Cell culture method and method for producing culture supernatant

Abstract

To provide a novel cell culture method and a method for producing a culture supernatant.SOLUTION: The present invention provides a method having a step for culturing animal cells in a medium having a glucose concentration of 4.5 g / L or more, wherein the content of useful components in a culture supernatant is increased compared to the case of culturing in a medium with a glucose concentration of 1 g / L.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing cells and a method for producing a culture supernatant, and more particularly to a method for producing a useful culture supernatant by cell culture. [Background technology]

[0002] Physiologically active substances that can be used in cosmetics and the like have been produced using cultured cells for some time (for example, Patent Document 1).

[0003] However, little research has been done on culture methods for increasing the concentration of target components, and there is a need to develop a method for producing a culture supernatant containing useful components at higher concentrations. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6286689 Summary of the Invention [Problem to be solved by the invention]

[0005] A novel cell culture method and a method for producing a culture supernatant are provided. [Means for solving the problem]

[0006] [Aspect 1] A method for increasing the content of useful components in a culture supernatant compared to when animal cells are cultured in a medium with a glucose concentration of 1 g / L, comprising the step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more. [Aspect 2] The method of embodiment 1, wherein said animal cell is an animal stem cell. [Aspect 3] The method of embodiment 1, wherein the useful ingredient is one or more components selected from the group consisting of keratinocyte proline-rich protein, type I keratinocyte cytoskeletal 9, type I keratinocyte cytoskeletal 10, type I keratinocyte cytoskeletal 14, type II keratinocyte cytoskeletal 1, type II keratinocyte cytoskeletal 3, type II keratinocyte cytoskeletal 5, type II keratinocyte cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. [Aspect 4] The method of embodiment 1 further comprises reducing the content of at least one selected from the group consisting of globin subfamily B member 1, mRNA turnover 4 protein homolog, guanine nucleotide-binding protein G(i) subunit alpha-3, ubiquilin-1, charged multivesicle protein 4a, merlin, and exportin-7 in the culture supernatant. [Aspect 5] A method for producing a culture supernatant, comprising the step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more to increase useful components in the culture supernatant. [Aspect 6] A method for producing a culture supernatant according to aspect 5, wherein the animal cells are animal stem cells. [Aspect 7] 6. The method for producing a culture supernatant according to claim 5, wherein the useful component is one or more components selected from the group consisting of keratinocyte proline-rich protein, type I keratinocyte cytoskeletal 9, type I keratinocyte cytoskeletal 10, type I keratinocyte cytoskeletal 14, type II keratinocyte cytoskeletal 1, type II keratinocyte cytoskeletal 3, type II keratinocyte cytoskeletal 5, type II keratinocyte cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. [Aspect 8] A method for producing a culture supernatant according to aspect 5, further comprising reducing the content of at least one selected from the group consisting of globin subfamily B member 1, mRNA turnover 4 protein homolog, guanine nucleotide-binding protein G(i) subunit alpha-3, ubiquilin-1, charged multivesicle protein 4a, merlin, and exportin-7 in the culture supernatant. [Aspect 9] A method for producing a culture supernatant according to Aspect 5, wherein the culture supernatant is for treating a deficiency in a living organism of a useful component selected from the group consisting of keratinocyte proline-rich protein, type I keratinocyte cytoskeletal 9, type I keratinocyte cytoskeletal 10, type I keratinocyte cytoskeletal 14, type II keratinocyte cytoskeletal 1, type II keratinocyte cytoskeletal 3, type II keratinocyte cytoskeletal 5, type II keratinocyte cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. [Aspect 10] A culture supernatant produced by the production method according to embodiment 5. [Aspect 11] A composition comprising the culture supernatant of embodiment 10. [Aspect 12] The composition of embodiment 11, wherein the composition is a cosmetic composition, a food composition, a medical composition, and / or a dental composition. [Effects of the Invention]

[0007] The present invention has the effect of providing a novel method for culturing animal cells and a culture supernatant with a novel composition. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a photograph of cells cultured by the culture method of the present invention. [Figure 2] FIG. 2 is a graph showing the number of cells cultured by the culture method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides a novel method for culturing animal cells and a novel culture supernatant obtained by the culture method. One embodiment of the present invention is to increase the amount of useful components in the culture supernatant and / or reduce the content of non-useful components by culturing animal cells in a medium with an increased glucose concentration.

[0010] That is, by culturing in a medium containing a high concentration of glucose of 4.5 g / L or more, more preferably 9.0 g / L or more, the present invention makes it possible to produce a culture supernatant containing a higher concentration of specific cosmetic ingredients than when culturing in a medium containing 1 g / L of glucose. Here, the medium containing 1 g / L of glucose is preferably the same as the medium containing 4.5 g / L or more of glucose except for the glucose concentration.

[0011] The culture method of the present invention is characterized by culturing in a medium containing a higher concentration of glucose than that of a typical animal culture medium when producing a culture supernatant. While typical media often contain approximately 1 g / L of glucose, the present invention uses a medium containing several to 10 times that amount of glucose. In the present invention, the preferred glucose concentrations in the medium are 2 g / L or more, 3 g / L or more, 4 g / L or more, 5 g / L or more, 6 g / L or more, 7 g / L or more, 8 g / L or more, 9 g / L or more, 10 g / L or more, 11 g / L or more, 12 g / L or more, 13 g / L or more, 14 g / L or more, 15 g / L or more, 16 g / L or more, 17 g / L or more, 18 g / L or more, 19 g / L or more, and 20 g / L or more. The preferred upper limit of the glucose concentration is 100 g / L or less, 50 g / K or less, 40 g / L or less, 30 g / L or less, or 25 g / L or less.

[0012] Preferably, after subculture, the cells are washed with PBS or the like and then cultured in a medium containing a high concentration of glucose.

[0013] Although it is thought that an excessively high glucose concentration may have an adverse effect on cells, in the present invention, it is preferable to increase the glucose concentration within a range that does not cause adverse effects.

[0014] In the present invention, glucose is used as the sugar, but other sugars may also be used, such as, but not limited to, sucrose, galactose, lactose, xylose, etc., as long as the sugar concentration is increased to increase the production amount of a physiologically active substance.

[0015] Animal cells are preferably used as cells suitable for the culture method of the present invention. Examples of animal cells include, but are not limited to, mouse, rat, hamster, rabbit, monkey, and human cells. Any animal cells that increase in useful components when glucose concentrations are increased can be used. Stem cells are more preferred, and human stem cells are even more preferred. Among stem cells, pluripotent stem cells are more preferably used. Pluripotent stem cells may be induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or other stem cells. Tissue stem cells can also be used in the culture method of the present invention. Human umbilical cord tissue-derived stem cells, bone marrow stem cells, adipose stem cells, or dental pulp stem cells may also be used.

[0016] Although the present invention can be used to culture cells directly in the medium of the present invention, it is generally preferred to use cells that have been subcultured in a general medium and then replace the medium with the medium of the present invention for culture. The number of passages in the subculture is preferably multiple, and it is preferable to use cells that have been subcultured two or more times. More preferred passages are 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. The upper limit for the number of subcultures is 30 or less, 25 or less, or 20 or less.

[0017] The method of the present invention may include a step of subculturing the animal cells or a step of washing the subcultured animal cells prior to the step of culturing the animal cells in a medium containing a glucose concentration of 4.5 g / L or more.

[0018] The culture time in a high-concentration glucose medium is 10 hours or more, more preferably 15 hours or more, even more preferably 20 hours or more, even more preferably 25 hours or more, even more preferably 30 hours or more, even more preferably 35 hours or more, even more preferably 40 hours or more, and most preferably 45 hours or more. The upper limit of the culture time in a high-concentration glucose medium is 50 hours or less, or 48 hours or less.

[0019] Culture vessels applicable to the present invention may be petri dishes, culture bottles, culture flasks, and the like commonly used in tissue culture of animal cells, or may be used for tank culture. Alternatively, cells may be embedded in a scaffold such as collagen and cultured. Furthermore, cells may be cultured in a bioreactor. In either case, culture is carried out at a carbon dioxide partial pressure of 4-10% and a temperature of 35-38°C. The culture rate and amount of cultured cells can be appropriately adjusted by supplying gases such as oxygen and hydrogen as needed.

[0020] As used herein, culture supernatant refers to the medium after culture. After the culture is completed, the culture supernatant may be sucked up using a pipette or the culture solution may be centrifuged to separate it from the cells. Alternatively, the culture supernatant obtained by the culture method of the present invention is preferably used after filtration. The pore size of the filter is preferably 1.0 μm, more preferably 0.45 μm, and even more preferably 0.22 μm. When purifying exosomes for use, it is preferable to further use ultrafiltration. The purified culture supernatant may be concentrated and used as a liquid, or it may be freeze-dried into a powder and mixed with various products for use.

[0021] The culture supernatant may be used to purify useful components using a column or the like. Columns include, but are not limited to, gel filtration, ion exchange resin, adsorption columns (such as antibody columns), and polar columns, and any column that can concentrate or purify useful components can be used. The column may be an open column, or may be used for high-performance liquid chromatography, gas chromatography, paper chromatography, or the like.

[0022] In this culture experiment in which the glucose concentration was increased, it was found that fibronectin (FN) and BMP increased due to the increased glucose concentration (Table 5). In addition, keratinocyte proline-rich protein, type I keratin cytoskeletal 9 (Keratin, type I cytoskeletal 9), type I keratin cytoskeletal 10 (Keratin, type I cytoskeletal 10), type I keratin cytoskeletal 14 (Keratin, type II cytoskeletal 1), type II keratin cytoskeletal 3 (Keratin, type II cytoskeletal 5), type II keratin cytoskeletal 2 epidermal (Keratin, type II cytoskeletal 2 epidermal), desmoglein-1, collagen alpha-1(II) chain (Collagen It was also found that increasing glucose concentrations increased collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, and desmocollin-2 (Table 5). These are components with medical and / or cosmetic benefits, as shown in Table 1.Therefore, the culture supernatant is preferably used to treat a deficiency in a living body of a useful component selected from the group consisting of keratinocyte proline-rich protein, type I keratinocyte cytoskeletal 9, type I keratinocyte cytoskeletal 10, type I keratinocyte cytoskeletal 14, type II keratinocyte cytoskeletal 1, type II keratinocyte cytoskeletal 3, type II keratinocyte cytoskeletal 5, type II keratinocyte cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. Here, examples of treatment include, for example, treatment and prevention. Treatment includes, for example, complete cure, alleviation of symptoms, improvement of prognosis, and prevention of recurrence. Examples of living organisms include locomotor systems such as bones, cartilage, skeletal muscles, tendons, and ligaments; sensory systems such as eyes, ears, nose, tongue, skin, mucous membranes, and muscle spindles; respiratory systems such as the mouth, nose, larynx, pharynx, trachea, bronchi, and lungs; circulatory systems such as the heart, spleen, bone marrow, and blood; digestive systems such as the mouth, teeth, larynx, pharynx, esophagus, stomach, small intestine, large intestine, anus, digestive glands, salivary glands, pancreas, liver, and gallbladder; nervous systems such as the brain, spinal cord, and nerves; reproductive systems such as the breast, ovaries, uterus, placenta, vagina, labia, clitoris, penis, scrotum, and testes; endocrine systems such as the hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal glands, ovaries, placenta, and testes; and urinary systems such as the kidneys, ureters, bladder, and urethra. Examples of living organisms include the living body of a target. Here, examples of targets include vertebrates. Vertebrates include mammals such as mice, rats, rabbits, pigs, cows, monkeys, and humans. The mammal is preferably a human. The subject can be of any age, including infants, juveniles, adolescents, adults, and the elderly.

[0023] [Table 1]

[0024] We also found that some components were reduced by increasing the glucose concentration (Table 6). Reducing components that have adverse effects can further enhance the effectiveness of useful components. Even in the case of components that do not have physiological activity, reducing unnecessary components may increase the content of useful components, so reducing components other than useful components may lead to the production of more useful culture supernatants.

[0025] Examples of uses for the culture supernatant include cosmetics (serums, creams, etc.), health foods, foods, and pharmaceuticals. In the case of serums, serums and cosmetics with a high content of collagen and other components can be provided. In the case of health foods, anti-aging and cartilage component supplements are conceivable. The preferred lower limit of the culture supernatant content in a product is 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. The preferred upper limit of the content is 99.9% or less, 99% or less, 95% or less, 90% or less, 80% or less, 70% or less, or 60% or less. [Example]

[0026] Example 1 Verification of the effect of different glucose concentrations when extracting culture supernatant The effect of different sugar concentrations during culture supernatant extraction was investigated. The sugar concentration added to the extraction medium was varied, and the resulting culture supernatant was analyzed. Cells: Human umbilical cord tissue-derived stem cells (passage number at the time of supernatant extraction: P8) Cell seeding density at passage: 5000 cells / cm 2

[0027] The cells were grown and cultured in Kohjin Bio's (ADSC-4) medium. When they reached confluence, they were detached using TrypLE Select and subcultured. At the 8th passage, the medium composition was changed to the one below, and culture supernatant was prepared. Medium composition: DEME (no-glucose), Gibco, #A1443001 Sugar type: Glucose (Nacalai Tesque, #16806-12) Mannitol (Nacalai Tesque, #11662-42) Sugar concentration added to the medium (Figure 1) 1. DMEM + Glucose (1.0 g / L) 2. DMEM + Glucose (4.5 g / L) 3. DMEM + Glucose (9.0 g / L) 4. DMEM + mannitol (10 mg / mL)

[0028] The morphology of the cultured cells is shown in Figure 1, and the cell count in Figure 2. As shown in Figure 1, there was no significant change in cell morphology when the glucose concentration in the medium was increased from 1 g / L to 4.5 g / L or 9.0 g / L. However, while the cell count did not differ significantly between the 1 g / L and 4.5 g / L glucose concentrations in the medium, it increased by approximately 20% at 9.0 g / L (Figure 2). Furthermore, even in the 1% mannitol addition group, there was almost no difference in cell count between the 1 g / L and 4.5 g / L glucose concentrations. In contrast, the cell count was significantly lower in the 0 g / L glucose and 100 mg / L trehalose groups.

[0029] Preparation of the supernatant At P8, when the cells reached confluence, the existing medium (ADSC-4) was removed and the cells were washed twice with PBS. The medium with the above composition was then added to each cell group and cultured for 48 hours. After culture, the culture supernatant was collected and filtered through a 0.22 μm filter to prepare the culture supernatant.

[0030] Preparation of samples for proteome analysis Sample preparation The prepared culture supernatant was subjected to the following pretreatment for proteome analysis. An equal volume of 400 mM Tris-HCl pH 8.5, 4% SDS was added to the sample solution, and the sample was treated with a closed-type ultrasonic homogenizer. The protein concentration of this sample was measured by BCA assay, and the protein concentration was adjusted to 1 μg / μL with 100 mM Tris-HCl pH 8.5, 2% SDS.

[0031] Next, to cleave the SS bonds of the protein, TCEP was added to the protein solution (protein amount 20 μg) to a final concentration of 20 mM and incubated for 30 minutes at 50°C. To alkylate the cysteine ​​residues, iodoacetamide (IAA) was added to a final concentration of 30 mM and incubated for 30 minutes at room temperature (protected from light).

[0032] Next, Cytiva's Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophylic) and Sera-Mag Carboxylate-Modified Magnetic Particles (Hydrophobic) were mixed 1:1 (v / v), washed three times with distilled water, and adjusted to 15 μg solids / μL with distilled water (SP3 beads). 20 μL of SP3 beads were added to the alkylated sample, and 2.5 volumes of ethanol were added and mixed at room temperature for 20 minutes. The beads were washed twice with 80% ethanol, followed by the addition of 100 μL of 50 mM Tris-HCl pH 8.0 and mixing. Next, 500 ng of Trypsin / Lys-C Mix (Promega) was added to fragment the proteins into peptides, and the mixture was incubated overnight at 37°C.

[0033] 20 μL of 5% TFA was added to this mixture, and the mixture was treated with a closed-type ultrasonic disintegrator. The resulting treated solution was desalted using a C18 spin column and then dried using a centrifugal evaporator. 3% ACN-0.1% formic acid was added to the dried mixture, and the peptide was dissolved using a closed-type ultrasonic disintegrator. The peptide concentration of the solution containing the dissolved peptide was measured using a BCA assay, and the peptide concentration was adjusted to 200 ng / μL with 2% ACN-0.1% TFA. The prepared sample was analyzed using the following nanoLC-MS / MS analysis conditions.

[0034] NanoLC analysis conditions Amount of injected peptide: 200 ng NanoLC used: UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific) Column size: 75 μm inner diameter x 120 mm length (Nikkyo Technos) Column temperature: 40℃ Solvent: A solvent: 0.1% formic acid added distilled water, B solvent: 0.1% formic acid added 80% CAN The gradient conditions are as shown in Table 2.

[0035] [Table 2]

[0036] MS analysis conditions MS used: Q Exactive HF-X (Thermo Fisher Scientific) Ionization method: ESI positive mode Measurement time: 40 minutes (gradient time 4 to 44 minutes) Type of MS analysis: Overlapping window DIA Scan event: Repeat events 1 to 4 below to acquire data MS analysis conditions MS used: Q Exactive HF-X (Thermo Fisher Scientific) Ionization method: ESI positive mode Measurement time: 40 minutes (gradient time 4 to 44 minutes) Type of MS analysis: Overlapping window DIA Scan event: Repeat events 1 to 4 in Table 3 below to acquire data.

[0037] [Table 3]

[0038] Full scan (MS1) measurement parameters for events 1 and 3 Resolution: 30,000 AGC target: 3e6 Maximum IT: 55 ms MS1 scan range: 495 to 785 m / z DIA(MS2) measurement parameters for events 2 and 4 Resolution: 15,000 AGC target: 3e6 Maximum IT: auto Loop count: 71 (for event 2), 70 (for event 4) MS2 scan range: 200 m / z or more Normalized Collision Energy: 28 Isolation window: 4.0 m / z Isolation window center m / zs: See Table 4 below

[0039] [Table 4]

[0040] Data analysis The MS data obtained was analyzed using Scaffold DIA under the following conditions to identify proteins and peptides and calculate quantitative values. The analysis results were then exported and compiled into an Excel file. Software used: Scaffold DIA (Proteome Software) Protein Sequence Database:HumanUniProtKB / Swiss-Prot database (UP000005640) Spectral Library: A library created using Prosit (https: / / www.proteomicsdb.org / prosit / ) from the above sequence database. Fragmentation:HCD Precursor Tolerance: 10 ppm Fragment Tolerance: 10 ppm Data Acquisition Type: Staggered DIA Digestion Enzyme: Trypsin Peptide Charge: 2-4 Max Missed Cleavages: 1 Fixed Modification: Carbamidomethylation [C] Peptide FDR: 1% or less Protein FDR: 1% or less

[0041] The useful components that increased and those that decreased when the glucose concentration was increased from 1 g / L to 4.5 g / L or 9.5 g / L are shown below (Tables 5 and 6).

[0042] [Table 5]

[0043] [Table 6]

[0044] The proteins whose values ​​more than doubled when the glucose concentration of the medium was increased from 1 g / L to 4.5 g / L are shown below. Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 14, Keratin, type II cytoskeletal 1, Retinol-binding protein 4, ADP-ribose pyrophosphatase, mitochondrial, Tenascin, Desmoplakin, Keratin, type II cytoskeletal 3, Hornerin, Keratin, type I cytoskeletal 10, Thrombospondin-3, Protein AHNAK2 AHNAK2, Keratin, type II cytoskeletal 2 epidermal, Multiple epidermal growth factor-like domains protein 8, Keratin, type II cytoskeletal 6Atype II cytoskeletal 6A, Cullin-3, Coiled-coil domain-containing protein 50, Mimecan, Immunoglobulin superfamily DCC subclass member 4, Collagen alpha-1(II) chain, Protein S100-A8, Epsin-1, Protocadherin Fat 1, Sterol carrier protein 2, Matrix Gla protein, Sec1 family domain-containing protein 1, Keratin, type II cytoskeletal 5), Phospholipase A-2-activating protein, Ubiquitin-conjugating enzyme E2 Z, Pre-mRNA-processing factor 19, Endoribonuclease LACTB2, Tryptophan-tRNA ligase, cytoplasmic, Legumin alpha-1,6-mannosyl-glycoprotein 2-beta N-acetylglucosamine transferase (Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, beta-enolase, MOB kinase activator 1B, MOB kinase activator 1B, MOB kinase activator 1A, immunity-related GTPase family Q protein, collagen alpha-3(V) chain, cathepsin F, epididymis-specific alpha-mannosidase, ubiquitin domain-containing protein UBFD1, epidermal growth factor receptor substrate 15-like 1, thimet oligopeptidase oligopeptidase, Disks large homolog 1, UPF0687 protein C20orf27, spermine synthase, Golgi-resident adenosine 3',5'-bisphosphate 3'-phosphatase, tubulin beta 8B, Ragulator complex protein LAMTOR3,

[0045] The proteins whose values ​​increased by more than two-fold when cultured with the glucose concentration in the medium increased from 1 g / L to 9 g / L are shown below. Keratin, type I cytoskeletal 9, TSC22 domain family protein 2, cold shock domain-containing protein E1, tenascin, hornerin, retinol-binding protein 4, Keratin, type I cytoskeletal 14, Eukaryotic translation initiation factor 3 subunit L, Multiple epidermal growth factor-like domains protein 8, Keratin, type II cytoskeletal 1, Thrombospondin-3, Keratin, type II cytoskeletal 3 Cytoskeletal 3, Desmoplakin, Annexin A7, Tissue alpha-L-fucosidase, Myosin-10, L-xylulose reductase, Phosphoribosyl formyl glycinamidine synthase, Thyroid receptor-interacting protein 6, Collagen alpha-1(II) chain, Sorting nexin-2, Phospholipase A-2-activating protein, Multiple coagulation factor deficiency protein 2factor deficiency protein 2, Matrix Gla protein, Signal recognition particle 54 kDa protein, Pre-mRNA-processing-splicing factor 8, Protocadherin Fat 1, Signal recognition particle receptor subunit alpha, Nuclear mitotic apparatus protein 1, 26S proteasome non-ATPase regulatory subunit 10, Immunoglobulin superfamily DCC subclass member 4, Cullin-3, Keratin, type II cytoskeletal 1b 1b), Mimecan, Epididymis-specific alpha-mannosidase, Pseudouridine-5'-phosphatase, Protein farnesyltransferase / geranylgeranyl transferase type-1 subunit alpha, Vinexin, Keratin, type II cytoskeletal 2 epidermal, Cystathionine gamma-lyase, Keratin, type I cytoskeletal 1010), high affinity cationic amino acid transporter 1, ragulator complex protein LAMTOR3, natural cytotoxicity trigger receptor 3 ligand 1, desmocollin-2, epsin-1, SEC23-interacting protein, immunoglobulin-binding protein 1, repulsive guidance molecule B, cathepsin F, small glutamine-rich tetratricopeptide repeat-containing protein beta, replication protein A 32 kDa subunit, serum amyloid A-2 protein amyloid A-2 protein, Bis(5'-nucleosyl)-tetraphosphatase [asymmetrical], Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyl transferase, Coiled-coil and C2 domain-containing protein 1A, Protein SOGA1, Immunity-related GTPase family Q proteinprotein, transmembrane protein 132A, ubiquitin-conjugating enzyme E2R2, keratin, type II cytoskeletal 5, endonuclease LACTB2, peptidyl-prolyl cis-trans isomerase FKBP14, CCR4-NOT transcription complex subunit 1, phosphopantothenate-cysteine ​​ligase, Golgi-resident adenosine 3',5'-bisphosphate 3'-phosphatase 3'-phosphatase, tryptophan-tRNA ligase, cytoplasmic, laminin subunit alpha-4, L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase, ATP-dependent DNA helicase Q1, protein unc-45 homolog A, glutamate-cysteine ​​ligase regulatory subunit, ubiquilin-2, complement component 3 (Complement C3), importin-9, vacuolar protein sorting associated protein 25 protein-sorting-associated protein25), ubiquitin domain-containing protein UBFD1, adenylyl cyclase-associated protein 2, ataxin-10, TRIO and F-actin-binding protein, thioredoxin-like protein 4A, keratin, type II cytoskeletal 73, mitochondrial Diablo homolog, beta-enolase, SPRY domain-containing protein 4, RNA-binding protein Raly, vitronectin, collagen alpha-3(V) chain chain, phenylalanine-tRNA ligase beta subunit, WD repeat-containing protein 82, anamorsin, retinoid-inducible serine carboxypeptidase, dipeptidyl peptidase 4, ethylmalonyl-CoA decarboxylase, dipeptidyl peptidase 9, thymidine phosphorylase, U8 snoRNA-decapping enzyme, Ran-binding protein 3, GTPase NasNRas, Leucyl-cystinyl aminopeptidase, Protein S100-A8, Legumain, 26S proteasome non-ATPase regulatory subunit 8, Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1, Integral membrane protein 2B, Vacuolar protein sorting-associated protein VTA1 homolog, Exportin-T, Ubiquitin-conjugating enzyme E2 Z, Bisphosphoglycerate mutase mutase, cell migration and invasion enhancer 1, transforming growth factor beta-1 proprotein, Kin of IRRE-like protein 1, inositol polyphosphate 1-phosphatase, CAD protein, mitochondrial enol-CoA hydratase, spermine synthase, glutathione S-transferase LANCL1, cellular retinoic acid-binding protein 2, acid ceramidaseceramidase, Cullin-2, Echinoderm microtubule-associated protein-like 2, Lysosomal acid lipase / cholesteryl ester hydrolase, Adaptin ear-binding coat-associated protein 2, Disintegrin and metalloproteinase domain-containing protein 17, Inosine-5'-monophosphate dehydrogenase 1, Phospholipid transfer protein, Pikachurin, Protein diaphanous homolog 1, Laminin subunit beta-2 subunit beta-2, prefoldin subunit 5, glucose 1,6-bisphosphate synthase, leucine-rich repeat-containing protein 47, extended synaptotagmin-2, GDH / 6PGL endoplasmic bifunctional protein, coatomer subunit zeta-2, protein AHNAK2, prostaglandin-H2 D-isomerase, cAMP-dependent protein kinase type II alpha regulatory subunitkinase type II-alpha regulatory subunit, Sec1 family domain-containing protein 1, isoamyl acetate-hydrolyzing esterase 1 homolog, programmed cell death protein 10, protein arginine N-methyltransferase 5, alpha-aminoadipic semialdehyde dehydrogenase, coiled-coil domain-containing protein 126, protein phosphatase 1F, smoothelin SUMO-activating enzyme subunit 1, epidermal growth factor receptor substrate 15-like 1 1), Ubiquitin-like conjugating enzyme ATG3, Protein YIPF3, L-lactate dehydrogenase A L-lactate dehydrogenase A-like 6B, beta-centractin, galactocerebrosidase, copine-3, putative peptidyl-tRNA hydrolase PTRHD1, alpha-N-acetylglucosaminidase, kinectin, astrocytic phosphoprotein PEA-15, inhibin beta A chain, epidermal growth factor receptor kinase substrate 8-like protein 2, STE20-like serine / threonine-protein kinase kinase LIM, LIM domain only protein 7, ceramide transfer protein, charged multivesicular body protein 4b, microfibril-associated glycoprotein 4, aspartyl aminopeptidase, E3 ubiquitin-protein ligase NEDD4-like, exocyst complex component 7, haptoglobin-related protein, ubiquitin-conjugating enzyme E2 Q1, and translin-associated protein X.X, calcium-binding protein 39-like, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, BAG family molecular chaperone regulator 2, apoptosis regulator BAX, protein phosphatase 1 regulatory subunit 7, transthyretin, vacuolar protein sorting-associated protein 35, and a disintegrin and metalloproteinase with thrombospondin motifs 4. 4), Beta-1,3-N-acetylglucosaminyltransferase lunaticfringe, N-2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Protein dpy-30 homolog, Fibroblast growth factor receptor 1, Phosphomannomutase 2

[0046] As shown above, it was found that increasing the glucose concentration increased the content of many proteins in the medium. From these data, it is possible to estimate the glucose concentration required to increase useful components. [Industrial Applicability]

[0047] The present invention can be used in the beauty industry, medical industry, etc.

Claims

1. A method for increasing the content of useful components in a culture supernatant compared to when animal cells are cultured in a medium with a glucose concentration of 1 g / L, comprising the step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more.

2. The method of claim 1 , wherein the animal cell is an animal stem cell.

3. The method of claim 1, wherein the useful ingredient is one or more components selected from the group consisting of keratinocyte proline-rich protein, type I keratin cytoskeletal 9, type I keratin cytoskeletal 10, type I keratin cytoskeletal 14, type II keratin cytoskeletal 1, type II keratin cytoskeletal 3, type II keratin cytoskeletal 5, type II keratin cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2.

4. The method of claim 1, further comprising reducing the content of at least one selected from the group consisting of globin subfamily B member 1, mRNA turnover 4 protein homolog, guanine nucleotide-binding protein G(i) subunit alpha-3, ubiquilin-1, charged multivesicle protein 4a, merlin, and exportin-7 in the culture supernatant.

5. A method for producing a culture supernatant, comprising the step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more to increase useful components in the culture supernatant.

6. The method for producing a culture supernatant according to claim 5, wherein the animal cells are animal stem cells.

7. 6. The method for producing a culture supernatant according to claim 5, wherein the useful component is one or more components selected from the group consisting of keratinocyte proline-rich protein, type I keratin cytoskeletal 9, type I keratin cytoskeletal 10, type I keratin cytoskeletal 14, type II keratin cytoskeletal 1, type II keratin cytoskeletal 3, type II keratin cytoskeletal 5, type II keratin cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2.

8. The method for producing a culture supernatant according to claim 5, further comprising reducing the content of at least one selected from the group consisting of globin subfamily B member 1, mRNA turnover 4 protein homolog, guanine nucleotide-binding protein G(i) subunit alpha-3, ubiquilin-1, charged multivesicle protein 4a, merlin, and exportin-7 in the culture supernatant.

9. The method for producing a culture supernatant according to claim 5, wherein the culture supernatant is for treating a deficiency in a living body of a useful component selected from the group consisting of keratinocyte proline-rich protein, type I keratinocyte cytoskeletal 9, type I keratinocyte cytoskeletal 10, type I keratinocyte cytoskeletal 14, type II keratinocyte cytoskeletal 1, type II keratinocyte cytoskeletal 3, type II keratinocyte cytoskeletal 5, type II keratinocyte cytoskeletal 2 epithelial cell, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, fibronectin, and desmocollin 2.

10. A culture supernatant produced by the production method according to claim 5.

11. A composition comprising the culture supernatant of claim 10.

12. The composition of claim 11 , wherein the composition is a cosmetic composition, a food composition, a medical composition, and / or a dental composition.

Images