Cell activator

Abstract

A cell activator comprising an isolated component isolated from a culture supernatant of deciduous tooth pulp stem cells, where the isolated component does not contain less than 3.5 kDa.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480035212.5 (22) Application Date 2024.05.10 (30) Priority Data 2023-086930 2023.05.26 JP (85) PCT International Application Entering National Phase Date 2025.11.26 (86) PCT International Application Application Data PCT / JP2024 / 017330 2024.05.10 (87) PCT International Application Publication Data WO2024 / 247651 JA 2024.12.05 (71) Applicant: Yufakto Co., Ltd. Address: Tokyo, Japan (72) Inventors: Shu Yujing, Hori Keigo, Seta Yasuhiro, Otake Kento, Teramura Yuji, Oba Yoshiro, Kuramochi Akiko (74) Patent Agency: Beijing Yingsaijia Hua Intellectual Property Agency Co., Ltd. 11204 Patent Attorneys: Wang Dazuo, He Ke (51) Int.Cl. A61K 35 / 28 (2015.01) A61P 43 / 00 (2006.01) (54) Invention Title: Cell Activator (57) Abstract: A cell activator comprising a separated component obtained from the culture supernatant of deciduous dental pulp stem cells, wherein the separated component does not contain a component less than 3.5 kDa. Claims 1 page, Description 10 pages, Drawings 26 pages, CN 121218999 A 2025.12.26 CN 1 21 21 89 99 A 1. A cell activator comprising a separated component obtained from the culture supernatant of deciduous dental pulp stem cells, wherein the separated component does not contain any component less than 3.5 kDa. 2. The cell activator of claim 1, wherein the separated component contains a component of 50 kDa to 100 kDa and does not contain any component less than 30 kDa. 3. The cell activator of claim 1 or 2, wherein the separated component does not contain any component greater than 100 kDa and / or a component of 30 kDa to 50 kDa. 4. The cell activator of any one of claims 1 to 3, wherein the deciduous dental pulp stem cells are primary cultured cells. 5. The cell activator of any one of claims 1 to 3, wherein the deciduous dental pulp stem cells are immortalized cells. 6. A method for activating cells in a subject, comprising administering the subject the cell activator according to any one of claims 1 to 5. 7. Use of a separated component obtained from the culture supernatant of deciduous dental pulp stem cells as a cell activator, wherein the separated component does not contain any component less than 3.5 kDa. 8. The use according to claim 7, wherein the separated component contains a component of 50 kDa to 100 kDa and does not contain any component less than 30 kDa.9. The use as described in claim 7 or 8, wherein the separated component does not contain components greater than 100 kDa and / or components of 30 kDa to 50 kDa. 10. The use as described in any one of claims 7 to 9, wherein the deciduous tooth pulp stem cells are primary cultured cells. 11. The use as described in any one of claims 7 to 9, wherein the deciduous tooth pulp stem cells are immortalized cells. Claims 1 / 1 page 2 CN 121218999 A Cell Activator Technical Field

[0001] This invention relates to a cell activator. Background Art

[0002] It is well known that the culture supernatant of mesenchymal stem cells (MSCs) contains a wide variety of growth factors and cytokines, and is currently expected to play a role in a wide range of fields from cosmetics to regenerative medicine. MSCs can be easily collected from a wide variety of tissues such as bone marrow, dental pulp, adipose tissue, and umbilical cord. In particular, deciduous tooth pulp stem cells, which can be collected from deciduous teeth, can be easily prepared from previously discarded deciduous teeth and have high proliferative capacity, and are therefore particularly expected to be useful MSCs.

[0003] To date, the culture supernatant of deciduous dental pulp stem cells has been reported to be useful for the repair of damaged tissues such as those from cerebral infarction and for the protection of skin tissue (Patent Documents 1 and 2). However, it is not yet clear which component in the culture supernatant is the effective component. In addition, the culture supernatant may also contain components that inhibit the effective component, but this is also not yet clear.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 2011 / 118795

[0007] Patent Document 2: International Publication No. 2014 / 126176 Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] The object of the present invention is to purify the culture supernatant of deciduous dental pulp stem cells and provide a composition with excellent cell activation effect.

[0010] Means for Solving the Problem

[0011] The inventors' in-depth research has resulted in the identification of isolated components with high cell activation activity and isolated components that inhibit cell activation activity in the culture supernatant of deciduous dental pulp stem cells, thereby achieving the present invention.

[0012] That is, the present invention provides a cell activator based on one embodiment, which comprises isolated components obtained from the culture supernatant of deciduous dental pulp stem cells, wherein the isolated components do not contain components less than 3.5 kDa.

[0013] In addition, the present invention provides, based on one embodiment, the use of isolated components obtained from the culture supernatant of deciduous dental pulp stem cells as a cell activator, wherein the isolated components do not contain components less than 3.5 kDa.

[0014] Preferably, the isolated components contain components of 50 kDa to 100 kDa and do not contain components less than 30 kDa.

[0015] Preferably, the separated components do not contain components greater than 100 kDa and / or components of 30 kDa to 50 kDa.

[0016] The deciduous tooth pulp stem cells can be primary cultured cells.

[0017] Alternatively, the deciduous tooth pulp stem cells can be immortalized cells.

[0018] In addition, the present invention provides a method for activating cells in a subject (object) based on one embodiment, which includes the step of administering the above-mentioned cell activator to the subject (object).

[0019] Effects of the Invention

[0020] The cell activator of the present invention can be easily and inexpensively prepared from the culture supernatant of deciduous tooth pulp stem cells, and does not contain components that inhibit cell activation activity, thus exhibiting excellent efficacy and safety and being useful. Brief Description of the Drawings

[0021] Figure 1 is a polyacrylamide gel electrophoresis image of SHED-CM and its separated components.

[0022] Figure 2 is a polyacrylamide gel electrophoresis image of IM-SHED-CM and its separated components.

[0023] Figure 3 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with SHED-CM isolates at different concentration ratios.

[0024] Figure 4 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with IM-SHED-CM isolates at different concentration ratios.

[0025] Figure 5 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a dilution of SHED-CM or its components adjusted according to protein concentration.

[0026] Figure 6 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a dilution of IM-SHED-CM or its isolates adjusted according to protein concentration.

[0027] Figure 7 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a 50kDa-100kDa isolate from SHED-CM, supplemented with an isolate greater than 100kDa or a dilution of an isolate from 30kDa-50kDa.

[0028] Figure 8 is a graph showing the WST assay results for evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a 50kDa-100kDa isolate from IM-SHED-CM, supplemented with an isolate greater than 100kDa or a dilution of an isolate from 30kDa-50kDa.

[0029] Figure 9 is a graph showing the WST assay results for NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or its isolates.Figure 10 is a graph showing the results of Western blot analysis comparing the expression level and phosphorylation of MAPK in NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or its isolated components.

[0031] Figure 11 is a graph showing the results of Western blot analysis comparing the expression level and phosphorylation of MAPK in NIH3T3 cells cultured with the same protein concentration of SHED-CM / IM-SHED-CM or its isolated components.

[0032] Figure 12 is a graph showing the results of Western blot analysis comparing the expression level and phosphorylation of Akt in NIH3T3 cells cultured with the same protein concentration of SHED-CM / IM-SHED-CM or its isolated components.

[0033] Figure 13 is a graph showing the results of a scratch assay for NIH3T3 cells cultured with IM-SHED-CM or its isolated components.

[0034] Figure 14 is a graph showing the WST assay results evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with IM-SHED-CM or its greater than 3.5 kDa fraction.

[0035] Figure 15 is a graph showing the scratch assay results of NIH3T3 cells cultured with IM-SHED-CM or its greater than 3.5 kDa fraction.

[0036] Figure 16 is a graph showing the WST assay results evaluating the intracellular dehydrogenase activity of HUVECs cultured with a dilution of SHED-CM or its 50 kDa to 100 kDa fraction adjusted according to protein concentration.

[0037] Figure 17 is a graph showing the WST assay results evaluating the intracellular dehydrogenase activity of HUVECs cultured with a dilution of IM-SHED-CM or its 50 kDa to 100 kDa fraction adjusted according to protein concentration.

[0038] Figure 18 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with 50kDa-100kDa isolates containing SHED-CM, or a mixture of isolates greater than 100kDa, 50kDa-100kDa, and 30kDa-50kDa.

[0039] Figure 19 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with 50kDa-100kDa isolates containing SHED-CM, or a mixture of isolates less than 30kDa.

[0040] Figure 20 shows the intracellular dehydrogenase activity of HUVECs cultured with 50kDa-100kDa fractions of IM-SHED-CM, or a mixture of fractions greater than 100kDa, 50kDa-100kDa, and 30kDa-50kDa fractions.

[0041] Figure 21 shows the intracellular dehydrogenase activity of HUVECs cultured with 50kDa-100kDa fractions of IM-SHED-CM, or a mixture of fractions of 50kDa-100kDa and less than 30kDa fractions.

[0042] Figure 22 shows the WST assay results evaluating the effect of IM-SHED-CM on inhibiting cell damage caused by hydrogen peroxide treatment.

[0043] Figure 23 shows the WST assay results evaluating the effect of IM-SHED-CM's 50kDa-100kDa fraction on inhibiting cell damage caused by hydrogen peroxide treatment.

[0044] Figure 24 is a graph showing the WST measurement results evaluating the inhibitory effect of the isolated component of IM-SHED-CM with a concentration greater than 100 kDa on cell damage caused by hydrogen peroxide treatment.

[0045] Figure 25 is a graph showing the WST measurement results evaluating the inhibitory effect of the isolated component of IM-SHED-CM with a concentration of 30 kDa to 50 kDa on cell damage caused by hydrogen peroxide treatment.

[0046] Figure 26 is a graph showing the results of quantitative analysis of the recovery of scratches in Figures 13 and 15. Detailed Description

[0047] Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described in this specification.

[0048] Based on the first embodiment, the present invention is a cell activator comprising isolated components obtained from the culture supernatant of deciduous dental pulp stem cells, wherein the isolated components do not contain components with a concentration less than 3.5 kDa.

[0049] In this embodiment, "cell activation" (and variations thereof) means normalizing or enhancing the functions of cell proliferation and metabolism. Therefore, "cell activation" in this embodiment may include, for example, the activation of enzymes related to cell proliferation and metabolism (various dehydrogenases, Akt, AMPK, etc.) and enzymes that inhibit oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), etc.). Here, "cell" can be the cell of any vertebrate, preferably the cell of mammals such as mice, rats, rabbits, pigs, cattle, monkeys, and humans, and particularly preferably human cells. In addition, there is no particular limitation on the type of cell, such as fibroblasts, endothelial cells, epithelial cells, nerve cells, stem cells, leukocytes, osteocytes, muscle cells, adipocytes, etc., preferably fibroblasts or endothelial cells.

[0050] The so-called "deciduous tooth pulp stem cells" are mesenchymal stem cells collected from the pulp of deciduous teeth. The deciduous tooth pulp stem cells of this embodiment can be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, pigs, cattle, monkeys, and humans, and particularly preferably from humans.

[0051] Methods for preparing deciduous tooth pulp stem cells have been established (e.g., International Publication No. 2011 / 118795), and deciduous tooth pulp stem cells can be isolated and prepared from the pulp of deciduous teeth according to methods known in the art.

[0052] The deciduous tooth pulp stem cells of this embodiment can be primary cultured cells or immortalized cells. "Immortalized cells" means cells that retain their proliferative capacity even after undergoing a certain number of divisions, i.e., cells have unlimited proliferative capacity. Methods for immortalizing cells have been established, and known methods can be used. For example, cells can be immortalized by introducing the SV40T antigen gene, telomerase reverse transcriptase (TERT) gene, etc.

[0053] Methods for culturing deciduous tooth pulp stem cells have been established (e.g., International Publication No. 2011 / 118795), CN 121218999 A, page 3 / 10 of the specification, and deciduous tooth pulp stem cells can be cultured according to methods known in the art. Specifically, deciduous tooth pulp stem cells can be cultured by seeding them at a concentration of, for example, 5 × 10⁴ to 2 × 10⁵ cells / mL in a culture medium obtained by appropriately adding serum such as FBS or serum substitutes such as KSR, glucose, amino acids, vitamins, etc., to a basal culture medium such as DMEM, IMDM, Ham's F-12, RPMI-1640, or mixtures thereof. The cell activator of this embodiment is preferably prepared from the culture supernatant obtained by culturing deciduous tooth pulp stem cells in a serum-free culture medium.

[0054] The cell activator of this embodiment does not contain components of less than 3.5 kDa derived from the culture supernatant of deciduous tooth pulp stem cells. Here, "free of" means that the component to be removed was not detected by conventional detection methods (such as immunochemical methods). Therefore, the cell activator of this embodiment can be a substance obtained by removing at least 85% by weight, 90% by weight or more, 95% by weight or more, 97% by weight or more, 98% by weight or more, or 99% by weight or more of the component less than 3.5 kDa contained in the unpurified culture supernatant. In other words, the cell activator of this embodiment can contain, at most, 15% by weight, less than 10% by weight, less than 5% by weight, less than 3% by weight, less than 2% by weight, or less than 1% by weight of the component less than 3.5 kDa contained in the unpurified culture supernatant. By removing the component less than 3.5 kDa from the culture supernatant of deciduous dental pulp stem cells, the cell activator of this embodiment shows an improved cell activation effect.

[0055] In this embodiment, "kDa" can be calculated, for example, based on SDS-PAGE, ultrafiltration, etc., and when the culture supernatant is purified using a commercially available ultrafiltration filter unit, it can be the nominal molecular weight cutoff (NMWL) or molecular weight cutoff (MWCO) value indicated on the product label.

[0056] The cell activator of this embodiment preferably contains a separation component containing 50kDa to 100kDa; on the other hand, it is preferable not to contain a separation component containing less than 30kDa. The cell activator of this embodiment is more preferably free of separation components containing more than 100kDa and / or separation components containing 30kDa to 50kDa. This is because components with 50kDa to 100kDa have higher cell activation activity, and on the other hand, there is also the situation where not only components less than 3.5kDa but also components greater than 100kDa, and components with 30kDa to 50kDa, can also adversely affect the cell activation activity of components with 50kDa to 100kDa.

[0057] For example, the cell activator of this embodiment can be prepared by using ultrafiltration membranes of 100,000 MWCO, 50,000 MWCO, 30,000 MWCO, and 3,500 MWCO in combination. For example, by using a 100,000 MWCO ultrafiltration membrane to remove components greater than 100 kDa from the culture supernatant, and by using a 50,000 MWCO ultrafiltration membrane to concentrate the obtained filtrate, a cell activator containing components of 50 kDa to 100 kDa from the culture supernatant of deciduous dental pulp stem cells and excluding components of 30 kDa to 50 kDa and components less than 30 kDa can be prepared.

[0058] The cell activator of this embodiment may consist only of the isolated components purified from the culture supernatant of deciduous dental pulp stem cells, but generally, it may also contain pharmaceutically permissible carriers and additives as optional components.

[0059] The cell activator of this embodiment can be prepared as a liquid or solid formulation, for example, it can be formulated into various dosage forms such as injections, gels, sprays, tablets, and granules, and is preferably prepared as a liquid formulation. When prepared as a liquid formulation, sterile water, physiological saline, glucose solution, etc. can be used as a carrier, and bactericides, isotonic agents, stabilizers, etc. can also be added as desired. When prepared as a solid formulation, additives such as starch, lactose, mannitol, and inorganic salts can be added, and binders, disintegrants, lubricants, etc. can also be added as desired.

[0060] When the total mass of the cell activator is set to 100%, the content of the 50kDa to 100kDa component (converted to protein weight) of the culture supernatant of deciduous tooth pulp stem cells in the cell activator of this embodiment can be, for example, 60kDa.The concentrations are 70% or more, 80% or more, 90% or more, and preferably 60-70% by weight.

[0061] The cell activator of this embodiment contains components with cell activating activity in the culture supernatant derived from deciduous tooth pulp stem cells and does not contain components that inhibit the activation. Therefore, the cell activator of this embodiment has a superior cell activation effect compared to the unpurified culture supernatant of deciduous tooth pulp stem cells (page 4 / 10, CN 121218999 A), and is useful.

[0062] According to the second embodiment of the present invention, it is a method for activating cells in a subject, which includes the step of administering the above-mentioned cell activator to the subject. In this embodiment, "activating cells" and "cells" are the same as those defined in the first embodiment.

[0063] In this embodiment, "subject" can be any vertebrate, preferably a mammal such as a mouse, rat, rabbit, pig, cow, monkey, or human, and is particularly preferably a human. The subject can be of any age, including infants, children, adolescents, young adults, adults, and elderly subjects.

[0064] The cell activator of the method of this embodiment can be an activator formulated into various dosage forms as defined in the first embodiment, and can be administered by any method suitable for the dosage form, such as oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, nasal administration, intraperitoneal administration, direct injection into the target tissue, inhalation administration, etc. The dosage of the cell activator can vary depending on the age, weight, health status, etc. of the subject, for example, it can be 0.1 mg / kg to 10 mg / kg (body weight), preferably 0.5 mg / kg to 2 mg / kg (body weight). The dosage can be administered once or in multiple doses.

[0065] According to the third embodiment of the present invention, a separated component obtained from the culture supernatant of deciduous dental pulp stem cells is used as a cell activator, the separated component containing a component of 50 kDa to 100 kDa and not containing a component of less than 30 kDa. In this embodiment, "deciduous dental pulp stem cells", "cell activator", "cell", "not containing", and "kDa" are the same as those defined in the first embodiment. According to the description in the first and second embodiments, a separated component of the culture supernatant derived from deciduous dental pulp stem cells containing 50kDa to 100kDa and not containing components less than 30kDa can be used as a cell activator.

[0066] Examples

[0067] The present invention will be further illustrated below by way of examples. In addition, these examples, etc., do not limit the present invention in any way.

[0068] <Materials and Reagents>

[0069] NIH3T3 cells were purchased from the JCRB cell bank. Human umbilical vein endothelial cells were purchased from Lonza.HUVEC cells, EBMTM-2 basal medium (CC-3156), and EGMTM-2 supplement kit (CC-4176) were used. Duchenne modified Eagle medium (DMEM) was purchased from GIBCO. Fetal bovine serum (hereinafter referred to as "CS") and antifungal agent solution (100×) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and 0.05% trypsin / EDTA solution were purchased from Invitrogen. Charcoal (powder, activated) was purchased from NACALAI TESQUE. Duchenne phosphate-buffered saline (PBS), RIPA buffer, and Quick CBB staining solution were purchased from Fujifilm and Koden Chemical Co., Ltd. Ultrafiltration units (Vivaspin Turbo 15 membranes, 30,000, 50,000, and 100,000 MWCO) were purchased from Sartorius. Millex-GV low-protein adsorption Durapore (PVDF) membranes (0.22 μm) were purchased from Merck Millipore. Spectra / Por™ 7 dialysis membranes (MWCO: 3.5 kD) were purchased from Repulgen. PD-10 columns were purchased from Cytiva. Micro BCA protein assay kits were purchased from Thermo Fisher Scientific. Cell Counting Kit-8 was purchased from Tongjin Chemical Research Institute. Acrylamide, protein standard labels, and Clarity Western ECL substrates were purchased from Bio-Rad. The protease inhibitor Complete (#11 697 498 001) was purchased from Roche. Anti-phospho p44 / 42 antibody (#9106) and anti-phospho Akt antibody (#9271) were purchased from Cell Signaling Technology. Anti-mouse IgG-HRP antibody (#62-6520) and anti-rabbit IgG-HRP antibody (#65-6120) were purchased from Invitrogen. Instructions for use, page 5 / 10, CN 121218999 A

[0070] < 1. Preparation of stem cell culture supernatant and its separated components derived from deciduous dental pulp>

[0071] (1-1) Preparation of stem cells (SHED) derived from human deciduous dental pulp

[0072] 5% chlorhexidine solution (Yamazen Pharmaceutical Co., Ltd.) or 10% povidone-iodine solution (Iwaki Pharmaceutical Co., Ltd.) were used to...After disinfection, the crowns of avulsed or extracted deciduous teeth were separated, and dental pulp tissue was recovered using a dental reamer. The pulp tissue was suspended in DMEM containing 10% FBS, with the addition of 2 mg / mL collagenase (Fujifilm and Hikari Pure Chemicals Co., Ltd.) and dispersant enzyme (Fujifilm and Hikari Pure Chemicals Co., Ltd.), and incubated at 37°C for 1 hour. The cells were centrifuged at 777×g for 5 minutes, the supernatant was removed, and the pulp cells were recovered. The pulp cells were then suspended in DMEM (4 mL) containing 10% FBS and 1% Gibco™ Antibiotic-Antimycotic and seeded into 6-well plates. The cells were cultured at 37°C and 5% CO2 until they reached a subconfluent state. Add 0.05% trypsin / EDTA solution, incubate at 37°C for 5 minutes, recover cells, and seed them into 10cm adherent cell trays (VIOLAMO). Subsequently, three passage cultures were performed to allow cell proliferation to reach approximately 1×10⁷ cells. Add 0.05% trypsin / EDTA solution, incubate at 37°C for 5 minutes, and the recovered cells were named "SHED (Stem cells from Human Exfoliated Deciduous teeth)".

[0073] (1-2) Preparation of stem cells derived from immortalized human deciduous tooth pulp (IM-SHED)

[0074] The immortalized SHED (IM-SHED) obtained by introducing the SV40 gene using a viral vector was commissioned to Applied Biological Materials for preparation.

[0075] (1-3) Preparation of Culture Supernatant

[0076] The SHED or IM-SHED prepared above were passaged in DMEM containing 10% FBS. In the preparation of culture supernatant, SHED after up to 8 passages was used, and IM-SHED after 50-55 passages was used. The culture medium was replaced with serum-free DMEM and cultured for 48 hours. Thereafter, the culture medium was recovered and filtered through a separation membrane (Stericup Quick Release-GP, PVDF, 0.22 μm; Merck Millipore) to obtain the culture supernatant (acclimation medium: CM). Hereinafter, the culture supernatant from SHED will be referred to as "SHED-CM", and the culture supernatant from IM-SHED will be referred to as "IM-SHED-CM".

[0077] (1-4) Separation and concentration of culture supernatant

[0078] CM was separated by centrifugation at 1000 rpm for 3 minutes at room temperature and by centrifugation at 500×g for 30 minutes at 4°C.The sample was centrifuged at 2000×g for 30 minutes at 4°C and filtered through a 0.22μm Millex-GV low protein adsorption and binding rate durapore (PVDF) membrane to remove impurities such as cells. Subsequently, the CM was fed to a 100000MWCO ultrafiltration unit and centrifuged at 2000×g for 20 minutes at 4°C. The filtrate (hereinafter referred to as "PT") was fed to a 50000MWCO ultrafiltration unit and centrifuged at 2000×g for 20 minutes at 4°C. The PT was then fed to a 30000MWCO ultrafiltration unit and centrifuged at 2000×g for 20 minutes at 4°C.

[0079] Furthermore, 10 mL of PBS was added to the aforementioned 100000MWCO ultrafiltration unit, and the sample was centrifuged at 2000×g for 20 minutes at 4°C. This operation was repeated 5 times to obtain a concentrated solution of the separated components with a concentration greater than 100 kDa. The PT obtained here was provided to the aforementioned 50000MWCO ultrafiltration unit and centrifuged at 2000×g for 20 minutes at 4°C to obtain a concentrated solution of the separated fractions of 50kDa to 100kDa. The PT was provided to the aforementioned 30000MWCO ultrafiltration unit and centrifuged at 2000×g for 20 minutes at 4°C to obtain a concentrated solution of the separated fractions of 30kDa to 50kDa. The PT was provided to a PD-10 column, and the buffer was replaced with PBS to obtain a separated fraction solution of less than 30kDa. After freeze-drying the concentrated solution of the separated fractions, it was dissolved in Milli-Q ultrapure water, and sample buffer (with or without SDS) was added. Electrophoresis was performed using a 12.5% ​​polyacrylamide gel.

[0080] Figure 1 shows the electrophoresis results of SHED-CM and its separated fractions, and Figure 2 shows the electrophoresis results of IM-SHED-CM and its separated fractions. It was confirmed that both SHED-CM and IM-SHED-CM contained a significant amount of protein components in the 50kDa-100kDa fraction.

[0081] < 2. Cell proliferation and viability activation using SHED-CM / IM-SHED-CM fractions (WST assay) >

[0082] Activated carbon-treated CS (hereinafter referred to as "CH-CS") was prepared by the following steps: 10mg of activated carbon was added to 1mL of CS, and a gentle vortex was performed for 15 seconds. After incubation at 37°C for 10 minutes, the cells were centrifuged at 12000rpm at 4°C for 20 minutes, and the supernatant was provided to a 0.22μm PVDF filter to obtain CH-CS. NIH3T3 cells were seeded into 96-well plates (VIOLAMO) (5000 cells / well) and cultured overnight in 10% CS / DMEM.After being subjected to serum starvation, the cells were washed once with DMEM, and then the culture medium was replaced with 0.4% CH-CS / DMEM and cultured for 24 hours. Subsequently, the isolated components prepared in step 1 were diluted with 0.4% CH-CS / DMEM and added, and the cells were cultured for 44 hours. After adding 10 μL / well of WST solution from Cell Counting Kit-8, the cells were cultured for another 4 hours, and the absorbance at 450 nm was measured.

[0083] The results are shown in Figures 3 and 4. The increase in absorbance at 450 nm indicates an increase in the activity of intracellular dehydrogenases, which can be interpreted as a higher absorbance at 450 nm indicating higher cell proliferation and survival. In the figures, "10%CS" means 10% CS / DMEM, and "CM×1 / 2 dil." means the culture medium after CM was diluted by 1 / 2 with 0.4% CH-CS / DMEM. Based on these results, it was shown that the 50kDa to 100kDa fractions of SHED-CM and IM-SHED-CM both contained components that activate cell proliferation and viability. The same results were obtained for HeLa cells derived from human cervical cancer cell lines, mesenchymal stem cells derived from human bone marrow, and human iPS cells (409B2 line) (data omitted).

[0084] Furthermore, Figures 5 and 6 show the results of comparing the activity of each fraction with the same protein concentration. In the figures, "calculated value" represents the value obtained by correcting the CM results based on the electrophoresis results in section 1 above and the protein concentration quantification results from the Micro BCA Protein Assay Kit. It was thus determined that only the 50kDa to 100kDa fractions of the fractions purified from SHED-CM and IM-SHED-CM contained components that activate cell proliferation and viability.

[0085] < 3. Inhibition of Cell Proliferation and Viability by SHED-CM / IM-SHED-CM Separation Components >

[0086] The 50kDa-100kDa separation component prepared in 1 above was prepared using 0.4% CH-CS / DMEM, a separation component greater than 100kDa, or a 30kDa-50kDa separation component diluted with the same steps as in item "2" above. Intracellular dehydrogenase activity was evaluated.

[0087] The results are shown in Figures 7 and 8. Intracellular dehydrogenase activity was reduced by mixing the 50kDa-100kDa separation component with a separation component greater than 100kDa or a 30kDa-50kDa separation component. In particular, the 30kDa-50kDa separation component derived from IM-SHED showed a greater inhibitory effect (Figure 8), suggesting the presence of inhibitory effects on cell proliferation and viability.Factors related to survival.

[0088] < 4. Activation of intracellular signal transduction in SHED-CM / IM-SHED-CM isolated components >

[0089] NIH3T3 cells were seeded into 12-well plates (6 × 10⁴ cells / well) and cultured overnight in 10% CS / DMEM. To induce serum starvation, the cells were washed once with DMEM, and the medium was replaced with 0.4% CH-CS / DMEM and cultured for 24 hours. Subsequently, the CM or its isolated components prepared in step 1 above were diluted with 0.4% CH-CS / DMEM and this dilution was added. 10% FBS or human TNFα (PeproTech) (50 ng / mL) was added to the control. After culturing for 30 minutes, the cells were washed twice with PBS and lysed using RIPA buffer with added protease inhibitors. The cell lysate was recovered into a microtube and shaken at 4°C for 30 minutes. The protein was centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was recovered. Sample buffer containing SDS was added, and the mixture was incubated at 95°C for 5 minutes, followed by electrophoresis on a 10% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk / TBST solution (room temperature, 1 hour). The nitrocellulose membrane was then incubated overnight at 4°C in a primary antibody solution. After removing the primary antibody solution, the nitrocellulose membrane was washed three times with TBST and incubated at room temperature for 1 hour in a secondary antibody solution. As primary antibodies, anti-phospho p44 / 42 antibody (1:2000) or anti-phospho Akt antibody (1:2000) was used; as secondary antibodies, anti-mouse IgG-HRP antibody (1:5000) or anti-rabbit IgG-HRP antibody (1:5000) was used. Clarity Western ECL substrate was used to detect the reaction.

[0090] The results are shown in Figures 9 and 10. Due to the addition of the 50kDa–100kDa isolate, the expression levels and phosphorylation of MAPK and Akt increased significantly. In addition, Figures 11 and 12 show the results of comparing CM prepared to the same protein concentration with the 50kDa–100kDa isolate. Compared with CM, an increase in phosphorylation of the 50kDa–100kDa isolate was confirmed. Based on these results, it is shown that the 50kDa–100kDa isolate contains components that activate intracellular phosphorylation signaling.

[0091] <5. Activation of cell migration by SHED-CM / IM-SHED-CM isolate (scratch assay)>

[0092] NIH3T3 cells were seeded into 48-well plates (3.75 × 10⁴ cells / well) and cultured overnight in 10% CS / DMEM. To induce serum starvation, the cells were washed once with DMEM, and the medium was replaced with 0.4% CH-CS / DMEM, and cultured for 24 hours. Scratches were created by damaging the cell monolayer in a straight line using a 200 μL pipette tip. After washing twice with DMEM, CM (using 0.4% CH-CS / DMEM, 1 / 2 dilution), 10% FBS / DMEM, or each of the isolated fractions (greater than 100 kDa, 50 kDa–100 kDa, or 30 kDa–50 kDa) were added (diluted with 0.4% CH-CS / DMEM to achieve a 5-fold protein concentration for each isolated fraction) and cultured for 24 hours.

[0093] The results are shown in Figure 13 (microscopic image). Furthermore, Figure 26 shows the results of numerical and quantitative analysis of cell area in the microscopic image using ImageJ software. It was confirmed that the 50kDa–100kDa fraction maintained wound healing activity equivalent to CM.

[0094] <6. Preparation and analysis of fractions of IM-SHED-CM with a concentration of 3.5kDa or higher>

[0095] IM-SHED-CM was encapsulated in a Spectra / Por™ 7 dialysis membrane (MWCO: 3.5kDa) and dialyzed against 100 times the volume of Milli-Q ultrapure water for 72 hours. The Milli-Q ultrapure water was replaced 8 times during this period. The dialyzed solution was freeze-dried, dissolved in 1 / 10 of the starting volume of PBS (on ice, for more than 30 minutes), and filtered using a 0.22μm PVDF filter. The activity of the obtained fractions with a concentration of 3.5kDa or higher against intracellular dehydrogenases and cell migration was evaluated using the same steps as in item “2” and item “5” above.

[0096] Figure 14 shows the results of the WST assay. The fraction prepared at a high protein concentration (100 μg / mL) of 3.5 kDa or higher activated intracellular dehydrogenases compared to CM prepared at the same protein concentration. Figure 15 shows the results of the scratch assay, and Figure 26 shows the results of its quantitative analysis. The fraction prepared at 3.5 kDa or higher significantly increased cell migration to the scratch. Based on these results, it is shown that by removing the fraction prepared at less than 3.5 kDa, cell activation effects equivalent to or greater than those of CM can be obtained.

[0097] < 7. Cell proliferation and viability activation of SHED-CM / IM-SHED-CM fraction (2) >

[0098] Using HUVEC instead of NIH3T3 cells, the cell proliferation and viability of cells prepared from SHED-CM were evaluated using the same steps as in item "2" above.Intracellular dehydrogenase activity of the 50kDa-100kDa fraction purified from IM-SHED-CM was activated. HUVECs were proliferated in 10cm dishes using EGMTM-2 basal medium (CC-3156) and EGMTM-2 supplemented medium (hereinafter referred to as "EGM-2 medium") until 80-90% confluence. Cells were then removed using 0.05% trypsin / EDTA solution. EGM-2 medium was added to suspend the cells, and cell number and viability were determined using a Countess2 automated cell counter and trypan blue. The cell suspension was prepared to a concentration of 25,000 cells / mL. Cells were seeded into 96-well plates (100μL / well). After one day, the medium was replaced with EGM-2 medium supplemented with CM or the 50kDa-100kDa fraction. After 3 days of culture, WST solution (10 μL / well) of Cell Counting Kit-8 was added, and after 3-4 hours of culture, the absorbance at 450 nm was measured.

[0099] The results are shown in Figures 16 and 17. In the figures, "calculated value" represents the value obtained by correcting the CM results based on the electrophoresis results in item "1" above and the protein concentration of each isolated fraction calculated from the protein concentration quantification results using the Micro BCA protein detection kit. Compared with CM at the same protein concentration, the 50kDa-100kDa isolated fractions from SHED-CM and IM-SHED-CM increased the intracellular dehydrogenase activity. According to this result, the 50kDa-100kDa isolated fractions from SHED-CM and IM-SHED-CM contain components that activate the proliferation and survival of HUVECs.

[0100] < 8. Inhibition of Cell Proliferation and Viability of SHED-CM / IM-SHED-CM Separation Components (2) >

[0101] Mixtures of separation components with values ​​greater than 100 kDa, 50 kDa to 100 kDa, and 30 kDa to 50 kDa (mixing ratio 1:1:1) and mixtures of separation components with values ​​of 50 kDa to 100 kDa and less than 30 kDa (mixing ratio 1:1) were prepared. The 50 kDa to 100 kDa separation components and the above mixtures were periodically diluted using EGM-2 medium, and intracellular dehydrogenase activity was evaluated using the same steps as in item "7" above.

[0102] Figures 18 and 20 show the results for the separation components and mixtures derived from SHED-CM, and Figures 19 and 21 show the results for the separation components and mixtures derived from IM-SHED-CM. In the figures, "mixtures greater than 100, 50, and 30 kDa" are used.The term "mixture of 50kDa, 30PT" refers to a mixture of isolated components with values ​​greater than 100kDa, 50kDa–100kDa, and 30kDa–50kDa. "Mixed 50kDa, 30PT" refers to a mixture of isolated components with values ​​between 50kDa and 100kDa and less than 30kDa, with "50kDa" referring specifically to isolated components between 50kDa and 100kDa. Compared to isolated components with values ​​between 50kDa and 100kDa, a significantly lower intracellular dehydrogenase activity was observed in the mixed solutions of isolated components greater than 100kDa, 50kDa–100kDa, and 30kDa–50kDa (Figures 18 and 20). Conversely, compared to isolated components with values ​​between 50kDa and 100kDa, a significantly lower intracellular dehydrogenase activity was observed in the mixed solutions of isolated components with values ​​between 50kDa and 100kDa and less than 30kDa (Figures 19 and 21). Based on these results, it was determined that the components in CM with a content less than 30 kDa inhibited the proliferation and survival of HUVECs.

[0103] < 9. Effect of IM-SHED-CM isolated components on inhibiting hydrogen peroxide-induced cell damage >

[0104] HUVECs were proliferated in 10 cm plates using EGM-2 medium until they reached 80-90% confluence. The cells were then removed using 0.05% trypsin / EDTA solution and recovered. EGM-2 medium was added to suspend the cells, and the cell count and cell viability were determined using a Countess2 automated cell counter and trypan blue. The concentration of the cell suspension was prepared to be 50,000 cells / mL. The cells were seeded into 96-well plates (100 μL / well). After 1 day, the medium was replaced with a staged dilution of IM-SHED-CM and its isolated components in EGM-2 medium, and the cells were cultured for 3 days. Hydrogen peroxide (final concentration 0.005–0.00031%) was added to the cells, and they were incubated at 37°C for 1 hour. The diluent was removed, and EGM-2 medium (100 μL / well) and WST solution from Cell Counting Kit-8 (10 μL / well) were added. After culturing for 3–4 hours, the absorbance at 450 nm was measured.

[0105] Figure 22 shows the results for cells treated with a staged dilution of IM-SHED-CM. Cells without IM-SHED-CM (control) showed a decrease in absorbance at 450 nm due to hydrogen peroxide treatment, with a further significant decrease in concentration-dependent hydrogen peroxide concentration. This result confirmed cell damage caused by hydrogen peroxide treatment. On the other hand, although the absorbance at 450 nm of cells treated with a staged dilution of IM-SHED-CM decreased in a concentration-dependent manner, the decrease in absorbance at 450 nm was inhibited compared to the control. This result confirms that IM-SHED-CM inhibits oxidative stress.Cell damage caused by oxidative stress.

[0106] Figure 23 shows the results for cells with staged dilutions of the 50kDa to 100kDa separation component containing IM-SHED-CM (page 9 / 10, CN 121218999 A), Figure 24 shows the results for cells with staged dilutions containing a separation component greater than 100kDa, and Figure 25 shows the results for cells with staged dilutions containing a separation component of 30kDa to 50kDa. For cells with the 50kDa to 100kDa separation component, the absorbance of the separation component at 450nm increased in a protein concentration-dependent manner, indicating that the 50kDa to 100kDa separation component concentration-dependently inhibited cell damage caused by oxidative stress. Instruction manual page 10 / 10, Figure 1 of CN 121218999 A, Figure 2 of CN 121218999 A, Figure 3 of CN 121218999 A, Figure 4 of CN 121218999 A, Figure 5 of CN 121218999 A, Figure 6 of CN 121218999 A, Figure 7 of CN 121218999 A, Figure 8 of CN 121218999 A, Figure 9 of CN 121218999 A, Figure 10 of CN 121218999 A. Figure 11 of 121218999 A, page 10 / 26; Figure 12 of 121218999 A, page 11 / 26; Figure 13 of 121218999 A, page 13 / 26; Figure 14 of 121218999 A, page 14 / 26; Figure 15 of 121218999 A, page 15 / 26; Figure 16 of 121218999 A, page 16 / 26; Figure 17 of 121218999 A, page 17 / 26; Figure 18 of 121218999 A, page 17 / 26; Figure 18 of 121218999 A, page 17 / 26.Page 18 / 26, 30 CN 121218999 A, Figure 19 (Instruction Manual Illustration), Page 19 / 26, 31 CN 121218999 A, Figure 20 (Instruction Manual Illustration), Page 20 / 26, 32 CN 121218999 A, Figure 21 (Instruction Manual Illustration), Page 21 / 26, 33 CN 121218999 A, Figure 22 (Instruction Manual Illustration), Page 22 / 26, 34 CN 121218999 A, Figure 23 (Instruction Manual Illustration), Page 23 / 26, 35 CN 121218999 A, Figure 24 (Instruction Manual Illustration), Page 24 / 26, 36 CN 121218999 A, Figure 25 (Instruction Manual Illustration), Page 25 / 26, 37 CN 121218999 A, Figure 26 (Instruction Manual Illustration), Page 26 / 26, 38 CN 121218999 A

Claims

1. A cell activator comprising a separated component obtained from the culture supernatant of deciduous dental pulp stem cells, wherein the separated component does not contain any component less than 3.5 kDa.

2. The cell activator as described in claim 1, wherein, The separated components contain components with a value of 50 kDa to 100 kDa and do not contain components with a value of less than 30 kDa.

3. The cell activator as described in claim 1 or 2, wherein, The separated components do not contain components with a value greater than 100 kDa and / or components with a value between 30 kDa and 50 kDa.

4. The cell activator according to any one of claims 1 to 3, wherein, The deciduous tooth pulp stem cells mentioned are primary cultured cells.

5. The cell activator according to any one of claims 1 to 3, wherein, The deciduous tooth pulp stem cells are immortalized cells.

6. A method for activating cells in a subject, comprising administering the subject the cell activator of any one of claims 1 to 5.

7. The use of a fraction isolated from the culture supernatant of deciduous dental pulp stem cells as a cell activator, wherein the fraction isolated does not contain any component less than 3.5 kDa.

8. The use as described in claim 7, wherein, The separated components contain components with a value of 50 kDa to 100 kDa and do not contain components with a value of less than 30 kDa.

9. The use as described in claim 7 or 8, wherein, The separated components do not contain components with a value greater than 100 kDa and / or components with a value between 30 kDa and 50 kDa.

10. The use as described in any one of claims 7 to 9, wherein, The deciduous tooth pulp stem cells mentioned are primary cultured cells.

11. The use as described in any one of claims 7 to 9, wherein, The deciduous tooth pulp stem cells are immortalized cells.

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