Edible microcarrier, composition, and method for producing same

Abstract

The present invention provides an edible microcarrier comprising an edible gel and plasma.

Description

Edible microcarriers, compositions and methods for producing the same

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 515,179, filed July 24, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to edible microcarriers, compositions and methods for making the same.

[0003] Global demand for meat is accelerating due to global population growth, economic development in emerging countries, and diversification of diets. However, livestock production is costly and livestock production is not unlimited, so it is predicted that meat supply will soon be unable to keep up.

[0004] Therefore, research and development of meat substitutes has been conducted as one solution to ensure a stable supply of meat.

[0005] There are many different types of meat substitutes produced from livestock, including imitation meat, plant-based meat, cultured minced meat, cultured steak meat, etc. Of these, cultured minced meat and cultured steak meat are called cultured meat, but while cultured minced meat is a collection of isolated muscle cells and has a texture inferior to that of meat, cultured steak meat is a meat substitute that reproduces the three-dimensional structure of muscle tissue and can be felt to have the texture of meat.

[0006] To reproduce the three-dimensional structure of muscle tissue, i.e., to construct three-dimensional tissue in vitro, it is necessary to culture cells in three dimensions. However, there are many technical challenges to overcome in culturing cells in three dimensions, such as how to fuse cells in three dimensions, how to construct culture media such as serum and scaffold materials, and whether these materials can be supplied in quantities and at costs that are suitable for industrial production.

[0007] Three-dimensional cell culture basically requires cells to adhere to and grow on a scaffold, which has traditionally been made from processed or purified extracellular matrix (ECM), or materials constructed to mimic the extracellular matrix (see Patent Document 1, etc.).

[0008] Re-tabled publication 2019 / 208831

[0009] When growing and mass-culturing cells or differentiating them to create three-dimensional tissues, special materials that allow cells to adhere, proliferate, and differentiate have been used when dealing with "adhesive cells" such as myoblasts, which require an adhesive surface for maturation. However, conventional materials are often expensive or not made of edible ingredients, making their price and edibility issues in the field of cultured meat, which involves producing meat from cells. Furthermore, even materials that can be used for cell culture using edible ingredients have issues such as poor processability and low cell adhesion and proliferation capabilities.

[0010] One of the main objects of the present invention is to provide an edible microcarrier that is easy to process and inexpensive.

[0011] The present invention provides the following edible microcarriers, compositions, and methods for producing the same. [1] An edible microcarrier comprising an edible gel and plasma. [2] The edible microcarrier according to [1], which is used for producing cultured meat. [3] The edible microcarrier according to [1], wherein the edible gel comprises at least one edible gelling agent selected from the group consisting of alginate, gelatin, collagen, pectin, xanthan gum, agar, methylcellulose, guar gum, iota-carrageenan, kappa-carrageenan, glucomannan, gum arabic, gellan gum, cellulose, cellulose nanofiber, mycoprotein, locust bean gum, psyllium seed gum, and tamarind seed gum. [4] The edible microcarrier according to [1], wherein the edible gel is alginate. [5] The edible microcarrier according to [1], wherein the edible microcarrier is a molded body. [6] The edible microcarrier according to [4], wherein the molded body is selected from the group consisting of fibers, beads, and sheets. [7] The edible microcarrier according to [1], having a plasma concentration of 0.1 to 99.9625% by mass. [8] The edible microcarrier according to [1], having an alginic acid concentration of 0.0375 to 99.9% by mass. [9] A composition comprising cells adhered to the surface of the edible microcarrier according to [1].

[10] The composition according to [9], wherein the cells comprise myoblasts.

[11] The composition according to [9] or

[10] , further comprising a culture medium.

[12] The composition according to

[11] , wherein the culture medium comprises CaCl2 and / or thrombin.

[13] A method for producing cultured meat, comprising culturing the composition according to any of [9] to

[12] in the presence of a culture medium.

[0012] The edible microcarriers of the present invention are constructed from edible ingredients and are highly processable materials that allow cells to adhere to the surface, grow, and differentiate.Compared to conventional carriers that culture cells inside the carrier, they are superior in that they enable mass cell culture and three-dimensional organization using edible, inexpensive ingredients.

[0013] In the edible microcarriers of the present invention, plasma provides cell adhesion, while alginate provides high processability. Plasma derived from bovine or other sources is an inexpensive component. In this invention, plasma derived from adult bovine or other sources was used as the component imparting cell adhesion. However, a disadvantage of plasma is its poor processability. For example, plasma gels with calcium ions, but it takes several tens of minutes to several hours for it to solidify. Among these, edible gels such as calcium alginate are known to be edible, inexpensive, and highly processable. However, cells do not adhere to edible gels such as calcium alginate. While cell adhesion is possible by introducing RGD sequences into edible gels, RGD-modified edible gels are expensive. Therefore, in this invention, by mixing both plasma and edible gel, we created a material capable of cell adhesion from inexpensive, edible components. When cell culture was performed using edible microcarriers made with this gel, the cell adhesion surface provided allowed adherent cells to proliferate, and, particularly when the edible microcarriers were in the form of fibers, differentiation of the cultured cells was also possible. The edible microcarriers of the present invention have cell adhesive properties and are highly processable, allowing cells to grow and differentiate, and can be made from edible ingredients.

[0014] The composition of the present invention, in which cells are attached to the surface of edible microcarriers, is suitable for the proliferation and differentiation of a wide range of cells and is therefore suitable for cell culture. For example, when the cells are myoblasts, cultured meat can be produced by culturing the composition in the presence of a medium.

[0015] Schematic manufacturing scheme for the edible microcarrier of the present invention containing calcium alginate and plasma. This figure shows the methods for collecting, freezing, and culturing bovine primary cells shown in Example 2, culturing a mouse skeletal muscle myoblast cell line (C2C12), generating plasma from adult bovine blood and preparing plasma-alginate gels, fabricating plasma-alginate gels with planar, fibrous, or beaded structures, culturing and evaluating cells on these gels for proliferation and differentiation, and fabricating three-dimensional skeletal muscle tissue by layering plasma-alginate gel fibers. The process of fabricating a plasma-alginate planar gel is shown. The scale bar is 3 cm. The plasma-alginate planar gel was photographed in an inverted position to demonstrate gelation. The scale bar is 5 mm. The process of culturing cells on the plasma-alginate planar gel is shown. The scale bar is 1 cm. Preparatory steps for evaluating cell growth and culture on the plasma-alginate planar gel are as follows: (a) The culture insert containing the stained plasma-alginate planar gel is inverted. (b) The culture insert is cut out with a scalpel. (c) Plasma-alginate planar gel is adhered to the plate. (d) If any membrane remains on the culture insert, it is removed. Scale bar: 1 cm. Assembled microfluidic device for fiber production. Scale bar: 1 cm. Setup for producing plasma-alginate gel fibers. Scale bar: 7 cm. (a) Handle and fiber fixing device that make up the differentiation device. (b) Connecting the handle to the fiber fixing device. Scale bar: 1 cm. Conceptual diagram of fixing plasma-alginate gel fibers. Conceptual diagram of constructing cultured meat, a three-dimensional tissue, using plasma-alginate gel fibers. Bead production device. Scale bar: 1 cm. Relationship between gel flow time and fiber length. Tested using plasma-alginate gel fibers made by mixing plasma and 1 wt% alginate at a volume ratio of 1:4 (n=3, mean ± SD). The lower limit of alginate concentration for gelation is 50% plasma by volume. Gel formation did not occur at an alginate concentration of 0.0188 wt%, but gelation occurred at alginate concentrations of 0.0375 to 0.15 wt%. C2C12 cell adhesion and proliferation on gel fibers. Plasma and 1 wt% alginate were mixed at a 1:1 ratio. Mouse skeletal muscle myoblast C2C12 cells were cultured at 1.57 x 106 The cells were seeded at 1000 cells / dish and cultured in growth medium. 2 3 wt% sucrose solution) was added to the medium. On Day 3 and Day 6, C2C12 cells adhered and proliferated on the surface of the gel fiber. Confirmation of C2C12 cell survival on the gel fiber. Cell number detection by ATP assay. Proliferation of C2C12 cells - Day 3 vs. Day 6. Adhesion and proliferation of bovine primary cells on the fiber. Seeding: 1 x 10 cells in a 6-well plate. 5 cells (75% plasma volume concentration of fiber) or 1.5 × 10 5 Bovine primary cells were seeded at 1 x 10 cells (87.5% plasma volume concentration in fiber). Bovine primary cells were seeded directly after freezing. Medium: Bovine growth medium supplemented with 10 mM CaCl2 and 3 wt% sucrose (final concentration). A total of 2 ml of medium was placed in each well. Culture conditions: Cultured with shaking. The entire medium was replaced every three days. Proliferation of bovine primary cells - Plasma concentration in fiber and number of days of culture Seeding: 1 x 10 cells in a 96-well plate 4 Culture medium for seeding bovine primary cells: Bovine growth medium supplemented with 10mM CaCl2 and 3wt% sucrose (final concentration). A total of 200μl of medium was added to each well. Culture conditions: Culture with shaking. All medium was replaced every three days. Fiber: Fiber mixed with plasma at a volumetric concentration of 50% or 20%. Confirmation of differentiation through fusion of C2C12 cells on plasma-alginate gel fiber. Myotubes (green) were observed due to the fusion of mouse skeletal muscle myoblasts (C2C12 cells). Green (MF20), blue (Hoechst). Application example of edible microcarriers. Plasma-alginate gel is edible, gels instantly (highly processable), and has cell adhesive properties. Beads: Bead-like microcarriers. Sheet: Cells adhere to the sheet and are layered to form a three-dimensional organization. Fiber: Fibre-shaped microcarriers. Fibers can also be bundled or rolled to form three-dimensional structures. Application example of multi-layered edible microcarriers. Plasma alginate gel is edible, instantly gels (highly processable), and has cell adhesive properties. Applications include not only bilayer but also multilayer structures.

[0016] The concentration of edible gel in edible microcarriers is preferably 0.0375-99.9% by mass, more preferably 0.075-10% by mass, and the plasma concentration is preferably 0.1-99.9625% by mass, more preferably 2-85% by mass. Edible microcarriers may be stored dry or in a water-containing state as used. The water concentration in water-containing edible microcarriers is preferably 0-99.8625% by mass, more preferably 5-85% by mass. In addition to edible gel, plasma, and water, edible microcarriers can contain components suitable for cell culture, such as medium components, nutrients, and buffers. Examples of water that can be used include sodium phosphate, sodium hydrogen phosphate, phosphate buffers such as PBS, citrate buffers, bicarbonate buffers such as sodium bicarbonate, and HEPES buffers. When using a buffer, the amount of water can be calculated by subtracting the amount of buffer components from the amount of buffer. When using a medium as water, the amount of water can also be calculated by removing the amount of solute components other than water. Addition of thrombin to the medium can promote the gelation of plasma, and it is particularly preferable to use plasma and thrombin in combination when using core-shell type edible microcarriers.

[0017] The edible gels used in the present invention include gels used in the food industry, such as gels prepared by gelling edible gel ingredients such as alginate, gelatin, collagen, pectin (LM pectin, HM pectin), xanthan gum, agar, methylcellulose, guar gum, iota carrageenan, kappa carrageenan, glucomannan, gum arabic, gellan gum, cellulose, cellulose nanofiber, mycoprotein, locust bean gum, psyllium seed gum, and tamarind seed gum, with alginate being preferred. Regarding alginic acid, examples of edible gel ingredients include alkali metal alginates such as sodium alginate and potassium alginate, and examples of edible gels include calcium alginate and magnesium alginate, with calcium alginate being preferred. Edible gel ingredients can be used alone or in combination. The gelling conditions for edible gel ingredients are known, and gel-like edible microcarriers can be prepared under conditions appropriate for the type of edible gel. The conditions for gelling the edible gel ingredients are not particularly limited, but include, for example, (i) contacting the edible gel ingredients with an aqueous solution containing cations such as calcium and magnesium, (ii) heating the solution containing the edible gel ingredients to a temperature suitable for gelling, (iii) adjusting the pH of the solution containing the edible gel ingredients to an acidic pH, and (iv) increasing the sugar content of the solution containing the edible gel. These can be used alone or in combination as appropriate. Alternatively, an edible gel can be produced by gelling two or more edible gel ingredients, such as xanthan gum and locust bean gum. Calcium alginate is particularly preferred as an edible gel because it gels instantly upon contact with alginic acid and calcium ions, and is highly processable.

[0018] Although plasma derived from adult bovine blood is preferably used, plasma derived from animals other than adult bovine blood, such as pig, chicken, sheep, tuna, salmon, and sea bream, can also be used.

[0019] Examples of the shape of edible microcarriers include fibers, sheets, beads, etc. (Example 2). Fibers may be hollow or solid, or may be core-shell fibers (Example 1). In the case of solid fibers or core-shell fibers, cells adhere to the fiber surface, while in the case of hollow fibers, cells adhere to the outer and / or inner surface. Furthermore, sheets and beads may have a two-layer or multi-layer structure combined with other materials, or may be hollow. While any shape of fiber, sheet, or bead is suitable for the proliferation of cells adhered to the surface, the fiber shape is particularly suitable for cell differentiation. Fibers can be bundled or wound to form a three-dimensional structure. Furthermore, sheets may be stacked to form a three-dimensional organization.

[0020] When cells, such as myoblasts, are seeded into a medium containing the edible microcarriers of the present invention, the myoblasts adhere to the surface of the edible microcarriers, allowing the production of a composition for cultured meat production. Medium components include DMEM (GIBCO, etc.), EMEM (GIBCO), MEM ALPHA (GIBCO), RPMI-1640 (Roswell Park Memorial Institute 1640 medium; GIBCO), etc., with DMEM being preferred. Furthermore, additive components typically used in medium can be appropriately blended into the medium components. Examples of additive components include antibiotics, vitamins, nucleic acids, amino acids, inorganic salts, sugars such as sucrose, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, etc. DMEM contains L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, D-glucose, folic acid, nicotinamide, riboflavin, vitamin B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine, iron, and the like.

[0021] When the edible gel raw material of the present invention is gelled by calcium ions, such as alginates (sodium alginate, potassium alginate, etc.), edible microcarriers may be produced by contacting a solution containing plasma and an edible gel with a solution containing cations such as calcium ions, particularly divalent or polyvalent cations, but preferably the edible microcarriers of the present invention are produced by contacting a solution containing the edible gel raw material with a solution containing calcium ions and plasma. The plasma-containing solution may contain at least one medium component, preferably DMEM, EMEM, MEM ALPHA, RPMI-1640, etc.

[0022] The following description will be given using as an example the case where the edible microcarrier of the present invention is a fiber containing calcium alginate and plasma, but edible microcarriers of other shapes or containing other components can be easily produced by those skilled in the art by referring to the following description and known techniques.

[0023] The fibrous edible macrocarriers of the present invention can be constructed by introducing a solution containing sodium alginate and plasma into an aqueous solution containing CaCl2, gelling the solution, and then ejecting it using a microfluidic device. The injection speed is not particularly limited, but when using a microfluidic device with a bore size of approximately 50 μm to 2 mm, the solution can be ejected at approximately 10 to 500 μ / min. The introduction rate into the aqueous solution containing calcium ions is also not particularly limited, but can be, for example, approximately 1 to 10 ml / min. The resulting fibers can be drawn into a silicone tube, and the gel stretched along the longitudinal axis of the tube for storage. While maintaining a straight gel is generally difficult when storing gelled microfibers in water or a buffer solution, placing the microfiber in water or a buffer solution and then immersing the tip of a silicone tube with an inner diameter of approximately 100 μm to several mm in the aqueous medium and then suctioning the silicone tube results in the microfiber being drawn into the silicone tube from the tip, stretched along the longitudinal axis and straightened. The gel can be stored in this state, and when it is ready for use, the silicone tube containing the microfibers can be cut to the appropriate length to prepare a gel of the desired length. For storage, appropriate chemicals such as preservatives, pH adjusters, and buffers can be added to the tube as needed.

[0024] The edible microcarriers of the present invention have excellent mechanical strength. For example, when the edible microcarriers are fibers, they can be suitably used to construct three-dimensional structures such as double-stranded or triple-stranded twisted structures, woven structures, cylindrical structures, helical structures, and tubular structures. The term "structure" used herein encompasses any structure obtained by molding a single microfiber or a single sheet, or any structure that can be constructed using two or more microfibers or two or more sheets. It should be interpreted in the broadest sense, including structures such as twisted structures that appear linear in appearance and sheets that appear flat in appearance, and these terms should not be interpreted restrictively in any sense. When a three-dimensional structure is specifically intended, it may be referred to as a "three-dimensional structure." Beads usually exist singly.

[0025] Furthermore, when the edible microcarrier of the present invention is a fiber, multiple fibers can be bundled together for use. For example, a sheet-like cell culture can be prepared by preparing an edible microfiber composition in which cells are attached to the surface of the fiber, bundling multiple microfibers laterally to form a sheet of microfibers in a single row, and culturing the sheet. Alternatively, a block-like cell culture can be prepared by stacking multiple sheets of the above-mentioned material to form a block and culturing the cells.

[0026] For example, to prepare a three-dimensional structure with a woven fabric structure, a microloom with warp threads spaced approximately 1 to 5 mm may be used to weave the above-mentioned fibrous edible microcarriers as warp and / or weft threads.

[0027] The fiber shape refers to a fibrous shape with an outer diameter of, for example, approximately 10 μm to 1 mm, but the outer diameter is not particularly limited to this range. The cross-sectional shape may be various, such as circular, elliptical, or polygonal, such as rectangular or pentagonal. A circular cross-sectional shape is preferred. The length of the microfiber is not particularly limited, but is approximately several millimeters to several tens of centimeters. (Method for producing a composition containing edible microcarriers having cells attached to their surface) The composition of the present invention containing edible microcarriers having cells attached to their surface can be produced by preparing edible microcarriers of any shape, such as fibers, sheets, or beads, under suitable conditions, such as adding a calcium-containing aqueous solution to a solution containing plasma and an edible gel or heating the solution to an appropriate temperature, and then seeding cells into the solution containing the edible microcarriers to adhere the cells to the surface of the edible microcarriers.

[0028] The cells to be cultured are not particularly limited in type as long as they are capable of adhering to the surface of an edible microcarrier, and may be human cells or insect cells. Examples include primary cultured cells such as myoblasts and tendon fibroblasts, pluripotent stem cells such as ES cells and iPS cells, and adhesive cells induced to differentiate from pluripotent stem cells, adipocytes, adipose stem cells, fibroblasts, smooth muscle cells, mesenchymal stem cells, hematopoietic stem cells, vascular endothelial cells, and established cell lines such as C2C12, CHO, HEK293, HL-60, HeLa, MDCK, NIH3T3, PC12, S2, and Vero.

[0029] Among these, myoblasts proliferate and become multinucleated or differentiate to become skeletal myoblasts in the form of myotubes (myotube cells) or muscle fibers. Fiber-based edible microcarriers are more preferred because they promote myotube formation and differentiation. Examples of myoblasts include those derived from vertebrates such as mammals (e.g., monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice); birds (e.g., ostriches, chickens, ducks, and sparrows); reptiles (e.g., snakes, crocodiles, lizards, and turtles); amphibians (e.g., frogs, newts, and salamanders); and fish (e.g., salmon, tuna, sharks, sea bream, and carp). Among these, when considering the possibility of using proliferated multinucleated skeletal myoblasts for food, myoblasts derived from mammals raised for livestock farming (e.g., cows, pigs, sheep, goats, and horses) are more preferred.

[0030] The preferred concentration of cells to be cultured varies depending on the concentration of the edible microcarrier, the composition of the medium, etc., but is preferably 1 x 10 4 cells / mL ~ 1 x 10 8 cells / mL, more preferably 5 x 10 4 cells / mL ~ 1 x 10 7 cells / mL, more preferably 1 x 10 ５ cells / mL ~ 1 x 10 6 It is desirable to blend the cells at a concentration that achieves a ratio of 100 cells / mL. If the number of cells is too small, it becomes difficult to form myotubes, while if the number of cells is too large, some cells may be unable to adhere to the surface of the edible microcarrier, which may reduce the efficiency of cell proliferation.

[0031] The culture temperature is preferably about 37° C. The culture time is, for example, about several days to several months, and more preferably about several days to several weeks.

[0032] The composition comprising the fibrous edible microcarriers and cells of the present invention may be used to culture cells using a fiber fixing device capable of applying tension. (Cell Production Method) The cell production method of the present invention involves culturing cells using the edible microcarriers or composition described above to obtain cells containing the desired proliferated and differentiated cells.

[0033] The cell culture may include edible microcarriers, cells, and other components commonly used in cell culture.

[0034] Examples of other components include serum. Examples of serum include adult bovine blood serum, horse serum, newborn calf serum, goat serum, rabbit serum, pig serum, chicken serum, and fetal bovine serum. Alternatively, serum-free serum may be used depending on the culture conditions. Among these, adult bovine blood serum, horse serum, and pig serum are preferred from the viewpoints that they can be easily obtained in amounts suitable for industrial production and are edible.

[0035] An example of a method for producing cells is to mix edible microcarriers with cells, allow the cells to adhere to the surface of the edible microcarriers to obtain a composition, and then culture the composition.

[0036] In cell culture, a method in which the culture temperature is about 37° C. and the carbon dioxide concentration is 5% is exemplified, but is not limited to these.

[0037] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. Although the present invention has been fully explained with reference to examples, it is understood that various changes and modifications may be made by those skilled in the art. Therefore, as long as such changes and modifications do not deviate from the scope of the present invention, they are included in the present invention.

[0038] Example 1: Fabrication of Core-Shell Fibers 1. Solution Preparation The shell of the core-shell fiber was prepared using plasma alginate, a mixture of plasma and 3.0 wt% sodium alginate at a mass ratio of 1:1. The sodium alginate solution was prepared by dissolving 3.0 wt% sodium alginate in purified water prepared using a Milli-Q filter. The calcium chloride solution was prepared by dissolving 100 mM calcium chloride and 3 wt% sucrose in purified water prepared using a Milli-Q filter. The citric acid solution, which was used to dissolve the sodium alginate solution in case it clogged the device, was prepared by dissolving 155 mM trisodium citrate dihydrate in purified water prepared using a Milli-Q filter. The citric acid solution, calcium chloride solution, and purified water were sterilized using a 0.22 μm filter.

[0039] 2. Fiber fabrication The device used to fabricate the core-shell fiber was ozone sterilized for 30 minutes, then the inside was washed with 70% ethanol, air was then passed through it using an empty syringe to dry the ethanol, and finally, any remaining ethanol and impurities were washed away with filter-sterilized pure water.

[0040] The calcium chloride solution, pure water, and citric acid solution were all sealed in a 10 mL Terumo syringe. After aspirating the solution to ensure no bubbles remained in the syringe, the air was removed by pushing it out from the tip. The alginate solution was prepared by placing 1.0x10 2C12 cells or bovine primary cells in a 2.5 mL Hamilton syringe. 7 The cells were added to a concentration of 1000 cells / mL, and 40 μL for C2C12 cells and 30 μL for bovine primary cells were sealed in a 250 μL Hamilton syringe using the same procedure.

[0041] The nozzle for core-shell fiber fabrication was fabricated using a stereolithography 3D printer (Sonic Mini 8K, Phrozen). After manually removing the support material from the printed nozzle, it was ultrasonically cleaned twice with ethanol. The first cleaning was 30 seconds, and the second cleaning was 2 minutes. After cleaning, the ethanol was evaporated with an air gun, followed by a 30-minute UV cure. After the nozzle was thoroughly dried, a 2 μm-thick parylene C (Cookson Electronics) coating was applied to the nozzle using a vacuum deposition system (LABOCOTER PDS2010, Japan Parylene). The prepared syringe and device were connected using silicone tubing, EFTE tubing, and a Luer-lock connector. To maximize cell recovery, the tube containing the core medium was left standing for 10 minutes in a vertical orientation. A syringe pump was used to extrude the syringe, and the flow rate was controlled using PC software. Core-shell fiber fabrication was performed according to the procedure shown in Table 1. First, pure water was flowed at 4.0 mL / min. After 2 seconds, calcium chloride solution was flowed at 3.0 mL / min, pure water at 1.0 mL / min, and alginate solution was also flowed. The alginate solution was flowed at 75 μL / min for 0.3 seconds to create a closed structure at the tip of the core-shell fiber, and growth medium was flowed for 40 seconds. The growth medium flow rate was 35 μL / min. 60 seconds after the growth medium flow began, the calcium chloride solution flow rate was changed to 0 mL / min and the pure water flow rate to 4 mL / min. 0.2 seconds later, the growth medium flow rate was changed to 0 μL / min, and 0.5 seconds later, the alginate solution flow rate was changed to 0 μL / min. To rinse the inside of the device, calcium chloride solution was flowed at 0 mL / min and pure water at 4.0 mL / min for 10 seconds. Finally, an appropriate amount of citric acid was flowed to completely remove any remaining alginate solution from the device.

[0042]

[0043] The fibers were cultured for 1-2 days in a growth medium containing thrombin at 1 unit / mL. After that, the medium was changed every 1-2 days, and 150 mg / mL of 6-aminocaproic acid was added to the growth medium at a volume ratio of 1:100.

[0044] PSA: Penicillin-Streptomycin-Amphotericin PS: Penicillin-Streptomycin

[0045] Example 2 2.1 Introduction In this example, we first describe methods for collecting, freezing, and culturing bovine primary cells, and for culturing a mouse skeletal muscle myoblast cell line (C2C12). Next, we describe methods for generating plasma from adult bovine blood and preparing plasma alginate gel. We then describe methods for producing plasma alginate gels with planar, fiber, and bead structures, as well as methods for culturing and evaluating cells on these gels for proliferation and differentiation. Finally, we describe a method for creating three-dimensional skeletal muscle tissue by layering plasma alginate gel fibers.

[0046] 2.2 Cell Culture 2.2.1 Method for Collecting and Freezing Bovine Primary Cells Bovine primary cells were isolated from fresh bovine cheek meat delivered approximately 1–2 days after slaughter. First, the surface of the cheek meat was disinfected, then trimmed using a sterilized knife and cutting board. The internal meat was minced and finely chopped, and then subjected to an enzymatic reaction to separate the cells. The enzymatic reaction was stopped by adding buffer, and the suspension was thoroughly dispersed mechanically. After mechanical dispersion, impurities were removed using a cell strainer, and the resulting solution was thoroughly centrifuged to obtain a precipitate. The precipitate was suspended in bovine growth medium and then seeded onto iMatrix-coated culture dishes for preplating. After preplating, non-adherent cells were collected and seeded onto culture dishes for cell culture. The culture dishes were washed with phosphate-buffered saline (PBS) between days 1 and 2 to prevent contamination. After culturing to an appropriate cell density of about subconfluent, the obtained cells were detached with a cell detachment solution, dispensed into cell stocks, and cryopreserved.

[0047] 2.2.2 Culturing method for bovine primary cells after freezing Frozen bovine primary cell cell stock was thawed in a 37°C water bath. After thawing, the stock solution was mixed with growth medium and centrifuged at 200 G and 25°C for 5 minutes. The supernatant was removed and the pellet was suspended in bovine growth medium. The cell suspension was then seeded into a culture dish and cultured. The cells used in the experiment were passaged less than two times.

[0048] 2.2.3 Culturing method for mouse skeletal myoblast cell line (C2C12) C2C12 cells were thawed in the same manner as bovine primary cells from a thawed frozen cell stock and seeded onto 10 cm cell culture dishes using C2C12 growth medium. Cells with a passage number of 10 or less were used in the experiments. C2C12 growth medium was DMEM (high glucose) supplemented with 10% FBS and 1% penicillin-streptomycin (PS) (P4333, Sigma-Aldrich). The cells were given a complete medium change every two days.

[0049] 2.2.4 Cell Detachment Method after Freezing and Thawing Cells cultured in 10 cm cell culture dishes were detached and collected using trypsin-EDTA solution. Because the enzymatic reaction of trypsin-EDTA solution is inhibited by serum components such as FBS in the culture medium, the culture medium was aspirated completely to remove the serum components, followed by two PBS washes. After the PBS wash, 2 mL of trypsin-EDTA solution was added and the cells were detached by incubation for five minutes in an incubator at 37°C with a 5% carbon dioxide concentration. If detachment was not sufficient, physical impact was applied to the dish to detach the cells. After detachment, 8 mL of growth medium appropriate for the cell type was added to stop the action of trypsin-EDTA. After adding the medium, the cells were suspended well and the cell suspension was collected in a centrifuge tube.

[0050] 2.3 Preparation of plasma alginate solution 2.3.1 Plasma purification method The plasma used in this experiment was isolated from adult bovine blood (Shibaura Organs) within a few days of slaughter. Adult bovine blood was purchased on the day of slaughter and stored in a 4°C refrigerator. Blood was collected using a Terumo syringe and then transferred to a 7 mL collection tube (VP-P070K30, Termo) using a needle. It was then centrifuged in PRP mode using a blood separation centrifuge (Medifuge, Corefront). After centrifugation, the blood was separated into a yellow plasma portion and a red blood cell portion. The supernatant was collected, taking care not to aspirate the blood cell components. The collected plasma was sterilized using a 5 μm filter and then sterilized using a 0.22 μm filter. The sterilized plasma was dispensed into Eppendorf tubes and frozen in a -30°C freezer for at least overnight.

[0051] 2.3.2 Solution preparation Sodium alginate solution Sodium alginate (194-13321, Fujifilm Wako Pure Chemical Industries) was added to PBS at concentrations of 1 wt%, 2 wt%, 4 wt%, and 6 wt%. Food coloring was then added in the range of 1 / 1000 to 1 / 2000 of the total weight. This solution was sterilized in an autoclave to dissolve the sodium alginate, and a sodium alginate solution colored with food coloring was prepared. Sodium alginate solution is prone to contamination, so it was stored in a refrigerator at 4°C. Plasma alginate solution Plasma alginate solution was prepared immediately before use. Plasma was thawed in a 37°C water bath and pipetted thoroughly. It was then mixed with sodium alginate solution and pipetted thoroughly to prepare plasma alginate solution.

[0052] 2.4 Preparation of plasma alginate gel and cell culture 2.4.1 Reagent preparation Sodium alginate is cross-linked by cations to form a gel. In this experiment, calcium chloride solution was selected to provide these cations. Calcium chloride solution was prepared by dissolving 100 mM calcium chloride (07057-00, Kanto Chemical) in purified water (Milli-Q). Additionally, a sucrose-containing calcium chloride solution was prepared by dissolving 3 wt% sucrose (196 00015, Fujifilm Wako Pure Chemical) in 100 mM calcium chloride solution for use in preparing gel fibers. Furthermore, a sodium citrate solution was prepared by dissolving 155 mM trisodium citrate dihydrate (37150-20, Kanto Chemical) in purified water to dissolve the gel if it clogs the device during gel preparation. These solutions were sterilized using a 0.22 μm filter after preparation.

[0053] 2.4.2 Preparation of plasma-alginate planar gels Planar plasma-alginate planar gels were prepared. First, a 12-well culture insert (353103, Corning) with a pore size of 1.0 μm was placed in a 12-well multiwell plate (3513, Corning), and 300 μL of plasma-alginate solution was added to the culture insert. The plasma-alginate planar gels used in this experiment had six different plasma concentrations: 75%, 50%, 25%, 12.5%, 6.25%, and 0% (negative control). The plasma-alginate solutions used had the compositions shown in Table 3. After adding 300 μL of the plasma alginate solution prepared as shown in Figure 2-1 to the culture insert, 1 mL of calcium chloride solution was added to the outer well plate portion and the insert was left standing in an incubator overnight or longer. During this time, calcium from the well plate portion of the insert diffused through the pores into the plasma alginate inside the insert, cross-linking and gelling the plasma alginate solution (Figure 2-2), creating a plasma alginate planar gel.

[0054]

[0055] 2.4.3 Cell proliferation culture on plasma alginate planar gels The method for seeding cells on plasma alginate planar gels is described below. First, the calcium chloride solution in the well plate was removed. Next, 1.0 × 10 cells were seeded on the planar gel formed on the culture insert. 4 The cells were prepared as follows: cultured C2C12 or bovine primary cells were harvested by trypsinization and then counted using a cell counter. Based on the counting results, a cell suspension was selected and centrifuged at 200 G for 5 minutes, and the pellet was diluted to 1.0 × 10 cells / well in growth medium appropriate for each cell type. 5 The cells were suspended at a concentration of 1.0 × 10 cells / mL. The cell count was confirmed again using a cell counter, and based on the results, the cells were added to a plasma-alginate plane gel at a concentration of 1.0 × 10 cells / mL. 4 Cells were seeded into the wells. After seeding the cell suspension, growth medium and calcium chloride solution appropriate for each cell type were added to prevent gel dissolution. The total volume of cell suspension, growth medium, and calcium chloride solution in the culture insert was adjusted to 500 μL. For C2C12 cells, calcium chloride solution was added to a final concentration of 10 mM (50 μL of calcium chloride solution added), and for bovine primary cells, calcium chloride solution was added to a final concentration of 2.5 mM (12.5 μL of calcium chloride solution added). The volume of growth medium was calculated by subtracting the volume of calcium chloride solution and cell suspension from 500 μL. After adding the cell suspension, growth medium, and calcium chloride solution to the culture insert, 1.8 mL of growth medium containing calcium chloride was added to the well plate. The calcium chloride concentration of the growth medium added to the well plate was adjusted to be equal to the calcium chloride concentration in the culture insert (10 mM calcium chloride solution for C2C12 cells, 2.5 mM calcium chloride solution for bovine primary cells). Figure 2-3 shows the appearance of the plasma alginate planar gel during culture prepared using the above method.

[0056] 2.4.4 Evaluation of cell proliferation culture on plasma-alginate planar gels Live / Dead assay and cell nucleus staining Cultured cells were subjected to a live / dead assay to stain the shape of live cells and the nuclei of dead cells, and cell nucleus staining. A staining solution was prepared by mixing PBS, calcium chloride solution, Hoechst 33342, Calcein-AM (L3224A, Thermo Fisher Scientific), and Ethidium Homodimer (L3224B, Thermo Fisher Scientific) in a ratio of 2000:20:2:2:1. After all the culture medium on the plasma-alginate planar gel and the culture medium in the well plate were aspirated with an aspirator, 100 μL of the staining solution was added to the plasma-alginate planar gel and stained for 10 minutes in an incubator at 37°C with a 5% carbon dioxide concentration. After staining, the staining solution was absorbed, and 100 μL of a washing solution made by mixing PBS and calcium chloride solution in a 100:1 ratio was added to the plasma alginate planar gel and absorbed, and this process was repeated three times.

[0057] Pre-observation steps: The cells adhered to the plasma-alginate planar gel. Even if the culture insert was placed directly on an inverted fluorescence microscope, the thick plasma-alginate gel between the lens and the cells would create significant noise, making it difficult to accurately observe the cell nuclei. Therefore, we performed pre-observation steps to facilitate gel observation, as shown in Figure 2-4. First, as shown in Figure 2-4(a), the culture insert with the plasma-alginate planar gel was inverted on a 6-well culture plate (5816-006, AGC Technoglass) or a 35-mm Petri dish. Then, as shown in Figure 2-4(b), the membrane on the bottom of the culture insert was carefully cut along the wall with a scalpel and pushed out with tweezers or a small medicine spoon, so that the cell-adhered surface of the plasma-alginate planar gel was positioned on the plate surface, making it easier to observe the cells (Figure 2-4(c)). If any membrane from the culture insert remained on the plasma-alginate planar gel, it was removed as shown in Figure 2-4(d). When placing the gel, care was taken to avoid the formation of air bubbles, and if any air bubbles were formed, the gel was lifted and pushed out using a medicine spoon or similar.

[0058] Cell number quantification by image analysis Images of cell nuclei at three different points on the plasma-alginate planar gel were taken using a 10x objective lens under an inverted fluorescence microscope (IX73, OLYMPUS). The images were converted to 16-bit images using Type in the Image menu, and then binarized by appropriately setting the threshold using the Threshold function in Adjust in the Image menu. Adjacent nuclei were then separated using the Watershed function in Binary in the Process menu, and cell nuclei were counted using the Analyze Particles function in the Analyze menu. The average value of the three different images was used as the cell number for that sample. The Turkey test was used as a statistical test for cell proliferation. Calculations were performed on Google Colaboratory using the pairwise_tukeyhsd function in the statsmodels package in Python.

[0059] 2.5 Preparation of plasma-alginate gel fibers 2.5.1 Preparation of fiber-fabrication microfluidic device Plasma-alginate planar gel fibers were prepared using a fiber-fabrication microfluidic device. Figure 2-5 shows the fiber-fabrication microfluidic device. This device consisted of a circular glass tube, a glass capillary, a three-way fixation device prepared using a 3D printer, a glass slide, an ETFE (Ethylene Tetra Fluoro Ethylene) tube, and a silicone tube. First, a circular glass tube (G-1, NARISHIGE) with an outer diameter of 1 mm and an inner diameter of approximately 0.6 mm was stretched at approximately 80°C using a puller (PC-10, NARISHIGE). The tips of the circular glass tubes were then polished to a diameter of 200 ± 10 μm using sandpaper with a grit of 1000 or 2000 while checking under a microscope. Additionally, glass capillaries (8100-100, Arte Glass Associates) were prepared to accommodate the circular glass tubes and cut to the appropriate size using a diamond pen. Air pressure was applied to the tips of the circular glass tubes and capillaries using an air gun or air duster to remove any adhering glass fragments and clean the interiors. These glass tubes and capillaries were then inserted into a three-way fixation device. An ETFE tube with an outer diameter of 2 mm and an inner diameter of 1 mm was then connected to the tip of the circular glass tube not containing the glass capillary via a silicone tube. Finally, the connected parts were placed on a glass slide and glued together with an adhesive made by mixing Araldite (AR-R30, Nichiban) base and hardener in a 1:1 ratio to prevent leakage of the solution. The parts were then left at room temperature overnight or longer.

[0060] 2.5.2 Preparation of plasma-alginate gel fibers The method for preparing plasma-alginate gel fibers is described below. First, syringes containing sucrose-containing calcium chloride solution, sterile water, plasma-alginate solution, and citric acid solution were prepared. Next, these syringes were connected to the microfluidic device for fiber fabrication using silicone tubing with an outer diameter of 2 mm and an inner diameter of 1 mm, ETFE tubing, and Y- or T-connectors. The syringe containing the citric acid solution was connected with a locking connector. This setup is shown in Figure 2-6. The device and tubing were sterilized with ozone and UV for more than 30 minutes before use. The solutions were delivered using a syringe pump, and the flow rate was controlled using PC software. The calcium chloride solution and sterile water were delivered at 3600 μL / min, and the plasma-alginate solution at 360 μL / min. To fabricate the fiber, pure water was first flowed for 1 second, after which the calcium chloride solution and plasma-alginate solution were simultaneously delivered into the device. When the plasma alginate solution came into contact with the calcium chloride solution inside the device, the calcium ions instantly gelled the plasma alginate solution, and it was extruded from the tip of the device in the form of a fiber. After the desired amount of fluid flow, pure water was again run for 5 seconds to extrude the gel inside the device. The formed fiber was collected in a 25 mL centrifuge tube containing a calcium chloride solution containing sucrose. To measure the length of the plasma alginate gel fiber, the fiber was transferred to a dish and dried in an incubator overnight or longer. The entire fiber was then photographed under a microscope, and its length was analyzed using Image J.

[0061] 2.6 C2C12 cell proliferation culture using plasma-alginate gel fibers 2.6.1 Preparation of plasma-alginate gel fibers for proliferation culture evaluation Because plasma takes several hours or more to completely gel, plasma-alginate gel fibers for cell culture were prepared by transferring the fibers prepared according to 2.5.2 to a dish containing calcium chloride solution and leaving it to stand in an incubator overnight or longer to crosslink. When evaluating proliferation using the ATP assay described below, fibers in which the solution had been flowed for 5 seconds were used. In addition, when preparing fibers with different plasma concentrations, plasma-alginate solutions were prepared by mixing plasma diluted in PBS with alginate, and fibers were prepared. Plasma-free fibers were prepared without adding plasma.

[0062] 2.6.2 Cell seeding on fibers for growth culture evaluation Fibers with different plasma concentrations obtained by flowing in a microfluidic device for 5 seconds were placed in a 96-well plate with a non-cell-adhesive coating. 1 × 10 C2C12 cells collected by the method described in 2.2.4 were seeded. 4 After seeding, the cells were cultured in a growth medium containing 20 mM calcium chloride. The medium was replaced with a new growth medium containing 20 mM calcium chloride every 2–3 days.

[0063] 2.6.3 Evaluation of proliferation culture using ATP assay Cell proliferation was evaluated using an ATP assay. After culturing, the entire proliferation medium was aspirated, and the fiber was transferred to a 96-well plate for measurement. Then, 100 μL of proliferation medium containing 20 mM calcium chloride and 100 μL of the ATP assay kit (CellTiter-Glo) were added. TM 100 μL of 3D Cell Viability Assay (Promega) was added. Following the manufacturer's protocol, the mixture was stirred for 5 minutes and incubated for 25 minutes, after which the luminescence intensity was measured. Since the luminescence intensity is proportional to the number of viable cells, this value was used to evaluate cell proliferation.

[0064] 2.6.4 Live / Dead Staining of C2C12 Cells on Plasma-Alginate Gel Fibers To assess cell viability, plasma-alginate fibers cultured with C2C12 cells were stained with Live / Dead staining. After washing once with PBS containing calcium chloride, the fibers were stained with Hoechst 33342, Calcein-AM, and Ethidium Homodimer diluted in PBS. Cell viability was assessed by staining cell nuclei, live cells, and dead cells.

[0065] 2.7 Growth and culture of bovine cells using plasma-alginate gel fiber 2.7.1 Preparation of bovine cells The bovine cells collected by the method in 2.21 were thawed from a frozen state, seeded on a 10 cm dish coated with iMatrix, and grown in bovine growth medium to prepare the cells.

[0066] 2.7.2 Preparation of plasma-alginate gel fiber Plasma-alginate solution was prepared by mixing 85% plasma and 15% 6 wt% alginate solution. The plasma-alginate solution was poured into the device as described in 2.5.2 and allowed to flow for 90 seconds to create gel fibers. Unlike C2C12, the calcium chloride solution used did not contain sucrose. The fabricated fibers were stored in the calcium chloride solution overnight or longer in an incubator. A negative control fiber without plasma was prepared by using PBS instead of 85% plasma.

[0067] 2.7.3 Culture on plasma-alginate gel fiber After gently washing the obtained fiber with growth medium, 2.5 × 10 cells were cultured. 5 After seeding, the cells were cultured in growth medium containing 10 mM calcium chloride and growth factors. The culture day was designated Day 0, and the entire medium was replaced on Day 3.

[0068] 2.7.4 Evaluation of cell number After cell proliferation, the fiber was washed twice with PBS. Then, to thoroughly dissolve the fiber and more accurately quantify the change in cell number, the fiber was decomposed in a solution of alginate-degrading enzyme lyase dissolved in PBS at 0.8 mg / mL. The decomposed solution was counted using a cell counter, and the cell number was quantified.

[0069] 2.8 C2C12 Differentiation Culture Using Plasma-Alginate Gel Fibers 2.8.1 Fabrication of a Differentiation Device A device was fabricated for differentiating cell-adhered plasma-alginate gel fibers. Myoblasts fuse with each other during differentiation to form myotubes and muscle fibers. This promotes tissue contraction, which can lead to tissue shorting during culture if the fibers are left unfixed, making evaluation difficult. To prevent this, a device was prepared that applies tension to both ends of the fiber to maintain its fixation. The fiber fixation device consisted of a fiber fixation device and a handle, and the handle was designed to be connectable and detachable (Figure 2-7). The design was performed using CAD software (Fusion 360, Autodesk). After printing the model using a 3D printer (AGILISTA-3100, Keyence), the model was removed from the stage with a spatula, the support removed, and ultrasonically cleaned in a solution containing tap water, ethanol, and detergent. After ultrasonic cleaning, the device was thoroughly dried to remove internal moisture. After drying, the fiber-fixed device was coated with parylene by depositing 1.6 g of parylene C dimer (DPX C, Cookson Electronics) on its surface in a vacuum deposition system (LABCOATER PDS2010, Japan Parylene LLC) to a thickness of 2 μm. The highly biocompatible parylene coating can reduce the adverse effects on cell culture caused by components derived from the resin used to create the 3D printer model.

[0070] 2.8.2 Differentiation culture The fiber fixation device described in the previous section was used for differentiation culture of plasma alginate gel fibers. In this experiment, two types of differentiation culture methods were performed depending on the calcium chloride concentration added to the medium.

[0071] Differentiation culture in a medium containing 10 mM calcium chloride. 1 × 10 6 Cells were cultured with growth medium containing 10 mM calcium chloride and plasma-alginate gel fibers. The total volume of the solution was 2 mL, and the volume of each solution was calculated as described in 2.6.3. Plasma-alginate gel fibers were prepared as described in 2.5.2 and then crosslinked overnight in a dish containing calcium chloride solution. Growth culture was performed for 4 days as described in 2.6.2. After growth culture, a fiber fixation device with a handle was placed near the plasma-alginate gel fiber to which the cells had adhered, and the fiber was hooked onto the device using tweezers. If the fiber was prone to breaking, calcium chloride solution was added. After the fiber was hooked, the handle was removed from the device, and both ends of the fiber were fixed to the device by dropping 1 wt% alginate solution and calcium chloride, as shown in Figure 2-8. The plasma-alginate fiber was then cultured in a 35 mm Petri dish with growth medium containing 10 mM calcium chloride for 2 days while still mounted on the fiber fixation device. A differentiation medium was prepared by adding 10% horse serum (HS) (1650-122, Thermo Fisher Scientific) and 1% PS to DMEM (low glucose). After proliferation culture, differentiation culture was carried out for 9 days in differentiation medium supplemented with calcium chloride solution to a final calcium chloride concentration of 10 mM. The differentiation medium was completely replaced every two days.

[0072] Differentiation culture in a medium containing 20 mM calcium chloride. 1 × 10 cells were cultured in a 35 mm Petri dish. 6Cells were cultured for proliferation in a proliferation medium containing 20 mM sucrose-containing calcium chloride solution, plasma alginate gel fibers, and the cells were then cultured for proliferation. The volume of the solution was calculated as in 2.6.3, except that the amount of sucrose-containing calcium chloride solution added was 400 μL. Plasma alginate gel fibers were prepared as in 2.5.2 and then crosslinked overnight in a sucrose-containing calcium chloride solution. After three days of proliferation culture as in the previous section, the cells were fixed to the fixation device by adding a drop of 1 wt% alginate solution and sucrose-containing calcium chloride. After two to three days of proliferation culture in a proliferation medium containing 20 mM sucrose-containing calcium chloride solution, differentiation culture was carried out in a differentiation medium containing 20 mM sucrose-containing calcium chloride solution for four to five days.

[0073] 2.8.3 Evaluation of differentiation potential Immunostaining was performed to evaluate the degree of cell differentiation in samples such as plasma alginate gel fibers and the prepared three-dimensional tissue. Differentiation potential evaluation was performed on samples differentiated by differentiation culture in a medium containing 10 mM calcium chloride (2.7.2).

[0074] When immunostaining tissue, it is necessary to fix it with paraformaldehyde (PFA), as described below. However, alginate gel cross-linked with calcium chloride dissolves when fixed with PFA, so the gel was insolubilized by replacing it with barium. First, a solution was prepared by dissolving barium chloride in MQ at a concentration of 100 mM. Next, barium chloride solution was added to the differentiation medium to a final concentration of 20 mM, and the tissue was immersed in the culture medium and left to stand in an incubator. The incubation time for plasma alginate gel fibers was 1 hour to 1 hour 20 minutes.

[0075] After barium substitution, the samples were fixed with PFA. First, the plasma alginate gel fiber was immersed in a 4% PFA solution (at room temperature) for 40 minutes. It was then washed three times with PBS. If the washed sample was not to be stained immediately, it was placed in a dish with the PBS immersed, wrapped in parafilm, and stored in a refrigerator at 4°C.

[0076] Because the reagents used in immunostaining have large molecular weights and cannot diffuse across cell membranes as is, the samples were subjected to membrane permeabilization after PFA fixation and washing. Triton X-100 (93443, Sigma-Aldrich) was diluted with PBS to a concentration of 0.2% to prepare a Triton solution. Next, the plasma-alginate gel fiber was immersed in Triton® solution for 10 minutes. After that, the Triton solution was removed, and the fiber was immersed in PBS for 5 minutes and then blotted, a process repeated three times.

[0077] After membrane permeabilization, the samples were subjected to a BSA blocking treatment using a BSA solution prepared by dissolving bovine serum albumin (BSA) (A2153, Sigma-Aldrich) in PBS at a concentration of 1 w / v % and sterilizing it with a 0.22 μm filter. This treatment prevents the antibodies used in immunostaining from binding to antigens other than the target antigen. The blocking treatment was performed by immersing the samples in the BSA solution and leaving them to stand overnight in a refrigerator at 4°C.

[0078] After blocking with BSA, the samples were stained with α-actinin and MF20. First, an α-actinin solution was prepared by mixing BSA solution with α-actinin antibody (A7811, Sigma-Aldrich) at a ratio of 400:1, and an anti-myosin heavy chain monoclonal antibody (MF20, Developmental Studies Hybridoma Bank) was prepared. The sample was then stained by dropping the solution onto it so that the entire sample was submerged and storing it in a refrigerator at 4°C overnight. The solution was then blotted off, and the sample was washed by immersing it in PBS for 5 minutes and blotted again three times.

[0079] After primary antibody staining, the samples were immersed in a solution containing a secondary antibody, which fluorescently labeled the results and allowed visualization under a fluorescence microscope. The secondary antibodies used were Alexa Fluor 568 goat anti-mouse IgG (H+L) antibody (A11004, Thermo Fisher Scientific) for α-actinin and Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody (A11001, Thermo Fisher Scientific) for MF20. The staining solution was prepared by mixing these secondary antibodies with BSA solution at a ratio of 1:400. The plasma alginate fibers stained with the primary antibody were immersed for approximately 1 hour, and then stained with the secondary antibody. After immersion in the staining solution, the fibers were removed and immersed in PBS for 5 minutes and blotted, three times. Nuclear staining was performed on the immunostained samples. A staining solution was prepared by mixing Hoechest 33342 and PBS at a ratio of 1000:1. The samples were immersed in this solution for approximately 10 minutes and then washed three times with PBS. After immunostaining, the samples were either stained with phalloidin as described in the next section, or fixed with PFA and washed again as described in this section, and then stored in a 4°C refrigerator in the dark while still in PBS.

[0080] 2.8.4 Evaluation of Orientation Evaluation of cell orientation in plasma-alginate gel fibers was performed using phase-contrast microscope images or phalloidin-stained confocal fluorescence microscope images. Phalloidin staining was performed on immunostained samples. Phalloidin stains F-actin, a cytoskeleton. A staining solution was prepared by mixing phalloidin solution (A12379, ThermoFisher Scientific) with BSA solution at a ratio of 1:400. Plasma-alginate gel fibers stained with α-actinin were immersed in this staining solution and allowed to react at room temperature for approximately 1 hour. The reaction was carried out in the dark. After the reaction, the staining solution was absorbed, and the fibers were washed by immersing them in PBS for 5 minutes and absorbing the liquid three times.

[0081] Orientation was calculated using Fiji or Image J. The images were cropped to a square size and converted to grayscale at 32 bits. A Hanning window was then generated and multiplied with the cropped image. A fast Fourier transform was performed on the multiplied image to evaluate the angle at which the cells were oriented.

[0082] 2.8.5 Construction of cultured meat Cultured meat was constructed by bundling differentiated plasma alginate gel fibers (Figure 2-9). Two methods were used for construction.

[0083] When bundling fibers with PFA fixation, fibers differentiated in a medium containing 20 mM calcium chloride were used to produce cultivated meat. The fibers were barium-substituted for approximately 1.5 hours in a differentiation medium containing 20 mM barium chloride, and then fixed with PFA for 1 hour. After washing twice with PBS, approximately 15 fibers were removed from the device and aligned in PBS. The PBS was then absorbed, and the fibers were bonded together using a 1 wt% alginate and calcium chloride solution. After bonding, the tissue was stained with food coloring.

[0084] When bundling fibers without fixation with PFA, fibers differentiated in a medium containing 10 mM calcium chloride were used to produce cultivated meat. After removing a total of approximately 25 fibers from the device, a 35 mm Petri dish containing 0.9 mL of differentiation medium and 0.1 mL of calcium chloride solution was prepared and the fibers were aligned in the dish. The fiber alignment was adjusted appropriately while viewing under a microscope. Plasma alginate gel fibers were adhered to the aligned fibers by dripping 1 wt% alginate solution and calcium chloride solution onto the aligned fibers, creating a three-dimensional tissue. For observation, the samples were barium substituted for at least one night and then fixed with PFA for at least one night before use.

[0085] 2.9 Preparation of plasma-alginate gel beads and cell culture 2.9.1 Preparation of a bead production device To prepare plasma-alginate gel beads, a bead production device was prepared. As shown in Figure 2-10, this device was made by assembling a circular glass tube, a glass tube fixing part, and a 1.5 mL Eppendorf tube. The circular glass tube used in this experiment was a glass tube (G-1.5, NARISHIGE) with an outer diameter of 1.5 mm and an inner diameter of approximately 0.9 mm, which was stretched at approximately 90°C using a puller. The tip was then carefully polished with 1000 or 2000 grit sandpaper while observing it under a microscope so that the inner diameter at the tip was 230 to 260 μm.

[0086] 2.9.2 Preparation of plasma alginate gel beads Calcium chloride solution was poured into the Eppendorf tube of the bead preparation device, so that it was at a suitable length from the tip of the circular glass tube. The plasma alginate solution was poured into the circular glass tube, and centrifuged at 25°C in a centrifuge (Hitachi) to prepare plasma alginate gel beads.

[0087] 2.9.3 Preparation and cultivation of beads for cell proliferation When using plasma-alginate gel beads for cell culture, we used a bead production device assembled using a round glass tube and glass tube fixing part that had been sterilized with ozone UV for more than 30 minutes, and a sterilized Eppendorf tube. We also used plasma-alginate gel beads prepared by placing 15 μL of a plasma-alginate solution (a 1:1 mixture of 6 wt% alginate solution and plasma) into a round glass tube and centrifuging it at 25°C, 300 G, and 30 seconds. To promote plasma gelation, the round glass tube and glass tube fixing part were removed from the Eppendorf tube used for preparation, and the tube was then capped. The beads were then crosslinked in calcium chloride solution by leaving the tube overnight or longer at 37°C in an incubator with a 5% carbon dioxide concentration.

[0088] For culturing, a 96-well plate (3474, Corning) was used, and the total volume of growth medium, calcium chloride solution, and cell suspension solution in each well was adjusted to 200 μL. 20 μL of calcium chloride was added to give a final concentration of 10 mM. To calculate the volume of each solution, a suspension of C2C12 cells detached by trypsin treatment was prepared, and 1 × 10 cells were placed in each well. 4 The amount of cell suspension required when seeding at a density of 1 × 10 cells was calculated. Based on this result, the amount of growth medium required was calculated. After the calculation was completed, the calcium chloride solution was gently pipetted into the Eppendorf tube containing the cross-linked plasma alginate gel beads, then spun down to precipitate the plasma alginate beads, and the calcium chloride solution was carefully removed so that only the beads remained. Then, the previously calculated amount of growth medium was added to the Eppendorf tube, and the entire volume was aspirated and transferred to a 96-well plate. After that, 1 × 10 C2C12 was added to the Eppendorf tube. 4 After all procedures were completed, the cells were left to stand in an incubator at 37°C with a carbon dioxide concentration of 5% for culture.

[0089] 2.9.4 Method for assessing cell proliferation Because the number of cells adhering to plasma-alginate gel beads cultured in 96-well plates was low, we performed the PrestoBlue assay, which allows for highly sensitive measurement of small numbers of viable cells. The PrestoBlue assay uses a cell-permeable resazurin-based solution that changes color using the reducing power of viable cells, allowing cell proliferation to be quantified. The growth medium was carefully removed from the 96-well plate during incubation, and 80 μL of growth medium, 10 μL of calcium chloride solution, and PrestoBlue (A13261, Thermo Fisher Scientific) were added. The plates were incubated at 37°C in a 5% CO2 incubator for 10 minutes. The fluorescence was then measured at 590 nm using a Cytation C10 (BioTek) with excitation at 560 nm.

[0090] 2.9.5 Evaluation of Orientation The degree of cell orientation in plasma alginate gel beads was evaluated using phalloidin-stained confocal fluorescence microscope images. Plasma alginate gel beads that had been cultured for 5 days were placed in growth medium containing 20 mM barium chloride in an incubator for over 1 hour to allow barium substitution. Subsequently, as in 2.7.4, PFA fixation, membrane permeabilization, and blocking with BSA were performed. PFA fixation took approximately 50 minutes. As in 2.7.5, a phalloidin solution was prepared and stained for approximately 3 hours, after which orientation evaluation was performed using Fiji.

[0091] 2.9.6 Preparation and Evaluation of Plasma-Alginate Gel Beads for Shape Evaluation 2-6 wt% alginate was prepared and mixed with an equal amount of plasma alginate to prepare solutions. A 4% amount of fluorescent beads was added to the total volume of each mixed solution, allowing for shape observation under a fluorescence microscope. The mixed solution was then placed in a 15 μL round glass tube and centrifuged at 25°C, 300 G, and 30 seconds in a centrifuge to produce plasma-alginate gel beads. Immediately after preparation, the beads were observed under a fluorescence microscope, and their shape was evaluated by image analysis.

[0092] 2.9.7 Experiment and Method for Fibronectin Immunostaining Evaluation Plasma was serially diluted with PBS and mixed with an equal volume of 6 wt% alginate to prepare a plasma-alginate solution. The final plasma concentration was adjusted to 50-6.25% of the plasma volume. Plasma-alginate gel beads were prepared by centrifugation at 25°C, 300 G, and 30 seconds. After preparation, the beads were stored overnight or longer in a 100 mM calcium chloride solution. The calcium chloride was then removed, and the alginate beads were subjected to barium substitution with growth medium containing 20 mM barium. After sufficient barium substitution, the growth medium was removed and the beads were fixed with PFA for 1 hour. After fixation, the beads were washed with PBS three times and then immersed in a BSA solution for overnight BSA blocking. The next day, an antibody solution containing BSA and fibronectin antibody (LB-1027, LSL, CO., LTD) was prepared at a ratio of 500:1. The beads were then added to the antibody solution, with the BSA solution removed, for primary staining. After two hours, the antibody solution was removed, and the beads were washed three times by dipping in PBS for five minutes and blotting. After washing, the beads were immersed in a secondary antibody solution corresponding to the fibronectin antibody for one hour, and secondary staining was performed for one hour. The secondary antibody solution was then aspirated, and the beads were washed three times by dipping in PBS for five minutes and blotting. The beads were then observed under a confocal microscope to confirm the presence of fibronectin. The plasma gel and alginate gel were prepared as follows: Plasma gel was formed by adding 20 mM calcium chloride to plasma in a dish and allowing it to crosslink in an incubator for one day. Alginate gel was prepared by adding a mixture of 6 wt% alginate and PBS to a 100 mM calcium chloride solution and incubating for one day. The immunostaining method was the same as for the beads.

[0093] 2.9.8 Bead Degradation / Solubility Experiment and Evaluation The beads prepared in the same manner as in 2.9.6 were washed twice with PBS. Then, trypsin-EDTA or trypsin-EDTA containing 0.8 mg / mL lyase was added and reacted. The process of the beads being dissolved by trypsin-EDTA or degraded by lyase was observed using a Citation and recorded as images. The obtained images were analyzed using Fiji, and degradability and solubility were evaluated as changes in the area where fluorescence was observed.

[0094] 2.9.9 Evaluation of cell recovery rate based on bead decomposition and solubility Beads prepared in the same manner as in 2.9.3 were used for 1 × 10 3 C2C12 cells were seeded in 96-well plates. The cells were cultured in 200 μL of growth medium containing 5 mM calcium chloride. The next day, the medium was removed and the plate was washed with PBS once. Then, 50 μL of trypsin-EDTA containing 0.8 mg / mL lyase was added. The beads were allowed to dissolve for 10 to 12 minutes, and 50 μL of growth medium was added to prepare a sample with degraded and dissolved beads. A separate sample without degraded or dissolved beads was prepared by adding 100 μL of growth medium after a PBS wash. Then, 100 μL of an ATP assay kit (G9683, Promega), which quantifies the number of viable cells as luminescence, was added to both samples, and the luminescence of both samples was measured using a Citation. Additionally, a calibration curve showing the relationship between luminescence and cell number was prepared by serially diluting C2C12 cells with known cell numbers and performing an ATP assay. Based on this calibration curve, the number of cells on the beads and the number of viable cells obtained after lysis and lysis were estimated from the luminescence values ​​of the samples. The cell recovery rate was calculated by dividing the estimated number of viable cells in the unlysed sample by the estimated number of viable cells in the lysed sample.

[0095] This invention can be applied not only in the field of cultivated meat but also in other fields such as biomedical engineering. Traditionally, bovine myoblasts have been used in research and development in the cultivated meat field, and adherent cells such as C2C12 cells have been used as model cell lines in biomedical engineering. However, there have been few materials made from edible and inexpensive ingredients that allow cell adhesion, proliferation, and differentiation. The practical application of cultivated meat requires mass cell culture. Using easily processable edible gels with high cell proliferation potential, edible microcarriers in the form of beads or fibers can be created. Cultivating cells on these microcarriers increases cell yield per unit volume compared to plate culture, making them applicable to mass cell culture. Furthermore, this technology can be applied to the creation of meat-like fibrous structures for cell proliferation and differentiation, creating cultivated meat structures with a meat-like texture that can be consumed directly. The cultivated meat market is expected to reach a future market size of 200 trillion yen, and a wide range of applications are anticipated, from cell culture to three-dimensional organization. At the same time, the biomedical engineering field is actively engaged in the construction of three-dimensional tissues that mimic the shape of organs. However, gels for tissue construction are expensive. The edible microcarriers used in the present invention are inexpensive, have high cell proliferation potential, and can be easily processed into various shapes. These advantages make them useful in the fields of biomedical engineering due to their convenience and competitive price.

Claims

1. Edible microcarriers, including edible gel and plasma.

2. The edible microcarrier of claim 1, which is for use in the production of cultured meat.

3. The edible microcarrier of claim 1, wherein the edible gel comprises at least one edible gelling agent selected from the group consisting of alginate, gelatin, collagen, pectin, xanthan gum, agar, methylcellulose, guar gum, iota carrageenan, kappa carrageenan, glucomannan, gum arabic, gellan gum, cellulose, cellulose nanofibers, mycoprotein, locust bean gum, psyllium seed gum, and tamarind seed gum.

4. The edible microcarrier of claim 1, wherein the edible gel is an alginate.

5. The edible microcarrier of claim 1, wherein the edible microcarrier is a molded body.

6. The edible microcarrier of claim 4, wherein the shaped body is selected from the group consisting of fibers, beads and sheets.

7. The edible microcarrier according to claim 1, having a plasma concentration of 0.1 to 99.9625% by mass.

8. The edible microcarrier according to claim 1, wherein the alginic acid concentration is 0.0375 to 99.9% by mass.

9. A composition comprising cells attached to the surface of the edible microcarrier of claim 1.

10. The composition of claim 9, wherein the cells comprise myoblasts.

11. The composition according to claim 9 or 10, further comprising a culture medium.

12. The composition of claim 11, wherein the medium contains CaCl2 and / or thrombin.

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