Method for operating bioreactor for cultured meat and corresponding bioreactor

Abstract

To provide a method that enables efficient parallel creation of elongated structures for cultured meat, especially large bundles comprising several tens, several hundreds, or even thousands of the elongated structures, a reactor for implementing the method with maximum efficiency and reliability, and a cultured meat product thus obtained.SOLUTION: A method for producing elongated structures for cultured meat, the method including: (a) extruding a paste via an extrusion plate 24 provided with at least one nozzle opening 44 and at least partly via an adjacent adhesion plate 22, preferably into a downstream space 58 of the adhesion plate; (b) subjecting the paste positioned within the adhesion plate and / or in the space to at least partial curing; and (c) continuing extrusion of the paste through the extrusion plate while increasing the distance between the extrusion plate and the adhesion plate, under formation of the elongated structures between the plates 22 and 24.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] The present invention relates to methods for producing elongated, typically fibrous, preferably microfibrillar structures for making cultured meat, and in particular to methods for efficiently producing large bundles of tens or hundreds or even thousands of such elongated, typically fibrous structures in parallel, as well as reactors for most efficiently and reliably implementing such methods. The present invention further relates to the corresponding cultured meat products obtained using the methods and / or the respective reactors. [Background technology]

[0002] Meat is an important source of protein in the human diet. With a growing global population and demand for meat products, animal welfare issues associated with the traditional meat industry have become controversial, making sustainable production alternatives essential. Cultured meat technology offers the opportunity to produce edible animal protein sources without the environmental impacts of livestock farming. In meat products, aligned fibrous / fibrillar structures exist at the tissue, cellular, and molecular scales, which contribute to the texture, tenderness, organoleptic properties, and nutritional characteristics of meat products. Currently, challenges in cultured meat production lie in the availability of scalable, cost-effective processes for producing tissues with the highly anisotropic fibrous structure characteristic of animal meat, and reactors capable of applying cyclic mechanical stimulation to cultivate such tissues at very high densities.

[0003] To replicate multiscale anisotropic structuring in cultured meat products, it is important to employ a scaffolding approach that produces a fibrous / fibrillar structure that guides muscle progenitor stem cells to differentiate into aligned muscle fibers. Additionally, applying cyclic mechanical stretch during culture can promote cell differentiation and anisotropic orientation. Similar to meat obtained from slaughtered animals, cultured meat products should be produced in very large quantities at a reasonably low cost. For these reasons, the scaffolds and culture approaches employed should not only be expected to be safe for human and non-human consumption, but also scalable and compatible with high production rates. With these points in mind, the prior art found in the patent and scientific literature is discussed in the following paragraphs.

[0004] Patent document 1 discloses a fibrous scaffold for cultivated meat production. The scaffold consists of a porous fiber mat. Cells are incorporated into the product by seeding a cell suspension onto the scaffold. The seeding step is a critical step and can last from several hours to several days, depending on the cells used. Seeding efficiency is controlled by the attachment kinetics of the cells and by the bioavailability of the attachment sites. Low seeding efficiency can also be experienced. Seeding is limited by the available surface area, which in turn limits the achievable cell density in the final product.

[0005] In Non-Patent Document 1, the preparation of fibrous gelatin scaffolds by immersion rotary jet spinning at high speeds (approximately 100 g / h, dry weight) has been reported; depending on the process conditions, this approach allows for the fabrication of edible scaffolds for cultivated meat production in a scalable manner. However, because the scaffolds rely on post-production cell seeding, the achievable seeding density is limited by the available surface area, as evidenced by the lack of mature muscle structure.

[0006] Patent Document 2 reports a bioengineered tissue substitute. This invention reports the creation of highly anisotropic muscle tissue consisting of an aligned microfiber array. In this invention, the microfibers must be individually prepared and then organized into an array of multiple fibers stacked parallel to each other in layers. In addition, cells must be seeded onto the structure. Therefore, the same drawbacks mentioned for other porous scaffolds that are seeded after fabrication also apply here. In addition, the disclosed invention requires equipment with a large footprint compared to the amount of fibers produced, and therefore is not suitable for large-scale production.

[0007] Non-Patent Document 2 reports a scaffold method based on a cell-laden gel. The reported method overcomes some of the seeding problems mentioned in the prior art. The gel formulation is based on fibrinogen cross-linked by thrombin, and embeds cells and collagen microfibers. Due to the composition of such formulations, the sol-to-gel transition has slow kinetics, and therefore fibers are produced by molding in a sacrificial gel. For these reasons, the disclosed method and composition are hardly scalable for producing large quantities of cultivated meat.

[0008] In Non-Patent Document 3, a gel paste formulation containing oxidized alginate and gelatin combined with shear stress was used to provide cell orientation and differentiation guidance. This prior art formulation allows for fast gelation compatible with high production capacity. Nevertheless, bioprinting is a slow process used to precisely deposit material in space, most often from a single nozzle at a time, and is therefore not a promising technology for mass production.

[0009] Non-Patent Document 4 reports a method for preparing aligned hydrogel fiber bundles. This method is used in a bioprinting setup by depositing fibers onto a substrate or in a molding process setup. Neither process is scalable for the production and culture of large amounts of biological tissue under sterile conditions.

[0010] In Non-Patent Document 5, the authors report a strategy for the generation of hydrogel microfibers in which internal alignment is induced by a combination of electrical and mechanical stretching. Cells can be loaded directly into the hydrogel formulation (i.e., no seeding step is required). Nevertheless, this process requires the use of high voltages (3000V-5000V). The use of high voltages in processing / manufacturing facilities poses a risk of electric arcs and brings special requirements in terms of electrical insulation of the machine. In addition to this, the reported process is relatively slow.

[0011] Patent Document 3 discloses an apparatus for producing tissue from cells. The apparatus includes an elongated body having at least one peripheral groove and operable to extend centrally through at least one trough in a tight-fitting relationship. The trough extends in a closed path such that at least one of the peripheral grooves opens to an inner end of the trough. Also disclosed is a process for producing tissue from cells via a cell-to-tissue transition intermediate.

[0012] Patent Document 4 discloses a cell growth system for culturing and growing cells in a hydrogel tube, which enables cell growth that can significantly reduce production time and costs while increasing production capacity.

[0013] Non-Patent Document 6 reports that a gel matrix culture environment provides a scaffold for tissue engineering and cues to guide cell differentiation. For many cell therapy applications, such as the production of islet clusters to treat type 1 diabetes, large-scale production is expected to be necessary. The throughput of commonly used nozzle-based devices for cell encapsulation is limited to approximately 0.5 L / h by the rate of droplet formation. This study describes a novel process for larger-scale batch immobilization of mammalian cells in an alginate-filled hollow fiber bioreactor (AHFBR). A methodology was developed in which: (1) alginate inhibition of medium flow in the intracapillary space was negligible, (2) alginate gelation outside the capillaries was complete, and (3) 83 ± 4% of the seeded and immobilized cells were recovered from the bioreactor. Chinese hamster ovary (CHO) cells, which grew from nearly single cells to islet-sized spheroids in 8 days of AHFBR culture, were used as a model aggregate-forming cell line. The growth and metabolic rates of CHO cells in the AHFBR were comparable to those of small-scale alginate slab controls. This process was then successfully applied to the culture of primary porcine neonatal pancreatic cells, with no significant difference in cell viability compared to the slab control. As expected, alginate-immobilized culture in the AHFBR increased the insulin content of these cells compared to suspension culture. The AHFBR process can be improved by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel matrix-assisted culture for cell therapy.

[0014] Non-Patent Document 7 reports that cultured meat aims to solve the current sustainability and environmental issues of traditional livestock farming by merging tissue engineering practices with food innovation science to replicate the composition and structure of animal-derived meat. To this end, various scaffolding technologies have been adopted and developed to support cell culture, growth, and differentiation using edible, low-cost, and sustainable materials and methods. Cell scaffold construction can benefit various processing strategies, including structuring approaches and additives, to generate the final cultured meat product. This paper details the key considerations for scaffold design for cultured meat applications, reviews major scaffolding technologies, and discusses current and potential applications for cultured meat engineering. It concludes that extensive research efforts in recent years have demonstrated the limitless potential of novel cultured meat scaffolds. Furthermore, various scaffolding technologies originally developed for tissue engineering can be adapted for cultured meat by using edible materials and avoiding toxic crosslinking agents and reagents throughout the development process, thus ensuring compliance with food and safety regulations. The combination of tissue engineering and food science technologies holds the promise of a technically and commercially viable scaffold for realizing the promise of cultivated meat.

[0015] Patent Document 4 relates to a cell growth system for culturing and growing cells in hydrogel tubes, which enables cell growth that can significantly reduce production time and costs while increasing production capacity.

[0016] Patent Document 5 discloses an anaerobic membrane bioreactor for wastewater treatment. The anaerobic membrane bioreactor includes a shell and a filtration material layer disposed in the shell. The anaerobic membrane bioreactor further includes a high-pressure gas backflush structure disposed on the filtration material layer, the high-pressure gas distribution structure and a plurality of high-pressure gas nozzles disposed on the high-pressure gas distribution structure, the openings of the high-pressure gas nozzles facing the filtration material layer. A low-pressure gas agitation structure disposed below the filtration material layer includes a low-pressure gas distribution structure and a plurality of low-pressure gas nozzles disposed on the low-pressure gas distribution structure, the openings of the low-pressure gas nozzles facing the filtration material layer. When the filtration material layer is clogged, high-pressure gas sprayed by the high-pressure gas nozzles is used to backwash the filtration material layer. When the filtration material layer is not clogged, the low-pressure gas nozzles are used to spray low-pressure gas near the filtration material layer, improving the fluidity and agitation of the wastewater near the filtration material layer. This improves the sewage purification effect in the filtration material layer, prolongs the clogging period of the filtration material layer, improves the treatment effect of the reactor, and reduces the backwash frequency.

[0017] Non-Patent Document 8 reports that since the first cultured meat burgers were born, the cultured meat market has been growing at an accelerated pace. Significant efforts have been made to reduce costs by eliminating serum in growth media and improving process efficiency by employing bioreactors. In parallel, efforts have also been made to innovate scaffolds to provide better cell proliferation, differentiation, and tissue development. To date, scaffolds used in cultured meat research have primarily been animal-derived collagen and gelatin. To meet the prospects of cell-based meat, namely, environmental conservation and animal welfare, plant-derived biomaterials for scaffolds have been actively explored. This paper reviews and discusses the advantages and disadvantages of scaffold materials and potential scaffolds relevant to scale-up approaches for cultured meat production.

[0018] No part of this discussion of the prior art should be construed as an admission of prior art. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] International Publication No. 2020 / 160533 [Patent Document 2] U.S. Patent No. 7,622,299 [Patent Document 3] International Publication No. 2019 / 211189 [Patent Document 4] U.S. Patent Application Publication No. 2021 / 0017485 [Patent Document 5] Chinese Patent Application Publication No. 112374607 [Non-patent literature]

[0020] [Non-Patent Document 1] MacQueen et al. (Muscle tissue engineering in fibrous gelatin: implications for meat analogs, npj Sci Food 3, 20 (2019)) [Non-patent document 2] Kang et al. (Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting, NATURE COMMUNICATIONS|(2021)12:5059) [Non-patent document 3] Distler et al. (3D printed oxidized alginate-gelatin bioink provides guidance for C2C12 muscle precursor cell orientation and differentiation via shear stress during bioprinting, Biofabrication 12 (2020) 045005) [Non-patent document 4] Kessel et al. (3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands, Adv. Sci. 2020, 7, 2001419) [Non-Patent Document 5] Zhang et al. (Creating polymer hydrogel microfibres with internal alignment via electrical and mechanical stretching, Biomaterials, Volume 35, Issue 10, March 2014, Pages 3243-3251) [Non-patent document 6] Hoesli et al. (A novel alginate hollow fiber bioreactor process for cellular therapy applications, Cell Culture and Tissue Engineering, Vol. 25(6), p. 1740-1751) [Non-Patent Document 7] Levi et al. (Trends in Food Science & Technology, Volume 126, August 2022, Pages 13-25) [Non-patent document 8] Seah et al. (CRITICAL REVIEWS IN BIOTECHNOLOGY, vol.42, no.2, 2022, pages 311-323) Summary of the Invention

[0021] Indeed, such multiscale, anisotropic, elongated structures characteristic of animal meat, made from muscle bundles (e.g., groups of aligned sarcovesicles, myofibers, and myofibrils), can only be achieved by fabricating scaffolds that mimic the aligned, elongated, typically fibrous structure of meat and then guiding the differentiation process of cells, usually stem cells, with the aid of an appropriate microenvironment, as well as topological and mechanical cues. Particularly good scaffolds for muscle cell growth incorporate anisotropic microstructures, in the sense of having an internal material structure with preferred axial orientation. The problem with providing such structures is that they require stringent manufacturing processes under sterile conditions, which limits the scalability of the process or increases the footprint of the equipment.

[0022] The present invention discloses methods and compositions that allow for the production of elongated structures, particularly fibrous meat bundles, with multiscale structural properties similar to those of animal meat, as well as a reactor capable of applying mechanical stimulation to cultivate such elongated structures, particularly fiber bundles, at high densities.

[0023] Such reactors are designed for cell seeding, for example, culturing porous fiber bundles or mats at high density, and optionally applying mechanical stretching.

[0024] The disclosed invention includes methods and reactors for the in-vitro production of elongated structures, and in particular for the in-vitro production of meat tissue or precursors thereof for cultivated meat production, which allows for the scalable production of large quantities of muscle bundles, particularly of high quality.

[0025] More specifically, the present invention discloses (i) methods for the preparation and optional culture of fibrous cell-laden gel structures, (ii) reactors for carrying out the corresponding methods, and (iii) products obtained using the methods / reactors. Long, thin structures, especially fibrous structures, support cell differentiation, e.g., into muscle bundles with a fibrous, highly anisotropic texture.

[0026] Therefore, according to a first aspect of the invention, the invention relates to a method for the production of elongated structures, preferably fibrous structures (including microfibrillar structures), for the production of cultured meat, preferably a method for the parallel production of a large number of such elongated structures, preferably fibrous structures, or bundles of such elongated structures, preferably fibrous structures.

[0027] The method is characterized by the steps defined in claim 1.

[0028] More specifically, according to the proposed method: (a) the paste is extruded through an extrusion plate having at least one nozzle opening (not only circular, but also elongated or specially shaped openings or a combination of openings of different shapes) and at least partially through and / or into an essentially adjacent attachment plate (preferably having aligned nozzle openings) (at least to an extent sufficient for adhesion after hardening for the drawing process, without passing completely through), preferably into the space downstream of the attachment plate, preferably in or behind the attachment plate, just enough paste is extruded through the two plates so that the front part enters the attachment plate, for example forming a layer of paste, or at least in or behind each nozzle opening or pore of the attachment plate, the paste is located and / or flows away to a diameter larger than the nozzle opening, (b) the paste located in the deposition plate and / or in the space downstream of the deposition plate is at least partially hardened, leading to a semi-automatic fixing or deposition of the paste in and / or behind the deposition plate, (c) The paste continues to be extruded through the extrusion plate while the distance between the extrusion plate and the attachment plate is continuously increased under the formation of the elongated structure between the plates.

[0029] In other words, in the final step (c), extrusion is carried out in parallel with moving the plates away from each other so that elongated structures, or in the case of multiple nozzle openings, bundles of elongated structures, are produced in a controlled manner.

[0030] Preferably, the rate of separation of the attachment plate from the extrusion plate is greater than the rate of extrusion so that there is some pulling of the elongated structures during extrusion.

[0031] Correspondingly, according to a first preferred embodiment, to form the elongated structure, extrusion is carried out at a discharge speed under the generation of a velocity gradient downstream of each nozzle opening in the extrusion plate by a separation speed between the extrusion plate and the attachment plate that is greater than the discharge speed, where the discharge speed is defined as the input flow rate of the paste (e.g., hydrogel) divided by the total nozzle opening area.

[0032] In step (c), the pulling speed, measured as the speed of the attachment plate relative to the push plate (the relative speed is important; the push plate can be stationary, the attachment plate can be stationary, or both can be moving relative to each other), is preferably in the range of 0.01 m / min to 100 m / min, more preferably 0.1 m / min to 10 m / min.

[0033] In order to ensure that the above-mentioned adhesion of the paste is established downstream of the adhesion plate, according to a further preferred embodiment, in at least one of steps (b), (c) and after step (c), the drawn / extruded paste is immersed in a hardening bath, preferably a hardening bath containing divalent or polyvalent cations, an acid bath or a bath having a different temperature than the gel paste, more preferably a hardening bath containing divalent cations.

[0034] the extrusion plate and / or the attachment plate preferably each comprise a plurality of nozzle openings, preferably at least 20, more preferably at least 100, most preferably at least 200, or in the range of 250 to 1000; and / or the nozzle openings cover 5% to 90% or 5% to 95%, more preferably 20% to 90% or 20% to 80%, most preferably 40% to 70% of the cross-sectional area of the plate; and / or nozzle opening is 1 nozzle / cm 2 ~5000 nozzles / cm 2 , preferably 5 nozzles / cm 2 ~400 nozzles / cm 2 The extrusion plate and / or the attachment plate are provided with a nozzle density in the range of

[0035] Preferably, the nozzle openings have a diameter, or in the case of non-circular openings a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm, and in particular the above nozzle specifications in terms of number, % and / or density are given in combination with nozzle sizes having nozzle openings with a diameter, or in the case of non-circular openings a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm.

[0036] The nozzle openings may be of circular cross section, elliptical, rectangular or square shape, and most preferably all have the same circular diameter, and if the attachment plate also has nozzle openings, they are preferably arranged in an overlapping manner in the two plates.

[0037] The attachment plate may be a mesh, a porous plate, or a plate with nozzle openings, preferably a plate with nozzle openings aligned with the nozzle openings of the extrusion plate.

[0038] In the case of a mesh or porous plate, the structure of the pores must be such that the paste extruded through the extrusion plate can partially penetrate into the attachment plate and harden in the pores of the attachment plate, for example by flooding the space downstream of the attachment plate with a hardener or by irradiation. In this way, it is possible to operate in a situation where, for example, the paste does not completely penetrate the attachment plate but only partially penetrates the pores of the attachment plate, and then hardens in the pores in the attachment plate, leading to the deposition of strings of paste for the subsequent extrusion process.

[0039] With regard to the nozzle openings in the extrusion plate, and if the deposition plate also contains corresponding nozzle openings in the same manner as these nozzles, these nozzle openings may be arranged in a density and pattern compatible with the desired bundle structure. For example, in the case of a particularly large extrusion / deposition plate, it is possible to have distinct areas with nozzle openings suitable and adapted to form fiber bundles corresponding to the desired shape of the meat product, e.g., strings of artificial muscle, while having areas without nozzle openings between these nozzle opening areas, thereby producing, in one process, several such fiber bundles similar to natural muscle strings, which can then be directly cultivated, if necessary and desired. The densities and percentages mentioned above preferably apply specifically only to such areas of nozzle openings.

[0040] Preferably, the nozzle opening has a diameter, or maximum lateral extension in the case of a non-circular opening, in the range of 10 μm to 5000 μm, more preferably 50 μm to 1000 μm.

[0041] Preferably, the nozzle openings are circular, most preferably all have the same circular diameter, and are arranged in an overlapping manner in the two nozzle plates.

[0042] According to yet another preferred embodiment, the extrusion in steps (b) and (c) is preferably carried out into a closed sterile reaction vessel (bioreactor, culture vessel), and following step (c) followed, if necessary, by a further step of hardening of the extruded elongated structures, the reaction vessel is filled with a culture growth medium and the elongated structures are used to grow meat cells seeded onto the structures and / or already contained in the paste.

[0043] According to a particularly preferred embodiment, the proposed method comprises: (a) the paste is extruded through the extrusion plate and through an adjacent deposition plate having at least partially aligned nozzle openings, preferably into a space downstream of the deposition plate; (b) slowing down or stopping the extrusion and hardening the paste located in the deposition plate and / or in the space downstream of the deposition plate by immersing the space downstream of the deposition plate in a hardening bath, preferably a hardening bath containing divalent or polyvalent cations, an acid bath, or a bath having a different temperature than the gel paste, preferably a calcium chloride bath, (c) if necessary, after removal of the hardening bath or in the presence of a hardening bath, continued extrusion of the paste through the extrusion plate while separating the attachment plates at a relative separation rate greater than the extrusion rate from the extrusion plate, followed by the formation of the elongated structures, preferably multi-scale anisotropic structures, between the nozzle openings; (d) slowing or stopping the spacing and extrusion and, if not already done so during step (c), hardening the paste in the form of microfibrils in the space between the two plates by immersing the space downstream of the deposition nozzle plate with a hardening bath, preferably a hardening bath containing divalent or polyvalent cations, an acid bath, or a protein cross-linking bath, preferably a calcium chloride bath, and optionally further hardening the paste in the space upstream of the extrusion plate; (e) replacing the space between the plates (typically the hardening or cross-linking bath) with culture medium and using the elongated structures, preferably fiber bundles (e.g., microfibrils), to grow meat cells seeded on the elongated structures and / or already contained in the paste; It is characterized by:

[0044] By controlling the composition of the medium, the temperature, the pH, the gas atmosphere of the supernatant, etc., the corresponding process can be controlled.

[0045] According to a further preferred embodiment, during the culture the two plates may be vibrated relative to each other to stimulate and influence the growth process on the anisotropic microfibril structure.

[0046] Preferably, the paste is for the production of cultured meat, comprising the following ingredients: (A) at least one polysaccharide capable of forming a solidified gel by the action of divalent or polyvalent cations, thermal gelation, photoinduced addition or condensation reactions, or a combination thereof, preferably at a concentration in the paste ranging from 0.01 g to 200 g per liter of component (D); (B) at least one protein, preferably at a concentration in the paste in the range of 0.001 g to 500 g per liter of component (D), preferably associated with the polysaccharides of component (A) via supramolecular interactions or covalent interactions or a combination thereof; (C) cells selected from mammalian cells, fish cells, crustacean cells, or a combination thereof, wherein the concentration in the paste is within the range of 0 to 300 billion cells per liter of component (D); (D) water or water-based culture medium; (E) An additive different from (A) to (D), preferably selected from the group consisting of a crosslinking kinetics property modifier having a concentration in the paste ranging from 0 mM to 500 mM, a flowability modifier having a concentration in the paste ranging from 0 g to 200 g per 1 L of component (D), or a combination thereof.

[0047] Indeed, preferably, pastes such as those described in European Patent Application No. 22164793.6 are preferably used in this method, the disclosure of which is incorporated herein by reference with respect to the pastes and methods of processing the pastes as defined therein and the corresponding products.

[0048] The main features of this paste and its use in the present method are described below: (i) The gel paste composition combined with the method of production allows for the fabrication of cell-laden composite gels characterized by unique anisotropic elongated structures, preferably fibrillar structures, at the microscale. (ii) The production process for developing such elongated structures, preferably fibrillar structures, is fast and scalable, and therefore compatible with the high production rates required for cultivated meat production. (iii) The elongated structure, preferably fibrillar structure, of the composite gel promotes superior differentiation of muscle progenitor stem cells into muscle tissue with characteristics typical of meat.

[0049] Advantages attributable to the main features of the present invention include the following aspects, taken alone or in combination: (1) The gel paste is composed of edible ingredients for the formation of a microfibril structure. (2) Differentiated cells assemble into muscle bundles with realistic sizes, with adjustable diameters ranging from 0.01 mm to 2 mm and lengths greater than 1 mm. (3) The size of the muscle bundles can be adjusted by utilizing different process parameters, which allows the production of muscle fibers with textures that mimic those of various meat species. (4) High cell densities comparable to those of animal muscle tissue can be achieved. 3 Cell densities of hundreds of millions of cells per well can be achieved. (5) Cell seeding is efficient (virtually 100% seeding efficiency) and homogeneous throughout the scaffold volume. (6) The composition and process parameters of the gel paste can be tailored to promote cell spreading and differentiation by meeting the biomechanical requirements of any particular cell type. (7) The fibrous gel embedded with cells can withstand culture conditions for several weeks. (8) The gel paste is transformed into a mechanically stable gel within a few hundred seconds, and cells are trapped and protected within it, making the gel fibers easy to handle and suitable for high production rates. The production of muscle bundles can be fully automated, and production can occur directly within the differentiation bioreactor. (9) As a result of the fibrous texture of the cell-entrapping gel, the resulting muscle bundles develop an excellent aligned fibrillar structure without active tension during culture. (10) As a result of the highly anisotropic gel microstructure and cell differentiation, a significant increase in protein content is observed upon differentiation. (11) Mild processing conditions. (12) Optionally, muscle bundles are physically clamped during production to directly apply external stimuli (e.g., mechanical stretch and / or electric fields) to further boost myofibril / myoglobin protein production. (13) Optionally, nonproteinaceous components (e.g., polysaccharide components) used as processing aids in the preparation of the gel paste may be degraded / dissolved at the end of the differentiation process to maximize the protein content and minimize the ingredient list. (14) Optionally, bioactive molecules may be immobilized in the gel to aid differentiation and diffusion. (15) Optionally, chromophores, flavor molecules, and aroma molecules may be added to the gel paste to improve the overall sensory properties of the meat produced. (16) Optionally, fibroblasts may be seeded onto and / or into the muscle bundles to deposit connective tissue and further modify the texture of the final meat product. (17) Optionally, the cross-linking bath used for gelling the gel paste may contain iron ions to increase the iron content of the final meat product. (18) Optionally, after cultivation, the muscle bundles may be glued together using microbial transglutaminase. Additionally, vegetable and animal proteins may be added to adjust the texture and protein content / source. (19) Optionally, the produced muscle bundles may be combined with vegetable fats, animal fats, and cultured fats to produce meat-based foods. (20) Optionally, micro / nanofibers may be added to the gel paste formulation to toughen the hydrogel and modify the texture.

[0050] Therefore, the present invention discloses methods and compositions for the preparation of cell-laden composite gels that can be formed on demand with a fibrillar structure. The invention is comprised of various components: a composition for the gel paste, a method for producing gel fibers from the gel paste, and its use to produce cultured meat products.

[0051] According to a preferred embodiment of such a paste, the at least one polysaccharide of component (A) is naturally crosslinkable and / or chemically modified to promote crosslinking, preferably the chemical modification comprises acrylate, methacrylate, epoxidation, allylation, or a combination thereof.

[0052] According to a further preferred embodiment of such a paste, the at least one polysaccharide of component (A) is selected from the group consisting of alginate, pectin, carrageenan, chondroitin sulfate, dermatan sulfate, heparin, heparin sulfate, and derivatives thereof and combinations thereof, preferably the polysaccharide component of component (A) is at least one alginate or alginate derivative.

[0053] Preferably, the polysaccharide(s) of component (A) are present in a concentration in the paste in the range of 0.1 g to 100 g per liter of component (D).

[0054] According to yet another preferred embodiment, the at least one protein of component (B) is in the form of a native protein, a denatured protein, a protein hydrolysate, or a combination thereof.

[0055] Preferably, at least one protein of component (B) is bioactive, capable of altering the function and / or properties of the cells of component (C), in particular promoting cell attachment by providing an integrin binding motif.

[0056] The at least one protein of component (B) may preferably be selected from the group consisting of gelatin, collagen, fibrinogen, fibrin, fibronectin, fibroin, elastin, laminin, basic albumin of plant origin, preferably napin, and preferably the protein component (B) is gelatin, more preferably type A gelatin.

[0057] Preferably, at least one protein of component (B) is chemically modified to facilitate crosslinking, and preferably the chemical modification comprises acrylate, methacrylate, epoxidation, allylation, or a combination thereof.

[0058] According to a preferred embodiment, the at least one protein of component (B) is preferably crosslinked by transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, photopolymerization, or a combination thereof, and preferably transglutaminase is used. When transglutaminase is used as a crosslinking agent, it is contained in an amount of 0 U to 50 U, more preferably 0.1 U to 10 U, per 1 g of protein.

[0059] Typically, the protein(s) of component (B) are present in a concentration in the paste in the range of 0.1 g to 250 g per litre of component (D).

[0060] According to yet another preferred embodiment, the cells of component (C) are present in the paste at a concentration in the range of 5 to 100 billion cells per liter of component (D).

[0061] The additive of component (E) preferably comprises at least one crosslinking kinetics modifier for slowing down the gelation kinetics of the crosslinkable polysaccharide by sequestering divalent or polyvalent cations.

[0062] Preferably, the additive of component (E) comprises at least one food-compatible compound selected from the group consisting of disodium phosphate, dipotassium phosphate, magnesium phosphate, ethylenediaminetetraacetic acid, ethylenediaminetetraacetate salts, or combinations thereof.

[0063] Typically, the additive of component (E) is present at a concentration of 1 mM to 100 mM.

[0064] The additive of component (E) may comprise at least one flowability modifier to impart shear thinning behavior to the gel paste, such that a high viscosity at rest mitigates cell sedimentation, while a low viscosity during draining allows processability; preferably, the flowability modifier is an edible filler in the form of micro / nanofibers, which is preferably insoluble under culture conditions and is preferably composed of proteins and / or polysaccharides different from the other components of the paste; such edible filler is preferably present in a concentration of 0 g to 200 g per liter of paste; more preferably, the edible polymer as flowability modifier may be selected from gums (including gellan gum, guar gum, xanthan gum), PEG, or combinations thereof; preferably, the edible polymer is present in a concentration of 0 g to 50 g per liter of gel paste.

[0065] Preferably, such paste is further supplemented with a microstructuring agent, preferably selected from the group consisting of bicarbonates, including sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, magnesium bicarbonate, or combinations thereof, for unfolding of the protein structure. Preferably, the microstructuring agent is added to the paste in an amount leading to a concentration ranging from 0.1 g to 300 g per liter of paste, preferably from 1 g to 200 g per liter of paste. The microstructuring agent is preferably added in the form of a powder. The microstructuring agent and the paste can also be in the form of a kit-of-parts that are joined together just before carrying out the method for producing the microfibrillar structure described below.

[0066] Preferably, the hardening bath contains Ca in water, preferably at a concentration in the range of 1 mM to 500 mM, more preferably 20 mM to 100 mM. 2+ , Mg 2+ , Fe 2+ , Fe 3+ , or a combination thereof.

[0067] The hardening bath of step (c) may be buffered at a pH ranging from 6 to 8, preferably by using a HEPES buffer, more preferably the HEPES buffer is used at a concentration comprised between 1 mM and 100 mM.

[0068] Preferably, the paste is extruded from a nozzle which preferably has a circular geometry at the point of discharge.

[0069] According to yet another preferred embodiment, the curing bath comprises a protein cross-linking agent or following step (b) and / or during step (c) the fibers are immersed in a protein cross-linking bath with such a cross-linking agent.

[0070] Preferably, the cross-linking agent is selected from the group consisting of transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, or a combination thereof, and is preferably transglutaminase, and when transglutaminase is used, is included in an amount of 1 U to 2000 U per mL of the cross-linking bath.

[0071] More preferably, crosslinking is carried out at a temperature in the range of 20°C to 45°C for a time period in the range of 10 to 120 minutes, more preferably 30 to 90 minutes.

[0072] Muscle bundles may be prepared by culturing cell-laden microfibrillated hydrogel fibers in an environment that promotes myogenesis.

[0073] Preferably, the resulting fibers have individual diameters preferably in the range of 0.1 μm to 50 μm, are preferably aligned ±40° from the same axis relative to the fiber axis, and are converted into muscle tissue, e.g., by culturing in a differentiation medium designed for the cell type used in step (e) defined above, or, if the fibers are produced from a cell-free paste, are seeded with cells before culturing. Preferably, the fibers, e.g., in the form of bundles, are crosslinked together to obtain a solid edible structure, and crosslinking of the fibers may be performed by one or more crosslinking agents selected from the group consisting of transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, or combinations thereof, preferably transglutaminase.

[0074] Typically, the resulting muscle tissue or aggregates are mixed with additional compositions to form a consumer cultured meat product.

[0075] According to yet another preferred embodiment, the extrusion in step (c) is carried out with a pull factor of at least 1.1, preferably at least 1.5 or at least 2, defined as the ratio of the discharge speed to the separation speed of the second nozzle plate relative to the first nozzle plate during step (c).

[0076] According to yet another aspect, the present invention relates to a reactor for carrying out the method as described above, which reactor preferably allows working under sterile conditions and comprises a closable reactor vessel of elongated shape along a main axis and of constant cross section, preferably cylindrical or rectangular / square or hexagonal, a (preferably stationary) extrusion plate at one end of the reactor (having the corresponding cross-sectional shape of the reactor vessel), at least one attachment plate (also having the corresponding cross-sectional shape of the reactor vessel), preferably movably mounted in said reactor vessel, elements for controlled supply of paste for its extrusion upstream of the extrusion plate, elements for controllably moving the attachment plate along the main axis, and means for supplying at least one of a culture medium, a hardening medium and a cross-linking medium to and from the interior of the reactor vessel, preferably also to the space upstream of the (stationary) extrusion plate.

[0077] According to a preferred embodiment of the reactor, a pusher plate is located at the bottom of the closable reactor vessel and the attachment plate is located above the pusher plate.

[0078] According to yet another preferred embodiment, the pusher plate can be moved in a contactless manner by said element, preferably by magnetic force; preferably for this purpose the pusher plate is mounted in and / or on a mounting structure having at least one magnet, and outside the reactor vessel there is a mobile magnetic element, preferably in the form of a ring having at least one magnet, which mobile magnetic element can be moved automatically, preferably by a motor.

[0079] the (stationary) extrusion plate and / or the deposition plate preferably each comprise a plurality of nozzle openings, preferably at least 20, more preferably at least 100, most preferably at least 200, or in the range of 250 to 1000 nozzle openings; and / or the nozzle openings cover 5 to 90% or 5% to 95%, more preferably 20% to 90% or 20% to 80%, most preferably 40% to 70% of the cross-sectional area of the plate; and / or nozzle opening is 1 nozzle / cm 2 ~5000 nozzles / cm 2 , preferably 5 nozzles / cm 2 ~400 nozzles / cm 2 The extrusion plate and / or the attachment plate are provided with a nozzle density in the range of

[0080] Preferably, the nozzle openings have a diameter, or in the case of non-circular openings a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm, and in particular the above nozzle specifications in terms of number, % and / or density are given in combination with nozzle sizes having nozzle openings with a diameter, or in the case of non-circular openings a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm.

[0081] The nozzle openings may be of circular cross section, elliptical, rectangular or square shape, and most preferably all have the same circular diameter, and if the attachment plate also has nozzle openings, they are preferably arranged in an overlapping manner in the two plates.

[0082] Preferably, the nozzle openings are circular, most preferably all have the same circular diameter, and are arranged in an overlapping manner in the two nozzle plates.

[0083] More preferably, said (stationary) pusher plate and / or said deposition plate further comprise at least one opening for controlled delivery of liquid through the respective plate.

[0084] According to yet another preferred embodiment of the reactor, the reactor comprises a control device for controlling, in particular, at least one of the extrusion rate of the paste, the separation rate of the attachment plates, the supply and removal of at least one of the culture medium, the crosslinking medium, the hardening medium.

[0085] According to another aspect of the invention, the invention relates to the use of the method described above for the production of a consumer cultured meat product, preferably involving the use of corresponding elongated structures, preferably in the form of bundles, in combination with: (i) fat and / or oil based ingredients, including cultured fat, vegetable fat or animal fat based ingredients and derivatives thereof, in particular in combination with separately cultured adipocytes and / or separately cultured adipocyte aggregates; (ii) and / or structuring agents, including hydrocolloids, and / or cellulose and its derivatives, and / or proteins of plant, animal, recombinant or cell culture origin; (iii) and / or connective tissue components, including animal-derived connective tissue, cultured connective tissue, especially in combination with cultured fibroblasts and / or chondrocytes, and / or aggregates of separately cultured fibroblasts and / or chondrocytes.

[0086] The ingredients may be associated to form compact bundles of fiber bundles, like pieces of meat.

[0087] Furthermore, the present invention relates to cultured meat obtained using a method as described above and / or obtained using a reactor as described above.

[0088] Further embodiments of the invention are defined in the dependent claims.

[0089] Preferred embodiments of the present invention will now be described with reference to the drawings, which are for the purpose of illustrating presently preferred embodiments of the invention and are not intended to limit the invention. [Brief explanation of the drawings]

[0090] [Figure 1a] FIG. 1 shows a bioreactor according to a first embodiment with low dead volume, in a) a perspective view with the upper extrusion plate in the mid-low position. [Figure 1b] FIG. 1 shows a bioreactor according to a first embodiment with low dead volume, in b) a first axial section in a plane through the closure mechanism is shown. [Figure 1c] FIG. 1 shows a bioreactor according to a first embodiment with low dead volume, where in c) a second axial section is shown in a plane perpendicular to that shown in b). [Figure 2a] FIG. 2 shows a second embodiment of a high dead volume bioreactor, in a) a perspective view with the upper pusher plate in the lowered position. [Figure 2b] FIG. 2 shows a bioreactor according to a second embodiment with high dead volume, in b) a first axial section in a plane through the closure mechanism is shown. [Figure 2c] FIG. 2 shows a bioreactor according to a second embodiment with high dead volume, where in c) a second axial section is shown in a plane perpendicular to that shown in b). [Figure 3] FIG. 3 shows an axial cut through the bioreactor according to the first embodiment, including the installation of the reactor, with the upper extrusion plate in the uppermost position. [Figure 4] FIG. 4 is an axial cut through a bioreactor according to a second embodiment in a position where the upper pusher plate is in its lowest position. [Figure 5] FIG. 5 is a perspective view of the bioreactor according to the first embodiment in a perspective representation, with the upper extrusion plate in an intermediate position, including the reactor and the mounting of further elements of the automation system. [Figure 6]FIG. 6 shows the bottom extrusion plate, in a) a perspective view, in b) a cutaway view along AA in c) and in c) a top view. [Figure 7] FIG. 7 shows the upper extrusion plate, in a) a perspective view from above, in b) a cutaway view along AA in d), in c) a perspective view from the bottom side, and in d) a top view. [Figure 8a] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in a) the step of pre-filling the gel is shown. [Figure 8b] FIG. 8 shows the sequence of operation of the proposed bioreactor, in b) the upper clamp is shown. [Figure 8c] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in c) extrusion is shown. [Figure 8d] FIG. 8 shows the sequence of operation of the proposed bioreactor, in which in d) the lower clamp is shown. [Figure 8e] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in e) the bridge filling is shown. [Figure 8f] FIG. 8 shows the sequence of operation of the proposed bioreactor, with the actual cross-linking shown in f). [Figure 8g] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in g) the medium filling and cross-linker draining are shown. [Figure 8h] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in h) medium filling is shown. [Figure 8i] FIG. 8 shows the sequence of operation of the proposed bioreactor, where in i) the cultivation is shown. [Figure 9]Figure 9 shows the materials and results of E.1: (A) a perforated attachment plate, (B) a 3D model of the attachment grid, and (C) an extruded fiber bundle transferred into a Petri dish (9 cm diameter). [Figure 10] Figure 10 shows the attachment plates used for E.2: attachment plates with densities of 11% (left), 22% (middle), and 45% (right). [Figure 11] Figure 11 shows the results of E.3. Microscopic images of extruded fibers encapsulating 20 million cells per mL (2a) and 60 million cells per mL (2b). The top image was taken from the section of the fiber just below the attachment plate, and the bottom image was taken from the section of the fiber just above the extrusion plate. [Figure 12] Figure 12 shows the results of E.4. (a) Extruded fiber clamped between the bottom extrusion plate and the top attachment plate of a differentiation bioreactor in cell culture medium after 4 days of culture; (b) optical microscope image of extruded fiber after harvest showing the microfibril texture; (c) calcein-AM image of harvested muscle fiber after 5 days in cell culture medium showing cell spreading and alignment along the fiber direction. [Figure 13] Figure 13 shows the results of E.5. Extruded and clamped fibers between the extrusion plate and the attachment plate in a myogenic differentiation bioreactor maintained in a saline crosslinking solution (A), ejection of the saline crosslinking solution after extrusion showing the stability of the clamped fibers (B), and extruded fiber bundles after harvest (C). DETAILED DESCRIPTION OF THE INVENTION

[0091] FIG. 1 shows a bioreactor 1 according to a first embodiment, with an insert 17 for providing a low dead volume, i.e. for producing bundles with a large length.

[0092] On the other hand, FIG. 2 shows a corresponding bioreactor 1 with an insert 17 for avoiding the use of too much process liquid when it is desired to produce a high dead volume, ie shorter microfibril bundles.

[0093] The embodiments according to Figures 1 and 2 are essentially the same, and correspondingly, reference signs in these two figures and other figures denote the same or equivalent elements unless specifically stated otherwise.

[0094] As can be seen from FIG. 1 a), which is a perspective representation from above, the reactor 1 comprises a cylindrical peripheral wall 2 which is supported along its main axis in the mid-section by an axial reinforcing structure 3 .

[0095] The reactor 1 is covered on the top by a top cover 4, which is provided with a first central inlet / outlet 5 and a second lateral inlet / outlet 6. The upper part of the reactor is based on a peripheral upper frame part 15 which is integral with the stem 3. This is followed by an upper peripheral closure extension 11 (see Figure 1b), which is attached to the upper frame part 15 by a lower upper closure bracket 8 which is hinged about hinge 10 and closed by a closure mechanism 13.

[0096] A peripheral seal 16 is provided between the upper frame portion 15 and the upper closure extension 11. On top of this upper closure extension 11 is the above-mentioned top cover 4, which is attached by another upper top closure bracket 7, hinged about an axis 9 and secured by a closure mechanism 12. Also here, a peripheral seal 14 is provided between the upper closure extension 11 and the top cover 4.

[0097] As can be seen in particular from the axial cutaway views in Figures 1b and 1c, the upper part is provided with an insert 17, which does not have the same outer diameter as the peripheral wall 2 but is smaller than it, so that there is a space between the insert and the wall, providing a passage 20 around the insert 17. The insert 17 surrounds a dead volume 18, into which no liquid can enter. The insert 17 is pierced at its top by an axially central vertical pipe 19 or channel attached to the first inlet / outlet 5 and, if necessary, sealed against said vertical pipe or channel by a protruding part 21 that contacts the lower opening of the first inlet 5. The second inlet / outlet 6, on the other hand, is connected to the aforementioned passage 20 and allows the circulation of liquid through the passage 20 into the space 58 below the insert.

[0098] The reactor as shown in Figure 1 has an attached upper plate 22, shown in a lower intermediate position. The attachment plate 22 is mounted on a circular mounting structure 23 and has a central opening 32 suitable and adapted to be penetrated by or connected / contacted at its lower end by a vertical pipe 19, for example, when the attachment plate 22 reaches its uppermost position. The attachment plate 22 is also provided with side openings 40. These openings 40 are provided to allow fluids to be supplied and / or removed through the corresponding plate 22, if necessary. Importantly, the attachment plate 22 is provided with a number of nozzle openings or holes 42, as will be further described below.

[0099] Below the attachment plate 22, a stationary extrusion plate 24 is positioned. This extrusion plate 24 is also provided with a number of holes 44, and in fact the holes in the two plates 22, 24 are arranged and mounted such that when the two plates 22, 24 are positioned adjacent to each other in flat contact, the holes in the two plates 22, 24 align and allow the extrusion material to pass through both plates 22, 24, so that the nozzle openings in the individual plates 22, 24 are combined with the nozzle openings of the double plate.

[0100] The extrusion plate 24 is provided with nozzle-opening-free surface areas 41 and 33 that align with the openings 40 and 32, respectively, in the attachment plate 22. As can be seen particularly in Figures 1b and 1c, around the mounting structures 23 of the attachment plate 22 there may be a layer of material 31, for example to hold magnets as described below.

[0101] Also in the bottom structure, it can be seen that there is a bottom plate 34, on which is a bottom side 37, which is attached to an upper peripheral bottom side 38, these two elements being attached to one another by a lower bottom closure bracket 26 using a corresponding closure mechanism 28. In the upward direction follows a lower frame portion 43, which is attached to the upper bottom side 38 by an upper bottom closure bracket 27 using a corresponding closure mechanism 29. Again, a peripheral sealing element 39 is provided between these elements.

[0102] There is a bottom inlet 25 which allows the extrusion material, i.e. the paste described above, to be fed initially into a horizontal continuous space 92 below the first lower extrusion plate 24, which space 92 is also provided with a side inlet / outlet 35. A lateral bottom inlet / outlet 30 is provided for feeding the space above the extrusion plate 24 and below the attachment plate 22, i.e. space 57. The extrusion plate 24 is sealed by one or more peripheral seals 36.

[0103] As can be seen from the representation in Figure 2, the insert 17 is here quite large in the axial direction and occupies a large part of the void volume of the reactor within the peripheral wall 2. In this sense, the space below the insert, i.e. space 58, combined with the space 57 between the second upper extrusion plate and the extrusion plate 24, is quite small. As indicated above, the use of this insert 17 is suitable and compatible with production processes in which short fiber bundles are to be produced.

[0104] Figure 3 shows an axial cutaway view of the bioreactor according to the first embodiment (Figure 1) in a position where the attachment plate 22 is in its uppermost position, and in addition shows the mounting of the reactor 1 and the means provided for moving the attachment plate 22 during the extrusion process.

[0105] The reactor 1 is mounted on a bottom mounting structure 48, which stands on several corresponding vertical legs 52. A housing 50 is provided which can accommodate or contain control elements for the movement of the plate 22 or the supply of liquid, and a corresponding support structure 51. This support structure 51 is particularly intended for mounting a vertical rail 46, on which a mounting structure 49, with attached transfer brackets 45, is movably mounted. On this transfer bracket 45, a peripheral transfer ring 53 is provided, which controls the axial position of the mounting structure 23 for the attachment plate 22. A motor 47 is provided which, by means of a corresponding belt or chain, makes it possible to vertically move the mounting structure 49 and, correspondingly, the transfer ring 53, depending on the process.

[0106] How this is done in a contactless manner to allow the interior of the reactor 1 to be sterile is best shown in FIG. 4. Here, a corresponding reactor 1 having a large insert with a slightly different top cover geometry, similar to the embodiment in FIG. 2, is shown with the attachment plate 22 in its lowest position, i.e., adjacent to and substantially in contact with the pusher plate 24. It can be seen that the transfer ring 53 is provided with magnets 54. The support structure 23 for the attachment plate 22 is also provided with a counter magnet 55 on a corresponding axial extension. Corresponding magnetic forces secure the transfer ring 53, the mounting structure 23, and the corresponding attachment plate 22 relative to one another, so that when the motor 47 begins to pull the mounting structure 49 upward, it correspondingly moves the transfer ring 53 upward, which, due to the magnetic forces, also pulls the mounting structure 23 and the corresponding attachment plate 22 vertically upward.

[0107] Yet another perspective view of the reactor setup with surrounding elements is shown in Figure 5. Here it can be seen that a glass housing 56 may be provided around the actual reactor, and at the bottom side various control and / or feed handling elements 93 may be provided.

[0108] Figure 6 shows the extrusion plate 24. Here, it can be seen that the plate is actually a nozzle plate with a large number of holes, in this particular example, it is provided with 6700 holes. As already indicated above, there is also a first area 33 on the axis of the plate 24 without openings, and two laterally offset areas 42 without openings. Most of the surface of the plate 24 is covered by these openings / nozzles / holes 42. The holes have a diameter of 0.7 mm.

[0109] 7 is a corresponding representation of a second upper extrusion plate 22. In this example, the plate 22 is provided with a peripheral rim 59 having attachment ribs 60 for attachment to the mounting structure 23.

[0110] Also in this example, the central opening 32 is provided with an upwardly projecting peripheral rim, as are the side openings 40 as described above.

[0111] Again, most of the surface of this plate 24 is covered with holes, which have the same distribution and geometric arrangement and the same size as the holes in the extrusion plate 24 shown in Figure 6. This is why, when the two plates 22, 24 are placed adjacent to each other and flat against each other, the openings will align to form the extrusion nozzles in the first stage of the fabrication process.

[0112] The sequence shown in Figure 8 a) to i) is used to explain a production method that is possible using the reactor 1 as described above.

[0113] In the context of Figure 8a) individual reference signs are mentioned and the same reference signs are used in the following Figures b) to i) to denote the same elements, but in the context of the following Figures only a description of the respective method steps is given.

[0114] 8 shows, on the left, a schematic representation of a reactor 1 in which the mounting structure 23 and the attachment plate 22 are shown in each case in three different positions: a bottom position 88, an intermediate position 89 and a top position 90. Depending on the corresponding process situation, only one of these positions is assumed.

[0115] There are three medium containers: the actual growth medium container 61 (M), a container 62 (CC) for calcium chloride which acts as a hardening agent, and a container 63 (CS) for the cross-linking solution.

[0116] Also provided is a container or reservoir together with a pump for the actual cell paste, designated by reference numeral 64. This reservoir 64 is attached by a valve 67 via a line 65 to the bottom inlet 25 of the reactor as shown above. To allow complete filling of the space 92 below the attachment plate 22 in the first step of the preparation illustrated in Figure 8a), an outlet 66 is also provided which can be opened or closed by a valve 68 to allow air and / or paste to exit the space 92 (see Figure 1b).

[0117] The medium container 61 is connected to the reactor by a line 70 controlled by a valve 71 which leads to a collection line 72. In this connecting line 72 there is a pump 69 and a valve 73 to control the supply and / or removal from the bottom inlet / outlet 30. There is also a cross line 91 connecting this collection line 72 to the upper branch of the tubing, which is also equipped with a corresponding valve 94.

[0118] Also connected to collection line 72 is calcium chloride container 62, which is connected to line 74 controlled by valve 75. Also connected to the collection line by line 76 and controlled by valve 77 is a container 63 containing further cross-linking solution.

[0119] In the upper branch there is an upper collection line 79 which is attached to a second inlet / outlet 6 provided in the top cover 4 of the reactor. This upper collection line 79 is connected by valve 80 to a growth medium container 61 by a recirculation line 81 controlled by valve 82, to a calcium chloride container 62 by a circulation line 83, and to a further crosslinking solution container 63 by a line 85 controlled by a valve 86. These lines are also provided with means for draining to collection or disposal means 78.

[0120] 8a) shows the step of pre-filling gel from reservoir 64. In this step, cell paste is pumped into the bioreactor. From reservoir 64, the paste is pumped through valve 67 and pipe 65 into space 92 below extrusion plate 24, which in this example is in bottom position 88. Extrusion is carried out until the cell paste passes through both adjacent extrusion plates 22, 24 or through the holes in these extrusion plates 22, 24, forming a layer of paste on attachment plate 22 or at least some spreading portions. To allow complete filling of space 92, line 66 is opened until this space is completely filled, and then the outflow is controlled by valve 68.

[0121] Once a layer (or spread) of gel has been achieved on the attachment plate 22, the next step, shown in FIG. 8b), i.e., the upper clamping step, is initiated. Here, the calcium chloride container 62 is connected, i.e., valves 75, 73 are opened and the pump 69 is activated, thereby filling the reactor volume with calcium chloride solution. This leads to the hardening of the cell paste layer on the second upper extrusion plate and to the automatic adhesion of the individual strings of paste located in and above the nozzle openings. During this stage, the pumping of the cell paste is interrupted.

[0122] In the next step, shown diagrammatically in FIG. 8c), the paste is extruded from the container 64, while the mounting structure 23 is continuously moved upward until it reaches the position shown in this figure, i.e., until the attachment plate 22 is in position 90, i.e., the uppermost position, at the end of the process. At this stage, the pumping of the cell paste is then turned on again, which causes the extrusion to occur, and the clamping plate, i.e., the attachment plate 22 with the top of the paste attached thereto, moves upward, forming a plurality of individual fibers from each or the corresponding hole in the attachment plate 22. During extrusion, these nascent fibers are in the hardening bath and are therefore successfully hardened simultaneously with extrusion. The speed of extrusion is chosen to be somewhat lower than the speed of the upward movement of the attachment plate 22, so that there is a certain degree of elongation during extrusion.

[0123] 8d) then follows, in which the pump of unit 64 is stopped and the movement of attachment plate 22 is also stopped, but now hardening solution is supplied by lines 74, 72, 66 and thus across valves 75, 73, 68 to space 92, so that the paste located in that intermediate space is also crosslinked.

[0124] This therefore leads to a situation where the bottom side of the fibres is also clamped by the continuous patch of paste located below the extrusion plate 24. Thus, at the end of this process stage there is a bundle of microfibrillar fibres clamped by two continuous patches of cross-linked or hardened paste, one of which is above the attachment plate 22 and the second of which is below the extrusion plate 24.

[0125] This is followed by what is shown in Figure 8e), where a second cross-linking solution CS, e.g. the actual cross-linking solution acting on the protein component of the paste, is introduced into the reaction cavity from a container 63. The calcium chloride solution is drained from the container during that step.

[0126] As shown in Figure 8f), the entire volume 94 of the reaction vessel is now filled with cross-linking solution from vessel 63, leading to the final cross-linking of the microfibril structure.

[0127] The crosslinking is then drained from the reactor by opening valve 30 and allowing the crosslinking solution to drain through line 87, as shown in FIG. 8g), while at the same time, culture medium M is pumped from medium container 61 into the container from above through lines 70, 72, 91 and 79 by opening valves 71 and 94 and operating pump 69.

[0128] This leads to the situation shown in Figure 8h), where reactor volume 94 is filled with growth medium, leading to the growth of cells in or on the fibril structures. If the paste does not already contain cells, the medium may be supplied with cells during or before this step, and a corresponding seeding of the microfibril structures may take place.

[0129] This is followed by the actual culture process, as shown in Figure 8i), whereby the reactor is kept at appropriate temperature and culture conditions leading to the corresponding growth of muscle cells in and / or on the microfibrils. Optionally, at this stage the attachment plate 22 may be moved up and down to create a cyclic mechanical stimulation of the fibers.

[0130] After this process is complete, the medium may be allowed to flow from the vessel and the microfibrillar structures may be removed and further processed to lead to a cultured meat product.

[0131] Experiments E1 and E2 provide experimental evidence for the production of fiber bundles. In both cases, the same method and the same extrusion prototype were used, but the nozzle density of the extrusion plate or the geometry of the attachment plate was changed.

[0132] device Syringe pump (Harvard Apparatus), general-purpose peristaltic pump, disposable syringes (Omnifix®), a small fiber extrusion mold (JAG Jakob AG, bottom containing inlet for hydrogel / paste, extrusion chamber, interchangeable extrusion plate, and cylindrical glass vessel (20 cm 2 × 10 cm), a perforated adhesion plate (JAG Jakob AG), and a 3D-printed adhesion grid (polyvinylidene fluoride (PVDF), MIRAI Foods AG).

[0133] Reagents, chemicals, and solutions Bovine acid bone gelatin (Gelita®), sodium alginate (Kimica), microstructuring agent (MSA) (MIRAI Foods AG), calcium chloride (Sigma Aldrich, C1016), MilliQ water.

[0134] Experiment 1 (E.1.) - Extrusion of fiber bundles using different attachment plate geometries overview In E.1., the fiber bundle is 15 cm long and contains 330 circular holes with a diameter of 0.7 mm. 2 The adhesive was extruded through an extrusion plate having a total area of ​​2.2 mm. Two different adhesive plates were tested: 1. An attachment plate with 330 circular holes that match the holes in the extrusion plate. 2.1mm 2 3D printed adherent grid with mesh size of .

[0135] method Step 1: Preparation of solutions: Saline crosslinking solution: 100 mM calcium chloride was dissolved in milliQ water. The pH was adjusted to 7.2. The solution was stored at room temperature. Hydrogel solution: 40 mg / mL gelatin and 25 mg / mL sodium alginate were completely dissolved in milliQ water under vigorous stirring at 58°C. The hydrogel solution was cooled to 37°C. 100 mg / mL of MSA was added to the hydrogel and mixed uniformly by vortexing.

[0136] Step 2: Fiber Extrusion: 1) The prototype was assembled according to the manufacturer's instructions. The assembled prototype was approximately 200 cm 2 The reactor had a small volume of 1000 sq. m. The bottom inlet was connected to a tube using a luer lock. 1. The attachment plate was attached to a metal rod by a magnet and placed on top of the extrusion plate so that the holes in the extrusion plate were aligned with the holes in the attachment plate. 2. The 3D printed adhesive grid was attached to a threaded rod and placed on top of the extrusion plate. 2) 30 mL of hydrogel was loaded into a disposable plastic syringe and placed on a syringe pump. 3) The hydrogel was extruded at a rate of 5 mL / min until a thin layer was formed on the adhesive grid. 4) Hydrogel extrusion was interrupted, and 200 mL of the crosslinking solution was injected into a small reactor vessel. 5) The syringe pump was restarted. 6) The attachment plate / grid was slowly pulled upwards in a steady motion while holding the rod to the end of the reaction vessel. 7) The extruded fiber bundle was transferred to a beaker containing the cross-linking solution.

[0137] Results and Conclusions Regardless of the attachment plate used, whether perforated or grid, fibers can be efficiently attached to the attachment plate and extruded throughout the entire length of the reactor vessel (Figure 9). The hydrogel volume lost during the attachment process is slightly greater when using a grid compared to a perforated attachment plate.

[0138] Experiment 2 (E.2.) - Extrusion of fiber bundles with different fiber densities overview In E.2., the fiber bundle is a 15 cm 2 The fiber bundles were extruded through an extrusion plate with a total area of ​​11%, 22%, and 45%. The extrusion of fiber bundles with varying fiber densities was evaluated using densities of 11%, 22%, and 45% open area relative to the total extrusion area (see Figure 10).

[0139] method Step 1: Preparation of solutions: Saline crosslinking solution: 100 mM calcium chloride was dissolved in milliQ water. The pH was adjusted to 7.2. The solution was stored at room temperature. Hydrogel solution: 40 mg / mL gelatin and 25 mg / mL sodium alginate were completely dissolved in milliQ water under vigorous stirring at 58°C. The hydrogel solution was cooled to 37°C. 100 mg / mL of MSA was added to the hydrogel and mixed uniformly by vortexing.

[0140] Step 2: Fiber Extrusion: 1) The prototype was assembled according to the manufacturer's instructions. The assembled prototype was placed in a small reactor approximately 200 cm 2 The bottom inlet was connected to a tube using a Luer lock. The 3D-printed adhesive grid was attached to a threaded rod and placed on top of the extrusion plate. 2) 30 mL of hydrogel was loaded into a disposable plastic syringe and placed on a syringe pump. 3) The hydrogel was extruded at a rate of 5 mL / min until a thin layer was formed on the adhesive grid. 4) Hydrogel extrusion was interrupted, and 200 mL of the crosslinking solution was injected into a small reactor vessel. 5) The syringe pump was started again with the following extrusion rate: a. 5 mL / min for an extrusion plate with 11% fiber density. b. 10 mL / min for an extrusion plate with a fiber density of 22% c. 20 mL / min for extrusion plates with 45% fiber density. 6) The attachment grid was slowly pulled upward in a steady motion, holding the threaded rod to the end of the reactor. 7) The extruded fiber bundle was transferred to a beaker containing the cross-linking solution.

[0141] Results and Conclusions Regardless of fiber density, homogeneous fiber bundles approximately 10 cm long could be extruded. Even at the highest fiber density, all fibers were efficiently crosslinked. However, as fiber density increased, a change of the crosslinking solution after fiber extrusion was required to fully crosslink the hydrogel. This indicates that the fiber extrusion method can be easily scaled up by increasing fiber density.

[0142] Overview of Experiments E3, E4, and E5 Experiments E3, E4, and E5 provide experimental evidence for the generation and cultivation of fiber bundles. In all three examples, the same fiber extrusion method and the same 9 L differentiation bioreactor were used.

[0143] device Syringe pump (Harvard Apparatus), general-purpose peristaltic pump, disposable syringes (Omnifix®), a 9 L differentiation bioreactor (manufactured by JAG Jakob AG according to Figure 1 and Figure 5, with interchangeable extrusion and attachment plates), and a 2 L benchtop bioreactor as a medium reservoir.

[0144] Reagents, Chemicals, Solutions Bovine acidic bone gelatin (Gelita®), sodium alginate (Kimica), microstructuring agent (MSA) (MIRAI Foods AG), calcium chloride (Sigma Aldrich, C1016), MilliQ water, MIRAI muscle growth medium (MIRAI Foods AG), microbial transglutaminase concentrate (mTGase)-2000U / g (BDF Ingredients), Chinese hamster ovary (CHO) cells (provided by the Zurich University of Applied Sciences (ZHAW)), MIRAI muscle cells (MIRAI Foods AG), calcein-AM (C3100MP, Fisher Scientific).

[0145] Experiment 3 (E.3.) - Fiber bundle extrusion in a 9 L differentiation bioreactor with varying cell densities overview In E.3, 20–60 × 10 per mL of hydrogel solution 6 Fiber bundles encapsulating cells are extruded, and the cell distribution along the fiber length is observed under a microscope.

[0146] method Step 1: Preparation of solutions: Saline crosslinking solution: 100 mM calcium chloride was dissolved in milliQ water. The pH was adjusted to 7.2. The solution was sterile filtered and stored at room temperature. Hydrogel solution: 80 mg / mL gelatin was completely dissolved in milliQ water under vigorous stirring at 58°C and then sterile filtered. 50 mg / mL sodium alginate was completely dissolved in milliQ water under vigorous stirring at 58°C and autoclaved at 110°C for 30 min. The gelatin solution and the alginate solution were mixed 1:1 using magnetic stirring under sterile conditions. The hydrogel solution was cooled to 37°C. 100 mg / mL of MSA was sterilized using UV irradiation (Spectronics, XL-1000 UV crosslinker) and added to the hydrogel solution.

[0147] Step 2: Encapsulation of cells An aliquot of Chinese hamster ovary (CHO) cells in suspension was 1) 400 million cells 2) 1.2 billion cells The cells were transferred to a 50 mL Falcon tube and centrifuged at 350 g for 10 minutes to obtain a cell count of 0.01. The supernatant was removed and the cells were resuspended in 20 mL of hydrogel solution, resulting in the following cell densities: 1) 20 million cells per mL 2) 60 million cells per mL

[0148] Step 2: Fiber Extrusion: 1) The bioreactor was assembled according to the manufacturer's instructions and autoclaved for 20 minutes at 120°C. The attachment plate was attached to a metal rod with a magnet and placed on top of the extrusion plate so that the holes in the extrusion plate overlapped with the holes in the attachment plate. 2) The following steps were performed under sterile conditions under laminar flow. 3) 30 mL of hydrogel was loaded into a disposable plastic syringe and placed on a syringe pump. 4) The hydrogel was extruded at a rate of 5 mL / min until a thin layer was formed on the adhesive grid. 5) Hydrogel extrusion was interrupted, and 200 mL of the crosslinking solution was injected into a small reactor vessel. 6) The syringe pump was restarted. 7) The attachment plate / grid was slowly pulled upwards in a steady motion while holding the rod to the end of the reaction vessel. 8) The extruded fiber bundle was transferred to a Petri dish containing the cross-linking solution and imaged using an optical microscope.

[0149] Results and Conclusions Extruded fibers are shown in Figure 11 with 20 million (Figure 11a) and 60 million (Figure 11b) cells per mL. Cell distribution is uniform along the fiber length. This experiment demonstrated that fiber bundles can be efficiently extruded with up to 60 million cells per mL.

[0150] Experiment 4 (E.4.) - Myofiber culture in a 9L differentiation bioreactor overview Using a 9 L differentiation bioreactor with a reactor volume of 9 L (Figure 2), myofibers were extruded and maintained in cell culture medium for 5 days. The goal of this experiment was to demonstrate that myofibers can be extruded, crosslinked, and cultured under sterile conditions within a single myofiber differentiation bioreactor.

[0151] method Step 1: Prepare the bioreactor: 1) A 9 L differentiation bioreactor was assembled according to the manufacturer's instructions and sterilized by autoclaving. The extrusion plate and attachment plate had matching hole patterns consisting of 390 circular holes with a diameter of 0.7 mm. The reactor was fully assembled with the attachment plate positioned at the bottom end. An insert (17 in Figure 2a) was incorporated into the reactor vessel as a volume blocker, reducing the volume to 4 liters. The hydrogel inlet / outlet (25 in Figure 1c) and medium perfusion inlets (5 and 30 in Figure 1c) were all equipped with weldable tubing. 2) For the medium reservoir, a benchtop bioreactor was autoclaved and equipped with weldable tubing.

[0152] Step 2: Preparation of solutions: Saline crosslinking solution: 100 mM calcium chloride was dissolved in milliQ water. The pH was adjusted to 7.2. The solution was sterile filtered and connected to the bottom inlet of the differentiation bioreactor (30 in Figure 1c) through a peristaltic pump. Enzyme crosslinking solution: 6.25 mg / mL mTGase powder was dissolved in MIRAI muscle growth medium, which was then sterile filtered and pre-warmed to 37°C. Hydrogel solution: 160 mg / mL gelatin was completely dissolved in milliQ water under vigorous stirring at 58°C and then sterile filtered. 100 mg / mL sodium alginate was completely dissolved in milliQ water under vigorous stirring at 58°C and autoclaved at 110°C for 30 minutes. Gelatin and alginate solutions were mixed 1:1 under sterile conditions using magnetic stirring. The hydrogel solution was cooled to 37°C. 15 mL of the hydrogel solution was transferred into a 50 mL Falcon tube under sterile conditions. 3 g of MSA was sterilized using UV irradiation (Spectronics, XL-1000 UV crosslinker), added to the hydrogel solution, and mixed by vortexing. The hydrogel / MSA mixture was kept at 37°C.

[0153] Step 3: Encapsulation of cells 200 million MIRAI myocytes were aliquoted into 50 mL Falcon tubes and centrifuged at 350 g for 10 min. The supernatant was removed and the cells were resuspended in 15 mL of MIRAI muscle growth medium. The resuspended cells were added to the hydrogel / MSA and mixed by vortexing until a homogenous paste was visible.

[0154] Step 4: Fiber Extrusion: 1) 30 mL of the hydrogel / cell mixture was evenly filled into two disposable syringes under sterile conditions, which were then connected to the differentiation bioreactor (35 in Figure 1) and loaded onto a syringe pump. 2) The hydrogel / cell mixture was extruded until a thin layer formed on top of the adherent grid and then discontinued. 3) The saline cross-linking solution was pumped into the bottom of the reactor vessel. 4) Once the hydrogel / cell layer was completely covered with the saline crosslinking solution, the syringe pump was restarted and the attachment plate was raised 10 cm at a rate of 2 mm / s. The syringe pump was stopped at 18 cm.

[0155] Step 4: Cross-linking the fibers 1) Fibers were cross-linked in a saline cross-linking solution for 10 minutes. The cross-linking solution was drained from the bottom of the bioreactor and replaced by an enzyme cross-linking solution through the top inlet. 2) The fibers were enzymatically cross-linked for 1.5 hours.

[0156] Step 5: Culturing muscle fibers 1) After enzymatic cross-linking, the cross-linking solution was drained from the bottom of the bioreactor and replaced with fresh MIRAI muscle growth medium from the top inlet. 2) 9 liters of MIRAI muscle growth medium (3 L in the medium reservoir and 4 L in the differentiation bioreactor) was perfused through the reactor for 5 days. 3) After 4 days of culture, the extruded fibers were stretched by 10% by moving the attachment plate upward by 10 mm.

[0157] Step 6: Fiber collection 1) On the fifth day, the medium was completely drained and the fibers were collected. 2) Calcein-AM imaging assessed cell viability, cell spreading, and cell distribution.

[0158] Results and Conclusions Thirty-micron-sized myofibers containing approximately 6 million cells per mL were successfully extruded 10 cm and maintained in culture for 5 days (Figure 12a). Due to some clogging of the extrusion plate, the extruded fibers exhibited some defects and bulging. Nevertheless, they could be clamped between the extrusion plate and the adhesion plate without rupture and could withstand 10% elongation for 5 days. Exchange of two crosslinking solutions and perfusion of medium within a single bioreactor could be managed without damaging the fibers, demonstrating the feasibility of maintaining and differentiating myofibers within a single bioreactor. As can be seen in Figure 12b, the myofibers possessed a microfibrillar structure. Cell viability was good, and the cells aligned along the fiber direction (Figure 12c).

[0159] Experiment 5 (E.5.) - High-density full-length myofiber extrusion in a 9L differentiation bioreactor overview Using a 9 L differentiation bioreactor (Figure 5), as many as 6700 acellular fibers with a length of 40 cm were successfully extruded and fixed in place.

[0160] method Step 1: Prepare the bioreactor: A 9-liter differentiation bioreactor was assembled according to the manufacturer's instructions. The extrusion plate and attachment plate had matching hole patterns consisting of 6700 circular holes with a diameter of 0.7 mm (Figure 6). The reactor was fully assembled with the attachment plate positioned at the bottom end. Peristaltic pumps were connected to the saline crosslinking inlet (30 in Figure 1) and the hydrogel inlet (25 in Figure 1).

[0161] Step 2: Preparation of solutions: Saline crosslinking solution: 100 mM calcium chloride was dissolved in milliQ water. The pH was adjusted to 7.2. The solution was connected to the bottom inlet of the differentiation bioreactor (30 in Figure 1) through a peristaltic pump. Hydrogel solution: 40 mg / mL gelatin and 25 mg / mL sodium alginate were completely dissolved in milliQ water under vigorous stirring at 58°C. The hydrogel solution was cooled to 37°C. 60 g of MSA was added to 0.6 liters of hydrogel solution and mixed by magnetic stirring until uniformly dispersed. The hydrogel / MSA mixture was kept at 37°C.

[0162] Step 4: Fiber Extrusion: The hydrogel solution was connected to the hydrogel injection point (30 in Figure 1) through a peristaltic pump. The hydrogel mixture was pumped until a thin layer formed on the attachment grid and then discontinued. The saline cross-linking solution was pumped into the bottom of the reactor vessel. Once the hydrogel layer was completely covered with the saline crosslinking solution, the syringe pump was restarted and the attachment plate was lifted 40 cm at a speed of 2 mm / s. Hydrogel injection was stopped at 38 cm. The fibers were cross-linked in a saline cross-linking solution for 10 minutes, then the solution was drained and the fibers were collected.

[0163] Results and Conclusions This experiment demonstrated the ability to extrude as many as 6,700 muscle fibers measuring 40 cm in length in less than 4 minutes. The fibers were successfully secured between the extrusion plate and the attachment plate (Figure 13A). The fiber bundle was successfully harvested, resulting in a thick fiber bundle weighing approximately 300 g (Figure 13C). [Explanation of symbols]

[0164] 1. Reactor 2.1 Surrounding Wall 3 Reinforcement structure 4 Top cover 5 First entrance / exit at 4 6 Second entrance / exit at 4 7 Upper Top Closure Bracket 8 Lower Top Closure Bracket 9 7 hinge 10 8 hinges 11 Upper closing extension 12 7 Closure Mechanism 13 8 Closure Mechanism 14 Sealing part between 4 and 11 15 Upper frame part 16 Sealing part between 11 and 15 17 Insert Dead volume at 18 17 Vertical pipe through 19 17 20 Passage around 17 21 19 protruding part 22 Adhesive Plate 23 Mounting structure for 22 24 Extrusion Plate 25 Bottom entrance 26 Lower Bottom Closure Bracket 27 Upper Bottom Closure Bracket 28 26 Closure Mechanism 29 27 Closure mechanism 30 bottom inlet / outlet 31 Sealing around 23 Central opening at 32 22 33 Surface area of ​​extrusion plate without nozzle openings 34 Bottom Plate 35 25 side entrance 36 Sealing around 24 37 Bottom side 38 Upper bottom side 39 Sealing part Side opening at 40 22 41 Surface area of ​​extrusion plate without nozzle openings Hole in 42 22 43 Lower frame part Hole in 44 24 45 Moving bracket 46 Rail 47 Motor 48 Bottom mounting structure Mounting structure for 49 45 50 Housing Support structure for 51 46 52 legs 53 Moving Ring 54 53 magnets 55 23 magnets 56 Glass housing 57 The space between 22 and 24 58 Space above 22 59 Peripheral Rim 60 Mounting rib 61 Culture medium container 62 Calcium chloride container 63 Container for cross-linking solution 64 Cell paste reservoir and pump 65 64 to 1 pipe 66 Exit from bottom area Valve in 67 65 Valve in 68 66 69 Pump 70 Line from medium container Valve in 71 70 72 Collection Line Valve in 73 72 74 Line from the calcium chloride container Valve in 75 74 76 Line from the container for cross-linking solution Valve in 77 76 78 Collection 79 Upper Collection Line Valve in 80 79 81 Recirculation line to medium container Valve in 82 81 83 Recirculation line to the calcium chloride vessel Valve in 84 83 85 Recirculation line to the container for the cross-linking solution Valve in 86 85 Exit line from 87 72 Bottom position of 88 22 89 22 midpoint 90 22 top position 91 Cross Line Space below 92 24 93 Supply / Control Elements 94 valves 95 reactor volume M medium CC Calcium chloride CS Further Crosslinking Solution

Claims

1. 1. A method of manufacturing elongated structures for the production of cultured meat, comprising: (a) the paste is extruded through an extrusion plate (24) having at least one nozzle opening (44) and at least partially through an adjacent deposition plate (22), preferably into a space (58) downstream of the deposition plate (22); (b) the paste located in the attachment plate (22) and / or in the space (58) is at least partially hardened; (c) the paste continues to be extruded through the extrusion plate (24) while increasing the distance between the extrusion plate (24) and the attachment plate (22) under the formation of the elongated structure between the plates (22, 24); The method.

2. 2. The method of claim 1, wherein the extrusion is carried out at a discharge velocity (Se) under the generation of a velocity gradient downstream of each nozzle opening (44) in the extrusion plate (24) by a separation velocity between the extrusion plate (24) and the attachment plate (22) that is greater than the discharge velocity (Se) to form the elongated fibrous structure.

3. 3. The method according to claim 1 or 2, wherein in at least one of step (b), step (c) and after step (c), the paste in the space (58) and / or in the intermediate space between the plates (22, 24) is immersed in a hardening bath, preferably a hardening bath comprising divalent or polyvalent cations, an acidic bath or a bath having a different temperature than the gel paste, more preferably a hardening bath comprising divalent cations.

4. the extrusion plate (24) and / or the attachment plate (22) comprise at least 20, preferably at least 100, more preferably at least 200, or in the range of 250 to 1000 nozzle openings; and / or the nozzle openings cover 5% to 90% or 5% to 95%, more preferably 20% to 90% or 20% to 80%, most preferably 40% to 70% of the cross-sectional area of ​​the plate; and / or the nozzle openings are 1 nozzle / cm ２ ~5000 nozzles / cm ２ , preferably 5 nozzles / cm ２ ~400 nozzles / cm ２ The nozzle density is within the range of and / or the nozzle opening has a diameter, or in the case of a non-circular opening, a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm; and / or the attachment plate is a mesh, a porous plate, or a plate with nozzle openings, preferably aligned with the nozzle openings of the nozzle plates (42, 44); The method according to any one of claims 1 to 3.

5. 5. The method of any one of claims 1 to 4, wherein the extrusion in steps (b) and (c) is carried out into a reaction vessel, and step (c) is followed, if necessary, by a further step of hardening the extruded elongated structures, and the reaction vessel is filled with culture growth medium and the elongated structures are used to grow meat cells seeded onto the structures and / or already contained in the paste.

6. (a) the paste is extruded through the extrusion plate (24) and the adjacent deposition plate (22) into a space (58) downstream of the deposition plate (22); (b) stopping or slowing down the extrusion and hardening the paste located in the space (58) by immersing the space downstream of the deposition plate (22) with a hardening bath, preferably a hardening bath containing divalent or polyvalent cations, an acid bath or a bath having a different temperature than the gel paste, preferably a calcium chloride (CC) bath, wherein preferably the paste forms a hardened gel volume immobilized in and / or on the deposition plate by geometric constraints; (c) continued extrusion of the paste through the extrusion plate (24) while increasing the distance between the extrusion plate and the attachment plate (22), preferably at a relative separation rate greater than the extrusion rate from the extrusion plate (24), under the formation of the elongated, preferably fibrous, structure between the nozzle openings (42, 44); (d) stopping the separation and extrusion and hardening the paste in the form of elongated structures, preferably fibers, in the space (57) between the two nozzle plates (22, 24) by immersing the space downstream of the attachment plate (22) with a hardening bath, preferably a hardening bath containing divalent or polyvalent cations, an acid bath, or a protein cross-linking bath, preferably a calcium chloride (CC) bath, and optionally hardening the paste in the space (92) upstream of the extrusion plate (24); (e) replacing the space (57) between the plates with a culture medium and using the elongated structures, preferably fibers, to grow meat cells seeded on the elongated structures, preferably fibers, and / or already contained in the paste. The method according to any one of claims 1 to 5.

7. The paste comprises the following components: (A) at least one polysaccharide capable of forming a solidified gel by the action of divalent or polyvalent cations, thermal gelation, photoinduced addition or condensation reactions, or a combination thereof, wherein the concentration of the at least one polysaccharide in the paste is in the range of 0.01 g to 200 g per liter of component (D); (B) optionally, one or more proteins, the concentration of which in the paste is in the range of 0 g to 500 g per liter of component (D), preferably the protein(s) associate with the polysaccharides of component (A) via supramolecular or covalent interactions, or a combination thereof; (C) cells selected from mammalian cells, fish cells, crustacean cells, or combinations thereof, wherein the concentration in the paste is within the range of 0 to 300 billion cells per liter of component (D); (D) water or a water-based culture medium; (E) an additive different from (A) to (D), selected from the group consisting of a crosslinking kinetics modifier having a concentration in the paste ranging from 0 mM to 500 mM, a flowability modifier having a concentration in the paste ranging from 0 g to 200 g per liter of component (D), or a combination thereof; A paste for producing cultured meat, comprising: and / or the hardening bath contains Ca in water, preferably at a concentration in the range of 1 mM to 500 mM, more preferably 20 mM to 100 mM. ２＋ , Mg ２＋ , Fe ２＋ , Fe ３＋ or a combination thereof, and / or the hardening bath is buffered at a pH ranging from 6 to 8, preferably by using a HEPES buffer, more preferably the HEPES buffer is used at a concentration comprised between 1 mM and 100 mM; and / or the hardening bath comprises a protein cross-linking agent or, following step (b), the fibers are immersed in a protein cross-linking bath with such a cross-linking agent, preferably the cross-linking agent is selected from the group consisting of transglutaminase, peroxidase, laccase, tyrosinase, lysyl oxidase, glutaraldehyde, genipin, citric acid, or combinations thereof, preferably transglutaminase, and when transglutaminase is used, it is present in an amount of 1 U to 2000 U per mL of cross-linking bath, and further preferably cross-linking is carried out at a temperature in the range of 20°C to 45°C, preferably for a time in the range of 10 minutes to 120 minutes, more preferably 30 minutes to 90 minutes; The method according to any one of claims 1 to 6.

8. 8. The method according to claim 1, wherein during the cultivation, the two nozzle plates are vibrated mechanically and / or electrically relative to each other, in particular to stimulate and influence the growth process on the anisotropic microfibril structure.

9. 9. The method according to any one of claims 1 to 8, wherein the extrusion in step (c) is carried out with a pull factor, defined as the ratio of the discharge rate to the separation rate, of at least 1.1, preferably at least 1.5, most preferably at least 2.

10. 10. A reactor for carrying out the method according to any one of claims 1 to 9, comprising a closable reactor vessel (1) of elongated shape along a main axis, preferably cylindrical in shape and of constant cross section, a preferably stationary extrusion plate (24), at least one attachment plate (22) movably mounted in the reactor vessel (1), elements for controlled supply of paste for extrusion thereof upstream of the extrusion plate (24), elements (45, 46, 47, 49, 53) for controllably moving the extrusion plate and / or the attachment plate (22) along the main axis, and means for supplying at least one of a culture medium, a hardening medium and a crosslinking medium to and from the interior of the reactor vessel (1), preferably also to the space upstream of the extrusion plate (24).

11. 11. The reactor according to claim 10, wherein the push-out plate (24) is located at one end of the closable reactor vessel, the attachment plate (22) is located adjacent to the push-out plate (24), and preferably the attachment plate (24) can be moved by the element (45-47, 49, 53) in a contactless manner, preferably by magnetic force, and preferably for this purpose the attachment plate (24) is mounted in and / or on a mounting structure (23) having at least one magnet (55), and there is a movable magnetic element on the outside of the reactor vessel (1), preferably in the form of a ring (53) having at least one magnet (54), and the movable magnetic element can be moved automatically, preferably by a motor (43).

12. the extrusion plate (24) and / or the attachment plate (22) comprise a plurality of nozzle openings (42, 44), preferably at least 20, more preferably at least 100, and most preferably at least 200, or in the range of 250 to 1000 nozzle openings; and / or the nozzle openings cover 5% to 90% or 5% to 95%, more preferably 20% to 90% or 20% to 80%, most preferably 40% to 70% of the cross-sectional area of ​​the plate; and / or the nozzle openings are 1 nozzle / cm ２ ~5000 nozzles / cm ２ , preferably 5 nozzles / cm ２ ~400 nozzles / cm ２ The nozzle density is within the range of and / or the nozzle opening has a diameter, or in the case of a non-circular opening, a maximum lateral extension, in the range of 10 μm to 5000 μm, more preferably 100 μm to 1500 μm; and / or said first extrusion plate (24) and / or said attachment plate (22) further comprise at least one opening (32, 33, 40, 41) for the controlled supply of liquid through the respective plate; 12. The reactor of claim 10 or 11.

13. 13. The reactor according to any one of claims 10 to 12, comprising a control device for controlling in particular at least one of the extrusion rate of the paste, the rate of separation between the extrusion plate and the attachment plate (22), the supply and removal of at least one of a culture medium, a crosslinking medium, a hardening medium.

14. Use of the method according to any one of claims 1 to 9 for the production of a consumer cultured meat product, preferably after culturing the corresponding elongated structures, preferably in the form of bundles, (i) fat and / or oil based ingredients, including cultured fat, vegetable fat or animal fat based ingredients and derivatives thereof, especially in combination with separately cultured adipocytes and / or separately cultured adipocyte aggregates; and / or (ii) structuring agents, including hydrocolloids, and / or cellulose and its derivatives, and / or proteins of plant, animal, recombinant or cell culture origin; and / or (iii) connective tissue components, including animal-derived connective tissue, cultured connective tissue, especially in combination with cultured fibroblasts and / or chondrocytes, and / or aggregates of separately cultured fibroblasts and / or chondrocytes; to form compact bundles of fiber bundles, particularly in pieces of meat.

15. Cultured meat obtainable using the method of any one of claims 1 to 9 and / or obtainable using the reactor of any one of claims 10 to 13.

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