosslinking agent in water is prepared in the range of 12.5 mg / mL to 50 mg / mL. This crosslinking agent can be selected from the group consisting of: transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, tannic acid, or a combination thereof, preferably transglutaminase. The crosslinking solution can be sterilized by filtration.

[0065] In step 1000, edible single-phase spherical micro-supports are immersed in a sufficient volume of the crosslinking solution and allowed to stand under agitation at 800–1000 rpm for 120–240 minutes. This allows the transglutaminase to bind to the surface of the edible single-phase spherical micro-supports.

[0066] In step 1100, the edible, single-phase spherical micro-supports are conditioned for cell culture by washing them with sterile water and then with a culture medium containing essential and non-essential amino acids, acids, vitamins, organic and inorganic compounds, hormones, growth factors, and trace elements. They are then incubated at 36–38°C under 4–6% CO2 conditions for 100–140 minutes to condition them to the cell culture conditions of 37°C and 5% CO2.

[0067] In stage 1200, mesenchymal stem cells or other cell lines suitable for producing cultured meat, such as embryonic stem cells, induced pluripotent stem cells, or others, whether immortalized or not, derived from animal tissues such as pig, bovine, poultry, or fish, are seeded onto conditioned, single-phase edible spherical micro-supports. These conditioned micro-supports are introduced into a stem cell suspension in a suitable culture medium containing essential and non-essential amino acids, acids, vitamins, organic and inorganic compounds, hormones, growth factors, and trace elements, at a concentration of 4,500 to 5,500 cells / cm³. 2 and are incubated at 36 to 38°C under CO2 conditions of 4 to 6% for a specified period (no longer than 7 days) to allow initial cell adhesion.

[0068] In stage 1300, cell culture and monitoring are carried out. Micro-supports seeded with stem cells are transferred to a bioreactor or three-dimensional culture system, maintaining optimal culture conditions (temperature, CO2, nutrients) according to the type of stem cells used. Periodic monitoring of cell growth and viability is performed using techniques such as cell counting, viability assays, and microscopy, adjusting culture conditions as needed to optimize cell growth and tissue formation. Finally, in stage 1400, the harvested cells are collected. Once the desired cell density is reached, the micro-supports with cultured tissue are harvested and carefully washed to remove residual culture medium.Micro-supports with cultured tissue are used according to the intended application, in this case for the development of cultured meat products such as 3D bioprinting, using the tissue micro-supports for the development of muscle and / or fat tissue bio-inks with which three-dimensional models can be printed simulating a traditional cut of meat or as functional ingredients for the development of food products such as sausages, hams, among others.

[0069] EXAMPLES OF IMPLEMENTATION OF THE INVENTION

[0070] The invention will now be described with respect to the following examples, which are solely for the purpose of illustrating how to implement the principles of the invention. The following examples are not intended to be an exhaustive representation of the invention, nor are they intended to limit its scope. EXAMPLE 1: BASIC SOLUTION

[0071] A first solution was prepared containing 0.75% w / v sodium alginate (85,000–250,000 g / mol) in a 0.9% w / v saline solution. For this, 0.75 g of sodium alginate was weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 2 hours. Subsequently, a second solution of 3% gelatin in a 0.9% w / v saline solution was prepared. Therefore, 3 g of gelatin with a Bloom number of 100 were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g of NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and heated to a temperature of 45–50°C until the gelatin was completely dissolved. Subsequently, a third solution of 0.25% cellulose in distilled water was prepared. In this way, 0.25 g of cellulose were dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 1 hour. Once the first, second, and third solutions were prepared, they were sterilized by autoclaving at 100–110°C for 10–15 minutes, and then refrigerated at approximately 4°C for 8 hours. Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first solution, the second solution, and the third solution were mixed homogeneously in a 50 / 40 / 10 ratio, that is, 10 mL of the homogeneous mixture were prepared, and for this purpose, 5 mL of the first solution were taken, then 4 mL of the second solution and 1 mL of the third solution were added.This homogeneous mixing was carried out in a biosafety chamber to avoid compromising sterilization.

[0072] EXAMPLE 2: SUBSTITUTION OF ALGINATE WITH CHITOSAN

[0073] A first solution was prepared containing 1.67% w / v chitosan of medium molecular weight (190,000–310,000 Da) in 0.4% w / v acetic acid. For this, 1.67 g of chitosan were weighed and dissolved in 100 mL of 0.4% w / v acetic acid (0.4 g of acetic acid in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 12 hours. Subsequently, a second solution of 3.35% gelatin in 0.9% w / v saline was prepared. Therefore, 3.35 g of gelatin with a Bloom number of 100 were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g of NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and heated to a temperature of 45–50°C until the gelatin was completely dissolved. Subsequently, a third solution of 0.25% cellulose in distilled water was prepared. In this way, 0.25 g of cellulose were dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 1 hour. Once the first, second and third solutions were prepared, they were subjected to a sterilization process by autoclave at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored under refrigeration at a temperature of around 4°C for 8 hours.

[0074] Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first, second, and third solutions were homogenously mixed in a 60 / 30 / 10 ratio; that is, 10 mL of the homogenous mixture was prepared by taking 6 mL of the first solution, then adding 3 mL of the second solution and 1 mL of the third solution. This homogenous mixing was carried out in a biosafety cabinet to avoid compromising sterilization.

[0075] EXAMPLE 3: REPLACEMENT OF CELLULOSE WITH MICROCRYSTALLINE CELLULOSE

[0076] A first solution was prepared containing 0.75% w / v sodium alginate (85,000–250,000 g / mol) in a 0.9% w / v saline solution. For this, 0.75 g of sodium alginate was weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 2 hours. Subsequently, a second solution of 3% gelatin in a 0.9% w / v saline solution was prepared. Therefore, 3 g of gelatin with a Bloom number of 100 were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g of NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and heated to a temperature of 45–50°C until the gelatin was completely dissolved. Subsequently, a third solution of 0.25% microcrystalline cellulose (particle size of 65 µm) in distilled water was prepared.Thus, 0.25 g of microcrystalline cellulose were weighed and dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 1 hour. Once the first, second, and third solutions were prepared, they were subjected to an autoclave sterilization process at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored under refrigeration at a temperature of around 4°C for 8 hours.

[0077] Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first, second, and third solutions were homogenously mixed in a 50 / 40 / 10 ratio; that is, 10 mL of the homogenous mixture was prepared by taking 5 mL of the first solution, then adding 4 mL of the second solution and 1 mL of the third solution. This homogenous mixing was carried out in a biosafety cabinet to avoid compromising sterilization.

[0078] EXAMPLE 4: SUBSTITUTION OF CELLULOSE WITH XANTHAN GUM

[0079] A first solution was prepared containing 0.75% w / v sodium alginate (85,000–250,000 g / mol) in a 0.9% w / v saline solution. For this, 0.75 g of sodium alginate was weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 2 hours. Subsequently, a second solution of 3% gelatin in a 0.9% w / v saline solution was prepared. Therefore, 3 g of gelatin with a Bloom number of 100 were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g of NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and heated to a temperature of 45–50°C until the gelatin was completely dissolved. Subsequently, a third solution of 0.25% xanthan gum in distilled water was prepared. In this way, 0.25 g of xanthan gum were dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and heated to a temperature of 45 to 50°C until the xanthan gum was completely dissolved. Once the first, second, and third solutions were prepared, they were subjected to an autoclave sterilization process at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored under refrigeration at a temperature of around 4°C for 8 hours.

[0080] Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first, second, and third solutions were homogenously mixed in a 50 / 40 / 10 ratio; that is, 10 mL of the homogenous mixture was prepared by taking 5 mL of the first solution, then adding 4 mL of the second solution and 1 mL of the third solution. This homogenous mixing was carried out in a biosafety cabinet to avoid compromising sterilization.

[0081] EXAMPLE 5: SUBSTITUTING GELATIN WITH PEA PROTEIN

[0082] A first solution was prepared containing 0.75% w / v sodium alginate (85,000–250,000 g / mol) in a 0.9% w / v saline solution. For this, 0.75 g of sodium alginate was weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 2 hours. Subsequently, a second solution of 3% pea protein isolate was prepared in a 0.9% w / v saline solution. Therefore, 3 g of pea protein isolate (aqueous medium extraction, 84% protein content) were weighed and sterilized by ultraviolet radiation for 10 minutes, then dissolved in 100 mL of sterile 0.9% weight / volume saline solution (0.9 g of NaCl in 100 mL of distilled water were mixed under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 36 hours. The solution was then centrifuged at 2000 rpm for 3 minutes. The supernatant, containing the most solubilized protein, was removed and set aside as a second sterile solution. Subsequently, a third solution of 0.25% cellulose in distilled water was prepared. 0.25 g of cellulose was weighed and dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 1 hour. Once the first and third solutions were prepared, they were subjected to an autoclave sterilization process at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored together with the second solution in refrigeration at a temperature of around 4°C for 8 hours.

[0083] Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first, second, and third solutions were homogenously mixed in a 50 / 40 / 10 ratio; that is, 10 mL of the homogeneous mixture was prepared by taking 5 mL of the first solution, then adding 4 mL of the second solution and 1 mL of the third solution. This mixture was subjected to a sonication process for 10 minutes (30-second on / off cycles) to improve the dispersion of the pea protein. This was carried out in a biosafety cabinet to avoid compromising sterilization. EXAMPLE 6: ALGINATE SUBSTITUTION WITH PECTIN

[0084] A first solution was prepared containing 2% w / v low-methoxyl pectin of medium molecular weight (60,000–90,000 g / mol) in a 0.9% w / v saline solution. For this purpose, 2 g of low-methoxyl pectin were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 2 hours. Subsequently, a second solution of 6% gelatin in a 0.9% w / v saline solution was prepared. Therefore, 6 g of gelatin with a Bloom number of 100 were weighed and dissolved in 100 mL of 0.9% w / v saline solution (0.9 g of NaCl in 100 mL of distilled water) under constant magnetic stirring at 400 rpm and heated to a temperature of 45–50°C until the gelatin was completely dissolved. Subsequently, a third solution of 0.25% cellulose in distilled water was prepared. In this way, 0.25 g of cellulose were dissolved in 100 mL of distilled water under constant magnetic stirring at 400 rpm and room temperature (22 ± 2°C) for 1 hour. Once the first, second, and third solutions were prepared, they were subjected to a sterilization process by autoclave at a temperature of 100 to 110°C for 10 to 15 minutes, and then stored under refrigeration at a temperature of around 4°C for 8 hours.

[0085] Subsequently, the first, second, and third solutions were tempered in a water bath at 35–37°C for at least 15–20 minutes. Then, the first, second, and third solutions were homogenously mixed in a 50 / 40 / 10 ratio; that is, 10 mL of the homogenous mixture was prepared by taking 5 mL of the first solution, then adding 4 mL of the second solution and 1 mL of the third solution. This homogenous mixing was carried out in a biosafety cabinet to avoid compromising sterilization.

[0086] EXAMPLE 7: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH BASE SOLUTION

[0087] Starting from the base solution described in Example 1, the preparation of micro-supports and cell culture was carried out as follows: a) Preparation of the micro-supports by means of electrospraying

[0088] The homogeneous mixture was subjected to an electro-spraying process using an infusion pump and a high-voltage power source. For this, the solution was drawn into a sterile syringe, which was then attached to an infusion pump and delivered at a flow rate of 0.9–1 mL / min under a voltage of 10–11 kV. A metal dish containing a 0.5–2% w / v calcium chloride solution was used as a collector for the micro-supports, allowing the formation of single-phase spherical micro-supports. During the electro-spraying or air micro-spraying process, the formation of aggregates on the surface must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes.The morphology of the micro-supports obtained is illustrated in Figures 3A and 3B by phase contrast microscope micrographs, Figure 3A being a 4x micrograph and Figure 3B a 10x micrograph. b) Preparation of the crosslinking solution:.

[0089] A microbial transglutaminase solution (enzyme activity: 100 U / g) was prepared at a concentration of 12.5–25 mg / mL in sterile distilled water. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Micro-support crosslinking:

[0090] The micro-supports obtained by electrospraying (average diameter: 203 ± 50 µm, as shown in Figures 3A and 3B) were immersed in the transglutaminase solution at a ratio of 1:10 (volume / volume). The mixture was stirred at 900 rpm for 180 minutes at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0091] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports. To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from porcine adipose tissue or avian muscle tissue, obtained from young animals between 3 and 6 months of age, were seeded at a cell density of 5000 cells / cm². 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low-adherence microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were monitored using phase-contrast microscopy, as shown in Figures 4A, 4B, 4C, and 4D. Cells were observed adhering to the micro-supports starting at 24 hours, and by day 7, some differentiated cells were visible on the micro-support surface. Cells adhering to the micro-supports were also analyzed at day 7 using fluorescence microscopy, stained with DAPI to visualize nuclei and phalloidin to visualize actin filaments, as shown in Figures 5A, 5B, and 5C.

[0092] EXAMPLE 8: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH SOLUTION EXAMPLE 2

[0093] Starting from the solution described in Example 2, the preparation of micro-supports and cell culture was carried out as follows: a) Preparation of the micro-supports by means of electro-spraying

[0094] The homogeneous mixture was subjected to an electrospraying process using an infusion pump and a high-voltage power source. For this, the solution was drawn into a sterile syringe, which was then attached to an infusion pump and delivered at a flow rate of 0.9–1 mL / min under a voltage of 11–20 kV. A metal dish containing a 5–10% w / v sodium tripolyphosphate solution was used as the microsupport collector, allowing the formation of single-phase spherical microsupports. During the electrospraying or air microspraying process, the formation of aggregates on the surface must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh sodium tripolyphosphate added every 30–60 minutes.The morphology of the micro-supports obtained is illustrated in Figures 6A and 6B by phase contrast microscope micrographs, Figure 6A being a 4x micrograph and Figure 6B a 10x micrograph. b) Preparation of the crosslinking solution:.

[0095] A microbial transglutaminase solution (enzyme activity: 100 U / g) was prepared at a concentration of 25 mg / mL in sterile distilled water. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Microsupport crosslinking: Microsupports obtained by electrospraying (average diameter: 228 ± 62 µm, as shown in Figures 6A and 6B) were immersed in the transglutaminase solution at a ratio of 1:10 (volume / volume). The mixture was stirred at 900 rpm for 180 minutes at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0096] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports

[0097] To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from pig adipose tissue or bird muscle tissue were used, obtained from young animals between 3-6 months of age, which were seeded at a cell density of 5000 cells / cm 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low-adherence microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were analyzed using phase-contrast microscopy, as shown in Figures 7A, 7B, 7C, and 7D, where cells were observed adhering after 24 hours. Cells adhering to the micro-supports were also analyzed after 7 days using fluorescence microscopy, stained with DAPI to visualize the nuclei and phalloidin to visualize the actin filaments of the cells adhering to the micro-supports, as shown in Figures 8A, 8B, and 8C.

[0098] EXAMPLE 9: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH SOLUTION EXAMPLE 3

[0099] Starting from the base solution described in Example 3, the preparation of micro-supports and cell culture was carried out as follows: b) Preparation of the micro-supports by means of electrospraying

[0100] The homogeneous mixture was subjected to an electro-spraying process using an infusion pump and a high-voltage power source. For this, the solution was drawn into a sterile syringe, which was then attached to an infusion pump and delivered at a flow rate of 0.9–1 mL / min under a voltage of 11–12 kV. A metal dish containing a 0.5–2% w / v calcium chloride solution was used as a collector for the micro-supports, allowing the formation of single-phase spherical micro-supports. During the electro-spraying or air micro-spraying process, the formation of aggregates on the surface must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes.The morphology of the micro-supports obtained is illustrated in Figures 9A and 9B by phase contrast microscope micrographs, Figure 9A being a 4x micrograph and Figure 9B a 10x micrograph. b) Preparation of the crosslinking solution:.

[0101] A microbial transglutaminase solution (enzyme activity: 100 U / g) was prepared at a concentration of 12.5–25 mg / mL in sterile distilled water. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Micro-support crosslinking:

[0102] The micro-supports obtained by electrospraying (average diameter: 234 ± 53 µm, as shown in Figures 9A and 9B) were immersed in the transglutaminase solution at a ratio of 1:10 (volume / volume). The mixture was stirred at 900 rpm for 180 minutes at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0103] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports

[0104] To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from pig adipose tissue or bird muscle tissue were used, obtained from young animals between 3-6 months of age, which were seeded at a cell density of 5000 cells / cm 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low-adherence microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were monitored using phase-contrast microscopy, as shown in Figures 10A, 10B, 10C, and 10D, where cells were observed adhering to the micro-supports starting at 24 hours. Cells adhering to the micro-supports were also analyzed at 7 days using fluorescence microscopy, stained with DAPI to visualize the nuclei and phalloidin to visualize the actin filaments of the cells adhering to the micro-supports, as shown in Figures 11A, 11B, and 11C. EXAMPLE 10: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH SOLUTION EXAMPLE 4

[0105] Starting from the base solution described in Example 4, the preparation of micro-supports and cell culture was carried out as follows: c) Preparation of the micro-supports by means of electrospraying

[0106] The homogeneous mixture was subjected to an electro-spraying process using an infusion pump and a high-voltage power source. For this, the solution was drawn into a sterile syringe, which was then attached to an infusion pump and delivered at a flow rate of 0.9–1 mL / min under a voltage of 17–19 kV. A metal dish containing a 0.5–2% w / v calcium chloride solution was used as a collector for the micro-supports, allowing the formation of single-phase spherical micro-supports. During the electro-spraying or air micro-spraying process, the formation of aggregates on the surface must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes.The morphology of the micro-supports obtained is illustrated in Figures 12A and 12B by means of phase contrast microscope micrographs, Figure 12A being a 4x micrograph and Figure 12B a 10x micrograph. b) Preparation of the crosslinking solution:.

[0107] A microbial transglutaminase solution (enzyme activity: 100 U / g) was prepared at a concentration of 25 mg / mL in sterile distilled water. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Micro-support crosslinking:

[0108] The micro-supports obtained by electrospraying (average diameter: 258 ± 52 µm, as shown in Figures 12A and 12B) were immersed in the transglutaminase solution at a ratio of 1:10 (volume / volume). The mixture was stirred at 900 rpm for 180 minutes at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0109] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports

[0110] To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from pig adipose tissue or bird muscle tissue were used, obtained from young animals between 3-6 months of age, which were seeded at a cell density of 5000 cells / cm 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low-adherence microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were monitored using phase-contrast microscopy, as shown in Figures 13A, 13B, 13C, and 13D, where cells were observed adhering to the micro-supports starting at 24 hours. Cells adhering to the micro-supports were also analyzed at 7 days using fluorescence microscopy, stained with DAPI to visualize the nuclei and phalloidin to visualize the actin filaments of the cells adhering to the micro-supports, as shown in Figures 14A, 14B, and 14C.

[0111] EXAMPLE 11: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH SOLUTION EXAMPLE 5

[0112] Starting from the base solution described in Example 5, the preparation of micro-supports and cell culture was carried out as follows: d) Preparation of the micro-supports by means of electrospraying

[0113] The homogeneous mixture was subjected to an electro-spraying process using an infusion pump and a high-voltage power source. For this, the solution was drawn into a sterile syringe, which was then attached to an infusion pump and delivered at a flow rate of 0.9–1 mL / min under a voltage of 10–16 kV. A metal dish containing a 0.5–2% w / v calcium chloride solution was used as a collector for the micro-supports, allowing the formation of single-phase spherical micro-supports. During the electro-spraying or air micro-spraying process, the formation of aggregates on the surface must be avoided. Depending on the type and size of the collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes.The morphology of the micro-supports obtained is illustrated in Figures 15A and 15B by means of phase contrast microscope micrographs, Figure 15A being a 4x micrograph and Figure 15B a 10x micrograph. b) Preparation of the crosslinking solution:.

[0114] A genipin solution was prepared at a concentration of 25 mg / mL in a 20% v / v ethanol solution in 0.9% w / v saline. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Micro-support crosslinking:

[0115] The micro-supports obtained by electrospraying (average diameter: 234 ± 97 µm, as shown in Figures 15A and 15B) were immersed in the genipin solution at a ratio of 1:10 (volume / volume). The mixture was kept under agitation at 900 rpm for 24–36 hours at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0116] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports

[0117] To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from pig adipose tissue or bird muscle tissue were used, obtained from young animals between 3-6 months of age, which were seeded at a cell density of 5000 cells / cm 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low-adherence microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were monitored using phase-contrast microscopy, as shown in Figures 16A, 16B, 16C, and 16D, where cells were observed adhering to the micro-supports starting at 24 hours. Cells adhering to the micro-supports were also analyzed at 7 days using fluorescence microscopy, stained with DAPI to visualize the nuclei and phalloidin to visualize the actin filaments of the cells adhering to the micro-supports, as shown in Figures 17A, 17B, and 17C.

[0118] EXAMPLE 12: PREPARATION OF MICRO-SUPPORTS AND CELL CULTURE WITH SOLUTION EXAMPLE 6

[0119] Starting from the base solution described in Example 6, the preparation of micro-supports and cell culture was carried out as follows: e) Preparation of the micro-supports by means of air microspraying

[0120] The homogeneous mixture was subjected to an air microspraying process using a coaxial device and a pressure controller connected to an external air source. The solution was loaded into a reservoir with microadapters connected to the pressure controller and flowed at a pressure of 150 mbar into the coaxial device through the core. Air at a pressure of 900 mbar was flowed through the shell portion to cut the solution droplets and form the microspheres. A metal plate containing a 0.5 to 2% w / v calcium chloride solution was used to collect the micro-supports, allowing the formation of single-phase spherical micro-supports. During the electrospraying or air microspraying process, the formation of aggregates on the surface must be avoided.Depending on the type and size of collector, the process should be stopped, the suspension recovered, and fresh calcium chloride added every 30 to 60 minutes. The morphology of the resulting micro-supports is illustrated in Figures 18A and 18B using phase-contrast micrographs, with Figure 18A being a 4x micrograph and Figure 18B a 10x micrograph. b) Preparation of the crosslinking solution:

[0121] A microbial transglutaminase solution (enzyme activity: 100 U / g) was prepared at a concentration of 12.5–25 mg / mL in sterile distilled water. The solution was filtered through a 0.22 µm filter to ensure sterility. c) Micro-support crosslinking:

[0122] The micro-supports obtained by electrospraying (average diameter: 185 ± 127 µm, as shown in Figures 18A and 18B) were immersed in the transglutaminase solution at a ratio of 1:10 (volume / volume). The mixture was stirred at 900 rpm for 180 minutes at room temperature (22 ± 2°C). d) Conditioning for cell culture:

[0123] The cross-linked micro-supports were washed three times with sterile water and subsequently with DMEM / F12 culture medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal (penicillin / streptomycin / amphotericin B). The micro-supports were incubated at 37°C (physiological temperature of mammals or birds) in a 5% CO2 atmosphere for 120 minutes. e) Cell culture and adhesion on the micro-supports

[0124] To evaluate cell adhesion on the fabricated micro-supports, mesenchymal stem cells derived from pig adipose tissue or bird muscle tissue were used, obtained from young animals between 3-6 months of age, which were seeded at a cell density of 5000 cells / cm 2Cells were attached to conditioned micro-supports, previously added to a 6-well ultra-low adhesion microplate. The microplate was incubated at 37°C and 5% CO2 for 7 days. Cell adhesion and growth on the micro-supports were monitored using phase-contrast microscopy, as shown in Figures 19A, 19B, 19C, and 19D. Cells were observed adhering to the micro-supports starting at 24 hours, and after 7 days, some differentiated cells were visible on the micro-support surface. Cells adhering to the micro-supports were also analyzed at 7 days using fluorescence microscopy, stained with DAPI to visualize nuclei and phalloidin to visualize actin filaments, as shown in Figures 20A, 20B, and 20C. EXAMPLE 13: CELL CULTURE OF MICRO-SUPPORTS WITH BASE SOLUTION IN A DYNAMIC SYSTEM a) Cell culture of micro-supports with base solution in a dynamic system:

[0125] Porcine muscle tissue-derived stem cells were seeded at a cell density of 5000 cells / cm² 2The micro-supports were prepared with the base solution by air microspraying and conditioned in a stirred bioreactor with a working volume of 125 mL. The bioreactor was maintained at 37°C and in a 5% CO2 atmosphere for 24 hours with intermittent stirring (3 minutes of stirring at 30 rpm every 60 minutes) to promote initial cell adhesion. It was then maintained under continuous stirring at 30 to 60 rpm. The culture medium was renewed every 48 hours. Cells attached to micro-supports were monitored for 14 days by phase-contrast and fluorescence microscopy using calcein-AM staining (live cells), ethidium homodimer staining (dead cells) to assess cell viability, and DAPI (nuclei) and Phalloidin (Actin), as shown in Figures 21A, 21B, 21C, 21D, 21E, 21F, 21G, 21H, 22 and 23.

[0126] EXAMPLE 14: CELL CULTURE OF MICRO-SUPPORTS WITH SOLUTION EXAMPLE 3 IN A DYNAMIC SYSTEM a) Cell culture of micro-supports with base solution in a dynamic system:

[0127] Porcine muscle tissue-derived stem cells were seeded at a cell density of 5000 cells / cm² 2The micro-supports were prepared with the base solution by air microspraying and conditioned in a stirred bioreactor with a working volume of 125 mL. The bioreactor was maintained at 37°C and in a 5% CO2 atmosphere for 24 hours with intermittent stirring (3 minutes of stirring at 30 rpm every 60 minutes) to promote initial cell adhesion. It was then maintained under continuous stirring at 30 to 60 rpm. The culture medium was renewed every 48 hours. Cells attached to micro-supports were monitored for 14 days by phase-contrast and fluorescence microscopy using calcein-AM staining (live cells), ethidium homodimer staining (dead cells) to assess cell viability, and DAPI (nuclei) and Phalloidin (Actin), as shown in Figures 24A, 24b, 24C, 24D, 24E, 24F, 24G, 24H, 25 and 26, respectively.EXAMPLE 15: CELL CULTURE OF MICRO-SUPPORTS WITH BASE SOLUTION IN A DYNAMIC SYSTEM AND ADIPOGENIC DIFFERENTIATION a) Cell culture of micro-supports with base solution in a dynamic system and adipogenic differentiation:

[0128] Porcine adipose tissue-derived stem cells were mixed with base solution to a cell density of 4x10 6Cells were collected at a concentration of 125 mL / mL, and micro-supports containing cells were prepared by electrospraying. These were then placed in a stirred bioreactor with a working volume of 125 mL. The bioreactor was maintained at 37°C in a 5% CO2 atmosphere and was continuously stirred at 30–60 rpm. The adipogenic differentiation culture medium was renewed every 48 hours. The differentiated cells on the micro-supports were monitored for 21 days using phase-contrast microscopy with oil red staining and fluorescence using lipid tox staining to assess lipid formation, as shown in Figure 27.

[0129] EXAMPLE 16: CHARACTERIZATION OF CELLS CULTURED ON MICRO-SUPPORTS

[0130] Mesenchymal stem cells derived from muscle and adipose tissue were analyzed using surface markers such as PAX 7, CD44, and CD90 by fluorescence microscopy. The results are presented in Figures 28 and 29.

[0131] The adipogenic differentiation capacity of the cells was also evaluated using oil red staining. The results of this characterization are presented in Figure 30.

[0132] EXAMPLE 17: USE OF CULTURED MICRO-SUPPORTS FOR THE DEVELOPMENT OF BIO-INKS AND THEIR APPLICATION IN 3D BIOPRINTING

[0133] Micro-supports cultured with pre-differentiated stem cells were harvested, and a bio-ink was prepared for muscle tissue and another for adipose tissue, which were used for bioprinting a fillet model. The results are shown in Figure 31.

[0134] Based on the embodiments described above, it is anticipated that modifications to the described embodiment environments, as well as alternative embodiment environments, will be considered obvious to a person skilled in the art of the technique described herein. Therefore, it is anticipated that the claims encompass such modifications and alternatives that are within the scope of the present invention or its equivalents.

Claims

CLAIMS Having described the invention as above, the following claims are claimed as property:

1. An edible spherical micro-support for three-dimensional stem cell culture for the preparation of a cultured meat product, the micro-support comprising a homogeneous mixture of 0.375 to 1.5% by weight of an edible ionic polysaccharide; 1 to 2.5% by weight of an edible protein; 0.025 to 0.1% by weight of an edible polymer; and 95.9 to 98.6% by weight of water; and wherein the micro-support is single-phase.

2. The micro-support according to claim 1, wherein it has a size of 50 to 300 micrometers.

3. The micro-support according to claim 1, wherein the ionic polysaccharide is selected from the group consisting of alginate, pectin, chitosan and combinations thereof.

4. The micro-support according to claim 3, wherein the crosslinked ionic polysaccharide is alginate of a molecular weight of 85,000 to 250,000.

5. The micro-support according to claim 1, wherein the edible protein is selected from the group consisting of gelatin, collagen, elastin, alpha-lactalbumin, peptides, soy protein, pea protein, laminin, and combinations thereof.

6. The micro-support according to claim 5, wherein the protein is gelatin with a molecular weight of 95,000-100,000 Da and a Bloom number of 90 to 300 g.

7. The micro-support according to claim 1, wherein the edible polymer is selected from the group consisting of cellulose, methylcellulose, microcrystalline cellulose, ethylcellulose, hydrocolloids, starch, and combinations thereof.

8. The micro-support according to claim 7, wherein the edible polymer is cellulose.

9. The micro-support according to claim 7, wherein the hydrocolloid is selected from the group consisting of xanthan gum, guar gum, carrageenans, locust bean gum, and combinations thereof.

10. The micro-support according to claim 1, wherein the micro-support has an average diameter of 185 to 258 micrometers when prepared by electro-spraying or air micro-spraying.

11. A method for preparing edible spherical micro-supports for stem cell culture of claim 1, the method comprising the steps of: preparing a first solution of at least one edible ionic polysaccharide in a saline solution; preparing a second solution of at least one edible protein in a saline solution; preparing a third solution of at least one edible polymer in water; homogeneously mixing the first solution, the second solution, and the third solution; subjecting the homogeneous mixture to electro-spraying with calcium chloride, allowing the formation of single-phase spherical micro-supports; and collecting the spherical micro-supports.

12. The method according to claim 11, wherein the step of preparing a first solution of at least one edible ionic polysaccharide in a saline solution is carried out under magnetic stirring, diluting 1 to 0.75% by weight of edible ionic polysaccharide from 98 to 99.25% by weight of saline solution at room temperature, wherein the ionic polysaccharide is selected from the group consisting of alginate, pectin, chitosan and combinations thereof.

13. The method according to claim 11, wherein the step of preparing a second solution of at least one edible protein in a saline solution is carried out by magnetic stirring at a temperature of 45 to 50°C, diluting 3 to 2% by weight of the edible protein in 97 to 98% by weight of saline solution at room temperature, wherein the edible protein is selected from the group consisting of gelatin, collagen, elastin, alpha-lactalbumin, peptides, soy protein isolate, pea protein isolate, laminin, and combinations thereof.

14. The method according to claim 11, wherein the step of preparing a third solution of at least one edible polymer in water is carried out by stirring, diluting 1 to 0.25% by weight of edible polymer in 99 to 99.75% by weight of filtered water at room temperature, wherein the edible polymer is selected from the group consisting of cellulose, methylcellulose, microcrystalline cellulose, ethylcellulose, hydrocolloids, starch, and combinations thereof.

15. The method according to claim 11, wherein the step of homogenizing the first solution, the second solution, and the third solution is performed by mixing a ratio of 50:40:10 to 60:30:10 of the first solution, the second solution, and the third solution, respectively, at room temperature.

16. The method according to claim 11, further comprising the step of resting the collected micro-supports in a calcium chloride solution at room temperature.

17. The method according to claim 10, wherein the crosslinking agent is used at a concentration of 12.5 to 50 mg / mL.

18. A method for culturing stem cells on the micro-supports of claim 1, comprising the steps of: preparing a crosslinking solution with a crosslinking agent; immersing the micro-supports in the crosslinking solution; conditioning the crosslinked micro-supports with culture medium; seeding stem cells at a density of 4,500 to 5,500 cells / cm² 2 ; and cultivate the cells under controlled temperature and CO2 conditions.

19. The method according to claim 18, wherein the crosslinking agent is selected from the group consisting of: transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, tannic acid and combinations thereof.

20. A method for manufacturing a cultured meat product comprising the steps of: cultivating stem cells on micro-carriers according to the method of claim 18; collecting the micro-carriers with the cultured cells; and processing the micro-carriers with cultured cells by 3D bioprinting to form a cultured meat product.

21. The use of the micro-supports of claim 1 in a 3D bioprinting process for the preparation of cultured meat products.

22. A cultured meat product comprising: micro-carriers of claim 1; and cultured stem cells selected from the group consisting of mesenchymal stem cells, cells of embryonic origin, induced pluripotent stem cells, and combinations thereof, derived from animal tissues selected from the group consisting of pig, bovine, poultry, fish, and combinations thereof.

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