PVA / PVP / alginate hydrogels, uses, and preparation thereof

Abstract

The present invention is directed to a composition including a plurality of capsules, wherein each capsule of the plurality of capsules includes polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell. Further provided are a method of preparing the composition of the invention, as well as using thereof, such as for feeding a subject.

Description

PVA / PVP / ALGINATE HYDROGELS, USES, AND PREPARATION THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U. S. Provisional Patent Application No. 63 / 714,165, filed October 31, 2024, titled “ENCAPSULATION AND GROWTH OF MICRO-ALGAE IN HYDROGEL CAPSULES”, the content of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention relates generally to the field of polymeric hydrogels, and use of same in the culturing of microalgae, cells, or both.BACKGROUND

[0003] Microalgae are microscopic aquatic organisms that have gained attention as potential sources for food, nutritional supplements, cosmetics, and medicines due to their rich content of proteins, antioxidants, polysaccharides, polyunsaturated fatty acids, and other valuable compounds. These organisms can grow faster than higher plants since each cell functions as a photosynthetic unit without energy- consuming tissues such as roots or flowers. Despite their potential, commercial cultivation of microalgae faces several challenges including high operating costs, susceptibility to pathogenic infections, contamination by other algae species, and energy-intensive harvesting processes that require separation of algae from water through methods such as centrifugation, flocculation, filtration, or sedimentation.

[0004] Similarly, cell cultivation for food production, particularly fish cells for cultured seafood applications, encounters difficulties related to bacterial and fungal infections when grown in conventional systems using plates and reactors. These cultivation methods typically require large amounts of expensive growth medium and present challenges in maintaining sterile conditions throughout the growth process.

[0005] Current cultivation systems for both microalgae and cells rely on open systems such as tanks and ponds, or closed systems including tanks, sleeves, pipes, and panels. Thesesystems often suffer from contamination issues, require substantial resources and energy consumption, and involve complex harvesting and separation processes that contribute to high production costs. The separation stage alone represents a resource-intensive process that consumes high levels of energy and affects the overall economic viability of large-scale production.

[0006] Hydrogels, which are three-dimensional hydrophilic and physical or covalent crosslinked polymeric networks with high water-swelling capacity, have found applications in various fields including drug delivery, tissue engineering, cosmetics, and agriculture. These materials can contain over 90% water and possess porous structures that allow for the diffusion of nutrients and other substances. Common hydrogel materials include polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol, and natural proteins such as collagen and gelatin.

[0007] There exists a need for improved cultivation methods that can address the challenges associated with conventional microalgae and cell cultivation systems, particularly with regard to contamination control, resource efficiency, and cost reduction in production processes.SUMMARY

[0008] According to one aspect, there is provided a composition comprising a plurality of capsules, wherein each capsule of said plurality of capsules comprises polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell.

[0009] In some embodiments, each capsule of said plurality of capsules comprises: (i) an outer surface comprising said PVA, PVP, and alginate, and wherein said alginate is crosslinked alginate; and (ii) an inner portion comprising a hydrogel comprising said PVA, PVP, alginate, and at least one cell, and wherein said alginate is non-cross-linked alginate.

[0010] In some embodiments, the at least one cell comprises a microalga cell, an animal cell, or a combination thereof.

[0011] In some embodiments, the animal cell is a muscle cell.

[0012] In some embodiments, the animal cell is a fish cell.

[0013] In some embodiments, the PVA and the PVP are crosslinked or non-crosslinked.

[0014] In some embodiments, said PVA and said PVP in a weight per weight ratio (w / w) ranging between 5: 1 and 1:1.

[0015] In some embodiments, the cross-linked alginate is in an amount of 1% to 8% per weight of the composition.

[0016] In some embodiments, each capsule of said plurality of capsules further comprises a growth medium.

[0017] In some embodiments, the at least one cell comprises a combination of cells comprising at least one microalgal cell and at least one animal cell, and said growth medium is suitable for culturing said combination of cells.

[0018] In some embodiments, the growth medium is devoid of a calcium-chelating amino acid. In some embodiments, the calcium-chelating amino acid is selected from the group consisting of: aspartic acid, glutamic acid, glycine, lysine, and any combination thereof.

[0019] In some embodiments, a water content of said hydrogel of the composition ranges between 10% to 90% w / w.

[0020] In some embodiments, the composition is an edible composition.

[0021] In some embodiments, the composition is formulated for oral administration.

[0022] In some embodiments, the composition is a meat analog composition.

[0023] According to another aspect, there is provided a method for preparing the composition of the present invention, the method comprising mixing a hydrogel comprising PVA, PVP, alginate, and at least one cell with an aqueous solution comprising calcium ions or a salt thereof, thereby preparing the composition.

[0024] In some embodiments, mixing is drop-wise mixing.

[0025] In some embodiments, the method further comprises a step before said mixing comprising obtaining said hydrogel, said step comprising mixing a pre-mix composition comprising PVA, PVP, and alginate with a suspension comprising at least one cell, thereby obtaining said hydrogel.

[0026] In some embodiments, said suspension comprises said at least one cell suspended in growth medium.

[0027] In some embodiments, said calcium ions are in the form of CaCl2.

[0028] In some embodiments, a concentration of said calcium ions or salt thereof in said aqueous solution ranges between 0.01% and 5% w / w.

[0029] In some embodiments, said pre-mix composition is obtained by mixing PVA, PVP, and alginate in a w / w / w ranging between 1:1:1 and 5:1:3.

[0030] In some embodiments, mixing said PVA, PVP, and alginate is under heat conditions comprising subjecting said PVA, PVP, and alginate to a temperature ranging between 80 °C and 100 °C.

[0031] In some embodiments, the method further comprises subjecting said composition comprising said plurality of capsules to conditions suitable for culturing said at least one cell.

[0032] According to another aspect, there is provided a method comprising feeding a subject, the method comprising orally administering an effective amount of the composition of the invention to a subject in need thereof.

[0033] In some embodiments, said subject is a human subject. In some embodiments, said subject is an aquatic animal. In some embodiments, said aquatic animal is a fish, a crustacean, or both. In some embodiments, said crustacean is selected from the group consisting of: crab, lobster, prawn, shrimp, crayfish, and any combination thereof.

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0035] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should beunderstood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0036] Fig. 1 includes photographs showing various shapes of PVA / PVP hydrogel: films (1), nursery trays for plant cultivation (2), capsules (alternative name: beads (3), long capsules (4), and emulsion (5).

[0037] Fig. 2 includes a scheme showing a non-limiting process for the preparation of PVA / PVP / A hydrogel.

[0038] Fig. 3 includes a photograph showing PVA / PVP / A capsules (alternative name: beads).

[0039] Figs. 4A-4B include photographs showing washed water-based PVA / PVP / A capsules (4A) immersed in algae growth medium (4B).

[0040] Fig. 5 includes a photograph showing PVA / PVP / A capsules immersed in algae growth medium (right) dipped in water (left).

[0041] Figs.6A-6D include a scheme and photographs showing preparation of PVA / PVP / A capsules including algae cells. (6A) A scheme showing a non-limiting illustration of a process for the preparation of PVA / PVP / A capsules preparation. (6B) PVA / PVP / A hydrogel. (6C) PVA / PVP / A / algae capsules at time 0. (6D) PVA / PVP / A / algae capsules after 21 days.

[0042] Fig. 7 includes a photograph showing algae cultures in growth medium after about 15 minutes of adding the PVA / PVP / A hydrogel to different concentrations (1%, 2%, and 3%) of CaCh solution (right to left).

[0043] Fig. 8 includes a vertical bar graph showing Chlorella growth in hydrogel capsules.

[0044] Fig.9 includes photographs showing Nannochloropsis algae after 30 days of growth in hydrogel capsules.

[0045] Figs. 10A-10D includes micrographs showing environmental scanning electron microscope (E-SEM) images of hydrogel capsules surface. (10A) Control (capsules without algae). (10B) Capsules with Nannochloropsis (growing algae for 35 days). (10C) Internal cross-section of the capsules containing Nannochloropsis algae. (10D) Internal cross-section of the capsules containing chlorella algae.

[0046] Figs. 11A-11G include LMD images of internal cross-section of hydrogel capsules with Nannochloropsis algae in different periods of time (T=days); (11A) T=0, (11B) T=3, (11C) T=7, (11D) T=14, (11E) T=21, (11F) T=28, and (11G) T=35.

[0047] Fig. 12 includes a graph showing comparison of the growth rate of Chlorella vulgaris (5a+5b) and Nannochloropsis (6a+6b) growing in capsules and Chlorella vulgaris (7a+7b)Nannochloropsis (8a+8b) growing in Erlenmeyer. All cultures were held in an air bubble and in light and temperature-controlled growth rooms.

[0048] Fig. 13 includes photographs showing confinement of Chlorella within hydrogel drops composed of PVA / PVP / A single components compared with Bristol and the hydrogel containing all the components.

[0049] Fig. 14 includes a graph showing the rate of growth of Chlorella within hydrogel drops composed of PVA / PVP / A single components compared with Bristol and the hydrogel containing all the components.

[0050] Fig. 15 includes a vertical bar graph showing the growth rate of Thalassiosira weissflogii algae encased in capsules stored in closed containers in the refrigerator or at room temperature.

[0051] Fig. 16 includes a vertical bar graph showing the rowth rate of Chlorella algae encapsulated within the capsules compared to non-encapsulated Chlorella algae.

[0052] Fig. 17 includes a scheme of a non-limiting process of extracting muscle cells from sea bass. 1 Anesthetizing the fish with clove oil; 2 Mechanical disassembly – using scalpel; 3 Enzymatic decomposition using Collagenase 1; 4 Incubation for 120 mm at 32 °C; 5 Filtration; 6 Centrifugation; and 7 Cell seeding including L15 +20%FBS + Laminine + FGF.

[0053] Figs. 18A-18B include a micrograph and a graph showing the establishment of a European sea bass Dicentrarchus labrax fish cell line. (18A) A Representative image ofthe European sea bass cell line at passage 24. (18B) A graph showing the average doubling time of the cell line up to passage 24.

[0054] Fig. 19 includes a photograph showing incubation of PVA / PVP / A capsules in L-15 medium containing 0% (1), 0.25% (2), 0.5% (3), and 1% CaCh (4).

[0055] Figs. 20A-20C include photographs showing PVA / PVP / A capsules (20A); immersed in a microalgae growth medium (20B); and the capsules after immersion in a microalgae growth medium (20C, 1) subsequently rinsed with water, becoming colorless again (2).

[0056] Figs. 21A-21B includes fluorescent micrographs and a vertical bar graph. (21A) Representative images of the European sea bass Dicentrarchus labrax) cells encapsulated in the capsules. European sea bass cells within capsules were stained with Calcein-AM for identification of living cells, propidium iodide (PI) for dead cells, and Hoechst for nuclear staining. The encapsulated cells were cultured in vitro for 2, 10, 23, and 31 days. At each time point, capsule samples were collected and analyzed using confocal microscopy at 40× magnification. (21B) A graph showing European sea bass nuclear cell growth kinetics within the capsules.

[0057] Fig. 22 includes representative fluorescent micrographs of Chlorella algae inside capsules. The encapsulated Chlorella algae were cultured in vitro for 2, 10, 23, and 1 days. At each time point, capsule samples were collected and analyzed using confocal microscopy at 40 x magnification.

[0058] Fig. 23 includes representative fluorescent micrographs of European sea bass Dicentrarchus labrax) cells and Chlorella algae inside Capsules. European sea bass cells were stained with Hoechst (blue) for nuclear staining, while Chlorella algae exhibited fluorescence at 680 nm (red). The capsules containing Chlorella algae and European sea bass cells were cultured in vitro for 2, 10, 23, and 31 days. At each time point, samples of the capsules were collected and analyzed using confocal microscopy at 40× magnification.DETAILED DESCRIPTION

[0059] According to the first aspect, there is provided a capsule comprising: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell.

[0060] In some embodiments, the capsule comprises: (i) an outer surface; and (ii) an inner portion.

[0061] In some embodiments, the outer surface is water insoluble. In some embodiments, the inner portion comprises or consists of a hydrogel. In some embodiments, the hydrogel is water soluble.

[0062] In some embodiments, the outer surface of the capsule of the invention comprises PVA, PVP, and alginate. In some embodiments, the alginate is or comprises cross-linked alginate. In some embodiments, the outer surface of the capsule of the invention comprises cross-linked alginate. In some embodiments, the outer surface of the capsule of the invention comprises Ca2+cross-linked alginate. In some embodiments, the inner portion of the capsule of the invention comprises a hydrogel comprising PVA, PVP, alginate, and at least one cell. In some embodiments, the alginate is a non-cross-linked alginate. In some embodiments, the inner portion of the capsule of the invention comprises non-cross-linked alginate.

[0063] According to another aspect, there is provided a capsule (bead) comprising a hydrogel comprising: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell. In some embodiments, the alginate is crosslinked alginate. In some embodiments, crosslinked alginate is Ca2+crosslinked alginate.

[0064] According to another aspect, there is provided a composition comprising the capsule (or bead) of the invention. In some embodiments, the composition comprises at least one capsule (or bead) of the invention. In some embodiments, the composition comprises one or more capsules (or beads) of the invention. In some embodiments, the composition comprises a plurality of capsules (or beads) of the invention.

[0065] The terms “bead” and “capsule” are used herein interchangeably.

[0066] As used herein, the term “capsule” refers to a discrete, enclosed structure that contains or encapsulates one or more components within a protective outer boundary, shell, or surface. A capsule may be formed from various materials including polymers, hydrogels, lipids, proteins, or other biocompatible substances, and may have a spherical, cylindrical, or other geometric configuration. The capsule may provide containment, protection, controlled release, or isolation of its contents from the surrounding environment, and may allow forselective permeability of certain substances such as nutrients, gases, or metabolites while maintaining structural integrity. In biological applications, capsules may serve to encapsulate cells, microorganisms, drugs, nutrients, or other bioactive compounds for various purposes including cultivation, delivery, or processing applications.

[0067] In some embodiments, alginate is a cross-linked alginate. In some embodiments, alginate is a non-cross-linked alginate.

[0068] According to another aspect, there is provided a composition comprising a plurality of capsules, wherein each capsule of the plurality of beads comprises polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell.

[0069] As used herein, the term “hydrogel” refers to a three-dimensional network of hydrophilic polymer chains that are cross-linked to form a gel-like structure capable of absorbing and retaining large amounts of water or aqueous solutions. Hydrogels may maintain their structural integrity while swelling in the presence of water, and may contain water content ranging from a few percent to over 90% of their total weight. The polymer networks may be formed through physical or chemical cross-linking mechanisms and may include natural polymers such as collagen, gelatin, alginate, chitosan, or agarose, synthetic polymers such as polyvinyl alcohol, polyethylene glycol, polyacrylamide, or polyvinyl pyrrolidone, or combinations thereof. Hydrogels may possess porous structures that allow for the diffusion of nutrients, gases, metabolites, and other substances while providing a supportive matrix for biological applications.

[0070] In some embodiments, PVA, PVP, alginate, and at least one cell are homogeneously distributed or dispersed in the capsule of the invention.

[0071] As used herein, the term “homogeneously dispersed or distributed” refers to a uniform arrangement or spread of particles, cells, or other components throughout a medium or matrix such that the concentration, density, or distribution pattern is substantially consistent across different regions or volumes of the system. In a homogeneously dispersed or distributed system, the components may be evenly scattered without significant clustering, aggregation, or concentration gradients, resulting in a relatively uniform spatial arrangement that may facilitate consistent access to nutrients, uniform growth conditions, or predictable material properties throughout the medium.

[0072] In some embodiments, the at least one cell comprises an animal cell, a plant cell, an alga cell, a bacterial cell, a fungal cell, or any combination thereof. In some embodiments, the at least one cell comprises any type of living cell. In some embodiments, the at least one cell comprises a microalga cell or is a microalga. In some embodiments, the at least one cell comprises or is an animal cell. In some embodiments, the at least one cell comprises a combination of a microalga cell and an animal cell.

[0073] In some embodiments, the animal cell comprises a muscle. In some embodiments, the animal cell is a muscle cell.

[0074] As used herein, the term “muscle cell” refers to a specialized cell type that has the capacity for contraction and is derived from muscle tissue. Muscle cells include skeletal muscle cells, cardiac muscle cells, or smooth muscle cells, and is characterized by the presence of contractile proteins such as actin and myosin. In the context of cell cultivation, muscle cells refer to cells isolated from animal muscle tissue that can be cultured and grown in vitro for various applications including food production, research, or therapeutic purposes. These cells retain their ability to proliferate, differentiate, and potentially form muscle-like structures when provided with appropriate growth conditions and nutrients.

[0075] In some embodiments, a muscle cell comprises myoblast, myocyte, myotube, cardiac myocyte, or any combination thereof.

[0076] In some embodiments, the animal is a fish. In some embodiments, the at least one cell is a fish cell. In some embodiments, the at least one cell is a fish muscle cell.

[0077] In some embodiments, PVA and PVP are non-crosslinked. In some embodiments, PVA and PVP are crosslinked.

[0078] In some embodiments, the capsule comprises PVA and PVP in a weight per weight ratio (w / w) ranging between 10: 1 and 1: 1, 9: 1 and 1: 1 (w / w), 8: 1 and 1: 1 (w / w), 7: 1 and 1: 1 (w / w), 6:1 and 1:1 (w / w), 5:1 and 1:1 (w / w), 4:1 and 1:1 (w / w), 3:1 and 1:1 (w / w), or 2:1. Each possibility represents a separate embodiment of the invention. In some embodiments, the composition comprises PVA and PVP in a weight per weight ratio (w / w) ranging between 10:1 and 1:1, 9:1 and 1:1 (w / w), 8:1 and 1:1 (w / w), 7:1 and 1:1 (w / w), 6:1 and 1:1(w / w), 5:1 and 1:1 (w / w), 4:1 and 1:1 (w / w), 3:1 and 1:1 (w / w), or 2:1. Each possibility represents a separate embodiment of the invention.

[0079] In some embodiments, the capsule comprises cross-linked alginate in an amount of 1% to 2%, 1% to 3%, 1% to 4%, 1% to 5%, 1% to 6%, 1% to 7%, or 1% to 8%, per weight of the bead. Each possibility represents a separate embodiment of the invention. In some embodiments, the outer surface of the capsule comprises cross-linked alginate in an amount of 1% to 2%, 1% to 3%, 1% to 4%, 1% to 5%, 1% to 6%, 1% to 7%, or 1% to 8%, per weight of the bead. Each possibility represents a separate embodiment of the invention. In some embodiments, the composition comprises cross-linked alginate in an amount of 1% to 2%, 1% to 3%, 1% to 4%, 1% to 5%, 1% to 6%, 1% to 7%, or 1% to 8%, per weight of the composition. Each possibility represents a separate embodiment of the invention.

[0080] In some embodiments, the capsule, the composition, or both, further comprise a growth medium. In some embodiments, the growth medium is a cell culture medium. In some embodiments, the growth medium is suitable for culturing a combination of cells comprising at least one microalga cell and at least one animal cell.

[0081] As used herein, the terms “growth medium” or “cell culture medium” are interchangeable and refer to a liquid or semi-solid nutrient solution that provides the essential components necessary for the growth, maintenance, and / or proliferation of cells in vitro. Growth medium contains a combination of nutrients including carbohydrates, amino acids, vitamins, minerals, salts, proteins, lipids, and other organic and inorganic compounds that support cellular metabolism, biosynthesis, and reproduction. The medium also includes buffering agents to maintain appropriate pH levels, osmotic regulators, and is supplemented with growth factors, hormones, or other bioactive compounds depending on the specific requirements of the cells being cultured. Growth medium is formulated as a defined medium with known concentrations of specific components, or as a complex medium containing undefined components such as serum or tissue extracts.

[0082] In some embodiments, the growth medium is devoid of calcium-chelating amino acid.

[0083] As used herein, the term “calcium-chelating amino acid” refers to any amino acid that has the ability to bind calcium ions through the formation of coordinate bonds,effectively sequestering or removing calcium from solution. Calcium-chelating amino acids contain functional groups such as carboxyl groups, amino groups, or other electron-donating moieties that can coordinate with calcium ions to form stable complexes. Examples include amino acids with multiple carboxyl groups or other chelating side chains that can form multidentate complexes with calcium ions, thereby reducing the availability of free calcium in the surrounding medium or environment.

[0084] In some embodiments, a calcium-chelating amino acid is selected from: aspartic acid, glutamic acid, glycine, lysine, or any combination thereof.

[0085] In some embodiments, water content of the capsule ranges between 10% to 80% w / w, 10% to 85% w / w, 10% to 90% w / w, 10% to 95% w / w, 20% to 95% w / w, 30% to 95% w / w, 40% to 95% w / w, 50% to 95% w / w, 60% to 95% w / w, 70% to 95% w / w, or 80% to 95%, of the bead. In some embodiments, water content of the capsule is at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, or at least 95% w / w of the bead, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, water content of the hydrogel ranges between 10% to 80% w / w, 10% to 85% w / w, 10% to 90% w / w, 10% to 95% w / w, 20% to 95% w / w, 30% to 95% w / w, 40% to 95% w / w, 50% to 95% w / w, 60% to 95% w / w, 70% to 95% w / w, or 80% to 95%, of the hydrogel. In some embodiments, water content of the hydrogel is at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, or at least 95% w / w of the hydrogel, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, water content of the inner portion of the capsule of the invention ranges between 10% to 80% w / w, 10% to 85% w / w, 10% to 90% w / w, 10% to 95% w / w, 20% to 95% w / w, 30% to 95% w / w, 40% to 95% w / w, 50% to 95% w / w, 60% to 95% w / w, 70% to 95% w / w, or 80% to 95%, of the hydrogel. In some embodiments, water content of the inner portion of the capsule of the invention is at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, or at least 95% w / w of the hydrogel, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, water content of the composition ranges between 10% to 80% w / w, 10% to 85% w / w, 10% to 90% w / w, 10% to 95% w / w, 20% to 95% w / w, 30% to95% w / w, 40% to 95% w / w, 50% to 95% w / w, 60% to 95% w / w, 70% to 95% w / w, or 80% to 95%, of the composition. In some embodiments, water content of the composition is at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, or at least 95% w / w of the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0086] In some embodiments, the plurality of capsules or the composition comprising thereof is characterized by an average bead size / diameter of at least 10 pm, 100 pm, 300 pm, 500 pm, 700 pm, 1 mm, 2 mm, 3 mm, 5 mm, 7 mm, or 10 mm, or any value and range therebetween. In some embodiments, the plurality of capsules or the composition comprising thereof is characterized by an average bead size / diameter ranging between 10 pm and 20 pm, 10 pm and 50 pm, 10 pm and 100 pm, 10 pm and 200 pm, 10 pm and 500 pm, 10 pm and 5 mm, 10 pm and 10 mm, 100 pm and 1 mm, 250 pm and 5 mm, 500 pm and 8 mm, 1 mm and 10 mm, or 700 pm and 5 mm. Each possibility represents a separate embodiment of the invention.

[0087] In some embodiments, the capsules and / or the composition comprise alginic acid and / or a salt thereof (e.g., Na-alginate). In some embodiments, the capsule and / or the composition comprises between 0.01 and 3% of calcium ions. In some embodiments, the capsule is substantially spherically shaped. In some embodiments, the capsule is solid or semi-solid. In some embodiments, the capsule has a spherical geometry or shape. In some embodiments, the capsule has a spherical shape, an elliptical shape, a quasi-spherical shape, a quasi-elliptical sphere, a deflated shape, a concave shape, an irregular shape, or any combination thereof.

[0088] In some embodiments, the composition is a stable composition. In some embodiments, the composition has increased stability. In some embodiments, increased stability is compared to a control. In some embodiments, stability relates to the survival, proliferation, or both, of the at least one cell. In some embodiments, a composition comprising at least one cell being a microalgal cell, an animal cell, or both, is stable for at least 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 10 months, 1 year, or 2 years, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0089] In some embodiments, stable comprises at least 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% survival, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0090] In some embodiments, stable refers to the ratio between the number of the at least one cell at the preparation point, e.g., the initial cell number, and the number of cells after culturing, according to the method of the invention.

[0091] In some embodiments, a stable composition is characterized by at least 5% more, 10% more, 20% more, 30% more, 50% more, 70% more, 90% more, 100% more, 200% more, 350% more, 500% more, 750% more, 900% more, or 1,000% more cells compared to the number of the at least one cell at the preparation point, e.g., the initial cell number, after at least 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 10 months, 1 year, or 2 years, of culturing, storage, or both, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0092] In some embodiments, the capsule is an edible bead. In some embodiments, the hydrogel is an edible hydrogel. In some embodiments, the composition is an edible composition.

[0093] In some embodiments, the capsule is formulated for oral administration. In some embodiments, the hydrogel is formulated for oral administration. In some embodiments, the composition is formulated for oral administration.

[0094] In some embodiments, the composition is a meat analog composition or a meat replacement.

[0095] In some embodiments, the composition further comprises an acceptable carrier, diluent, or excipient.

[0096] As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or a vehicle administered together with the active ingredient. In some embodiments, the carrier improves the stability of the active ingredient in a living organism. In some embodiments, the carrier improves the stability of the active ingredient within the pharmaceutical composition. In some embodiments, the carrier enhances the bioavailability of the active ingredient.

[0097] In some embodiments, carriers are sterile liquids such as water-based liquids; oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like; solvents such as polyethylene glycols, glycerin, propylene glycol, ethanol or other synthetic solvents. In some embodiments, the composition further comprises sodium benzoate.

[0098] In some embodiments, preservatives such as benzyl alcohol, benzoic acid (including any salt thereof, such as sodium benzoate) and / or methyl parabens; antioxidants (such as ascorbic acid, propyl gallate, tocopherols, tertiary butylhydroquinone, butylated hydroxyanisole, sodium pyrosulfite, potassium pyrosulfite, and butylated hydroxytoluene, or sodium bisulfite); and agents for the adjustment of tonicity (such as sodium chloride or dextrose) are also envisioned.

[0099] In some embodiments, the carrier comprises, in total, from about 0.1% to about 99.99999% by weight of the composition presented herein.

[0100] Other non-limiting examples of carriers for include but are not limited to: cocoa butter (relates to a mixture of triglycerides of saturated and unsaturated fatty acids, such as stearic, palmitic, oleic, lauric, and linoleic acids), a cocoa butter substitute (relates to vegetable oils modified by esterification, hydrogenation, etc., including hydrogenated vegetable oil and hard fat), glycerinated gelatin, a polyethylene glycol-based carrier, and a surfactant (such as polyoxyethylene sorbitan fatty-acid esters and polyoxyethylene stearates) or any combination thereof.

[0101] As used herein, the terms “meat analog” and “meat replacement” are interchangeable and refer to a food product that is designed to mimic, resemble, or substitute for conventional animal-derived meat in terms of appearance, texture, flavor, nutritional profile, or culinary functionality. Meat analogs are produced from alternative protein sources such as plant-based materials, cultured cells, microorganisms, or other non-animal derived components, and are formulated to replicate the sensory characteristics, cooking properties, and consumer experience associated with traditional meat products. These products serve as substitutes for beef, pork, poultry, seafood, or other animal proteins in various food applications and culinary preparations.

[0102] According to another aspect, there is provided a method for preparing the hydrogel, the capsule, the composition, or any combination thereof, of the invention.

[0103] In some embodiments, the method comprises mixing a hydrogel or a mixture comprising PVA, PVP, alginate, and at least one cell with an aqueous solution comprising divalent cations, thereby preparing the capsule, composition, or any combination of the invention.

[0104] In some embodiments, mixing comprises or is drop-wise mixing. In some embodiments, mixing comprises or is steering. In some embodiments, mixing comprises or is sonication or sonicating.

[0105] In some embodiments, the method further comprises a step before the mixing comprising obtaining a hydrogel. In some embodiments, the step before the mixing comprises mixing a pre-mix composition comprising PVA, PVP, and alginate with a suspension comprising the at least one cell, thereby obtaining a hydrogel.

[0106] In some embodiments, a hydrogel comprises PVA, PVP, and alginate. In some embodiments, a hydrogel comprises PVA, PVP, alginate, and at least one cell. In some embodiments, there is provided a plurality of hydrogels. In some embodiments, a first hydrogel comprises PVA, PVP, and alginate. In some embodiments, a second hydrogel comprises PVA, PVP, alginate, and at least one cell.

[0107] In some embodiments, the suspension comprises the at least one cell suspended in a growth medium, e.g., such as disclosed herein.

[0108] In some embodiments, the divalent cations are derived from or obtained by solvation of a divalent metal salt. In some embodiments, divalent cations comprise calcium ions. In some embodiments, the calcium ions are derived or obtained by solvation of calcium salt. In some embodiments, calcium salt comprises CaCl2.

[0109] In some embodiments, the concentration of calcium ions in the aqueous solution ranges between 0.01% and 5% w / w, 0.1% and 5% w / w, 1% and 5% w / w, 2% and 5% w / w, 3% and 5% w / w, 4% and 5% w / w, 0.01% and 2% w / w, 0.01% and 3% w / w, 0.01% and 4% w / w, 0.1% and 1.5% w / w, 0.5% and 2.5% w / w, or 1% and 4% w / w, of the aqueous solution. Each possibility represents a separate embodiment of the invention.

[0110] In some embodiments, the pre-mix composition is obtained by mixing PVA, PVP, and alginate in a w / w / w ranging between 1:1:1 and 5:1:3, 1:1:1 and 4:1:3, 1:1:1 and 3:1:3, 1:1:1 and 2:1:3, 1:1:1 and 1:1:3, 1:1:1 and 5:1:2, 1:1:1 and 5:1:1, 1:1:1 and 4:1:2, 1:1:1 and 3:1:2, or 1:1:1 and 2:1:2. Each possibility represents a separate embodiment of the invention.

[0111] In some embodiments, mixing PVA, PVP, and alginate is under heat conditions comprising subjecting PVA, PVP, and alginate to a temperature ranging between 80 °C and 100 °C, 85 °C and 100 °C, 90 °C and 100 °C, 95 °C and 100 °C, 80 °C and 95 °C, 80 °C and 90 °C, or 85 °C and 95 °C. Each possibility represents a separate embodiment of the invention.

[0112] In some embodiments, the method further comprises subjecting the composition comprising the plurality of capsules to conditions suitable for culturing the at least one cell.

[0113] According to another aspect, there is provided a method for feeding a subject. In some embodiments, the method is a non-therapeutic method. In some embodiments, the subject is a subject in need of feeding.

[0114] In some embodiments, the method comprises feeding the composition of the invention to a subject. In some embodiments, the method comprises orally administering the composition of the invention to a subject. In some embodiments, the method comprises orally administering the subject with the composition of the invention.

[0115] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the mammal is a human subject. In some embodiments, the subject is an animal. In some embodiments, the subject is an aquatic animal. In some embodiments, an aquatic animal comprises a fish, a crustacean, or both. In some embodiments, a crustacean comprises a decapod crustacean. In some embodiments, the decapod crustacean is selected from: crab, lobster, prawn, shrimp, crayfish, and any combination thereof.General

[0116] As used herein the term “about” refers to ± 10 %. Further, all numerical values, e.g. when referring to the amounts or ranges of the elements constituting the formulation are approximations which are varied (+) or (-) by up to 10% of the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term “about”.

[0117] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0118] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0119] The term “consisting of means “including and limited to”.

[0120] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. The term “consisting essentially of’ is used to define formulations which include the recited elements but exclude other elements that may have an essential significance on the formulation.

[0121] The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0122] The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict. The word “optionally” and the word "further" are used herein interchangeably.

[0123] The terms, film / films and layer / layers are used herein interchangeably. As used herein, the term "coat" refers to the combined layers disposed over the substrate, excluding the substrate, while the term "substrate" refers to the part of the compositestructure supporting the disposed layer / coating. In some embodiments, the terms "layer", "film" or as used herein interchangeably, refer to a substantially uniform -thickness of a substantially homogeneous substance.

[0124] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0125] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0126] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0127] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specificallyembraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0128] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.EXAMPLESMaterials and methods

[0129] All materials were of analytical grade and purchased from commercial sources. Calcium chloride, alginic acid sodium salt from brown algae, PVA (99+% hydrolyzed and average Mw 89-98 kDa), PVP K-30 and TREION double distilled water (DDW) were obtained from Sigma Aldrich.

[0130] Three species of microalgae were used in the experiments: Chlorella vulgaris (C. vulgaris) a green microalga of fresh water, Nannochloropsis salina - (N. salina) a green microalga of sea water, and Thalassiosira weissjlogii (TW). Microalgae were grown in Bristol and F / 2 media, respectively in 2-liter Erlenmeyer flasks with air agitation; In growth rooms at a constant temperature of 21 °C and under constant LED lighting with an intensity of 70 pmol quanta m’2s-1.

[0131] The optical density of the cultures was measured at a wavelength of 680 nm in a Synergy H1 spectrophotometer (Agilent Technologies, Inc., USA) in a plate of 96 bars in triplicate. C. vulgaris culture at a density of 1.25+0.18 and N. salina culture at a density of 1.2+0.03 were transferred to the continuation of the experiment to prepare hydrogel capsules containing micro-algae for growth.Environmental scanning electron microscope (E-SEM) characterization of the macro-beads

[0132] The hydrogel capsules surface morphology and internal cut was characterized using scanning electron microscope (E-SEM, Quanta FEG 250). The samples undergocryopreservation via immersion in liquid nitrogen followed by lyophilization for a duration of 12 h. All gel capsules were sputter-coated with gold layer for 90 s before measurement. Laser microscope surface analysis

[0133] PVA / PVP / SA / Algae surface microscale analysis was conducted by laser-micro-dissection apparatus (LMD-DMI18; Leica microsystem). An internal section was extracted from each sample and affixed onto a microscope glass slide, then overlaid with a coverslip. After covering, the sample was pressed to remove excess liquid.Binocular vision

[0134] Employing a binocular apparatus for imaging involves positioning the sample under the microscope’s objective lens. Adjustments are made to focus the image, using both oculars to achieve a three-dimensional view. Light intensity and contrast may be modified for optimal visualization. Once adjustments are complete, images are captured through an attached camera or observed directly through the eyepieces.Laser microscope surface analysis

[0135] Optical density determination involves measuring the absorbance of light by a sample. The absorbed light intensity is then detected, allowing for the calculation of optical density. The algae contain chlorophyll that absorbs wavelengths at 680 nm. Chlorophyll optical density measured by SynergyHl microplate reader (BioTek) equipment by direct UV light onto the sample. This method provides insights into the concentration of chlorophyll present, aiding in various scientific analyses such as photosynthetic efficiency assessments and health evaluations.ResultsEXAMPLE 1Preparation of PVA / PVP / A hydrogel capsules

[0136] Table 1 illustrates the content of each component used to prepare the PVA •+• PVP +■ Alginate (PVA / PVP / A) hydrogel capsules.Table 1. The content of each component used in the current study to prepare PVA / PVP / A hydrogel capsules / beads.PVA [g] PVP [g] Alginate [g] H? O [g] 2.215 0.85 0.525 21.5

[0137] A hydrogel aqueous solution was prepared in which the total content of polymers is 14%, the weight ratio between PVA / PVP is 2.6, and the weight percentage of alginate out of all the polymers is ~15%. The PVA (MW 89-98 kDa, 98% hydrolyzed) and PVP (MW=40-80 kDa) were dissolved in water at high temperature (80-90 °C), and then added to an aqueous solution of alginate (A) until a uniform solution was obtained. After that, the solution was heated to 100 °C to sterilize the hydrogel. After spontaneous cooling of the hydrogel to room temperature, the entire mixture was transferred to a burette under sterile conditions and dripped uniformly and regularly into an aqueous solution containing 3% CaCb to obtain uniform hydrogel capsules that are stabilized by crosslinking bonds between the carboxyl groups of the alginate and the calcium ions. These capsules were then washed with water to remove excess CaCh.

[0138] The penetration of the microalgae growth medium (Bristol and F / 2) into the capsules was tested. It was demonstrated that when the water-based capsules were soaked in the growth medium, they turned pink, while when they were washed in water, they became transparent again, which indicates the penetration and exit of the medium's components into / out of the capsules, and vice versa (Figs 4-5). Figs. 4-5 indeed illustrate on the porous structure of the PVA / PVP / A capsules, and the ability to transfer different substances from the capsules to the aqueous continuous phase and vice versa.EXAMPLE 2Microalgae cultivation in PVA / PVP / A hydrogel capsules

[0139] Encapsulation of Chlorella vulgaris microalgae and Nannochloropsis salina in the PVA / PVP / A capsules was done similarly to the preparation of the capsules from PVA / PVP / A, as described above. The algae culture was added to the hydrogel and the mixture was dropped into the cross-linking solution of calcium ions dispersed in the appropriate growth medium (Bristol for Chlorella vulgaris and F / 2 for Nannochloropsis salina to obtain PVA / PVP / A capsules containing the microalgae (Fig.6).

[0140] At the same time, for the purpose of studying the effect of the presence of the calcium ions on the algae, the hydrogel aqueous solution containing the algae grown in Bristol medium was added drop by drop to different concentrations (1%, 2%, and 3%) of CaCh dispersed in the growth medium, or water, the sedimentation of the hydrogel capsules containing the algae was observed almost immediately (Fig. 7). After about two weeks, it was still possible to notice a green and healthy culture of the algae dispersed in the hydrogel capsules.

[0141] In addition, hydrogel capsules containing a low concentration of algae (OD 0.02) were prepared. Optical density measurements of the chlorophyll extraction, after 7 and 30 days is illustrated in Figs. 8-9. The absorption peak of the chlorophyll (at a wavelength of 680 nm) increases with time and indicates the algae culture inside the hydrogel capsules. The control PVA / PVP / A capsules w'ere almost transparent. The transparency is slightly decreased by entrapping low concentration of micro-algae (OD 0.02).

[0142] It should be noted that the encapsulation process of the different algae could be in water only, e.g., the continuous phase of the hydrogel and the CaCh may be pure water only. However, for growing the niicroalgae in the capsules it is recommended that they should be dispersed in an appropriate growth medium, e.g., Bristol for Chlorella vulgaris and F / 2 for Nannochloropsis salina, since usually the microalgae growth process in an appropriate medium is improved relative to water. This can be accomplished by various w-ays, e.g., washing the microalgae encapsulated hydrogel capsules dispersed in water with appropriate medium, or dropping the algae encapsulated hydrogel aqueous dispersion into CaCh dissolved in medium aqueous solution.

[0143] N-15 medium (Sigma, Cat: LI 518) was also found suitable and efficient for microalgae growth within the PVA / PVP / A capsules.EXAMPLE 3E-SEM images of the capsules

[0144] E-SEM measurements were conducted on two distinct samples: one devoid of microalgae (Fig. 10A) and another incubated with algae for a duration of 35 days (Fig. 10B).An observable morphological change between the two samples is clearly illustrated. The presence of the microalgae probably induced changes on the surface of the capsule. The surface of the algae-free capsule exhibits remarkable smoothness devoid of any cracks, whereas the algae-incubated capsule surface demonstrates roughness.

[0145] Scanning E-SEM images of the internal cross-section (Figs. 10C-10D) illustrate the presence of the microalgae within the hydrogel. Figs 10C-10D illustrate that the diameter of both capsules containing Nannochloropsis microalgae and Chlorella microalgae is significantly lower than that of the wet hydrogel capsules (about 1 cm) since they are in the dry state. Also, the diameter of the dry capsules containing Nannochloropsis microalgae is slightly higher than that containing the Chlorella microalgae.EXAMPLE 4LMD imaging of the capsules

[0146] Micrographs of the inner cross-section of the capsules were captured over a 35 -day period. Images were obtained at weekly intervals to monitor temporal changes. Fig. 11A represents the initial time-point (t=0), coinciding with the encapsulation of microalgae in the capsules. Fig. 11A presents a solitary microalgal cell yet to undergo division, and vacant spaces are discernible. Fig. 11B presents the picture after t=3 days of incubation, signs of microalgal division became apparent. Subsequent images illustrate robust microalgal proliferation, forming dense clusters. Notably, the absence of vacant spaces in the final image underscores the prolific growth (Figs. 11D-11G). These observations verify the resilience, reproductive capacity, and algae growth in the hydrogel capsules.EXAMPLE 5Microalgae growth rate

[0147] The measurements were made to determine the growth rate of algae (Chlorella vulgaris and Nannochloropsis) by assessing their concentration. Measurements were taken every 7 days over a 35-day growth period by measuring the chlorophyll absorption light at680 nm. Growing algae in capsules compared to Erlenmeyer was done in duplicate, and measurements of each sample were done in triplicate, as shown in Fig. 12.

[0148] Fig. 12 indeed illustrates that the growth rate of both algae Chlorella vulgaris and Nannochlor opsis in the capsules is significantly higher compared to the algae that grew in Erlenmeyer.

[0149] The current preliminary results show that the freshwater microalgae Chlorella vulgaris continues to thrive in the capsules for a few months. The cultivation of microalgae was done in capsules kept in an aqueous solution using conventional growing mediums. The penetration of the microalgae growth medium (Bristol and F / 2) into the capsules was found to be feasible.EXAMPLE 6The effect of capsule components on Chlorella vulgaris microalgae growth rate

[0150] In a preliminary 21 -day study, the effect of the hydrogel components on the growth rate of the freshwater algae Chlorella vulgaris was evaluated. As part of this experiment, the survival and growth of Chlorella vulgaris in Erlenmeyer was tested in the presence of the hydrogel capsule components: PVA, PVP, and Alginate individually or in combination with all the hydrogel components together, e.g., PVA+PVP+A. This was compared with the standard growing medium (Bristol) without these components. Figs. 13-14 illustrate clear differences in microalgae density and distribution within the different hydrogel capsule compositions. It can clearly be seen that microalgae grown in all the hydrogel components (PVA+PVP+A) developed a uniform and strong green color faster than the control(s), suggesting high photosynthetic activity and higher cell density than cells grown in the Bristol control medium. On the other hand, the separate components of the hydrogel such as Alginate, PVA, and PVP alone showed an increase in microalgae that were in a scattered form.EXAMPLE 7Microalgae encased in capsules have increased shelf-life

[0151] In an initial experiment, the rearing of Thalassiosira weissflogii (TW) microalgae maintained in capsules versus free TW algae was compared. The microalgae inside the capsules were kept without medium refreshing, under two conditions: 1. At room temperature; and 2. In a refrigerator (in the dark at 4 °C). After 3 days, a pungent odor was detected in the vessel containing the free TW algae, which indicated the decomposition of the microalgae grown under both conditions; on the other hand, this odor was not detected m the microalgae that were enclosed in capsules under both conditions. In order to ascertain whether the encapsulated microalgae actually grew inside the capsules under the different conditions, a measurement of chlorophyll absorption was carried out in samples of capsules at time zero and after three weeks. The results showed that the encapsulated TW niicroalgae grew' faster when refrigerated compared to room temperature, as was noted for a similar diatom, Thalassiosira pseudonana^ as shown in Fig. 15. Similar stabilization effect is shown in Fig. 16 for Chlorella microalgae encapsulated within the capsules compared to non¬ encapsulated Chlorella microalgae.EXAMPLE 8Fish ceil cultivation in PVA / PVP / A hydrogel capsules with or without microalgae Materials and methodsCell development

[0152] To develop primary European sea bass (Dicentrarchus labrax) cell culture as shown in Fig. 17, young fish were obtained from Maagan Michael fish farm, Israel. The fish were euthanized using clove oil, wiped with 70% ethanol, and transferred to a tissue culture hood under sterile conditions. Muscle tissues were excised using a scalpel, and the skin was removed. The isolated muscle tissue was sterilized by immersion in iodine solution (IOD) for 3 minutes, followed by treatment with 1% penicillin / streptomycin / amphotericin B (P / S / AmphoB ) in PBS for another 3 minutes. The tissue was mechanically dissociated into ~1 mnri fragments using a scalpel and transferred to a digestion solution containing 0.1% collagenase type I (Worthington Biochemical, Cat: LS004196) in Leibovitz’s L-l 5 medium (L-15). Digestion w'as carried out at 32CC for 2 hours, with trituration every’ 20 minutes to enhance dissociation. The resulting cell suspension was filtered sequentially through 70 umand 40 pm cell strainers, then centrifuged at 300 x g for 5 minutes. The pellet was washed with PBS and centrifuged again under the same conditions. Cells were then resuspended in 3 ml of growth medium, composed of Leibovitz’s L-15 medium (Sigma, Cat: LI 518), 20% fetal bovine serum (Sigma, Cat: F7524), 1 ng / niL recombinant human fibroblast growth factor (FGF-basic 154 a.a., PeproTech #100-18B), 1% GlutaMAX (Gibco, Cat:35050-038), 1% a-MEM NEAA (Gibco, Cat: 11140-035), 1% penicillin (10,000 lU / mL), streptomycin (10 mg / mL), and amphotericin B (25 pg / mL) (MPB, Cat: 091674049), and 10 pg / mL gentamicin (Sigma Aldrich #G1397). The cell suspension was seeded onto 1 pg / cm2Laminin (Sigma, Cat: L2020)-coated 6-well plates and incubated at 27 °C without CO2. The first medium change was performed after 24-48 hours, and subsequently, the medium was replaced every 3-4 days.Cell division and freezing

[0153] When the cells reached approximately 80% confluence, they were passaged using TrypLE (Gibco, Cat: 12563-029) and either seeded at a density of 20,000 cells / cm2in fresh growth medium or cryopreserved using NutriFreez (Sartorius, 05-713-1B) for long-term storage.Doubling time measurements

[0154] In order to measure cell doubling time we count the cells and use the doubling time formula:Culture time (h) X ln(2)Doublinq time (h) = - 77: — 7 - - — -k, F inai concentration,initial concentration' Fluorescence staining — live / dead cells

[0155] Sections (1 mm thick) were taken from both the center and periphery of PVA / PVP / A capsules containing Dicentrarchus lab rax cells, Chlorella microalgae, or a combination thereof,

[0156] For identifying live cells, samples containing only Dicentrarchus labrax cells were incubated in 4 pM Calcein-AM (Cayman chemical company, Cat: 14948) diluted in 0.9% NaCl saline for 15 minutes to obtain green color. To detect dead cells, sections were rinsed with saline and then immersed in 1 pg / mL Propidium Iodide (PI) (Biolegend Cat: 421301)diluted in saline for 3 minutes to obtain red color. After another saline wash, the sections were stained with 5 pg / mL Hoechst 33258 (Cayman chemical company, Cat: 16756-50) diluted in saline for 5—10 minutes to visualize cell nuclei (blue color).

[0157] F or samples containing both European sea bass (Dicentrarchus labrax) cells and Chlorella microalgae, only Hoechst staining was performed. AH stained samples were examined using Leica confocal stellaris microscopy.ResultsEstablishment of continuous European sea bass (Dicentrarchus labrax) cell culture

[0158] Muscle tissue from European sea bass Dicentrarchus labrax) fish was dissociated using Collagenase I, following the procedure outlined hereinabove. The resulting primary cells were continuously cultured until they underwent spontaneous immortalization. As illustrated in Fig. 18A, the cells were passaged using TrypLE once they reached 80% confluence, continuing through passage P24. The average doubling time of the cultured cells was 77.8 ± 22 hours (Fig. 18B).EXAMPLE 9PVA / PVP / A hydrogel and capsules for cell growthP VA / P VP / A hydrogel prepara ti on

[0159] PVA / PVP / A hydrogel solution was prepared with a total polymer content of 14%, maintaining a P VA / P VP weight ratio of 2.5 and an alginate weight percentage of 5.6% relative to the entire hydrogel components. To ensure cell compatibility, the hydrogel was formulated using a saline solution (0.9% NaCl) as the base. Sequentially, 7.17 g of PVA, 2.85 g of PVP, and 4 g of alginate were added to 86 mL of saline under continuous heating (95 °C) and stirring until a uniform solution was obtained. After that, the solution was heated to 100 °C to sterilize the hydrogel. The solution was then allowed to cool naturally to room temperature under sterile atmosphere.PVA / PVP / A hydrogel capsules preparation.

[0160] The capsules were prepared as follows: Saline was incorporated into the hydrogel, adjusting the total polymer concentration to 9.1%. The mixture was then transferred to aburette or to a peristaltic pump under sterile conditions and dripped uniformly and consistently into a saline solution containing 3% CaCh. This process enabled the formation of uniform hydrogel capsules, stabilized through crosslinking interactions between the carboxyl groups of alginate and calcium ions. After 20 min, the resulting capsules were washed with a medium to remove any CaCh excess. The dripping process can also be automated using a peristaltic pump.

[0161] The encapsulation of cells, either alone or in combination with algae, within the PVA-PVP / A capsules was conducted using the same procedure described above and illustrated in Fig. 6A. The cell culture, either alone or in combination with algae, was added to the hydrogel, and the mixture was dripped into the cross-linking saline solution containing 3% CaCh. After 20 minutes, the capsules were washed with the N-15 growth medium and then transferred to a 24- well plate containing medium with 0.5% CaCh and incubated at 27 °C in the dark. All work was done under sterile conditions.Stability of PVA / PVP / A hydrogel capsules in L-15 medium

[0162] The stability of the PVA / PVP / A capsules incubated in L-15 medium containing various concentrations of CaCh was tested. As shown in Fig. 19, the capsules incubated in L-15 only became weakened after 30 days. However, incubation in L-15 containing 0.25%, 0.5%, and 1% CaCh provided stable capsules over time. The 0.5% concentration was chosen for further experiments. Similar results were obtained substituting the L-15 medium for Bristol and F / 2.

[0163] The penetration of the cell medium into the capsules was evaluated. It was observed that when the water-based capsules were immersed in the L-15 medium, they exhibited a pink coloration. Conversely, upon rinsing with water, they reverted to a transparent state. This color change indicates the bidirectional diffusion of medium components into and out of the capsules (Fig. 20).Encapsulation of European sea bass cells in PVA / PVP / A capsules

[0164] European sea bass (Dicentrarchus labrax) cells were encapsulated in PVA / PVP / A capsules following the procedure described hereinabove. To assess cell survival within the capsules, sections were stained with Calcein-AM (live cells), Propidium Iodide (PI) (deadcells), and Hoechst (nuclear staining) and analyzed using confocal microscopy. As shown in Fig. 22, the cells remained alive after 31 days. The cells appear alive and multiplied throughout the incubation period. Additionally, the red fluorescence intensity of dead cells remained unchanged throughout the observation period, indicating stable cell persistence within the capsules (Figs. 21A-21B).Encapsulation of Chloreila algae in PVA / PVP / A capsules

[0165] C ’hlorella cells encapsulated in PVAZPVP / A capsules exhibit natural fluorescence at 680 nm (red), enabling their visualization through confocal microscopy.. As illustrated in Fig. 22, a marked increase in red fluorescence intensity was detected by day 31 compared to day 2, confirming the growth and proliferation of Chloreila within the capsules.Encapsulation of European sea bass cells in combination with Chloreila microaigae in PVA / PVP / A capsules

[0166] The encapsulation of cells alongside Chloreila within PVAZPVP / A capsules is illustrated in Fig. 23. The cells were stained with Hoechst, while Chloreila was identified by its intrinsic red fluorescence. Over 2, 10, 23 and 31 days, the fluorescence intensity of the cells remained stable, whereas the intensity of Chloreila increased significantly, indicating its proliferation. Additionally, Chloreila growth within the capsules occurred in clusters.

[0167] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a plurality of capsules, wherein each capsule of said plurality of capsules comprises polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), alginate, and at least one cell.

2. The composition of claim 1, wherein each capsule of said plurality of capsules comprises: (i) an outer surface comprising said PVA, PVP, and alginate, and wherein said alginate is cross-linked alginate; and (ii) an inner portion comprising a hydrogel comprising said PVA, PVP, alginate, and at least one cell, and wherein said alginate is non-cross-linked alginate.

3. The composition of claim 1 or 2, wherein said at least one cell comprises a microalga cell, an animal cell, or a combination thereof.

4. The composition of claim 3, wherein said animal cell is a muscle cell.

5. The composition of claim 3 or 4, wherein said animal cell is a fish cell.

6. The composition of any one of claims 1 to 5, wherein said PVA and said PVP are crosslinked or non-crosslinked.

7. The composition of any one of claims 1 to 6, comprising said PVA and said PVP in a weight per weight ratio (w / w) ranging between 5: 1 and 1:1.

8. The composition of any one of claims 2 to 7, comprising said cross-linked alginate in an amount of 1% to 8% per weight of the composition.

9. The composition of any one of claims 1 to 8, wherein each capsule of said plurality of capsules further comprises a growth medium.

10. The composition of claim 9, wherein said at least one cell comprises a combination of cells comprising at least one microalgal cell and at least one animal cell, and said growth medium is suitable for culturing said combination of cells.

11. The composition of claim 10, wherein said growth medium is devoid of a calcium -chelating amino acid.

12. The composition of claim 11, wherein said calcium-chelating amino acid is selected from the group consisting of: aspartic acid, glutamic acid, glycine, lysine, and any combination thereof.

13. The composition of any one of claims 2 to 12, wherein a water content of said hydrogel ranges between 10% to 90% w / w.

14. The composition of any one of claims 1 to 13, being an edible composition.

15. The composition of any one of claims 1 to 14, being formulated for oral administration.

16. The composition of any one of claims 1 to 15, being a meat analog composition.

17. A method for preparing the composition of any one of claims 1 to 16, the method comprising mixing a hydrogel comprising PVA, PVP, alginate, and at least one cell with an aqueous solution comprising calcium ions or a salt thereof, thereby preparing said composition.

18. The method of claim 17, wherein said mixing is drop- wise mixing.

19. The method of claim 17 or 18, further comprising a step before said mixing comprising obtaining said hydrogel, said step comprising mixing a pre-mix composition comprising PVA, PVP, and alginate with a suspension comprising at least one cell, thereby obtaining said hydrogel.

20. The method of claim 19, wherein said suspension comprises said at least one cell suspended in growth medium.

21. The method of any one of claims 17 to 20, wherein said calcium ions are in the form of CaCl2.

22. The method of any one of claims 17 to 21, wherein a concentration of said calcium ions or salt thereof in said aqueous solution ranges between 0.01% and 5% w / w.

23. The method of any one of claims 17 to 22, wherein said pre-mix composition is obtained by mixing PVA, PVP, and alginate in a w / w / w ranging between 1:1:1 and 5:1:3.

24. The method of claim 23, wherein mixing said PVA, PVP, and alginate is under heat conditions comprising subjecting said PVA, PVP, and alginate to a temperature ranging between 80 °C and 100 °C.

25. The method of any one of claims 17 to 24, further comprising subjecting said composition comprising said plurality of capsules to conditions suitable for culturing said at least one cell.

26. A method comprising feeding a subject, the method comprising orally administering an effective amount of the composition of any one of claims 1 to 16 to a subject in need thereof.

27. The method of claim 26, wherein said subject is a human subject.

28. The method of claim 26, wherein said subject is an aquatic animal.

29. The method of claim 28, wherein said aquatic animal is a fish, a crustacean, or both.

30. The method of claim 29, wherein said crustacean is selected from the group consisting of: crab, lobster, prawn, shrimp, crayfish, and any combination thereof.

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