Multi-Layer Film Comprising Nutrient Material for Cell Culture, Method for Preparing Same, and Use of Same

Abstract

The present application relates to a multi-layer film comprising a nutrient material for cell culture, a method for preparing same, and the use of same. The multi-layer film of the present application can be utilized for cell culture for preparing cultured meat.

Claims

1. A multi-layer film for cell culture comprising: (a) a substrate; and (b) a multi-layer structure which is formed by alternately stacking, on the substrate, a cationic material having an amine group in the side chain thereof, and an anionic material having a carboxyl group in the side chain thereof, and contains pores, and in which the cationic material and the anionic material are cross-linked to each other.

2. The multi-layer film of claim 1, further comprising nutrient materials for cell culture which are incorporated into the pores of the multi-layer structure.

3. The multi-layer film of claim 1, wherein the cationic material having an amine group in the side chain thereof is one selected from the group consisting of chitosan, gelatin, collagen, fibrinogen, silk fibroin, casein, elastin, laminin, fibronectin, polydopamine, polyethyleneimine, poly-l-lysine, poly(vinylamine)hydrochloride, and poly(amino acid).

4. The multi-layer film of claim 1, wherein the anionic material having a carboxyl group in the side chain thereof is one selected from the group consisting of hyaluronic acid, poly-l-lactic acid, alginic acid, dextran sulfate, lignin, tannic acid, chondroitin sulfate, cellulose-based polymers, fucoidan, and heparin.

5. The multi-layer film of claim 1, further comprising a capping layer formed on the multi-layer structure.

6. The multi-layer film of claim 5, wherein the capping layer is prepared by polysaccharides, and the polysaccarides comprises at least one selected from the group consisting of agarose, carageenan, fucoidan, laminaran, cellulose, hemicellulose, and lignan.

7. (canceled)

8. The multi-layer film of claim 2, wherein the nutrient material for cell culture is C-phycocyanin; growth factors selected from the group consisting of IGF-1, BMP-2, and FGF-1; amino acids; or ascorbic acid.

9. The multi-layer film of claim 1, wherein the multi-layer film has a thickness of 0.1-10 μm.

10. The multi-layer film of claim 1, wherein the multi-layer structure has a bilayer (BL), which is a stack unit comprising a layer of cationic material and a layer of anionic material, and the BL is in the range of 10-70 BLs.

11. The multi-layer film of claim 1, wherein the multi-layer structure has a porosity of 10-26% or wherein the multi-layer structure has a degree of cross-linking of 50-80%.

12. (canceled)

13. A composition for cell culture comprising: (i) the multi-layer film of claim 1; and (ii) serum components.

14. The composition of claim 13, wherein the serum component content is less than 10 wt % in the composition for cell culture.

15. An apparatus for cell culture comprising the multi-layer film of claim 1.

16. A method for preparing cultured meat comprising bringing the multi-layer film of claim 1 into contact with a cell medium containing cells.

17. The method of claim 16, wherein the cells are animal-derived muscle cells.

18. A method for reducing serum components in a cell culture medium, the method comprising bringing the multi-layer film of claim 1 into contact with the cell culture medium containing cells.

19. A method for preparing a multi-layer film for cell culture comprising the steps of: (a) preparing a first solution by adjusting the pH of a solution containing a cationic material having an amine group in the side chain thereof to a predetermined range, and preparing a second solution by adjusting the pH of a solution containing an anionic material having a carboxyl group in the side chain thereof to a predetermined range; (b) alternately contacting the first solution and the second solution on a substrate to alternately stack a layer of cationic material and a layer of anionic material to form a multi-layer structure; and (c) cross-linking cationic material and anionic material in the multi-layer structure.

20. The method of claim 19, further comprising, after step (c), (d) a step of incorporating nutrient materials for cell culture in the pores formed by the cross-linking.

21. The method of claim 19, wherein the first solution and the second solution have a pH of 3-5.

22. The method of claim 20, further comprising, after step (d), (e) a step of forming a capping layer on the multi-layer structure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0098] FIG. 1 shows a process of preparing a multi-layer film based on chitosan (CHI) and carboxymethylcellulose (CMC) by a layer-by-layer assembly (LbL assembly).

[0099] FIG. 2 is a graph showing that the thickness of a multi-layer film increases according to the number of stacking CHI/CMC bilayer in the LbL assembly.

[0100] FIG. 3 is a graph showing a decrease in the thickness of a multi-layer thin film according to exposure time in a physiological saliva environment.

[0101] FIG. 4 shows the result that there is a difference in the released amount and release behavior of C-PC according to the thickness of the multi-layer film.

[0102] FIG. 5 is an SEM observation image of a multi-layer film (CHI/CMC).sub.40 sample before cross-linking, a multi-layer film (X-linked) sample after cross-linking, and a multi-layer film (C-PC Loaded) sample after C-PC incorporation.

[0103] FIG. 6 is a graph showing the results of analyzing a change in porosity in a thin film after C-PC is incorporated inside a cross-linked multi-layer film.

[0104] FIG. 7 is a graph showing the results of analyzing the C-PC release behavior of the film before and after the formation of the capping layer in the cross-linked multi-layer film.

[0105] FIG. 8 is a photograph of a C-PC incorporated multi-layer film sample coated on an OHP film.

[0106] FIG. 9 shows improvement in the proliferation of C2C12 cells (left graph) by a multi-layer film for C-PC delivery and cell counting results (right graph) by Cell Counting Kit-8 (CCK-8) analysis.

MODE FOR CARRYING OUT THE INVENTION

[0107] Hereinafter, the present application will be described in detail with reference to examples. However, the following examples specifically illustrate the present application, and the contents of the present application are not limited by the following examples.

EXAMPLES

Example 1: Production of Polysaccharide-based Multi-layer Film

[0108] A polysaccharide-based multi-layer film was prepared by a layer-by-layer assembly (LbL assembly) using mutual attraction between materials constituting the layers (FIG. 1).

[0109] First, an aqueous solution of chitosan (CHI, Sigma-Aldrich) having a concentration of 1 mg/mL and an aqueous solution of carboxymethylcellulose (CMC, Sigma-Aldrich) having a concentration of 1 mg/mL were prepared, and the pH of the aqueous solutions was adjusted to 4 using 1 M NaOH and 1 M HCl (step 1). Chitosan and carboxymethylcellulose are present in the pH 4 aqueous solution with at least 80% thereof ionized. Subsequently, an oxygen plasma-treated substrate (Si-wafer) was prepared, supported on an aqueous solution of chitosan positively charged for 15 minutes, and then washed twice in tertiary distilled water (step 2). Subsequently, the substrate was supported in an aqueous solution of carboxymethylcellulose negatively charged for 10 minutes, and then washed twice in tertiary distilled water (step 3).

[0110] The CHI/CMC layer was formed by electrostatic attraction between chitosan and carboxymethylcellulose in the above-described steps 2 and 3, and the above-described steps 2 and 3 were repeated to prepare a CHI/CMC multi-layer film. The film prepared by repeating one cycle of the steps 2 and 3 is referred to as a 1 bilayer (BL) film, and as the number of repeating cycles increases, a film having a higher number of BLs was formed (FIG. 2). As shown in FIG. 2, an increase in the thickness of the film from 3 BLs to 19 BLs was confirmed, and a relatively linear growth pattern was observed between the number of stacks and the thickness of the multi-layer thin film, and the thickness of 19 BLs was measured to be about 1.8 μm.

Example 2: Cross-linking and C-PC Incorporation of Polysaccharide-based Multi-layer Film

[0111] In order to incorporate C-phycocyanin (C-PC), which is a nutrient material, in the prepared multi-layer film, cross-linking was introduced to the multi-layer film to impart porosity. The substrate coated with the multi-layer film was allowed to stand in a cross-linking solution containing 0.2 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5 mM of N-hydroxysuccinimide (NHS) in 0.05 M of a 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution for 30 minutes to perform primary cross-linking and then washed with 1× PBS (step 4). Subsequently, the primarily cross-linked multi-layer film was further rinsed with distilled water and left in a 2.5% glutaraldehyde (Sigma-Aldrich) solution for 45 minutes without a drying process to perform secondary cross-linking (step 5). After the cross-linked multi-layer film was added to 2 mg/mL of an aqueous C-PC (Sigma-Aldrich) solution, incubation was performed in a dark environment for 12 hours to induce incorporation of C-PC into the porous multi-layer film (step 6).

Example 3: Formation of Capping Layer of Polysaccharide-based Multi-layer Film

[0112] After the incorporation of C-PC, in order to prevent the initial burst release of C-PC, an agarose solution (0.25 w/v %) was applied on the surface of the multi-layer film to form a capping layer (step 7).

Example 4: Analysis of Multi-layer Film Characteristics

[0113] An analysis of the multi-layer characteristics was performed as follows: The change in mass per unit area was measured by measuring changes in frequency of a quartz crystal microbalance (QCM) device in the process of preparing the coating using an electrode for QCM as a coating substrate by means of the QCM, and the total mass of each material forming the multi-layer film was calculated from the measured change in mass per unit area. The thickness of the multi-layer film was measured using a profilometer, and the porosity, pore size, and volume of the multi-layer film were analyzed using a porosimeter. SEM was used to observe the roughness and porous fine shape of the multi-layer film. The release analysis of nutrient materials was performed by detecting C-PC released from the multi-layer film with photoluminescence spectroscopy (PL) to quantify the release amount over time and analyze the release behavior. C-PC released from the multi-layer film was detected through a micro reader, the release amount over time was quantified and the release behavior was analyzed.

1. Measurement of Effect of Inhibiting Decomposition of Multi-Layer Film by Cross-linking

[0114] The effect of inhibiting the decomposition of the multi-layer film by cross-linking in the multi-layer film was confirmed by measuring the thickness reduction rate. In the physiological artificial saliva environment, the degree of decomposition of the multi-layer film which was not subjected to the cross-linking was 30% or less, but the degree of decomposition of the multi-layer film which was subjected to the cross-linking was 10% or less, and thus the decomposition of the multi-layer film was reduced by the cross-linking (FIG. 3). This may mean that the effect of the multi-layer film may be maintained for a long period of time, particularly during the process of preparing cultured meat.

2. Measurement of Amount of C-PC Released According to Thickness of Multi-Layer Film

[0115] As the thickness of the multi-layer thin film increases, the portion in which C-PC can be incorporated not only increases, but also the formation of small-sized pores in the multi-layer thin film may be promoted by cross-linking, thereby increasing the final amount of C-PC released. Since the increase in the film thickness promotes the formation of small pores in the film, the rate of increase in release was measured to be higher than the rate of increase in thickness. There was a significant difference in the tendency of C-PC released according to a precursor layer/core layer/out-layer of the multi-layer film. That is, as a result of observing the release behavior of C-PC, a burst release due to the diffusion of C-PC from the surface was initially shown, followed by the release due to the C-PC diffusion from the small pores inside the film, which is smaller than the pore size of the film surface. At least 100 μm/mL of C-PC was released from the 40 BL film, and this release amount was identified as the most effective concentration for long-term proliferation and differentiation of C2C12 cells (FIG. 4).

3. Shape Analysis of Cross-linked Polysaccharide-Based Multi-Layer Film by C-PC Incorporation

[0116] Due to the chain reassembly by the cross-linking, the internal shape of the multi-layer film has an increase in porosity and exhibits rough characteristics, and thus the area in which C-PC can be incorporated inside the multi-layer film is increased, thereby increasing the loading amount. After loading C-PC on the cross-linked multi-layer film, it was observed that pores inside the film were covered by C-PC (FIG. 5). The cross-linked (CMC/CHI).sub.40 film has an apparent pore structure and has a porosity of about 18% with respect to the whole, and the total porosity was reduced to about 5.8% after loading C-PC into the pores (FIG. 6 and Table 1). A sharp decrease in the intra-particle porosity (%) indicates that C-PC has been incorporated up to the inside of the film.

TABLE-US-00001 TABLE 1 Total Total Total interparticle intraparticle porosity porosity (%) porosity (%) (%) (CMC/CHI) 8.6018 8.4423 18.0442 film (CMC/CHI) 5.5061 1.3827 5.887 film/CPC

4. Measurement of Amount of C-PC Released According to Preparation of Capping Layer on Surface of Multi-Layer Thin-Film.

[0117] It can be confirmed that the burst release of C-PC was inhibited by the capping layer, and continuous release of C-PC occurred until 96 hours (FIG. 7). Therefore, it has been suggested that a method of physically inhibiting the release using the capping layer may be effective in order to prevent the burst release that occurs initially.

5. Evaluation of Cell Proliferation of C-PC Incorporated Cross-linked Polysaccharide-Based Multi-layer Film

[0118] Cell proliferation effects were evaluated by applying the C-PC incorporated multi-layer film to cell culture. The C-PC incorporated multi-layer film sample was prepared according to the method described in Example 1, by preparing a 40 BL multi-layer film using an OHP film as a substrate, and then incorporating C-PC. It was observed that the color of the film was also blue due to the incorporation of the blue-colored C-PC (FIG. 8).

[0119] In order to confirm the effect of enhancing the proliferation of C2C12 myoblasts by the multi-layer film for C-PC delivery, C2C12 cells were cultured for 7 days in control groups and an experimental groups, respectively. C2C12 myoblasts (Passage 6) was seeded into each well of a 24 well-plate at 3.5×10.sup.3 each. The culture was performed using a culture medium in which 1% penicillin/streptomycin antibiotic (Gibco® Life Technologies) and 10% or 5% FBS (WELGENE) are contained in Dulbecco's Modified Eagle Medium (DMEM, Gibco® Life Technologies).

[0120] Control group 1 was cultured using a culture medium (FBS 10%) containing 10% FBS, Control group 2 was cultured using a culture medium (FBS 5%) containing 5% FBS, Experimental group 1 was cultured using a culture medium (Film_CPC) prepared by adding a multi-layer film, in which C-PC was incorporated, to a culture medium containing 5% FBS, Experimental group 2 was cultured using a culture medium (Capping_CPC) prepared by adding a multi-layer film, in which C-PC was incorporated and to which an agarose capping layer was applied, to a culture medium containing 5% FBS, and Experimental group 3 was cultured using a culture medium (Exo_CPC) in which C-PC was added to a 5% FBS culture medium at a concentration of 100 μm/mL. As a result of the experiments, the Film_CPC group showed a cell proliferation degree that was almost similar to that of the FBS 10% group, and it was observed that the Capping_CPC group to which the agarose capping layer was applied had a cell proliferation degree higher than the FBS 10% group (FIG. 9). Through this, it may be confirmed that FBS can be reduced by the multi-layer film of the present application. It is thought that these resulted from that the agarose, which is a saccharide component, improves the thermal stability of C-PC and at the same time inhibits the burst release from the multi-layer film.

[0121] Although the representative embodiments of the present application have been exemplarily described, the scope of the present application is not limited to the specific embodiments as described above, and a person skilled in the art can change the present application within the scope described in the claims of the present application.