Cell activator

Abstract

This cell activator comprises a fraction isolated from a culture supernatant of dental pulp stem cells of milk teeth, wherein the fraction does not contain a component of less than 3.5 kDa.

Description

Cell activators

[0001] The present invention relates to a cell activating agent.

[0002] Mesenchymal stem cell (MSC) culture supernatant is known to contain various growth factors and cytokines, and is currently expected to be useful in a wide range of fields, from cosmetics to regenerative medicine. MSCs can be easily extracted from various tissues, such as bone marrow, dental pulp, adipose tissue, and umbilical cord. In particular, deciduous dental pulp stem cells, which are MSCs extracted from deciduous teeth, are considered particularly promising as they can be easily prepared from deciduous teeth that have traditionally been discarded, and have high proliferation potential.

[0003] It has been reported that culture supernatants of deciduous dental pulp stem cells are useful for repairing damaged tissues such as those caused by cerebral infarction and for protecting skin tissues (Patent Documents 1 and 2). However, it is unclear which components in the culture supernatant are the active ingredients. Furthermore, the culture supernatant may contain components that act inhibitoryly against the active ingredients, but these have not been clarified.

[0004] International Publication No. 2011 / 118795 International Publication No. 2014 / 126176

[0005] The present invention has been made with the aim of purifying the culture supernatant of deciduous dental pulp stem cells and providing a composition with excellent cell activation effects.

[0006] As a result of extensive research, the inventors have identified fractions from the culture supernatant of deciduous dental pulp stem cells that have high cell activation activity and fractions that inhibit cell activation activity, thereby completing the present invention.

[0007] That is, according to one embodiment, the present invention provides a cell activation agent comprising a fraction isolated from the culture supernatant of deciduous dental pulp stem cells, wherein the fraction does not contain components less than 3.5 kDa.

[0008] Furthermore, according to one embodiment, the present invention provides the use of a fraction isolated from the culture supernatant of deciduous dental pulp stem cells as a cell activating agent, wherein the fraction does not contain components less than 3.5 kDa.

[0009] Preferably, the fraction contains components of 50 to 100 kDa and does not contain components of less than 30 kDa.

[0010] Preferably, said fraction does not contain components greater than 100 kDa and / or components between 30 and 50 kDa.

[0011] The deciduous dental pulp stem cells may be primary culture cells.

[0012] Alternatively, the deciduous dental pulp stem cells may be immortalized cells.

[0013] Furthermore, according to one embodiment, the present invention provides a method for activating cells in a subject, the method comprising the step of administering the above-described cell activation agent to the subject.

[0014] The cell activation agent of the present invention can be easily and inexpensively prepared from the culture supernatant of deciduous dental pulp stem cells, and does not contain any components that inhibit cell activation activity, making it highly effective, safe, and useful.

[0015] FIG. 1 is a polyacrylamide gel electrophoresis image of SHED-CM and its fractions. FIG. 2 is a polyacrylamide gel electrophoresis image of IM-SHED-CM and its fractions. FIG. 3 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with various concentrations of SHED-CM fractions. FIG. 4 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with various concentrations of IM-SHED-CM fractions. FIG. 5 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with dilutions of SHED-CM or its fractions adjusted based on protein concentration. FIG. 6 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with dilutions of IM-SHED-CM or its fractions adjusted based on protein concentration. Figure 7 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a dilution of the 50-100 kDa fraction derived from SHED-CM, the >100 kDa fraction, or the 30-50 kDa fraction. Figure 8 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with a dilution of the 50-100 kDa fraction derived from IM-SHED-CM, the >100 kDa fraction, or the 30-50 kDa fraction. Figure 9 shows the results of Western blot analysis comparing the expression levels and phosphorylation of MAPK in NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or fractions thereof. Figure 10 shows the results of Western blot analysis comparing the expression levels and phosphorylation of Akt in NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or fractions thereof. Figure 11 shows the results of Western blot analysis comparing the expression levels and phosphorylation of MAPK in NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or fractions thereof at the same protein concentrations.Figure 12 shows the results of Western blot analysis comparing the expression levels and phosphorylation of Akt in NIH3T3 cells cultured with SHED-CM / IM-SHED-CM or fractions thereof at the same protein concentration. Figure 13 shows the results of a scratch assay for NIH3T3 cells cultured with IM-SHED-CM or fractions thereof. Figure 14 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of NIH3T3 cells cultured with IM-SHED-CM or its >3.5 kDa fraction. Figure 15 shows the results of a scratch assay for NIH3T3 cells cultured with IM-SHED-CM or its >3.5 kDa fraction. Figure 16 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of HUVECs cultured with dilutions of SHED-CM or its 50-100 kDa fraction adjusted based on protein concentration. Figure 17 is a graph showing the results of a WST assay evaluating the intracellular dehydrogenase activity of HUVECs cultured with dilutions of IM-SHED-CM or its 50-100 kDa fraction adjusted based on protein concentration. Figure 18 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with the 50-100 kDa fraction of SHED-CM or a mixture of the >100 kDa, 50-100 kDa, and 30-50 kDa fractions. Figure 19 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with the 50-100 kDa fraction of SHED-CM or a mixture of the 50-100 kDa and <30 kDa fractions. Figure 20 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with the 50-100 kDa fraction of IM-SHED-CM or a mixture of the >100 kDa, 50-100 kDa, and 30-50 kDa fractions. Figure 21 is a graph showing the intracellular dehydrogenase activity of HUVECs cultured with the 50-100 kDa fraction of IM-SHED-CM or a mixture of the 50-100 kDa and <30 kDa fractions. Figure 22 is a graph showing the results of a WST assay evaluating the inhibitory effect of IM-SHED-CM on cell damage caused by hydrogen peroxide treatment.Figure 23 is a graph showing the results of a WST assay evaluating the inhibitory effect of the 50-100 kDa fraction of IM-SHED-CM on cell damage caused by hydrogen peroxide treatment. Figure 24 is a graph showing the results of a WST assay evaluating the inhibitory effect of the >100 kDa fraction of IM-SHED-CM on cell damage caused by hydrogen peroxide treatment. Figure 25 is a graph showing the results of a WST assay evaluating the inhibitory effect of the 30-50 kDa fraction of IM-SHED-CM on cell damage caused by hydrogen peroxide treatment. Figure 26 is a graph showing the results of a quantitative analysis of scratch recovery in Figures 13 and 15.

[0016] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0017] According to a first embodiment, the present invention provides a cell activator comprising a fraction isolated from a culture supernatant of deciduous dental pulp stem cells, wherein the fraction does not contain components less than 3.5 kDa.

[0018] In this embodiment, "cell activation" (and grammatical variations thereof) refers to normalizing or improving functions such as cell proliferation and metabolism. Therefore, "cell activation" in this embodiment may include, for example, activation of enzymes related to cell proliferation and metabolism (such as various dehydrogenases, Akt, and AMPK) and enzymes that suppress oxidative stress (such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)). Here, the "cells" may be cells of any vertebrate, preferably mammalian cells such as mice, rats, rabbits, pigs, cows, monkeys, and humans, with human cells being particularly preferred. The type of cells is also not particularly limited, and may be, for example, fibroblasts, endothelial cells, epithelial cells, nerve cells, stem cells, leukocytes, bone cells, muscle cells, adipocytes, and the like, preferably fibroblasts or endothelial cells.

[0019] "Decorative tooth pulp stem cells" are mesenchymal stem cells collected from the dental pulp of a deciduous tooth. The deciduous tooth pulp stem cells in this embodiment may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, pigs, cows, monkeys, and humans, and particularly preferably from humans.

[0020] Methods for preparing deciduous dental pulp stem cells have already been established (for example, International Publication No. 2011 / 118795), and stem cells can be isolated and prepared from deciduous dental pulp according to methods known in the art.

[0021] In this embodiment, the deciduous dental pulp stem cells may be primary cultured cells or immortalized cells. "Immortalized cells" refer to cells that remain proliferative even after a certain number of divisions, i.e., cells that have the ability to proliferate indefinitely. Methods for immortalizing cells have already been established, and known techniques can be used. For example, cells can be immortalized by introducing the SV40 T antigen gene or the telomerase reverse transcriptase (TERT) gene into the cells.

[0022] Methods for culturing deciduous dental pulp stem cells have already been established (e.g., International Publication No. 2011 / 118795), and deciduous dental pulp stem cells can be cultured according to methods known in the art. Specifically, for example, deciduous dental pulp stem cells can be cultured at a concentration of, for example, 5 × 10 in a medium containing, as a basal medium, DMEM, IMDM, Ham's F-12, RPMI-1640, or a mixture thereof, appropriately supplemented with serum such as FBS or serum substitutes such as KSR, glucose, amino acids, vitamins, etc. ４ ~2 x 10 ５ The cell activating agent of this embodiment is preferably prepared from a culture supernatant obtained by culturing the deciduous dental pulp stem cells in a serum-free medium.

[0023] The cell activator of this embodiment does not contain components less than 3.5 kDa derived from the culture supernatant of deciduous dental pulp stem cells. Here, "free" means that the components to be removed are not detected by commonly used detection methods (e.g., immunochemical techniques). Therefore, the cell activator of this embodiment may be one in which 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 components less than 3.5 kDa contained in the crude culture supernatant have been removed. In other words, the cell activator of this embodiment may contain up to 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 components less than 3.5 kDa contained in the crude culture supernatant. The cell activator of this embodiment exhibits improved cell activation effects due to the removal of components less than 3.5 kDa from the culture supernatant of deciduous dental pulp stem cells.

[0024] In this embodiment, "kDa" may be calculated based on, for example, SDS-PAGE or ultrafiltration, and when the culture supernatant is purified using a commercially available ultrafiltration filter unit, it may be the nominal molecular weight cutoff (NMWL) or molecular weight cut-off (MWCO) value indicated on the product.

[0025] The cell activator of this embodiment preferably contains a fraction containing a component of 50 to 100 kDa; on the other hand, it preferably does not contain a fraction containing a component of less than 30 kDa. It is more preferable that the cell activator of this embodiment does not contain a fraction containing a component of more than 100 kDa and / or a fraction containing a component of 30 to 50 kDa. This is because the 50 to 100 kDa component has higher cell activating activity, while not only the components less than 3.5 kDa but also the components over 100 kDa and the 30 to 50 kDa components may adversely affect the cell activating activity of the 50 to 100 kDa component.

[0026] The cell activator of this embodiment can be prepared, for example, by combining ultrafiltration membranes with 100,000 MWCO, 50,000 MWCO, 30,000 MWCO, and 3,500 MWCO. For example, by removing components of more than 100 kDa from a culture supernatant using a 100,000 MWCO ultrafiltration membrane and concentrating the resulting filtrate using a 50,000 MWCO ultrafiltration membrane, a cell activator can be prepared that contains 50 to 100 kDa components from the culture supernatant of deciduous dental pulp stem cells, but does not contain 30 to 50 kDa components or components less than 30 kDa.

[0027] The cell activation agent of this embodiment may consist solely of a fraction purified from the culture supernatant of deciduous dental pulp stem cells, but generally may further contain pharmaceutically acceptable carriers and additives as optional components.

[0028] The cell activator of this embodiment may be prepared as a liquid or solid, and may be formulated into various dosage forms such as injections, jellies, sprays, tablets, and granules, but is preferably prepared as a liquid. When prepared as a liquid, sterile water, physiological saline, an aqueous glucose solution, or the like may be used as a carrier, and if desired, a bactericide, an isotonic agent, a stabilizer, or the like may be further blended. When prepared as a solid, additives such as starch, lactose, mannitol, and inorganic salts may be further blended, and if desired, a binder, a disintegrant, a lubricant, or the like may be further blended.

[0029] In the cell activator of this embodiment, the content (in terms of protein weight) of the 50 to 100 kDa component derived from the culture supernatant of deciduous dental pulp stem cells may be, for example, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more, and preferably 60 to 70% by weight, when the total mass of the cell activator is taken as 100%.

[0030] The cell activator of this embodiment contains components derived from the culture supernatant of deciduous dental pulp stem cells that have cell activating activity, but does not contain components that inhibit this activity. Therefore, the cell activator of this embodiment has a superior cell activating effect and is useful compared to unpurified culture supernatant of deciduous dental pulp stem cells.

[0031] According to a second embodiment, the present invention provides a method for activating cells in a subject, the method comprising administering the cell activation agent to the subject. In this embodiment, the terms "activating cells" and "cells" are defined as in the first embodiment.

[0032] The "subject" in this embodiment may be any vertebrate, but is preferably a mammal such as a mouse, rat, rabbit, pig, cow, monkey, or human, and is particularly preferably a human. The subject may be of any age, including infants, juveniles, adolescents, adults, and elderly subjects.

[0033] The cell activator in the method of this embodiment may be formulated into various dosage forms as defined in the first embodiment, and may be administered by any administration method appropriate for the dosage form, for example, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, nasal administration, intraperitoneal administration, direct injection into target tissue, inhalation administration, etc. The dosage of the cell activator may vary depending on the age, weight, health condition, etc. of the subject, but may be, for example, 0.1 to 10 mg / kg (body weight), preferably 0.5 to 2 mg / kg (body weight). The dosage may be administered once or multiple times.

[0034] According to a third embodiment, the present invention provides use of a fraction isolated from the culture supernatant of deciduous dental pulp stem cells as a cell activating agent, wherein the fraction contains components of 50 to 100 kDa and does not contain components of less than 30 kDa. In this embodiment, the terms "deciduous dental pulp stem cells," "cell activating agent," "cells," "free," and "kDa" are the same as those defined in the first embodiment. In accordance with the contents described in the first and second embodiments, a fraction derived from the culture supernatant of deciduous dental pulp stem cells containing components of 50 to 100 kDa and not containing components of less than 30 kDa can be used as a cell activating agent.

[0035] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0036] Materials and Reagents: NIH3T3 cells were purchased from the JCRB Cell Bank. Human umbilical vein endothelial cells (HUVECs), EBM ＴＭ -2 basal medium (CC-3156) and EGM ＴＭ The α-2 supplement kit (CC-4176) was purchased from Lonza. Dulbecco's Modified Eagle Medium (DMEM) was purchased from GIBCO. Bovine Calf Serum (hereinafter referred to as "CS") and antimycotic solution (100x) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and 0.05% trypsin / EDTA solution were purchased from Invitrogen. Charcoal (powder, activated) (hereinafter referred to as "activated charcoal") was purchased from Nacalai Tesque. Dulbecco's phosphate buffer solution (PBS), RIPA buffer, and quick CBB staining solution were purchased from Fujifilm Wako Pure Chemical Industries. Ultrafiltration units (Vivaspin Turbo 15 membrane 30,000, 50,000, 100,000 MWCO) were purchased from Sartorius. Millex-GV low protein binding durapore (PVDF) membrane 0.22 μm was purchased from Merck Millipore. Spectra / Por ＴＭ　7 Dialysis Membrane (MWCO: 3.5 kD) was purchased from REPLIGEN. PD-10 columns were purchased from Cytiva. Micro BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 was purchased from Dojindo Laboratories. Acrylamide, protein standard markers, and Clarity Western ECL Substrate were purchased from Bio-Rad. 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, and anti-mouse IgG-HRP antibody (#62-6520) and anti-rabbit IgG-HRP antibody (#65-6120) were purchased from Invitrogen.

[0037] <1. Preparation of Deciduous Dental Pulp-Derived Stem Cell Culture Supernatant and Its Fraction> (1-1) Preparation of Human Deciduous Dental Pulp-Derived Stem Cells (SHED) After detached or extracted human deciduous teeth were disinfected with 5% chlorhexidine solution (Yamazen Pharmaceuticals) or 10% povidone-iodine solution (Iwaki Pharmaceuticals), the crowns were divided, and dental pulp tissue was collected using a dental reamer. The dental pulp tissue was suspended in DMEM containing 10% by volume FBS, and 2 mg / ml collagenase (Fujifilm Wako Pure Chemical Industries) and dispase (Fujifilm Wako Pure Chemical Industries) were added, followed by incubation at 37°C for 1 hour. The mixture was centrifuged at 777 x g for 5 minutes, the supernatant was removed, and dental pulp cells were collected. The supernatant was then added to 10% by volume FBS and 1% by volume antibiotic-antimycotic agent (Gibco ＴＭ　 Dental pulp cells were suspended in DMEM (4 mL) supplemented with Antibiotic-Antimycotic and seeded onto a 6-well plate. The cells were cultured at 37°C and 5% CO2 until they became subconfluent. 0.05% trypsin / EDTA solution was added and incubated at 37°C for 5 minutes, after which the cells were collected and seeded onto a 10 cm dish (Violamo) for adherent cells. They were then subcultured three times, resulting in a cell population of approximately 1 x 10 7The cells were grown until they became cells. A 0.05% trypsin / EDTA solution was added, and the cells were incubated at 37°C for 5 minutes. The collected cells were named "SHED (Stem cells from Human Exfoliated Deciduous Teeth)."

[0038] (1-2) Preparation of Immortalized Human Deciduous Dental Pulp-Derived Stem Cells (IM-SHED) SHED (IM-SHED) immortalized by introducing the SV40 gene from a viral vector were prepared by Applied Biological Materials.

[0039] (1-3) Preparation of culture supernatant The SHED or IM-SHED prepared above were subcultured in DMEM containing 10% by volume FBS. For the preparation of culture supernatant, SHED cells were used after up to 8 passages, and IM-SHED cells were used after 50 to 55 passages. The medium was replaced with serum-free DMEM and cultured for 48 hours. Thereafter, the medium was collected and filtered through a separation membrane (Stericup Quick Release-GP, PVDF, 0.22 μm; Merck Millipore) to obtain a culture supernatant (conditioned medium: CM). Hereinafter, the culture supernatant derived from SHED is referred to as "SHED-CM," and the culture supernatant derived from IM-SHED is referred to as "IM-SHED-CM."

[0040] (1-4) Fractionation and Concentration of Culture Supernatant The CM was centrifuged at room temperature at 1,000 rpm for 3 minutes, at 500 × g for 30 minutes at 4 °C, and at 2,000 × g for 30 minutes at 4 °C, and then filtered through a Millex-GV low protein binding durapore (PVDF) membrane (0.22 μm) to remove impurities such as cells. The CM was then passed through a 100,000 MWCO ultrafiltration unit and centrifuged at 2,000 × g for 20 minutes at 4 °C. The filtrate (hereinafter referred to as "PT") was passed through a 50,000 MWCO ultrafiltration unit and centrifuged at 2,000 × g for 20 minutes at 4 °C. The PT was subjected to a 30,000 MWCO ultrafiltration unit and centrifuged at 2,000 xg for 20 minutes at 4°C.

[0041] Furthermore, 10 mL of PBS was added to the 100,000 MWCO ultrafiltration unit and centrifuged at 2,000 x g at 4 ° C for 20 minutes. This process was repeated five times to obtain a >100 kDa fraction concentrate. The PT obtained here was subjected to the 50,000 MWCO ultrafiltration unit and centrifuged at 2,000 x g at 4 ° C for 20 minutes to obtain a 50-100 kDa fraction concentrate. The PT was then subjected to the 30,000 MWCO ultrafiltration unit and centrifuged at 2,000 x g at 4 ° C for 20 minutes to obtain a 30-50 kDa fraction concentrate. The PT was then subjected to a PD-10 column, the buffer was replaced with PBS, and a <30 kDa fraction was obtained. The concentrated fraction solution was lyophilized, dissolved in Milli-Q water, and a sample buffer (with or without SDS) was added, followed by electrophoresis on a 12.5% ​​polyacrylamide gel.

[0042] The results of electrophoresis of SHED-CM and its fractions are shown in Figure 1, and the results of electrophoresis of IM-SHED-CM and its fractions are shown in Figure 2. It was confirmed that both SHED-CM and IM-SHED-CM contained many protein components in the 50-100 kDa fraction.

[0043] 2. Activation of Cell Proliferation and Viability by SHED-CM / IM-SHED-CM Fractions (WST Assay) Activated charcoal-treated CS (hereinafter referred to as "CH-CS") was prepared as follows: 10 mg of activated charcoal was added to 1 mL of CS and gently vortexed for 15 seconds. After incubation at 37°C for 10 minutes, the mixture was centrifuged at 12,000 rpm at 4°C for 20 minutes, and the supernatant was filtered through a 0.22 μm PVDF filter to obtain CH-CS. NIH3T3 cells were seeded in a 96-well plate (Violamo) at 5,000 cells / well and cultured overnight in 10% CS / DMEM. To serum-starve the cells, they were washed once with DMEM, then the medium was replaced with 0.4% CH-CS / DMEM, and the cells were cultured for 24 hours. The fraction prepared in step 1 above was then diluted with 0.4% CH-CS / DMEM and added to the wells, followed by incubation for 44 hours. Cell Counting Kit-8 WST solution (10 μL / well) was added, and the wells were further incubated for 4 hours, after which the absorbance at 450 nm was measured.

[0044] The results are shown in Figures 3 and 4. An increase in absorbance at 450 nm indicates increased activity of intracellular dehydrogenases, and higher absorbance at 450 nm is interpreted as indicating higher cell proliferation and viability. In the figures, "10% CS" refers to 10% CS / DMEM, and "CM x 1 / 2 dil." refers to a medium in which CM was diluted 1 / 2 with 0.4% CH-CS / DMEM. These results indicate that the 50-100 kDa fraction of both SHED-CM and IM-SHED-CM contains components that activate cell proliferation and viability. Similar results were also obtained with HeLa cells, a human cervical cancer-derived cell line, human bone marrow-derived mesenchymal stem cells, and human iPS cells (409B2 line) (data not shown).

[0045] Furthermore, the results of comparing the activity of each fraction at the same protein concentration are shown in Figures 5 and 6. In the figures, "Calculated value" indicates the value obtained by correcting the CM result by the protein concentration of each fraction calculated from the electrophoresis results in 1 above and the protein concentration quantification results using the Micro BCA Protein Assay Kit. Of the fractions purified from SHED-CM and IM-SHED-CM, only the 50-100 kDa fraction was found to contain components that activate cell proliferation and viability.

[0046] <3. Inhibition of cell proliferation and viability by SHED-CM / IM-SHED-CM fractions> Dilutions of the 50-100 kDa fraction prepared in 1 above with 0.4% CH-CS / DMEM, the >100 kDa fraction, or the 30-50 kDa fraction were prepared, and intracellular dehydrogenase activity was assessed using the same procedure as in 2 above.

[0047] The results are shown in Figures 7 and 8. Mixing the 50-100 kDa fraction with the >100 kDa fraction or the 30-50 kDa fraction reduced intracellular dehydrogenase activity. In particular, the inhibitory effect of the 30-50 kDa fraction derived from IM-SHED was significant (Figure 8), suggesting the presence of a factor that inhibits cell proliferation and viability.

[0048] 4. Activation of Intracellular Signaling by SHED-CM / IM-SHED-CM Fractions NIH3T3 cells were seeded in a 12-well plate (6 × 10 ４ Cells (cells / well) were cultured overnight in 10% CS / DMEM. To serum-starve the cells, they were washed once with DMEM, then the medium was replaced with 0.4% CH-CS / DMEM and cultured for 24 hours. Then, the CM prepared in step 1 or its fractions was diluted with 0.4% CH-CS / DMEM and added. As controls, 10% FBS or human TNFα (PeproTech) (50 ng / mL) was added. After 30 minutes of culture, the cells were washed twice with PBS and lysed in RIPA buffer supplemented with protease inhibitors. The cell lysate was collected in a microtube and shaken for 30 minutes at 4°C. The cells were centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was collected. SDS-containing sample buffer was added, and the cells were incubated at 95°C for 5 minutes, followed by electrophoresis on a 10% polyacrylamide gel. After transferring the proteins to a nitrocellulose membrane, the membrane was blocked with 5% skim milk / TBST solution (room temperature for 1 hour). The membrane was then incubated overnight at 4°C in the primary antibody solution. The primary antibody solution was removed, and the membrane was washed three times with TBST before being incubated in the secondary antibody solution at room temperature for 1 hour. The primary antibodies used were anti-phospho p44 / 42 antibody (1:2000) or anti-phospho Akt antibody (1:2000), and the secondary antibodies used were anti-mouse IgG-HRP antibody (1:5000) or anti-rabbit IgG-HRP antibody (1:5000). Clarity Western ECL Substrate was used for detection.

[0049] The results are shown in Figures 9 and 10. Addition of the 50-100 kDa fraction significantly increased the expression and phosphorylation of MAPK and Akt. Figures 11 and 12 show the results of comparing CM and the 50-100 kDa fraction, both prepared at the same protein concentration. Compared to CM, the 50-100 kDa fraction demonstrated increased phosphorylation. These results indicate that the 50-100 kDa fraction activates intracellular phosphorylation signaling.

[0050] 5. Activation of cell migration by SHED-CM / IM-SHED-CM fractions (scratch assay) NIH3T3 cells were seeded in a 48-well plate (3.75 × 10 ４ Cells were cultured overnight in 10% CS / DMEM (cells / well). To serum-starve the cells, the medium was replaced with 0.4% CH-CS / DMEM and cultured for 24 hours. A 200 μL pipette tip was used to create a linear scratch on the cell monolayer. After washing twice with DMEM, CM (diluted 1 / 2 with 0.4% CH-CS / DMEM); 10% FBS / DMEM; or fractions of >100 kDa, 50-100 kDa, or 30-50 kDa (diluted with 0.4% CH-CS / DMEM to a 5-fold increase in protein concentration relative to the original solution) were added and cultured for 24 hours.

[0051] The results (microscopic images) are shown in Figure 13. Furthermore, the cell areas in the microscopic images were digitized using ImageJ software, and the results of quantitative analysis are shown in Figure 26. It was confirmed that the 50-100 kDa fraction retained wound healing activity equivalent to that of CM.

[0052] 6. Preparation and Analysis of ≥3.5 kDa Fraction of IM-SHED-CM> IM-SHED-CM was analyzed by Spectra / Por ＴＭ　 The resulting fraction was sealed in a 3.5 kDa MWCO (MWCO) dialysis membrane and dialyzed against 100 times the volume of Milli-Q water for 72 hours. The Milli-Q water was exchanged eight times during this period. The dialyzed solution was lyophilized, dissolved in 1 / 10 the starting volume of PBS (on ice for >30 minutes), and filtered through a 0.22 μm PVDF filter. The resulting ≥3.5 kDa fraction was evaluated for its intracellular dehydrogenase and cell migration activity using the same procedures as in steps 2 and 5 above.

[0053] The results of the WST assay are shown in Figure 14. The ≥3.5 kDa fraction prepared at a high protein concentration (100 μg / mL) activated intracellular dehydrogenases more effectively than CM prepared at the same protein concentration. The results of the scratch assay are shown in Figure 15, and the results of the quantitative analysis are shown in Figure 26. The ≥3.5 kDa fraction significantly increased cell migration to the scratch. These results indicate that removing the <3.5 kDa fraction can achieve cell activation effects equal to or greater than those achieved with CM.

[0054] 7. Activation of Cell Proliferation and Viability by SHED-CM / IM-SHED-CM Fractions (2)> Using HUVECs instead of NIH3T3 cells, the activation of intracellular dehydrogenases by the 50-100 kDa fractions purified from SHED-CM and IM-SHED-CM was evaluated by the same procedure as in 2 above. HUVECs were cultured in a 2000-well platelet-free medium (EBM). ＴＭ -2 basal medium (CC-3156) and EGM ＴＭ Cells were grown in 10-cm adherent cell dishes to 80-90% confluence using medium prepared with the EGM-2 supplement kit (hereafter referred to as "EGM-2 medium"). Cells were then detached with 0.05% trypsin / EDTA solution and harvested. EGM-2 medium was added to suspend the cells. Cell count and viability were measured using a Countess 2 automated cell counter and trypan blue. The cell suspension was adjusted to a concentration of 25,000 cells / mL. Cells were seeded in a 96-well plate (100 μL / well). One day later, the medium was replaced with EGM-2 medium supplemented with CM or the 50-100 kDa fraction. After 3 days of culture, WST solution (10 μL / well) from Cell Counting Kit-8 was added, and the absorbance at 450 nm was measured after 3-4 hours of culture.

[0055] The results are shown in Figures 16 and 17. In the figures, "Calculated Value" indicates the value obtained by correcting the CM results by the protein concentration of each fraction calculated from the electrophoresis results in 1 above and the protein concentration quantification results using the Micro BCA Protein Assay Kit. Both the 50-100 kDa fractions derived from SHED-CM and IM-SHED-CM increased intracellular dehydrogenase activity more than CM at the same protein concentration. These results indicated that the 50-100 kDa fractions derived from SHED-CM and IM-SHED-CM contain components that activate the proliferation and viability of HUVECs.

[0056] 8. Inhibition of Cell Growth and Viability by SHED-CM / IM-SHED-CM Fractions (2) Mixtures of the >100 kDa, 50-100 kDa, and 30-50 kDa fractions (mixing ratio 1:1:1), and a mixture of the 50-100 kDa and <30 kDa fractions (mixing ratio 1:1) were prepared. The 50-100 kDa fraction and the above mixture were serially diluted with EGM-2 medium, and intracellular dehydrogenase activity was assessed using the same procedure as in 7 above.

[0057] The results for the SHED-CM-derived fractions and their mixtures are shown in Figures 18 and 20, and the results for the IM-SHED-CM-derived fractions and their mixtures are shown in Figures 19 and 21. In the figures, "Mix >100, 50, 30 kDa" refers to a mixture of the >100 kDa, 50-100 kDa, and 30-50 kDa fractions, "Mix 50 kDa, 30 PT" refers to a mixture of the 50-100 kDa and <30 kDa fractions, and "50 kDa" refers to the 50-100 kDa fraction. The mixed solution of the >100 kDa, 50-100 kDa, and 30-50 kDa fractions did not show a significant decrease in intracellular dehydrogenase activity compared to the 50-100 kDa fraction (Figures 18 and 20). In contrast, the mixed solution of the 50-100 kDa and <30 kDa fractions showed a significant decrease in intracellular dehydrogenase activity compared with the 50-100 kDa fraction (Figs. 19 and 21). These results demonstrated that components less than 30 kDa in CM inhibited the proliferation and viability of HUVECs.

[0058] 9. Inhibitory Effect of IM-SHED-CM Fractions on Hydrogen Peroxide-Induced Cytotoxicity. HUVECs were grown in EGM-2 medium in 10-cm adherent cell dishes until 80-90% confluent, then detached with 0.05% trypsin / EDTA solution and harvested. EGM-2 medium was added to suspend the cells. Cell count and viability were measured using a Countess 2 automated cell counter and trypan blue, and the cell suspension was adjusted to a concentration of 50,000 cells / mL. Cells were seeded in a 96-well plate (100 μL / well). After one day, the medium was replaced with serial dilutions of IM-SHED-CM and its fractions in EGM-2 medium and cultured for three days. Hydrogen peroxide (final concentration: 0.005-0.00031%) was added to the cells, and they were incubated at 37°C for one hour. The dilution solution was removed, and EGM-2 medium (100 μL / well) and WST solution of Cell Counting Kit-8 (10 μL / well) were added, and after 3 to 4 hours of culture, the absorbance at 450 nm was measured.

[0059] The results for cells treated with serially diluted IM-SHED-CM are shown in Figure 22. In cells without IM-SHED-CM (control), hydrogen peroxide treatment reduced absorbance at 450 nm, and the reduction was even greater in a hydrogen peroxide concentration-dependent manner. These results confirmed cell damage caused by hydrogen peroxide treatment. On the other hand, in cells treated with serially diluted IM-SHED-CM, absorbance at 450 nm decreased in a hydrogen peroxide concentration-dependent manner, but the reduction in absorbance at 450 nm was suppressed compared to the control. These results confirmed that IM-SHED-CM suppressed cell damage caused by oxidative stress.

[0060] The results for cells to which serial dilutions of the 50-100 kDa fraction of IM-SHED-CM were added are shown in Figure 23, the results for cells to which serial dilutions of the >100 kDa fraction were added are shown in Figure 24, and the results for cells to which serial dilutions of the 30-50 kDa fraction were added are shown in Figure 25. In cells to which the 50-100 kDa fraction was added, the absorbance at 450 nm increased in a protein concentration-dependent manner, indicating that cell damage due to oxidative stress was suppressed in a concentration-dependent manner by the 50-100 kDa fraction.

Claims

1. A cell activating agent comprising a fraction isolated from a culture supernatant of deciduous dental pulp stem cells, said fraction not containing components less than 3.5 kDa.

2. The cell activating agent according to claim 1, wherein the fraction contains a component of 50 to 100 kDa and does not contain a component of less than 30 kDa.

3. The cell activating agent according to claim 1 or 2, wherein the fraction does not contain components of more than 100 kDa and / or components of 30 to 50 kDa.

4. The cell activation agent according to any one of claims 1 to 3, wherein the deciduous dental pulp stem cells are primary cultured cells.

5. A cell activation agent according to 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 the step of administering to the subject a cell activation agent according to any one of claims 1 to 5.

7. Use of a fraction isolated from the culture supernatant of deciduous dental pulp stem cells as a cell activating agent, wherein the fraction does not contain components less than 3.5 kDa.

8. The use according to claim 7, wherein the fraction contains components of 50 to 100 kDa and does not contain components of less than 30 kDa.

9. The use according to claim 7 or 8, wherein the fraction does not contain components above 100 kDa and / or components between 30 and 50 kDa.

10. The use according to any one of claims 7 to 9, wherein the deciduous dental pulp stem cells are primary cultured cells.

11. The use according to any one of claims 7 to 9, wherein the deciduous dental pulp stem cells are immortalized cells.

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