Cell culture method and method for producing culture supernatant

Abstract

The present invention provides a novel cell culture method and a method for producing a culture supernatant.　The present invention provides a method having a step for culturing animal cells in a medium containing 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.

Description

Technical Field

[0001] The present invention relates to a method for culturing cells and a method for producing a culture supernatant. More specifically, it relates to a method for producing a useful culture supernatant by cell culture.

Background Art

[0002] It has been conventionally practiced to produce physiologically active substances that can be used in cosmetics and the like using cultured cells (for example, Patent Document 1).

[0003] However, regarding the culture method for increasing the concentration of the target component, there has been little research, and there has been a demand for the development of a method for producing a culture supernatant containing a higher concentration of useful components.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] To provide a novel method for culturing cells and a method for producing a culture supernatant.

Means for Solving the Problems

[0006] [Aspect 1] A method for increasing the content of useful components in a culture supernatant, comprising a step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more, which is higher than the case of culturing in a medium with a glucose concentration of 1 g / L. [Aspect 2] The method according to Aspect 1, wherein the animal cells are animal stem cells. [Aspect 3] The method according to aspect 1, wherein the active ingredient is at least one component 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, 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 according to aspect 1, further 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, ubiquitin-1, charged multivesicular body protein 4a, merlin, and exportin-7 in the culture supernatant. [Aspect 5] A method for producing a culture supernatant, comprising a step of culturing animal cells in a medium containing a glucose concentration of 4.5 g / L or more to increase the active ingredient in the culture supernatant. [Aspect 6] The method for producing a culture supernatant according to aspect 5, wherein the animal cells are animal stem cells. [Aspect 7] The method for producing a culture supernatant according to aspect 5, wherein the active ingredient is at least one component 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, desmoglein 1, collagen alpha-1 (II) chain, bone morphogenetic protein 4, collagen alpha-1 (XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. [Aspect 8] Furthermore, a method for producing the culture supernatant of Aspect 5, which reduces 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, ubiquitin-1, charged multivesicular body protein 4a, merlin, and exportin-7 in the culture supernatant. [Aspect 9] The method for producing the culture supernatant of Aspect 5, wherein the culture supernatant is for treating the deficiency in a living body of a useful component 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, 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 Aspect 5. [Aspect 11] A composition containing the culture supernatant according to Aspect 10. [Aspect 12] The composition of Aspect 11, wherein the composition is a cosmetic composition, a food composition, a medical composition, and / or a dental composition. [Advantages of the Invention]

[0007] According to the present invention, there is an effect that a novel method for culturing animal cells and a culture supernatant of a novel composition can be obtained. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The present invention provides a novel method for culturing animal cells and a novel culture supernatant obtained by the culturing 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 with an increased glucose concentration in the medium.

[0010] That is, the present invention can produce a culture supernatant with an increased amount of specific cosmetic components, etc., 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, compared to culturing in a medium containing 1 g / L of glucose. Here, the medium containing 1 g / L of glucose is preferably the same medium as the medium containing 4.5 g / L or more of glucose except for the glucose concentration.

[0011] The culturing method of the present invention is characterized in that when producing the culture supernatant, it is cultured in a medium containing a higher concentration of glucose than a normal animal culture medium. Usually, a normal medium often contains about 1 g / L of glucose, but in the present invention, it is cultured using a medium containing several times to 10 times or more of glucose. In the present invention, the preferred glucose concentration in the medium is preferably 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, 20 g / L or more. The upper limit of the preferred glucose concentration is 100 g / L or less, 50 g / K or less, 40 g / L or less, 30 g / L or less, 25 g / L or less.

[0012] Preferably, after subculture, it is preferably cultured in a medium containing a high concentration of glucose after washing the cells with PBS or the like.

[0013] Although it is conceivable that increasing the glucose concentration too much may have an adverse effect on cells, in the present invention, it is preferable to increase the glucose concentration within a range where no adverse effect occurs.

[0014] In the present invention, glucose is used as the sugar, but other sugars may also be used. Examples of such sugars include sucrose, galactose, lactose, xylose, etc., but are not limited thereto. Any sugar that can increase the production amount of the physiologically active substance by increasing the sugar concentration is not particularly limited.

[0015] As the cells suitable for the culture method of the present invention, animal cells are preferably used. Examples of animal cells include mouse, rat, hamster, rabbit, monkey, human, etc., but are not limited thereto. Any animal cell that can increase the useful component by increasing the glucose concentration can be used. More preferably, stem cells, and even more preferably human stem cells can be mentioned. Among stem cells, pluripotent stem cells are more preferably used. The types of pluripotent stem cells may be induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or other stem cells. Tissue stem cells are also 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 be used.

[0016] The present invention can also be cultured directly in the medium of the present invention, but usually, it is preferable to use cells subcultured in a general medium and then replace them with the medium of the present invention for culture. The range of the number of subcultures is preferably multiple times, and it is preferable to use cells subcultured two or more times. More preferable numbers of subcultures are three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times. The upper limit number of subcultures is within 30 times, within 25 times, within 20 times.

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

[0019] The culture vessel applicable to the present invention may be a petri dish, a culture bottle, a culture flask, etc. generally used for tissue culture of animal cells, or may be a tank culture. Alternatively, cells may be embedded in a scaffold such as collagen for culture. Further, cells may be cultured using a bioreactor. In any case, the culture is carried out at a carbon dioxide partial pressure of 4 to 10% and a temperature of 35 to 38°C. If necessary, the culture rate and the amount of cultured cells can be appropriately adjusted by supplying gases such as oxygen and hydrogen.

[0020] In this specification, the culture supernatant refers to the medium after culture. After the culture is completed, the culture supernatant may be sucked up with a pipette or the like, 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 filtration filter is preferably 1.0 μm, more preferably 0.45 μm, and still more preferably 0.22 μm. When exosomes are purified and used, it is more preferable to use ultrafiltration. The purified culture supernatant may be concentrated and used as a liquid, or freeze-dried into a powder and mixed with various products for use.

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

[0022] In the present culture experiment with increased glucose concentration, it was found that fibronectin (FN) and BMP increased due to the increase in glucose concentration (Table 5). Furthermore, Keratinocyte proline-rich protein, Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 10, Keratin, type I cytoskeletal 14, Keratin, type II cytoskeletal 1, Keratin, type II cytoskeletal 3, Keratin, type II cytoskeletal 5, Keratin, type II cytoskeletal 2 epidermal, Desmoglein-1, Collagen alpha-1(II) chain, Bone morphogenetic protein 4, Collagen alpha-1(XVIII) chain, Desmoplakin, and Desmocollin-2 were also found to increase by increasing the glucose concentration (Table 5). These are components that are medically and / or cosmetically useful as shown in Table 1.Therefore, the culture supernatant is preferably for treating the deficiency in a living body of a useful component 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 epithelial 2, desmoglein 1, collagen alpha-1 (II) chain, bone morphogenetic protein 4, collagen alpha-1 (XVIII) chain, desmoplakin, fibronectin, and desmocollin 2. Here, the treatment includes, for example, in addition to treatment, prevention, etc. The treatment includes, in addition to complete cure, relief of symptoms, improvement of prognosis, prevention of recurrence, etc. The living body includes, for example, locomotor organs such as bone, cartilage, skeletal muscle, tendon, and ligament; sensory organs such as eye, ear, nose, tongue, skin, mucosa, and muscle spindle; respiratory organs such as mouth, nose, larynx, pharynx, trachea, bronchus, and lung; circulatory organs such as heart, spleen, bone marrow, and blood; digestive organs such as mouth, teeth, larynx, pharynx, esophagus, stomach, small intestine, large intestine, anus, digestive gland, salivary gland, pancreas, liver, and gallbladder; nervous system such as brain, spinal cord, and nerve; genital organs such as breast, ovary, uterus, placenta, vagina, labia, clitoris, penis, scrotum, and testis; endocrine organs such as hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland, ovary, placenta, and testis; urinary organs such as kidney, ureter, bladder, and urethra, etc. The living body is, for example, the living body of a subject. Here, the subject includes, for example, vertebrates. The vertebrates include, for example, mammals such as mouse, rat, rabbit, pig, cow, monkey, and human. The mammal is preferably human. The subject can be of any age including infants, juveniles, youths, adults, and the elderly.

[0023]

Table 1

[0024] In addition, by increasing the glucose concentration, components that decreased were also found (Table 6). If the components that have an adverse effect are reduced, the effects of the useful components can be enhanced further. Especially in the case of components that do not have physiological activity, since the content of the useful components may increase due to the reduction of unnecessary components, the reduction of components other than the useful components may lead to the production of a more useful culture supernatant.

[0025] Examples of the usage methods of the culture supernatant include cosmetics (such as skin lotions and creams), health foods, foods, and pharmaceuticals. In the case of skin lotions, skin lotions and cosmetics with a high content rate of collagen and the like can be provided. In the case of health foods, anti-aging, supplements of cartilage components, and the like can be considered. The lower limit of the preferable content in the product of the culture supernatant 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, 50% or more. The upper limit of the preferable content is 99.9% or less, 99% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less.

Example

[0026] (Example 1) Verification of the influence due to the difference in glucose concentration at the time of extracting the culture supernatant The influence due to the difference in sugar concentration at the time of extracting the culture supernatant was investigated. The sugar concentration added to the extraction medium was changed, and the obtained culture supernatant was analyzed. · Cells: Human umbilical cord tissue-derived stem cells (the passage number at the time of supernatant extraction was P8) · Cell seeding density at the time of passage: 5000 cells / cm 2

[0027] The cells were proliferated and cultured in a medium from Cosmo Bio Co., Ltd. (ADSC-4). At the stage of reaching confluence, the cells were detached by TrypLE Select and passage operations were performed. At the timing of the total passage number 8, the medium composition was switched to the following, and a culture supernatant was prepared. · Culture medium composition: DEME (no-glucose), Gibco, #A1443001 · Type of sugar: Glucose (Nacalai Tesque, #16806-12) Mannitol (Nacalai Tesque, #11662-42) Sugar concentration added to the culture 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 is shown in Figure 2. As shown in Figure 1, even when the glucose concentration in the culture medium was increased from 1 g / L to 4.5 g / L or 9.0 g / L, the cell morphology did not change significantly. However, the cell count was not significantly different between 1 g / L and 4.5 g / L of glucose in the culture medium, but at 9.0 g / L, the cell count increased by about 20% (Figure 2). Also, in the group with 1% mannitol added, there was almost no difference in the cell count between 1 g / L and 4.5 g / L of glucose. In contrast, in the group with 0 g / L of glucose and 100 mg / L of trehalose, the cell count was significantly lower.

[0029] Method for preparing the supernatant When the cells reached confluence at the P8 time point, the existing culture medium (ADSC-4) was removed and the cells were washed twice with PBS. Then, the culture medium with the above composition was added to each cell group and cultured for 48 hours. After culturing, the culture supernatant was collected and adjusted to obtain the culture supernatant by filtering through a 0.22 μm filter.

[0030] Preparation of samples for proteome analysis Sample preparation For the prepared culture supernatant, the following treatments were performed as pretreatment for proteome analysis. An equal volume of 400 mM Tris-HCL pH8.5 and 4% SDS was added to the sample solution, and the sample was treated with a sample-sealed ultrasonic crusher. The protein concentration of this material was measured by BCA assay and adjusted with 100 mM Tris-HCL pH8.5 and 2% SDS to a protein concentration of 1 μg / μL.

[0031] Next, to cleave the S-S bonds of the protein, TCEP was added to the protein lysate (20 μg of protein amount) to a final concentration of 20 mM and incubated at 50 °C for 30 minutes. To alkylate the cysteine residues, iodoacetamide (IAA) was added to a final concentration of 30 mM and incubated at room temperature (in the dark) for 30 minutes.

[0032] Next, Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophylic) and Sera-Mag Carboxylate-Modified Magnetic Particles (Hydrophobic) from Cytiva were mixed at 1:1 (v / v), washed three times with distilled water, and prepared with distilled water to 15 μg solids / μL (SP3 beads). 20 μL of SP3 beads was added to the sample alkylated above, and after adding 2.5 times the volume of the sample solution of ethanol, it was mixed at room temperature for 20 minutes. The beads were washed twice with 80% ethanol, 100 μL of 50 mM Tris-HCL pH8.0 was added and mixed. Next, 500 ng of Trypsin / Lys-C Mix (Promega) was added to fragment the protein into peptide fragments and incubated overnight at 37 °C.

[0033] To this, 20 μL of 5% TFA was added and processed using a sample-sealed ultrasonic disruptor. The obtained processed solution was desalted using a C18 spin column and then dried to solid using a centrifugal evaporator. To the dried solid, 3% ACN - 0.1% formic acid was added, and the peptide was dissolved using a sample-sealed ultrasonic disruptor. The peptide concentration of the solution in which the peptide was dissolved was measured by BCA assay and adjusted with 2% ACN - 0.1% TFA to a peptide concentration of 200 ng / μL. The prepared sample was measured under the following nanoLC-MS / MS analysis conditions.

[0034] Regarding the analysis conditions of nanoLC Amount of injected peptide: 200 ng Used nanoLC: UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific) 0Column size: inner diameter 75 μm × length 120 mm (Nikkai Technos) Column temperature: 40°C Solvent: Solvent A - distilled water added with 0.⁠1% formic acid, Solvent B - 80% CAN added with 0.⁠1% formic acid The gradient conditions are as shown in Table 2

[0035]

Table 2

[0036] Regarding the analysis conditions of MS Used MS: Q Exactive HF-X (Thermo Fisher Scientific) Ionization method: ESI positive mode Measurement time: 40 minutes (measuring the gradient time from 4 to 44 minutes) Type of MS analysis: Overlapping window DIA Scan event: Repeatedly acquire data for the following events 1 - 4 Regarding the analysis conditions of MS 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 obtained MS data was analyzed using Scaffold DIA under the following conditions to identify proteins and peptides and calculate quantitative values. After exporting the analysis results, the data was compiled into an Excel file. Software used: Scaffold DIA (Proteome Software) Protein Sequence Database: HumanUniProtKB / Swiss-Prot database (UP000005640) Spectral Library: A library was created from the above sequence database by Prosit (https: / / www.proteomicsdb.org / prosit / ) 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] By increasing the glucose concentration from 1 g / L to 4.5 g / L or 9.5 g / L, the increased useful components and decreased components are shown below (Tables 5 and 6).

[0042] [Table 5]

[0043] [Table 6]

[0044] By increasing the glucose concentration in the medium from 1 g / L to 4.5 g / L, the proteins whose values became more than twice 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, 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, Legumain alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, Beta-enolase, 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, 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] When cultured with the glucose concentration in the medium increased from 1 g / L to 9 g / L, the proteins whose values increased by 2-fold or more 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, 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 coagulationfactor 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, typeII cytoskeletal 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 triggering 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, 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 Qprotein), Transmembrane protein 132A, Ubiquitin-conjugating enzyme E2 R2, Keratin, type II cytoskeletal 5, Endoribonuclease LACTB2, Peptidyl-prolyl cis-trans isomerase FKBP14, CCR4-NOT transcription complex subunit 1, Phosphopantothenate--cysteine ligase, Golgi-resident adenosine 3',5'-bisphosphate 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 C3, Importin-9, Vacuolar protein-sorting-associated protein25), ubiquitin domain-containing protein UBFD1 (Ubiquitin domain-containing protein UBFD1), adenylyl cyclase-associated protein 2 (Adenylyl cyclase-associated protein 2), ataxin-10 (Ataxin-10), TRIO and F-actin-binding protein (TRIO and F-actin-binding protein), thioredoxin-like protein 4A (Thioredoxin-like protein 4A), keratin, type II cytoskeletal 73 (Keratin, type II cytoskeletal 73), diablo homolog, mitochondrial (Diablo homolog, mitochondrial), beta-enolase (Beta-enolase), SPRY domain-containing protein 4 (SPRY domain-containing protein 4), RNA-binding protein Raly (RNA-binding protein Raly), vitronectin (Vitronectin), collagen alpha-3(V) chain (Collagen alpha-3(V) chain), phenylalanine--tRNA ligase beta subunit (Phenylalanine--tRNA ligase beta subunit), WD repeat-containing protein 82 (WD repeat-containing protein 82), anamorsin (Anamorsin), retinoid-inducible serine carboxypeptidase (Retinoid-inducible serine carboxypeptidase), dipeptidyl peptidase 4 (Dipeptidyl peptidase 4), ethylmalonyl-CoA decarboxylase (Ethylmalonyl-CoA decarboxylase), dipeptidyl peptidase 9 (Dipeptidyl peptidase 9), thymidine phosphorylase (Thymidine phosphorylase), U8 U8 snoRNA-decapping enzyme (snoRNA-decapping enzyme), Ran-binding protein 3 (Ran-binding protein 3), GTPase Nas (GTPaseNRas), 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, Migration and invasion enhancer 1, Transforming growth factor beta-1 proprotein, Kin of IRRE-like protein 1, Inositol polyphosphate 1-phosphatase, CAD protein, Enoyl-CoA hydratase, mitochondrial, Spermine synthase, Glutathione S-transferase LANCL1, Cellular retinoic acid-binding protein 2, Acidceramidase), 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, 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 subunit (cAMP-dependent protein(kinase 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, Ubiquitin-like-conjugating enzyme ATG3, Protein YIPF3, L-lactate dehydrogenase A -like 6B (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 LIM, LIM domain only protein 7, Ceramide transfer protein, Charged multivesicular body protein 4b, Microfibril-associated glycoprotein 4, Aspartyl aminopeptidase, NEDD4-like E3 ubiquitin-protein ligase, Exocyst complex component 7, Haptoglobin-related protein, Ubiquitin-conjugating enzyme E2 Q1, Translin-associated protein XX), 39-like calcium-binding protein, 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, a disintegrin and metalloproteinase with thrombospondin motifs 4, beta-1,3-N-acetylglucosaminyltransferase lunatic fringe, 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, the glucose concentration for increasing the useful components can be estimated.

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 the case of culturing in a medium with a glucose concentration of 1 g / L, the method comprising the step of culturing human umbilical cord tissue-derived stem cells in a medium containing a glucose concentration of 4.5 g / L or more and 25 g / L or less for 10 hours or more and 50 hours or less, wherein the useful components are 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 type, desmoglein 1, collagen alpha-1(II) chain, bone morphogenetic protein 4, collagen alpha-1(XVIII) chain, desmoplakin, and desmocollin 2.

2. A method for decreasing the content rate of components in a culture supernatant compared to the case of culturing in a medium with a glucose concentration of 1 g / L, the method comprising the step of culturing human umbilical cord tissue-derived stem cells in a medium containing a glucose concentration of 4.5 g / L or more and 25 g / L or less for 10 hours or more and 50 hours or less, and decreasing the content rate of at least one component 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, ubiquitin-1, charged multivesicular body protein 4a, merlin, and exportin-7 in the culture supernatant.

3. A method for producing a culture supernatant, comprising a step of culturing human umbilical cord tissue-derived stem cells for 10 hours or more and 50 hours or less in a medium containing a glucose concentration of 4.5 g / L or more and 25 g / L or less to increase useful components in the culture supernatant, wherein the useful components are 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, desmoglein 1, collagen alpha-1 (II) chain, bone morphogenetic protein 4, collagen alpha-1 (XVIII) chain, desmoplakin, and desmocollin 2, and the method is characterized in that the useful components are one or more components included in the group consisting of these components.

4. A method for producing a culture supernatant, comprising a step of culturing human umbilical cord tissue-derived stem cells for 10 hours or more and 50 hours or less in a medium containing a glucose concentration of 4.5 g / L or more and 25 g / L or less to reduce the content rate of components in the culture supernatant, and reducing the content rate of at least one component 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, ubiquitin-1, charged multivesicular body protein 4a, merlin, and exportin-7 in the culture supernatant.

5. The method for producing a culture supernatant according to claim 3, 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 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, desmoglein 1, collagen alpha-1 (II) chain, bone morphogenetic protein 4, collagen alpha-1 (XVIII) chain, desmoplakin, and desmocollin 2.

6. A culture supernatant produced by the production method according to claim 3 or 4.

7. A composition comprising the culture supernatant according to claim 6.

8. The composition according to claim 7, wherein the composition is a cosmetic composition, a food composition, a medical composition, and / or a dental composition.

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