Gelatin microparticles containing nutrients for cell culture, their preparation and use

Abstract

The present application relates to gelatin microparticles containing nutrients for cell culture, a method for producing the same, and uses thereof. The gelatin microparticles of the present application can be useful in cell culture for the production of cultivated meat.

Description

[Technical field]

[0001] The present application relates to gelatin microparticles containing nutrients for cell culture, their preparation method and use. [Background technology]

[0002] Cultured meat is artificial meat produced by mass culturing cells without slaughtering livestock, and is produced in an ethical and environmentally friendly manner, unlike real meat, which requires the raising and slaughtering of livestock and the use of antibiotics. The manufacturing process of cultured meat involves the following steps: First, muscle satellite cells are extracted from livestock and separated, and the separated cells are then mass-grown in a culture medium containing amino acids, vitamins, sugars, inorganic salts and minerals, and various growth factors in a bioreactor. The produced cells are then transferred to a specific support or differentiated into muscle tissue in a differentiation culture medium through self-assembly between cells.

[0003] Although many companies around the world are trying to enter the cultured meat business, the reality is that it is difficult to commercialize due to the complicated production process and high production costs. The cost of the culture medium used in the mass proliferation process of muscle cells extracted from livestock accounts for more than 70% of the total production cost of cultured meat, so research to reduce this cost is essential. The culture medium used for conventional cell proliferation contains fetal bovine serum (FBS), which contains various growth factors, cytokines, and proteins that are essential for cell growth. However, since the use of animal-derived ingredients does not conform to the original purpose of cultured meat, there is currently a need to develop technology that can eliminate or reduce FBS. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Published Patent US2012 / 0225483 [Patent Document 2] Korean Patent Publication No. 10-2019-0054425 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have made research efforts to develop a nutrient delivery platform that can replace or reduce animal-derived serum during cell culture for the production of cultured meat. As a result, they have experimentally proven that by preparing crosslinked gelatin microparticles, indenting the pores of the prepared gelatin microparticles with nutrients for cell culture, and applying the resulting gelatin microparticles to a cell culture medium, a superior cell growth effect can be achieved compared to the use of serum due to the sustained and stable release of nutrients, leading to the completion of this application.

[0006] It is therefore an object of the present application to provide gelatin microparticles for nutrient transfer to cell cultures.

[0007] Another object of the present application is to provide a composition for cell culture comprising gelatin microparticles.

[0008] Another object of the present application is to provide a method for producing cultured meat comprising culturing cells in a cell culture medium that includes gelatin microparticles.

[0009] Another object of the present application is to provide a method for reducing serum components in a cell culture medium comprising culturing cells in the cell culture medium containing gelatin microparticles.

[0010] Another object of the present application is to provide a method for preparing gelatin microparticles for nutrient transfer to cell cultures.

[0011] Another object of the present application is to provide a method for controlling the release of cell culture nutrients from gelatin microparticles. [Means for solving the problem]

[0012] In order to achieve the above objective, One aspect of the present application provides a gelatin microsphere comprising a cross-linked gelatin microsphere and a nutrient substance for cell culture embedded within the particle.

[0013] Another aspect of the present application provides a composition for cell culture comprising said gelatin microparticles.

[0014] Another aspect of the application provides a method for producing cultured meat comprising culturing cells in a cell culture medium that includes the gelatin microparticles.

[0015] Another aspect of the present application provides a method for reducing serum components in a cell culture medium comprising culturing cells in a cell culture medium containing said gelatin microparticles.

[0016] Another aspect of the present application provides a method for producing said gelatin microparticles.

[0017] Another aspect of the present application provides a method for controlling the release of cell culture nutrients from gelatin microparticles by adjusting the degree of crosslinking of the gelatin microparticles.

[0018] The present application will now be described in detail. In one aspect, the present application provides gelatin microspheres formed by crosslinking gelatin and containing nutrient substances for cell culture.

[0019] Gelatin Microparticles In the present application, gelatin microparticles may be prepared by an emulsification method, for example, a water-in-oil type emulsification method in which a solution in which gelatin is dissolved is dispersed in oil.

[0020] The gelatin microparticles produced by the emulsification method may be subjected to a drying step. The dried gelatin microparticles may be crosslinked by a crosslinking reaction.

[0021] Crosslinking of gelatin microparticles The gelatin microparticles produced may be crosslinked.

[0022] In one embodiment, cross-linking of the gelatin microparticles may be performed in the presence of a cross-linking agent, which may be genipin.

[0023] Genipin may be a compound represented by the following formula 1:

[0024] [ka]

[0025] The crosslinking may be carried out by adding the gelatin microparticles produced by the emulsification method to a crosslinking agent solution prepared by dissolving a crosslinking agent in a solvent, and then allowing the mixture to react.

[0026] The degree of cross-linking of the gelatin microparticles may be adjusted by adjusting the cross-linking reaction, for example, by adjusting the concentration of the cross-linking agent, the concentration of the microparticles, or the reaction time.

[0027] In the present application, the cross-linking degree of gelatin microparticles may vary depending on the cross-linking reaction time and may have a cross-linking degree of 10-60%, 12-58%, 14-56%, 16-54%, 18-52%, 20-50%, 22-48%, 24-46%, 26-44%, 28-42%, or 30-40%.

[0028] In this application, the cross-linking degree of gelatin microparticles may be expressed in terms of cross-linking reaction time.

[0029] In one embodiment, the degree of crosslinking of the gelatin microparticles may be a degree of crosslinking obtained when a crosslinking reaction solution containing 90% ethanol solvent, 0.5 w / v% genipin, and 50 mg / mL dried gelatin microparticles is reacted for a reaction time of 2-60 hours, 3-58 hours, 4-56 hours, 5-54 hours, 5-52 hours, 5-50 hours, 5-49 hours, 5-48 hours, or 6-48 hours, more specifically, 6-16 hours, 4-8 hours, 5-7 hours, 10-14 hours, 11-13 hours, 14-18 hours, 15-17 hours, 22-26 hours, 23-25 ​​hours, 46-40 hours, or 47-49 hours.

[0030] In the present application, since the loading amount and release characteristics of the nutrients for cell culture embedded in the gelatin microparticles vary depending on the degree of crosslinking of the gelatin microparticles, the loading amount and release characteristics of the nutrients may be adjusted by adjusting the degree of crosslinking of the microparticles.

[0031] In one embodiment, crosslinking is performed by a crosslinking agent, and the surface of the gelatin microparticles may have a higher level of crosslinking agent than the interior of the gelatin microparticles. The presence of the crosslinking agent on the surface of the gelatin microparticles may affect the loading and release rate of nutrients.

[0032] In this application, the size of the gelatin microparticles produced by the emulsification method and crosslinking may be 1-200 μm, 3-170 μm, 5-150 μm, 5-120 μm, 5-110 μm, 5-100 μm, 5-90 μm, 5-80 μm, 5-70 μm, 6-70 μm, 7-70 μm, 8-70 μm, or 10-50 μm.

[0033] Invagination of nutrients for cell culture In the present application, gelatin microparticles cross-linked with a cross-linking agent may be added to a solution in which a nutrient substance for cell culture is dissolved, to allow the nutrient substance to be invaginated into the microparticles.

[0034] The cell culture nutrient material to be embedded in the gelatin microparticles may be any nutrient material that can form a complex with gelatin through electrostatic attraction, ionic bonds, or hydrogen bonds.

[0035] The cell culture nutrient substances that can be incorporated into the gelatin microparticles can be high molecular weight substances having a molecular weight of 1 kDa-50 kDa, 3 kDa-47 kDa, 5 kDa-45 kDa, 7 kDa-43 kDa, 10 kDa-40 kDa, 13 kDa-37 kDa, 15 kDa-35 kDa, 17 kDa-33 kDa, or 20 kDa-30 kDa.

[0036] Additionally, the cell culture nutrient material that can be embedded in the gelatin microparticles may be a low molecular weight material having a molecular weight of 1 kDa or less, specifically, 0.01 kDa-1 kDa, 0.03 kDa-1 kDa, 0.05 kDa-1 kDa, 0.07 kDa-1 kDa, 0.07 kDa-0.95 kDa, 0.10 kDa-0.9 kDa, 0.15 kDa-0.90 kDa, 0.2 kDa-0.85 kDa, 0.25 kDa-0.8 kDa, or 0.3 kDa-0.75 kDa.

[0037] Nutrients for cell culture that can be incorporated into gelatin microparticles may include hormones, growth factors, amino acids, or vitamins required for cell culture, such as, but not limited to, C-phycocyanin, albumin, a growth factor selected from the group consisting of IGF-1, BMP-2, FGF-1, and VEGF, amino acids, or ascorbic acid.

[0038] The gelatin microparticles of the present application described above may be used for cell culture.

[0039] In one embodiment, the cells may be muscle cells of animal origin.

[0040] In another aspect, the present application provides a composition for cell culture comprising: (i) gelatin microparticles formed by crosslinking gelatin and having a nutrient substance for cell culture embedded therein; and (ii) a serum component.

[0041] In the cell culture composition of the present application, the crosslinking may be performed by genipin.

[0042] In the cell culture composition of the present application, the nutrient substance for cell culture may be C-phycocyanin, albumin; a growth factor selected from the group consisting of IGF-1, BMP-2, FGF-1, and VEGF; an amino acid; or ascorbic acid.

[0043] In the cell culture composition of the present application, the cross-linking degree of the gelatin microparticles may be 10-60%.

[0044] In the cell culture composition of the present application, the crosslinking degree of the gelatin microparticles may be a crosslinking degree obtained when a crosslinking reaction solution containing 90% ethanol solvent, 0.5 w / v% genipin, and 50 mg / mL dried gelatin microparticles is reacted for a reaction time of 2-60 hours, 3-58 hours, 4-56 hours, 5-54 hours, 5-52 hours, 5-50 hours, 5-49 hours, 5-48 hours, or 6-48 hours, more specifically, 6-16 hours, 4-8 hours, 5-7 hours, 10-14 hours, 11-13 hours, 14-18 hours, 15-17 hours, 22-26 hours, 23-25 ​​hours, 46-40 hours, or 47-49 hours.

[0045] In the present application, the composition for cell culture may further include other components that may be included in a cell culture medium in addition to the gelatin microparticles containing the nutrients for cell culture. The additional components may be, for example, but are not limited to, a carbon source (e.g., glucose), inorganic salts, amino acids, vitamins, lipids, serum, serum-derived components, or a combination of one or more of these.

[0046] The cell culture composition of the present application may contain reduced amounts of serum components.

[0047] In one embodiment, the content of the serum component may be less than 10% by weight of the cell culture composition, more specifically less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, or less than 4% by weight.

[0048] The lower limit of the content of the serum component in the cell culture medium may be 0.001% by weight, 0.01% by weight, or 0.1% by weight.

[0049] In one embodiment, the serum component may be fetal bovine serum, and the content of the fetal bovine serum may be less than 10% by weight of the composition for cell culture, more specifically less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, or less than 4% by weight, and the lower limit of the content of the fetal bovine serum may be 0.001% by weight, 0.01% by weight, or 0.1% by weight of the composition for cell culture.

[0050] Even if the composition of the present application contains a serum component at a content lower than 10% by weight, which is the content of serum components (e.g., fetal bovine serum) contained in a typical cell culture medium, when it contains the above-mentioned gelatin microparticles of the present application, it is possible to achieve the same or higher levels of cell proliferation, more specifically, muscle cell proliferation.

[0051] The cell culture composition of the present application may further comprise a serum-free medium, for example, Dulbecco's Modified Eagle's Medium (DMEM), Eagle's MEM (Minimal Essential Medium), RPMI 1640, Ham's F12, or SF 12 serum-free medium, or may further comprise some of the components of these media.

[0052] In the cell culture composition of the present application, the cells may be cells for the production of cultivated meat.

[0053] In the cell culture composition of the present application, the cells for the production of cultured meat may be muscle cells derived from an animal, and the muscle cells may include myoblasts, muscle satellite cells, myotube cells, myotubes, myofibers, or mature myofibers.

[0054] According to yet another aspect, the application provides a method for producing cultured meat that includes culturing cells in a cell culture medium containing gelatin microparticles formed by cross-linking gelatin and invaginated with a nutrient substance for cell culture.

[0055] In the method of producing cultured meat of the present application, the crosslinking may be crosslinked with genipin.

[0056] In the method of producing cultured meat of the present application, the nutrient substance for cell culture may be C-phycocyanin, albumin; a growth factor selected from the group consisting of IGF-1, BMP-2, FGF-1, and VEGF; an amino acid; or ascorbic acid.

[0057] In the cultured meat production method of the present application, the cross-linking degree of the gelatin microparticles may be 10-60%.

[0058] In the method for producing cultured meat of the present application, the degree of crosslinking of the gelatin microparticles may be the degree of crosslinking obtained when a crosslinking reaction solution containing a 90% ethanol solvent, genipin at a concentration of 0.5 w / v%, and dried gelatin microparticles at a concentration of 50 mg / mL is reacted for a reaction time of 2-60 hours, 3-58 hours, 4-56 hours, 5-54 hours, 5-52 hours, 5-50 hours, 5-49 hours, 5-48 hours, or 6-48 hours, and more specifically, the degree of crosslinking obtained when the crosslinking reaction solution is reacted for 6-16 hours, 4-8 hours, 5-7 hours, 10-14 hours, 11-13 hours, 14-18 hours, 15-17 hours, 22-26 hours, 23-25 ​​hours, 46-40 hours, or 47-49 hours.

[0059] In the method of producing cultured meat of the present application, the cells may be muscle cells of animal origin, and the muscle cells may comprise myoblasts, muscle satellite cells, myotube cells, myotubes, myofibers, or mature myofibers.

[0060] According to yet another aspect, the present application provides a method for reducing serum components in a cell culture medium, comprising culturing cells in a cell culture medium containing gelatin microparticles formed by crosslinking gelatin and having a nutrient substance for cell culture embedded therein.

[0061] In the method for reducing serum components of the present application, the cross-linking may be performed by genipin.

[0062] In the method for reducing serum components of the present application, the nutrient substance for cell culture may be C-phycocyanin, albumin; a growth factor selected from the group consisting of IGF-1, BMP-2, FGF-1, and VEGF; an amino acid; or ascorbic acid.

[0063] In the method for reducing serum components of the present application, the cross-linking degree of the gelatin microparticles may be 10-60%.

[0064] In the method for reducing serum components of the present application, the degree of crosslinking of the gelatin microparticles may be a degree of crosslinking obtained when a crosslinking reaction solution containing 90% ethanol solvent, 0.5 w / v% genipin, and 50 mg / mL dried gelatin microparticles is reacted for a reaction time of 2-60 hours, 3-58 hours, 4-56 hours, 5-54 hours, 5-52 hours, 5-50 hours, 5-49 hours, 5-48 hours, or 6-48 hours, more specifically, 6-16 hours, 4-8 hours, 5-7 hours, 10-14 hours, 11-13 hours, 14-18 hours, 15-17 hours, 22-26 hours, 23-25 ​​hours, 46-40 hours, or 47-49 hours.

[0065] According to yet another aspect, the present application provides a method for preparing gelatin microparticles for cell culture, comprising the steps of: (a) preparing gelatin microparticles by an emulsification method; (b) reacting and cross-linking the prepared gelatin microparticles in the presence of a cross-linking agent; and (d) invaginating the cross-linked gelatin microparticles with nutrients for cell culture.

[0066] In the method for producing gelatin microparticles for cell culture of the present application, the crosslinker may be genipin.

[0067] According to yet another aspect, the present application provides a method for controlling the release of nutrients for cell culture, comprising: (a) a step of preparing gelatin microparticles by an emulsification method; (b) a step of reacting and crosslinking the prepared gelatin microparticles in the presence of a crosslinking agent; and (c) a step of encapsulating nutrients for cell culture in the crosslinked gelatin microparticles, wherein the release of the nutrients for cell culture from the gelatin microparticles is controlled by controlling the degree of crosslinking in the step (b).

[0068] In the above-mentioned other aspects of the present application, namely, the composition for cell culture, the method for producing cultured meat, the method for reducing serum components in a cell culture medium, the method for producing gelatin microparticles, and the method for controlling the release of nutrients for cell culture, the contents related to gelatin microparticles are the same as those described in the gelatin microparticles, which is one aspect of the present application, and therefore will not be described again. Effect of the Invention

[0069] The gelatin microparticles of the present application can stably deliver nutrients necessary for cell culture.

[0070] The gelatin microparticles of the present application allow the use of nutritional substances that can replace animal-derived serum, thus resolving the problems of ethics, high cost, and disease induction caused by the use of serum.

[0071] The degree of cross-linking of the gelatin microparticles of the present application can be easily adjusted, allowing control of the amount of nutritional substance entrapment, release amount, and release rate.

[0072] This application uses gelatin and crosslinking agents that are derived from living organisms, edible, and safe, making it suitable for the production of artificial cultured meat.

[0073] However, the effects of the present application are not limited to the effects described above, and other effects not mentioned herein will be clearly understood by those skilled in the art from the following description. [Brief description of the drawings]

[0074] [Figure 1] 1 shows optical microscope images showing the morphology and size of gelatin microspheres (GMS) depending on the degree of crosslinking. [Figure 2a] 1 shows the mechanism by which gelatin particles are cross-linked by Genipin. [Figure 2b]1 is a graph showing changes in Raman spectroscopy due to the formation of heterocyclic amines and aromatic amides in response to crosslinking with genipin. [Figure 2c] 1 shows scanning electron microscope (SEM) images of the change in morphology of GMS depending on the crosslinking time. [Diagram 3] The degradation rate of GMS samples crosslinked for 12 and 24 hours was measured. [Figure 4a] The graph on the right shows the total accumulated release amount of C-PC in the GMS samples that were crosslinked for 6 hours, 16 hours, 24 hours, and 48 hours, and the graph on the left shows the release behavior of C-PC. [Figure 4b] 1 is a graph analyzing the release behavior of IGF-1 from GMS crosslinked for 12 hours (12h_GMS). [Figure 4c] This is a graph showing the analysis of the amount of IGF-1 released from GMS samples that were crosslinked for bare, 6 hours, 16 hours, 24 hours, and 48 hours. [Diagram 5] FIG. 1 is a schematic diagram of an experiment in which gelatin microparticles containing nutrients are applied to cell culture to evaluate the cell proliferation effect. [Figure 6] This shows the results of evaluating the proliferation of C2C12 cells by C-PC-involved GMS. [Figure 7] This shows the results of evaluating the proliferation of C2C12 cells by IGF-1-incorporated GMS. [Figure 8] 1 is a graph showing the results of analyzing the release behavior of hyaluronic acid (HA) and FITC released from GMS in which HA or FITC has been invaginated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The present application will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present application, and the content of the present application is not limited to the following examples. EXAMPLES

[0076] Example 1: Preparation of gelatin microparticles (GMS) and nutrient inclusion complexes Gelatin microparticles were prepared by the water / oil emulsion method. First, 11.1 wt% gelatin (Gelatin, Sigma-Aldrich) solution preheated to 45°C was added dropwise to olive oil (Sigma-Aldrich) and stirred at 500 rpm for 10 minutes. After continuous stirring, the mixture was slowly cooled to 4°C to gel, and after 30 minutes, 100 ml of chilled acetone was added, mixed and stirred. After 1 hour, 100 ml of chilled acetone was added and stirred at 1000 rpm for 5 minutes. The produced microparticles were collected by filtration, washed with acetone to remove residual oil, and then air-dried. A 0.5 w / v% genipin (genipin, Sigma-Aldrich) crosslinking solution was prepared using 90% ethanol (Sigma-Aldrich) as a solvent, and the dried microparticles (50 mg / mL) were added to crosslink the mixture. The degree of crosslinking was adjusted by setting the crosslinking time to 6, 12, 16, 24, or 48 hours. The crosslinked gelatin microparticles (GMS) were washed with ethanol three times and then dried in an oven at 80°C for 3 hours. The dried GMS was placed in an IGF-1 solution (0.5μg / mL) or C-PC (C-phycocyanin) solution (2mg / mL) at a concentration of 10mg / mL and reacted for 12 hours to allow the invagination of nutrients.

[0077] Example 2: Evaluation experiment of the nutrient delivery efficacy of gelatin microparticles The gelatin microparticles containing nutrients were applied to a cell culture environment to evaluate the efficacy of nutrient delivery. C2C12 myoblasts (Passage 6) were cultured in each well of a 24-well plate at 3.5 x 10 3Cells were seeded one by one. For cell culture, Dulbecco's Modified Eagle Medium (DMEM, Gibco® Life Technologies) containing 1% penicillin / streptomycin antibiotics (Gibco® Life Technologies) and 10% or 5% FBS (WELGENE) was used. In the experiment of cell proliferation improvement for GMS invaginated with C-PC, control group 1 used a culture medium containing 10% FBS, control group 2 used a culture medium containing 5% FBS, experimental group 1 used a culture medium containing GMS invaginated with C-PC for 6 hours (GMS_6), experimental group 2 used a culture medium containing GMS invaginated with C-PC for 16 hours (GMS_16), experimental group 3 used a culture medium containing GMS invaginated with C-PC for 24 hours (GMS_24), and experimental group 4 used a culture medium containing GMS invaginated with C-PC for 48 hours (GMS_48). The cell proliferation enhancement experiment for IGF-1-incorporated GMS was also carried out in the same manner as the experiment for C-PC-incorporated GMS, with a control group and an experimental group.

[0078] Example 3: Characterization method of gelatin microparticles The characteristics of GMS were analyzed as follows: SEM was used to observe the microscopic morphology of roughness and porosity of GMS; Optical microscope (OM) was used to observe the color, morphology, and swelling ratio of GMS due to crosslinking; Photoluminescence spectroscopy (PL) and a microreader were used to detect C-PC released from GMS, quantify the amount released over time, and analyze the release behavior; ELISA was used to quantify growth factor (IGF-1) released from GMS.

[0079] Example 4: Analysis of the morphology, size and degradation rate of gelatin microparticles according to the degree of cross-linking The size of the produced GMS was observed to be 10-50 μm, and as the crosslinking time increased, the blue color of the GMS became darker and aggregation between GMS was observed (Figure 1). As the crosslinking progressed through the Raman spectrum data, the peak at 1550 cm was observed due to the formation of heterocyclic amines and aromatic amides. -1 A new peak forms near 1680 cm -1 Nearby amide 2 The occurrence of a peak shift confirmed that crosslinking had proceeded successfully (Figures 2a and 2b). In addition, the change in GMS morphology depending on the crosslinking time was observed (Figure 2c).

[0080] The decomposition rate of each GMS was evaluated by measuring the weight loss rate of GMS for 12 days in a cell culture environment. GMS cross-linked for 12 hours (12h_GMS) lost a significant amount of weight within 48 hours, with more than 90% decomposing over the final 12 days. Considering that a total of 12 days of cell culture is required for the production of cultured meat, with 7 days required for mass cell proliferation and 5 days required for cell differentiation, the degree of decomposition of 12h_GMS was suitable for the production of cultured meat. GMS cross-linked for 24 hours (24h_GMS) lost a significant amount of weight within 96 hours, with approximately 50% decomposing over the final 12 days. About half of GMS was decomposed over the total 12-day cell proliferation and differentiation period, indicating that nutrients remain in GMS even after 12 days (Figure 3).

[0081] The experimental results demonstrated that the decomposition rate of GMS can be controlled according to the degree of crosslinking, and therefore the amount and rate of release of the incorporated nutrients can also be controlled. Since the decomposition rate of GMS is closely related to the release of nutrients, it is expected that as the degree of crosslinking increases, the decomposition rate will decrease and the release of nutrients will occur more slowly and continuously.

[0082] Example 5: Analysis of inclusion and release of nutritional substances in gelatin microparticles (GMS) according to the degree of cross-linking 1) Analysis of C-PC loading efficiency in GMS depending on the degree of crosslinking The loading amount of C-PC was analyzed according to the degree of crosslinking of GMS. According to the experimental results shown in Table 1 below, the loading amount and efficiency of C-PC were in the order of 6h>Bare>24h≧16h>48h. As the degree of crosslinking increased, the density of GMS increased and the swelling ratio decreased, which reduced the C-PC loading space. The loading efficiency measurement results showed that the loading efficiency of the bare and 6h samples was over 30%, while the remaining samples were 17-20%, indicating that crosslinking of the internal structure of GMS occurred rapidly between 6h and 16h. The difference in loading efficiency between 6h and 16h was 16% despite only a 10-hour difference, but the difference in loading efficiency between the 16h and 48h samples was only 3.3% despite a 32-hour difference, so there was no significant difference. This is presumed to be the result of genipin molecules accumulating in the outer layer of GMS and C-PC binding to the outer part. The loading efficiency was higher in 6h GMS than in bare. A low degree of cross-linking increased the viscoelasticity of GMS, making it easier to load C-PC and minimizing the decomposition of GMS during the loading process of C-PC.

[0083] [Table 1]

[0084] 2) Analysis of the release behavior of C-PC from GMS GMS samples were prepared in 4 mg portions after crosslinking for 6, 16, 24, and 48 hours, and C-PC was invaginated as a nutrient. The release behavior was analyzed for 7 days. Crosslinking gives the inside of GMS a denser microstructure, which is expected to affect the invagination and release of 30 kDa C-PC. The first stage of C-PC release from GMS is when a large amount of material is released quickly due to initial diffusion caused by a concentration gradient. The second stage of release is when the invaginated material is released gradually and continuously due to the decomposition of GMS.

[0085] As a result of the experiment, the release amount (μg) was in the order of Bare>16h≧6h>24h>48h. Bare GMS was saturated after 24h, with C-PC diffusion and GMS decomposition occurring. 6h_GMS had a slower C-PC release rate than bare due to the reduced internal diffusion space caused by slight crosslinking, but 95% was released after 48h and was almost saturated by 96h. In the case of 16h-48h GMS, there was no significant difference in the loading amount between samples, and the release behavior was also almost similar, but the release amount showed a clear difference and was higher in the order of 48h<24h<16h. When the crosslinking time was longer than 24 hours, not only was the inner structure of GMS crosslinked, but genipin began to accumulate in the outer layer, strengthening the interaction between the GMS surface and C-PC, or between C-PC and C-PC, thereby slowing down the release rate (Figure 4a).

[0086] 3) Analysis of IGF-1 release behavior in GMS The amount and rate of IGF-1 release were evaluated by quantitative ELISA analysis. In the first 24 hours, rapid release occurred due to diffusion caused by the concentration gradient inside / outside GMS, and after 24 hours, IGF-1 was gradually released due to the slow decomposition of GMS. IGF-1 release continued even after 72 hours, with the maximum release amount being 256ng / mL, which met the effective concentration for improving the proliferation of C2C12 cells (Figure 4b). The amount of IGF-1 release (ng / mL) according to the degree of crosslinking was as follows: Bare<6h<24h<48h≒168h (Figure 4c).

[0087] Example 7: Evaluation of the efficacy of nutrient-loaded gelatin microparticles on cell proliferation The nutrient-loaded gelatin microparticles were applied to cell culture to evaluate their effect on cell proliferation (Figure 5).

[0088] Cell culture media is usually supplemented with 10% FBS (Fetal Bovine Serum). In this example, in order to evaluate the effect of FBS reduction through the delivery of nutrients, a conventional medium (10% FBS), a medium with reduced FBS (5% FBS), and a medium with GMS containing C-PC with different crosslinking degrees added to a 5% FBS medium were prepared, and cell proliferation was evaluated. The experimental results showed that the most significant effect was observed when C-PC was released from GMS crosslinked for 16 hours (GMS_16), and despite the use of a medium with reduced FBS, the cell number increased by 1.6 times compared to the conventional medium (10% FBS). In addition to GMS_16, the cell number and density in all groups supplemented with GMS were higher than those in the FBS-reduced medium (5% FBS) (Figure 6).

[0089] Using the same method as the method for evaluating the proliferation of C2C12 cells using the C-PC-involved GMS sample, we prepared conventional medium (10% FBS), FBS-reduced medium (5% FBS), and medium containing IGF-1-involved GMS manufactured with different degrees of cross-linking in 5% FBS medium, and evaluated cell proliferation. The experimental results showed that the cell density, cell number, and proliferation rate in the GMS-infused group were significantly improved compared to the FBS 5% group, and in particular, the 6h_GMS, 24h_GMS, and 48h_GMS groups had 1.4 times more cell numbers than the FBS 10% group (Figure 7).

[0090] From the above experimental results, it was confirmed that the amount of FBS used can be reduced by using GMS containing nutrients.

[0091] Example 8: Analysis of the release behavior of gelatin microparticles encapsulated with polymers or small molecules GMS allows for the inclusion of a variety of substances due to its swelling properties and charged amino acid chains. In fact, low molecular weight substances other than C-PC and IGF-1 and various types of growth factors may be used in the production of cultured meat. In addition to IGF-1 and C-PC as substances to be invaginated into GMS, hyaluronic acid (HA) as a polymeric substance and FITC (fluorescein isothiocyanate) as a low molecular weight substance were selected as model substances. The release behavior of model substances invaginated for 72 hours in C2C12 cell culture medium was analyzed for GMS samples invaginated with HA or FITC.

[0092] HA was mixed with rhodamine B dye-conjugated HA in a ratio of 1:9, and the release behavior was analyzed through photoluminescence (PL) measurement. HA has a relatively high negative charge density in neutral solution conditions, so it is possible that a strong electrostatic attraction occurs between HA and positively charged GMS. As a result, the GMS / HA interaction was greater than the diffusion rate, and the area affected by diffusion decreased, and HA was gradually released due to GMS decomposition. FITC is a low molecular weight substance model, unlike IGF-1, C-PC, and HA, and its release behavior was analyzed through PL fluorescence analysis. In the case of FITC, the molecular weight is much smaller than GMS, so the fast release occurred depending on the diffusion force rather than the interaction with GMS (Figure 8).

[0093] These results demonstrate that nutrients with many functional groups or molecular weights required for the production of cultured meat can be incorporated into edible GMS and efficiently released.

[0094] The above describes exemplary embodiments of the present application, but the scope of the present application is not limited to the specific embodiments described above, and a person having ordinary knowledge in the relevant field can make appropriate modifications within the scope of the claims of the present application.

Claims

1. A composition for muscle cell culture comprising: crosslinked gelatin microparticles formed by crosslinking gelatin with genipin, the crosslinked gelatin microparticles having a size of 10-50 μm and C-phycocyanin embedded therein; and a serum component, the gelatin microparticles being obtained by subjecting a crosslinking reaction solution containing genipin and dried gelatin microparticles in an ethanol solvent to a crosslinking reaction for 14-18 hours, and the serum component being contained in the composition for muscle cell culture in an amount of less than 10% by weight.

2. The composition for muscle cell culture according to claim 1, wherein the gelatin microparticles are obtained by reacting a crosslinking reaction solution containing 0.5 w / v% genipin and 50 mg / mL dried gelatin microparticles in an ethanol solvent for 14-18 hours.

3. The composition for muscle cell culture according to claim 1 , wherein the serum component is contained in the composition for muscle cell culture in an amount of 0.001% by weight to less than 9% by weight.

4. The composition for muscle cell culture according to claim 1 , wherein the muscle cells are myoblasts, satellite cells, myotubes, myofibers, or mature muscle fibers.

5. A method for culturing muscle cells for producing cultured meat, comprising culturing muscle cells in a composition for culturing muscle cells comprising crosslinked gelatin microparticles formed by crosslinking gelatin with genipin, the crosslinked gelatin microparticles having a size of 10-50 μm and containing C-phycocyanin, and a serum component, the gelatin microparticles being obtained by crosslinking a crosslinking reaction solution containing genipin and dried gelatin microparticles in an ethanol solvent for 14-18 hours, and the serum component being contained in the composition for culturing muscle cells in an amount of less than 10% by weight.

6. The method for culturing muscle cells according to claim 5, wherein the crosslinking degree of the gelatin microparticles is obtained by reacting a crosslinking reaction solution containing 0.5 w / v% genipin and 50 mg / mL dried gelatin microparticles in an ethanol solvent for 14-18 hours.

7. The method for culturing muscle cells according to claim 5, wherein the serum component is contained in the composition for culturing muscle cells in an amount of 0.001% by weight to less than 9% by weight.

8. The method for culturing muscle cells according to claim 5 , wherein the muscle cells are myoblasts, muscle satellite cells, myotubes, myofibers, or mature muscle fibers.

9. A method for reducing serum components in a muscle cell culture medium, comprising culturing muscle cells in the composition for muscle cell culture, the composition comprising crosslinked gelatin microparticles formed by crosslinking gelatin with genipin, the crosslinked gelatin microparticles having a size of 10-50 μm and containing C-phycocyanin, the gelatin microparticles being obtained by subjecting a crosslinking reaction solution containing genipin and dried gelatin microparticles in an ethanol solvent to a crosslinking reaction for 14-18 hours.

10. The method for reducing serum components in a muscle cell culture medium according to claim 9, wherein the gelatin microparticles have a degree of crosslinking obtained by reacting a crosslinking reaction solution containing 0.5 w / v % genipin and 50 mg / mL dried gelatin microparticles in an ethanol solvent for 14-18 hours.

11. The method for reducing serum components in a muscle cell culture medium according to claim 9, wherein the serum components are contained in a content of 0.001% by weight to less than 9% by weight in the composition for muscle cell culture.

12. The method for reducing serum components in a muscle cell culture medium according to claim 9, wherein the muscle cells are myoblasts, muscle satellite cells, myotubes, myofibers, or mature muscle fibers.

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