HYDROGELS FOR CULTURED MEAT PRODUCTION

Abstract

The invention is directed to a modified polysaccharide hydrogel, comprising a low molecular weight alginate with a specific M/G ratio. The modified polysaccharide is modified with a specific peptide, preferably comprising a cell-adhesion peptide. The modified polysaccharide hydrogel may be used as a hydrogel for the growth of cultured meat preferably as a sacrificial biopolymer.

Claims

1. A modified polysaccharide hydrogel, comprising a low molecular weight alginate having a M.sub.w of 10 to 50 kDa and a M/G ratio of 0.8 to 1.5, wherein said alginate is conjugated with one or more cell-adhesion peptides, for use in cultured meat applications.

2. The modified polysaccharide hydrogel according to claim 1, said one or more cell-adhesion peptides are animal-free.

3. The modified polysaccharide hydrogel according to claim 1, wherein said cell-adhesion peptide comprises an integrin-binding ligand, which preferably comprises RGD.

4. The modified polysaccharide hydrogel according to claim 1 wherein said alginate is crosslinked by cations, preferably divalent cations, more preferably calcium (Ca.sup.2+) cations.

5. The modified polysaccharide hydrogel according to claim 1, wherein the concentration of the cations which are used for crosslinking is 0.05-0.5 M.

6. The modified polysaccharide hydrogel according to claim 1, wherein the concentration of the cations which are used during culture is 0-50 mM.

7. The modified polysaccharide hydrogel according to claim 1 wherein said alginate, further comprises one or more further specific peptides, different from said first specific peptide.

8. The modified polysaccharide hydrogel according to claim 7, wherein the one or more further specific peptide comprises a cell-adhesion peptide, preferably an integrin-binding ligand, which more preferably comprises RGD.

9. A method of producing a modified polysaccharide hydrogel comprising a low molecular weight alginate having a M.sub.w of 10 to 50 kDa and a M/G ratio of 0.8 to 1.5, wherein said alginate is conjugated with one or more cell-adhesion peptides.

10. The method for producing a modified polysaccharide hydrogel according to claim 1 comprising the steps of: providing a modified alginate with Mw of 10-50 kDa and M/G ratio of 0.8-1.5; conjugate said modified alginate with said first specific peptide, using a reaction which is selected from one or more of the following: carbodiimide chemistry-based reaction; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/imidazole based coupling; oxidation of said alginate to create reactive aldehyde groups, and consequently react with amine-, hydrazide-, or aminooxy-terminated peptides to form imine, hydrazone or oxime bonds, respectively; coupling of maleimide-functionalities to carboxylates on the polysaccharide backbone followed by maleimide reaction with thiol-groups of cysteine-comprising peptides via a Michael type addition.

11. The method according to claim 8, further comprising a crosslinking step wherein said modified alginate is crosslinked with cations, preferably divalent cations, more preferably calcium (Ca.sup.2+) cations, wherein the concentration of the cations is preferably 0.05-0.5 M when used as crosslinker, and 0-50 mM during culture.

12. The method according to claim 10, further comprising a step of modification of the modified alginate with one or more further specific peptides.

13. The modified alginate obtainable by the method of claim 10.

14. The method of claim 9 wherein the alginate is a sacrificial biopolymer.

Description

[0017] FIG. 1 is a schematic overview of a RGD modified-alginate allowing cells to find each other, spread and form aligned morphologies.

[0018] FIG. 2 shows microscope images that depict the change in cellular shape and dispersion in 3D between unmodified alginate and RGD-modified alginate after 2 days.

[0019] FIG. 3 illustrates a pillar used to form compacted hydrogels, which result in the formation of muscle tissue.

[0020] FIG. 4 is a microscope image showing the alignment of myosatellite cells in the compacted hydrogel.

[0021] FIG. 5 shows immunofluorescent images indicating the alignment of myosatellite cells, formation of multinucleated cells and the expression of myosin, filamentous-actin, desmin and nuclei in RGD-modified alginates.

[0022] FIG. 6 shows the increased biodegradability and swelling of low molecular weight alginate and resulting effect on cellular alignment.

[0023] FIG. 7 shows the effect of enzymatic degradation, using alginate lyase, on the degree of compaction of the hydrogels.

[0024] Thus, in the first aspect, the present invention is directed to a modified polysaccharide hydrogel comprising a low molecular weight alginate with a molecular weight of 10 to 50 kDa and a M/G ratio of 0.8 to 1.5, wherein the alginate is conjugated with one or more cell-adhesion peptides.

[0025] The low molecular weight of the alginate typically allows for increased biodegradability which may be beneficial for removal through metabolic functions e.g. the kidneys. More importantly, the low molecular weight of the modified alginate is typically preferred as it allows for a faster stress relaxation, due to the shorter chain length. A faster stress relaxation generally allows myosatellite cells to spread within the gel, as can be seen in FIG. 6. The faster stress relaxation further typically allows for the myosatellite cells to form cell-cell contacts and myotubes. Accordingly, the low molecular weight alginate may provide a hydrogel with structural and mechanical properties that is typically an optimal environment for the production of muscle tissue, and therefore cultured meat.

[0026] In accordance with the invention, the modified polysaccharide hydrogel, comprising alginate, is used to encapsulate cells. Alginate is isolated from seaweed and different seaweed species, each resulting in a specific molecular weight and composition. In accordance with the invention the alginate is typically from food grade sources, to allow for use for cultured meat for consumption. With food grade sources is meant any material safe for human consumption complying to the Food Chemicals Codex or any equivalent standard. Further, the alginate is conjugated with one or more cell-adhesion peptides, such as RGD. In the hydrogel of the present invention the cells may differentiate and mature.

[0027] In a preferred embodiment the alginate is modified with a first specific peptide, which is preferably animal-free. Preferably, the specific peptide comprises a cell-adhesion peptide. The cell-adhesion peptide is capable of binding to a receptor on the cell to encourage several processes, such as cell migration, spreading, guidance, proliferation and differentiation. Cell adhesion peptides may attach to various integrin receptors on the cell surface. They induce attachment, signaling and remodeling through cleavage.

[0028] It is also possible to provide a plurality of different cell-adhesion peptides in the hydrogel. Accordingly, the alginate may be modified by two or more different cell-adhesion peptides such as RGD and another functional peptide such as GGGGDGEA, which is considered relevant for satellite cell interaction. In this peptide sequence, the independent letters correspond to a specific amino acid (e.g. G is glycine). The use of two or more cell-adhesion peptides typically allows for a plurality of binding sites, which may result in increased encouragement of migration, spreading, guidance, proliferation and differentiation. Moreover, an additional carrier or support affects the chemical, topographical and mechanical properties of the modified alginate, which is related to the final function of the tissue.

[0029] More preferably, the specific peptide comprises an integrin-binding ligand. Integrins, upon binding to integrin-binding ligands, activate signal transduction pathways that mediate cellular signals, including regulation of the cell cycle. Regulation of the cell cycle includes processes such as cell spreading, migration, guidance, proliferation, apoptosis. Integrins are moreover responsible for tissue organization, hemostasis, inflammation, target recognition of lymphocytes, differentiation of cells by the interaction of the integrin with the environment.

[0030] Examples of suitable integrin-binding ligands are for instance given by Humphries et al. (J. Cell Sci. 119 (2006) 3901-3903) and comprise fibronectin, osteopontin, laminin, collagen, ADAM family members, COMP, connective tissue growth factor, Cyr61, E-cadherin, fibrillin, fibrinogen, ICAM-4, LAP-TGFβ, MMP-2, nephronectin, L1, plasminogen, POEM, tenascin, thrombospondin, VEGF-C, VEGF-D, vitronectin, heparin and combinations thereof.

[0031] Preferably the specific peptide comprises cell-adhesion peptides, more preferably RGD. RGD is naturally found in the extracellular matrix and it is considered the most common motif responsible for cell adhesion. FIG. 1 is a schematic overview of a preferred embodiment wherein a RGD modified-alginate allows cells to find, spread and form aligned morphologies. FIG. 2 shows micrographs where the difference of cell shape after two days between the cells in an unmodified alginate hydrogel and a RGD modified alginate hydrogel according to a preferred embodiment of the present invention is visualized.

[0032] In a preferred embodiment of the invention, the alginate is crosslinked. Crosslinking is achieved via cations, as the alginate is an anionic polymer. The concentration in which the cations are present determines the crosslinking density. Preferably, the concentration of the cations for crosslinking during preparation of the hydrogels is between 0.05 to 0.5 M. When the hydrogels are used with a cell culture it may also be desirable to have these cations present, in which case the concentration of cations is preferably between 0 to 50 mM. The crosslinking density is in part responsible for the rigidity of the system, thereby having an influence on the chemical, topographical and mechanical properties of the modified-polysaccharide hydrogel. The chemical, topographical and mechanical properties determine the final function of the tissue. The preferred concentration of the cations and thus the degree of crosslinking typically presents suitable chemical, topographical and mechanical properties for myosatellite cells to migrate, spread, align, proliferate and differentiate into muscle tissue. Preferably, the cations are divalent cations. More preferably, the divalent cations are calcium ions (Ca.sup.2+).

[0033] In a preferred embodiment, the alginate present in the modified polysaccharide hydrogel comprises one or more further specific peptides, wherein said one or more further specific peptides are different from the other specific peptide. The further specific peptide may function as an additional carrier and/or support for the cells.

[0034] Preferably, the further specific peptide also comprises a cell-adhesion peptide.

[0035] More preferably the further specific peptide comprises an integrin-binding ligand. Integrins are transmembrane receptors that, upon binding to integrin-binding ligands, activate signal transduction pathways that mediate cellular signals, including regulation of the cell cycle. Processes such as cell spreading, migration, guidance, proliferation, and apoptosis are all directly or indirectly related to the regulation of the cell cycle. Integrins are moreover responsible for tissue organization, hemostasis, inflammation, target recognition of lymphocytes, and differentiation of cells by the interaction of the integrin with the environment.

[0036] As mentioned above, suitable examples of integrin-binding ligands are fibronectin, osteopontin, laminin, collagen, ADAM family members, COMP, connective tissue growth factor, Cyr61, E-cadherin, fibrillin, fibrinogen, ICAM-4, LAP-TGFβ, MMP-2, nephronectin, L1, plasminogen, POEM, tenascin, thrombospondin, VEGF-C, VEGF-D, vitronectin, heparin (Humphries et al. (J. Cell Sci. 119, (2006), 3901-3903)).

[0037] Most preferably the specific peptide comprises RGD.

[0038] It may be appreciated that the modified polysaccharide hydrogel may be used for the promotion of muscle tissue regeneration, preferably as a sacrificial biopolymer. The term sacrificial is used herein to describe the possibility to selectively remove the biopolymer from the tissue. Selective removal may be achieved by dissolving the polysaccharide either via diffusion, using a chelator (e.g. EDTA) and/or enzymatic degradation (e.g. Alginate Lyase) of the polysaccharide. FIG. 7 shows the effect of alginate lyase on the degree of compaction and accordingly illustrates the increase of cell-matrix density via selective degradation of alginate. As a result of the selective degradation, the degree of compaction may be controlled. The modified polysaccharide hydrogel—typically provides an environment for several processes such as cell guidance, spreading, migration, proliferation and differentiation. The processes are necessary during the regeneration process of cells and thus for the regeneration of tissue. A damaged tissue may be encouraged to regenerate by the modified polysaccharide hydrogel. The modified polysaccharide hydrogel according to the present invention thus typically provides a suitable environment for the regeneration process of tissue.

[0039] The modified polysaccharide hydrogel according to the present invention, more importantly, further provides an ideal environment for the production of cultured meat. The modified polysaccharide hydrogel may accordingly be used in the production of cultured meat suitable for consumption, preferably as sacrificial biopolymer.

[0040] As the cultured meat is suitable for consumption it is typically required that there are substantially no toxic compounds present in the modified polysaccharide hydrogel. Toxic is herein defined as considered non-safe for human consumption. Therefore, it is typically preferred that the purity of the modified polysaccharide is at least complying to such as, but not limited to, the purity criteria in a European Council Directive, such as the Directive concerning food additives other than colours and sweeteners authorized for use in foodstuffs for human consumption. Further, it may be beneficial to add nutrients to the hydrogel such as vitamins and/or minerals that would add nutritional value to the cultured meat product.

[0041] The production of cultured meat is based upon the principle that muscle tissue can be grown from a myosatellite cell. The myosatellite cell is of non-human animal origin, preferably from non-human mammal origin, more preferably from bovine, sheep, pigs, and the like. The myosatellite cell may be obtained via a non-sacrificial and animal-friendly method, e.g. via a small biopsy. Cultured meat as referred to herein is suitable for human consumption. The modified polysaccharide hydrogel according to the present invention typically provides a suitable environment for myosatellite cell guidance, spreading, migration, proliferation and differentiation to muscle tissue. The muscle tissue may be harvested and may be sold as cultured meat.

[0042] The modified polysaccharide hydrogel according to the present invention may be produced by the provision of a modified alginate with a Mw of 10 to 50 kDa and/or a M/G ratio of 0.8 to 1.5, modified with a first specific peptide. The modification may involve a chemical coupling reaction that covalently binds the specific peptide to the alginate. For example, conventionally carbodiimide chemistry may be used for RGD-modification, herein the amine-functionality of RGD is coupled to carboxylates to form amide bonds.

[0043] Another approach to chemically couple the cell-adhesion peptides to the polysaccharide is by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/imidazole based coupling. Furthermore, alginates can also be oxidized to create reactive aldehyde groups, and consequently react with amine-, hydrazide, or aminooxy-terminated peptides to form imine, hydrazone or oxime bonds, respectively (Xu et al., Molecules 24 (2019) 3005).

[0044] Other approaches may include the coupling of maleimide-functionalities to carboxylates on the polysaccharide backbone. Consequently, maleimide can then readily react with thiol-groups of cysteine-comprising peptides via a Michael type addition (see e.g. Ravasco et al., Chem. Eur. J. (2019), 25, 43-59).

[0045] In a preferred embodiment, the modified polysaccharide hydrogel may be produced by the provision of a modified alginate with a Mw of 10 to 50 kDa and a M/G ratio of 0.8-1.5 modified with a first specific peptide and further crosslinked with cations. FIG. 6 shows the increased degradation and swelling of this preferred sacrificial polysaccharide hydrogel compared to alginates outside this range. Additionally, due to the faster degradation the hydrogel swelling is increased. As a result of this, satellite cells embedded inside the gel show a more spread out morphology in alginate with a Mw of 10 to 50 kDa and a M/G ratio of 0.8-1.5. The spread out morphology is accompanied by increased hydrogel compaction and myosin expression. The polysaccharide hydrogel, according to the present invention, thus typically provides a suitable environment for the regeneration process of muscle tissue.

[0046] Preferably, the modified polysaccharide hydrogel is produced by the provision of a modified alginate with a Mw of 10 to 50 kDa and a M/G ratio of 0.8 to 1.5 modified with a first specific peptide and modified with a further specific peptide. The modification may involve a chemical coupling reaction that covalently binds the specific peptide to the alginate. For example, conventional carbodiimide chemistry may be used for RGD-modification, herein the amine-functionality of RGD is coupled to carboxylates to form amide bonds.

[0047] FIG. 3 is an image of a pillar used to form a modified polysaccharide hydrogel according to the present invention. The location of the compacted hydrogel is indicated by the arrow.

[0048] FIG. 4 is a microscope image showing the alignment of myosatellite cells in a compacted hydrogel according to the present invention. The direction of the cellular alignment is indicated by the arrow.

[0049] FIG. 5 shows immunofluorescence images of the alignment of the myosatellite cells and fusion into multinucleated cells. Additionally, the expression of myosin, filamentous-actin and desmin, is included inside the RGD modified alginate hydrogels according to a preferred embodiment of the present invention. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.