Method for producing cell culture supernatant liquid, cell culture supernatant liquid, and use thereof

Abstract

The present invention addresses the problem of preparing a culture supernatant solution having better quality than conventional two-dimensional culture methods. Provided as a means for solving the problem is a method for producing a cell culture supernatant liquid, the method including a step for obtaining a cell culture supernatant liquid by adhesively culturing adhesive cells on fibers.

Description

Method for producing cell culture supernatant, cell culture supernatant, and use thereof

[0001] This invention relates to a method for producing cell culture supernatant, cell culture supernatant, and the use thereof.

[0002] Stem cell culture supernatant is the liquid secreted during stem cell culture and was traditionally discarded. However, in recent years, it has become clear that the supernatant is rich in many growth factors, cytokines, and exosomes, attracting attention in the fields of regenerative medicine, cosmetics, and healthcare. In particular, unlike cell-based therapies, the supernatant does not contain cells, so there is no risk of immune rejection or tumor formation, and transplantation procedures are unnecessary, which is expected to reduce costs, making it a promising alternative technology to stem cell therapy. There are already examples of its application in tissue repair, chronic wounds, nerve damage, arthritis, and heart disease, and its effectiveness has been confirmed.

[0003] However, the composition of the culture supernatant can vary depending on the culture conditions, and several challenges remain in its quality control and clinical application. Specifically, the following issues can be cited: (1) Variation in the composition of the culture supernatant: Because the composition of the cell culture supernatant may differ depending on the culture conditions and the type of stem cells used, the establishment of a standardized manufacturing process and quality evaluation is required. (2) Unclear aspects of the components: The composition and mechanism of action of physiologically active substances and secreted factors contained in the culture supernatant have not been fully elucidated, and improving reliability in clinical application is a challenge. (3) Limitations of the culture environment: Conventional 2D cell culture methods cannot reproduce the microenvironment in vivo, and the amount and type of secreted factors in the obtained cell culture supernatant may differ from those actually found in the body.

[0004] Patent No. 7556627

[0005] To solve the above problems, standardization of the manufacturing process is necessary. Furthermore, it is important to establish methods for labeling and evaluating substances in the culture supernatant. This invention aims to establish optimal culture conditions that mimic the in vivo microenvironment in order to improve cell function in the culture environment.

[0006] To date, cell culture techniques using devices such as sponges and microparticles have been developed to mimic the microenvironment within living organisms and have been applied to mesenchymal stem cells. However, even with such culture techniques, cells adhere to the gaps in the sponge or the surface of the microparticles, so this simply increases the surface area of ​​cell adhesion and still differs from the true in vivo environment. In particular, these culture techniques are not suitable for cells that require close intercellular communication, such as cardiomyocytes and myoblasts. On the other hand, there are tools that create cell aggregates (spheres) using microwells and bioreactors, but even with culture using these tools, problems remain such as spheres easily fusing together, having non-uniform sizes, and being difficult to manipulate.

[0007] The inventors have utilized micro- and nano-fabrication technologies to develop devices for maintaining undifferentiated cells, transplantation, and drug response for various cell types (iPS cells, cardiomyocytes, nerve cells, etc.), including iPS-derived cardiomyocytes. They succeeded in constructing myocardial tissue fused with oriented fibers, achieving a thickness of 1 mm in vitro and a residual thickness of 0.8 mm after in vivo transplantation, reaching world-leading levels. The tissue constructed using this technology exhibits high functionality, and the types and quantities of factors secreted from the tissue are also high. When this tissue was actually transplanted into an animal model of myocardial infarction, angiogenesis was promoted by the paracrine effect of factors secreted from the cells, and the recovery of cardiac function was clearly improved. These results suggest that using scaffold materials with a three-dimensional structure can produce a higher quality culture supernatant than conventional two-dimensional culture methods. The culture supernatant obtained by this method is expected to have therapeutic effects on myocardial infarction and skin damage. This invention was completed by further investigation based on these findings and includes the following embodiments.

[0008] [Item 1] A method for producing a cell culture supernatant, comprising the step of obtaining a cell culture supernatant by adhering and culturing adherent cells on a fiber. [Item 2] The method for producing a cell culture supernatant according to Item 1, wherein in the step, a cell tissue with a thickness of 50 μm or more, including the adherent cells, is formed. [Item 3] The method for producing a cell culture supernatant according to Item 1 or 2, wherein the fiber is a microfiber and / or a nanofiber. [Item 4] The method for producing a cell culture supernatant according to any one of Items 1 to 3, wherein the adherent cells are at least one adherent cell selected from the group consisting of cardiomyocytes, fibroblasts, smooth muscle cells, skeletal muscle cells, skin cells, liver cells and mesenchymal stem cells, and the fiber is a microfiber. [Item 5] The method for producing a cell culture supernatant according to any one of Items 1 to 3, wherein the adherent cells are at least one adherent cell selected from the group consisting of nerve cells, iPS cells and ES cells, and the fiber is a nanofiber. [Item 6] The method for producing a cell culture supernatant according to any one of Items 1 to 5, wherein the fiber is oriented or randomly oriented. [Item 7] The method for producing a cell culture supernatant according to any one of Items 1 to 6, wherein the fiber is fixed to a culture substrate. [Item 8] Cell culture supernatant obtained by the manufacturing method described in any one of items 1 to 7. [Item 9] A cosmetic composition comprising the cell culture supernatant described in item 8. [Item 10] A hair growth composition comprising the cell culture supernatant described in item 8.

[0009] According to the present invention, a higher quality culture supernatant can be produced than with conventional two-dimensional culture methods.

[0010] This is a photograph showing a specific example of the method for producing the cell culture supernatant of the present invention. This is a photograph showing the analysis results of the cell culture supernatant of the present invention. "2D" refers to culture using only a normal culture dish, and "2.5D" refers to culture using the fiber of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. "2D" refers to culture using only a normal culture dish, and "2.5D" refers to culture using the fiber of the present invention. The asterisk indicates a factor with particularly high expression levels (the same applies below). This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. This shows the analysis results of the cell culture supernatant of the present invention. The results of the analysis of the cell culture supernatant of the present invention are shown. The results of the analysis of the cell culture supernatant of the present invention are shown. The results of the analysis of the cell culture supernatant of the present invention are shown.

[0011] 1. Method for producing cell culture supernatant The method for producing cell culture supernatant of the present invention includes the step of obtaining cell culture supernatant by adhering and culturing adherent cells on a fiber.

[0012] The fibers may be either microfibers or nanofibers. The fibers may also be a combination of microfibers and nanofibers.

[0013] The fibers can be appropriately selected depending on the type of adherent cells. For example, when the adherent cells are cardiomyocytes, fibroblasts, smooth muscle cells, skeletal muscle cells, skin cells, liver cells, and mesenchymal stem cells, using microfibers is preferable because it yields a high-quality cell culture supernatant.

[0014] For example, when the adherent cells are nerve cells, iPS cells, or ES cells, using nanofibers is preferable because it allows for the production of high-quality cell culture supernatant.

[0015] The fiber diameter of the microfiber is preferably 1 μm to 10 μm, more preferably 1 μm to 5 μm, and even more preferably 1 μm to 3 μm.

[0016] The fiber diameter of the nanofiber is preferably 10 nm to 1000 nm, more preferably 100 nm to 500 nm, and even more preferably 150 nm to 250 nm.

[0017] The fibers are preferably used as aggregates. The weight of the fibers per unit area is 10 μg / cm³. ２ ~10000 μg / cm³ ２ It can be done as 100 μg / cm³ ２ ~1000 μg / cm³ ２ It is preferable to do so.

[0018] The fibers may be oriented or randomly oriented.

[0019] The material of the fiber is not particularly limited. Examples of fiber materials include polystyrene (PS), polycaprolactone (PCL), and poly(lactic acid-glycolic acid) (PLGA). The fiber may be made of a single material or of multiple materials. The surface of the fiber may be processed as needed. It is preferable that the fiber be sterilized by an appropriate method such as gamma ray or UV irradiation.

[0020] The fibers may be fixed to the culture substrate. In this case, since the fibers, which serve as a scaffold for cell culture, do not float in the culture medium, cell culture can be performed in a more stable state. Preferably, the fibers are fixed to the bottom of the culture substrate.

[0021] The fiber may be processed so that it can be easily immobilized on an existing culture substrate. For example, as shown in FIG. 1, it may be processed into a form having an outer frame, containing fibers within the outer frame, and capable of fixing the outer frame to the culture substrate. The shape and size of the outer frame are not particularly limited and can be appropriately set according to the shape and size of the culture substrate to be fixed, as well as the culture purpose, etc. For example, when fixing to a circular culture substrate (culture dish) as shown in FIG. 1, the shape of the outer frame can also be circular.

[0022] By culturing adherent cells using the fiber as a scaffold, it is preferable that a cell tissue having a thickness of 50 μm or more and containing adherent cells is formed. It is more preferable that a cell tissue having a thickness of 50 μm to 500 μm and containing adherent cells is formed, and it is even more preferable that a cell tissue having a thickness of 100 μm to 300 μm and containing adherent cells is formed.

[0023] The cell tissue obtained by culturing adherent cells using the fiber as a scaffold tends to have a good binding state between cells. In terms of the binding state between cells, particularly when the cell density is 0.1 to 4×10 ６ / cm ２ it is preferable, and when it is 1 to 3×10 ６ / cm ２ it is more preferable.

[0024] The cell culture conditions do not need to be changed from the normal culture conditions of the adherent cells used and can be appropriately set.

[0025] For example, when using mesenchymal stem cells as adherent cells, the culture conditions can be as follows. Culture conditions: Coat the bottom surface of the culture dish with 1% Laminin in advance and treat it in an incubator at 37°C for 30 minutes to 1 hour. Seed a suspension of 1x10e6 to 5x10e6 / cm 2 of mesenchymal stem cells on the scaffold. After the cells adhere to the scaffold, further add medium. After culturing for 2 days, change the culture medium to DMEM basal medium. After culturing for another 1 day, collect the supernatant.

[0026] A wide range of known cell culture media can be used. The cell culture medium contains amino acids, vitamins, inorganic salts, and other compounds. Vitamins include fat-soluble vitamins and / or water-soluble vitamins. Inorganic salts typically include calcium, magnesium, potassium, and sodium salts, as well as components of the carbonate buffer system, which plays a role in maintaining the pH of the medium. Other compounds may include phenol red, a pH indicator, and D-glucose, etc. Antibiotics, serum, etc., may be added as needed.

[0027] 2. Cell Culture Supernatant The cell culture supernatant of the present invention is obtained by the method for producing the cell culture supernatant of the present invention.

[0028] The cell culture supernatant of the present invention tends to contain more beneficial factors, such as at least three types of factors, compared to cell culture supernatant obtained by conventional culture methods, including GRO (growth-regulating oncology), HGF (hepatocyte growth factor), and VEGF (vascular endothelial growth factor). GRO primarily has effects such as regulating inflammatory responses (promoting neutrophil migration), cell proliferation, and wound healing (promoting fibroblast migration and tissue regeneration). HGF has effects such as treating hepatitis and cirrhosis, recovering from liver damage, regenerating damaged tissue, wound healing, neuroprotection, and promoting nerve regeneration. VEGF has effects such as angiogenesis, blood vessel formation, and repair, and also has therapeutic effects on age-related macular degeneration, diabetic retinopathy, and cardiovascular diseases.

[0029] Therefore, the cell culture supernatant of the present invention can be used as a cosmetic composition or a hair growth composition.

[0030] The cosmetic composition or hair growth composition may further contain, in addition to the cell culture supernatant of the present invention, components that are typically included in cosmetic compositions or hair growth compositions, to the extent that they do not interfere with the effects of the present invention.

[0031] Other such ingredients include, for example, antioxidants, preservatives and disinfectants, oily components, surfactants, and thickeners.

[0032] Examples of antioxidants include vitamin E, butylhydroxytoluene, erythorbic acid, dilauryl thiodipropionate, sodium metabisulfite, tea extracts, ascorbyl palmitate, ascorbyl stearate, octyl gallate, ascorbic acid derivatives, and the like.

[0033] Examples of preservatives and bactericides include phenoxyethanol, coconut alkyl PG-dimonium chloride phosphate Na, propiolyl butylcarbamate, benzoic acid, cresol, hinokitiol, benzalkonium chloride, sorbic acid, butyl paraben, propyl paraben, benzyl paraben, and the like.

[0034] Examples of oily components include higher alcohols, vegetable oils, and plant sterols (such as phytosterols).

[0035] Examples of higher alcohols include aliphatic alcohols having 6 or more carbon atoms, more preferably 6 to 30 carbon atoms. The oily component is preferably solid at normal temperature (25°C). Specifically, cetyl alcohol, behenyl alcohol, myristyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, isostearyl alcohol, hexadecyl alcohol, lanolin alcohol, and the like can be mentioned. These higher alcohols may be used alone or in combination of two or more.

[0036] Examples of vegetable oils include macadamia seed oil, jojoba seed oil, canina bala fruit oil, coconut oil, avocado oil, olive oil, almond oil, kukui nut oil, grape seed oil, safflower oil, sweet almond oil, corn germ oil, sunflower oil, hazelnut oil, jojoba wax, and rose hip oil, and the like. These vegetable oils may be used alone or in combination of two or more.

[0037] Phytosterols are a mixture present in the cell membranes of plants, including sitosterol with double bonds, stigmasterol, campesterol, brassicasterol, fucosterol, and sterols without double bonds. The phytosterols used in the present invention are not particularly limited. Examples include grains such as soybeans, rice, wheat, and sesame; fruits and vegetables such as radishes, cabbages, apples, and lettuces; and extracts and purified products from other sources such as sunflowers, rapeseeds, coconuts, cotton seeds, and tree bark. The type of plant raw material to be extracted is not particularly limited, but preferably includes phytosterols obtained by separating from scum and the like produced in the deodorization process of vegetable oils, and sterols obtained as by-products during the production of pulp.

[0038] Surfactants include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants. Nonionic surfactants are preferred. Specifically, polyglyceryl-6 laurate, polyglyceryl-6 myristate, polyglyceryl-6 stearate, polyglyceryl-10 laurate, polyglyceryl-10 myristate, polyglyceryl-10 stearate, glyceryl myristate, glyceryl stearate, and glyceryl oleate can be mentioned.

[0039] Thickeners include carboxyvinyl polymer, acrylic acid / acrylic acid stearyl copolymer, (acrylates / acrylic acid alkyl (C10-30)) cross-polymer, carboxymethyl cellulose Ca, xanthan gum, tamarind gum, and the like.

[0040] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples and the like.

[0041] <Example 1> As shown in FIG. 1, a device serving as a scaffold was attached to the surface of a conventional culture dish to prepare a culture substrate. When observed from the vertical side (site view), the scaffold was adhered to the culture substrate.

[0042] Microfibers (material: PCL; average fiber diameter: 2 μm) were used as the scaffolding. Pre-irradiation with UV light was performed, resulting in a weight of 179.1 μg ± 16.63 μg / cm² per unit area. ２ It was used in such a way that it resulted in the following.

[0043] We compared two cases: one where the same microfibers were fixed to the culture substrate with orientation, and another where they were fixed to the culture substrate with random orientation. A circular outer frame was provided, and only the outer frame was fixed to the bottom of the culture dish with adhesive. Human adipose-derived mesenchymal stem cells were cultured under normal culture conditions.

[0044] In both oriented and random orientations, cells adhere closely to each other, forming a multilayer structure, resulting in the same number of cells (3 × 10⁻¹⁶). ６ cells / cm ２ Comparing the area required for culturing (using only a standard culture dish): 60 cm² ２ When using fiber: 1 cm ２ That was the case.

[0045] The same number of cells (3 x 10 ６ cells / cm ２ The types and amounts of factor components contained in the cell culture supernatant obtained using only a standard culture dish and when using a fiber were analyzed. The results are shown in Figures 2 and 3. In particular, it was revealed that the expression levels of GRO, HGF, and VEGF were significantly increased when a fiber was used.

[0046] <Example 2> Next, we investigated how the thickness and state of the multilayer structure constructed when using fibers change by varying the cell density. The results are shown in Figure 4. Regarding the state of cell-to-cell bonding, 1 to 3 × 10 ６ cells / cm ２ The density is good, and is 5 × 10 ６ cells / cm ２ Beyond a certain point, it was found that the binding force between cells weakened, resulting in poor organizational structure.

[0047] <Example 3> Furthermore, the types and amounts of factor components in the cell culture supernatant obtained when cultured by the method of the present invention at the optimized cell density as described above were analyzed. The results are shown in Figures 5 to 7. Regarding HGF, the secretion concentration in the culture supernatant was approximately 24 times higher compared to normal culture, the secretion amount per cell was approximately 2.4 times higher, and the secretion amount per unit area was approximately 144 times higher. Furthermore, when the amount of HGF secretion per unit area was measured as the cell density increased, a tendency to increase in proportion to the cell density was confirmed, as shown in Figure 8. Furthermore, when VEGF was also investigated in the same way, as shown in Figure 9, the secretion concentration in the culture supernatant was approximately 24 times higher compared to normal culture, the secretion amount per cell was approximately 2.6 times higher, and the secretion amount per unit area was approximately 184 times higher. Also, similar to HGF, a tendency to increase in proportion to the cell density was confirmed, as shown in Figure 10. Similarly, when GRO was investigated, as shown in Figure 11, the secretion concentration in the culture supernatant was approximately 172 times higher compared to conventional culture, the secretion amount per cell was approximately 18.3 times higher, and the secretion amount per unit area was approximately 1309 times higher. Furthermore, as with HGF and VEGF, a tendency for it to increase with cell density was confirmed, as shown in Figure 12. In addition, as shown in Figure 13, the expression of the gene Cx43, which constitutes gap junctions, was similarly investigated, and the result showed that it was approximately 7 times higher in the culture method of the present invention compared to conventional culture. Similarly, the expression of the gene VEGF, which is associated with angiogenesis, was similarly investigated, and the result showed that it was approximately 1.7 times higher in the culture method of the present invention compared to conventional culture.

[0048] <Example 4> In scaffolds with orientation and random structure, the types and amounts of factor components in the cell culture supernatant obtained when cultured by the method of the present invention at the optimized cell density as described above were analyzed. As shown in Figure 14, the secretion concentration of VEGF was almost the same despite the orientation and random structure. The amount of HGF secreted was slightly higher in scaffolds with orientation than in those with random structure.

[0049] <Example 5> When the culture supernatant was analyzed in the same manner as above using various mesenchymal stem cells of different origins (adipose-derived (AD), bone marrow-derived (BM), and umbilical cord blood-derived (UC)), similar results were obtained regardless of the cell type used (Figure 15).

[0050] <Example 6> When the culture supernatant was analyzed using human dermal fibroblasts (HDF) in the same manner as above, an increase in factors different from those found in mesenchymal stem cells was confirmed, as shown in Figures 16 and 17.

[0051] <Example 7> Regarding VEGF, as shown in Figure 19, the secretion concentration in the culture supernatant was approximately 36 times higher compared to normal culture, the secretion amount per cell was approximately 2.7 times higher, and the secretion amount per unit area was approximately 466 times higher. Similarly, when GRO was investigated, as shown in Figure 19, the secretion concentration in the culture supernatant was approximately 71 times higher compared to normal culture, the secretion amount per cell was approximately 5.26 times higher, and the secretion amount per unit area was approximately 900 times higher.

Claims

1. A method for producing a cell culture supernatant, comprising the step of obtaining a cell culture supernatant by adhering and culturing adherent cells on a fiber.

2. The manufacturing method according to claim 1, wherein in the above step, a cell tissue containing the adherent cells with a thickness of 50 μm or more is formed.

3. The manufacturing method according to claim 1 or 2, wherein the fiber is a microfiber and / or a nanofiber.

4. The manufacturing method according to claim 1 or 2, wherein the adherent cells are at least one adherent cell selected from the group consisting of cardiomyocytes, fibroblasts, smooth muscle cells, skeletal muscle cells, skin cells, liver cells, and mesenchymal stem cells, and the fibers are microfibers.

5. The manufacturing method according to claim 1 or 2, wherein the adherent cells are at least one type of adherent cell selected from the group consisting of nerve cells, iPS cells, and ES cells, and the fibers are nanofibers.

6. The manufacturing method according to claim 1 or 2, wherein the fiber is oriented or randomly oriented.

7. The manufacturing method according to claim 1 or 2, wherein the fiber is fixed to the culture substrate.

8. Cell culture supernatant obtained by the manufacturing method described in claim 1 or 2.

9. A cosmetic composition comprising the cell culture supernatant described in claim 8.

10. A hair growth composition comprising the cell culture supernatant described in claim 8.