Bfgf production method

Abstract

The present invention addresses the problem of providing a method for producing bFGF without depending on a gene recombination method. The present invention is a basic fibroblast growth factor (bFGF) production method, the production method comprising: a step in which immortalized dental pulp stem cells are cultured to prepare a culture supernatant; and a step in which bFGF is purified from the culture supernatant.

Description

Method for producing bFGF

[0001] The present invention relates to a method for producing basic fibroblast growth factor (bFGF or FGF2).

[0002] bFGF is known to have angiogenic effects as well as the ability to promote benign granulation formation. Granulation tissue plays an important role in the early stages of wound healing, and bFGF is therefore believed to be effective in treating pressure ulcers and skin ulcers. Trafermin, which contains human recombinant bFGF as its main ingredient, is currently available as a topical treatment, Fiblast (registered trademark).

[0003] In addition, various growth factors other than bFGF are known to be effective in treating many diseases. In recent years, mesenchymal stem cell (MSC) culture supernatant has attracted attention as a source of growth factors and other cytokines. MSCs are pluripotent cells with self-renewal and differentiation capabilities, and are known to produce various growth factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGF), platelet-derived growth factor (PDGF), and transforming growth factor β (TGF-β). Therefore, mesenchymal stem cell culture supernatant, which is rich in these growth factors, has been reported to be useful for treating inflammation and injury sites, as well as cancer (Patent Documents 1, 2, and 3).

[0004] As mentioned above, it is known that the culture supernatant of mesenchymal stem cells contains many growth factors, but the quantities are not very large. In particular, there have been no reports of bFGF being detected in the culture supernatant of mesenchymal stem cells, at least in primary cultures.

[0005] International Publication No. 2011 / 118795 International Publication No. 2014 / 126176 Japanese Patent Application Laid-Open No. 2017-160264

[0006] An objective of the present invention is to provide a method for producing bFGF without relying on genetic recombination methods, for example, a method for producing bFGF that has undergone post-translational modification (e.g., glycosylation) using the culture supernatant of the above-mentioned mesenchymal stem cells.

[0007] The present inventors have attempted to prepare bFGF using the culture supernatant of mesenchymal stem cells, which are a valuable source of cytokines, but found that almost no bFGF was secreted into the culture supernatant of primary cultured mesenchymal stem cells. However, when immortalized mesenchymal stem cells, specifically dental pulp-derived stem cells, were cultured, they found that significantly larger amounts of bFGF were secreted into the culture supernatant compared to the culture supernatant of primary cultured dental pulp-derived stem cells.

[0008] That is, the present invention relates to the following (1) to (8). (1) A method for producing basic fibroblast growth factor (bFGF), comprising the steps of culturing immortalized dental pulp stem cells to prepare a culture supernatant and purifying bFGF from the culture supernatant. (2) The method according to (1) above, wherein the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells. (3) The method according to (1) or (2) above, wherein the dental pulp is deciduous dental pulp. (4) A method for producing a culture supernatant containing 100 pg / mL or more of bFGF, comprising the steps of culturing immortalized dental pulp stem cells to prepare a culture supernatant and recovering the culture supernatant. (5) The method according to (4) above, wherein the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells. (6) The method according to (4) or (5) above, wherein the dental pulp is deciduous dental pulp. (7) A pharmaceutical composition for treating or preventing wounds, comprising a culture supernatant of immortalized dental pulp stem cells containing at least 100 pg / mL or more of bFGF. (8) The pharmaceutical composition according to (7), characterized in that the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells. In this specification, the symbols "to" indicate a numerical range including the values ​​on either side of the symbol.

[0009] The culture supernatant of immortalized dental pulp-derived stem cells contains significantly more bFGF than the culture supernatant of primary cultured dental pulp-derived stem cells, making it possible to easily prepare non-recombinant, biologically derived bFGF.

[0010] Figure 1 shows a schematic diagram of the preparation method for standards used in quantitative cytokine ELISA assays. Figure 2 shows the results of quantitative ELISA assays of various cytokine concentrations in SHED-CM and IM-SHED-CM. Figure 3 shows the effect of the passage number of IM-SHED on the secretion of bFGF. Figure 4 shows the changes in bFGF secretion during long-term culture of 50-passage IM-SHED in an automated culture system. Figure 5 shows the results of Western blotting of bFGF in IM-SHED-CM. Figure 6 shows the results of Western blotting of bFGF with different apparent molecular weights separated by ultrafiltration in IM-SHED-CM. Figure 7 shows the results of examining the presence or absence of cell migration activity of bFGF in IM-SHED-CM. The results of scratch assays performed at 0 h and 24 h after the addition of various bFGF fractions are shown. FIG. 8 shows the results of quantifying the wound area after 24 hours in FIG.

[0011] Hereinafter, embodiments of the present invention will be described. Note that, unless otherwise specified, the term "the present embodiment" refers to all embodiments described in this specification.

[0012] The first embodiment is a method for producing basic fibroblast growth factor (bFGF or FGF2), which comprises the steps of culturing immortalized dental pulp stem cells to prepare a culture supernatant, and purifying bFGF from the culture supernatant.

[0013] Furthermore, a second embodiment is a method for producing a culture supernatant containing bFGF, the culture supernatant containing at least 100 pg / mL or more of bFGF, the method comprising the steps of culturing immortalized dental pulp stem cells to prepare the culture supernatant, and recovering the culture supernatant.

[0014] In this embodiment, "immortalized dental pulp stem cells" refer to cells derived from primary cultured "dental pulp stem cells" that have acquired the ability to continuously divide. Furthermore, the culture supernatant of "immortalized dental pulp stem cells" refers to the supernatant of the culture medium used to culture the immortalized dental pulp stem cells, and is a substance that does not contain impurities such as cells.

[0015] In this embodiment, the animal species from which dental pulp stem cells are derived is not particularly limited, and may include humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses, among others, with humans being the preferred animal. Furthermore, among human-derived dental pulp stem cells, deciduous tooth dental pulp stem cells are particularly preferred.

[0016] Methods for immortalizing mesenchymal stem cells such as dental pulp stem cells include the method disclosed in U.S. Patent No. 10,494,606 B2. This method involves introducing four genes, hTERT, bmi-1, E6, and E7, into primary cultured cells obtained by initial culturing mesenchymal stem cells such as dental pulp stem cells, thereby immortalizing the cells.

[0017] Another immortalization method is the method disclosed in U.S. Patent No. 6,146,888. This method involves introducing the SV40 gene into primary cultured mesenchymal stem cells to immortalize the cells. Another suitable immortalization method is to introduce the telomerase reverse convertase (TERT) gene into primary cultured mesenchymal stem cells.

[0018] In this embodiment, a preferred method for immortalizing dental pulp-derived stem cells is to introduce the SV40 gene into primary cultured dental pulp-derived stem cells to immortalize the cells.

[0019] In this embodiment, the culture supernatant of immortalized dental pulp stem cells preferably does not contain animal serum to enhance safety. Removal of serum and other substances from the culture supernatant can be easily carried out by dialysis, solvent substitution, or the like. The culture supernatant in this embodiment may be in the form of a frozen or lyophilized product, or may be a solution prepared by dissolving the lyophilized product in an appropriate solvent. As described above, the "culture supernatant of immortalized dental pulp stem cells" in this embodiment is preferably a culture supernatant of immortalized deciduous dental pulp stem cells, and most preferably a culture supernatant of immortalized human deciduous dental pulp stem cells.

[0020] The culture medium used to produce the culture supernatant in this embodiment may be any culture medium that can be used to culture mesenchymal stem cells, and is not particularly limited. Examples of such culture medium include a basal culture medium such as DMEM (Dulbecco's Modified Eagle Medium), αMEM (alpha Modified Eagle Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), Ham's F-12, RPMI (Roswell Park Memorial Institute)-1640, or a mixture thereof, and a serum such as fetal bovine serum (FBS) or Knockout Reagents (KSR). TM The culture medium may be supplemented with serum substitutes such as serum replacement (e.g., serum replacement), glucose, amino acids, vitamins, antibiotics, etc. as appropriate.

[0021] In this embodiment, two types of culture medium are preferably prepared: a serum-containing culture medium (when DMEM is used as the basal medium, this may also be referred to as "FBS-DMEM"), and a serum-free culture medium for recovering culture supernatant (when DMEM is used as the basal medium, this may also be referred to as "CM-DMEM"). FBS-DMEM may contain DMEM, 5 to 20% by volume of FBS, and antibiotics, etc. CM-DMEM is a culture medium in which antibiotics, etc. are added to DMEM, and preferably contains little or no serum.

[0022] In this embodiment, bFGF contained in the culture supernatant of immortalized dental pulp stem cells can be detected using a bFGF-specific antibody, and the concentration of bFGF contained in the culture supernatant can be easily measured, for example, by using a quantitative ELISA method, etc. The existence of several variants of bFGF has been confirmed, and as long as the protein can be recognized by an anti-bFGF antibody (i.e., binds to an anti-bFGF antibody), these proteins are also included in the bFGF of this embodiment, even if they have different apparent molecular weights.

[0023] The culture supernatant of immortalized dental pulp stem cells containing bFGF is preferably a culture supernatant obtained by culturing immortalized dental pulp stem cells that have been passaged at least five times for, for example, 24 to 72 hours, preferably about 48 hours. The culture supernatant obtained by culturing immortalized dental pulp stem cells that have been passaged at least five times contains 100 pg / mL or more of bFGF, while the culture supernatant of immortalized dental pulp cells that have been passaged about 40 to 80 times contains about 200 pg / mL to about 600 pg / mL of bFGF.

[0024] Purification of bFGF from the culture supernatant of immortalized dental pulp stem cells can be easily carried out using methods known in the art. For example, contaminants are first removed from the collected culture supernatant by centrifugation or filtration. The desired bFGF can then be isolated from the resulting culture supernatant by an appropriate combination of known separation and purification methods. These methods include methods that utilize solubility (e.g., salting out or solvent precipitation), methods that primarily utilize molecular weight differences (e.g., dialysis, ultrafiltration, gel filtration, SDS-PAGE), methods that utilize charge differences (e.g., ion exchange chromatography), methods that utilize specific affinity (e.g., using resins bound to anti-bFGF antibodies), methods that utilize hydrophobicity (e.g., reverse-phase high-performance liquid chromatography), and methods that utilize isoelectric point differences (e.g., isoelectric focusing). The degree of bFGF purification at each purification step can be confirmed by SDS-PAGE and Western blotting using anti-bFGF antibodies.

[0025] The third embodiment is a pharmaceutical composition for wound treatment (hereinafter also referred to as "the pharmaceutical composition of this embodiment") containing a culture supernatant produced by the method of the second embodiment. That is, the third embodiment is a pharmaceutical composition for wound treatment or prevention, which contains a culture supernatant of immortalized dental pulp stem cells containing at least 100 pg / mL or more of bFGF. The pharmaceutical composition of the third embodiment contains a culture supernatant of immortalized dental pulp stem cells as an active ingredient, and contains a large amount of bFGF and other cytokines, making it effective for treating or preventing wounds such as pressure sores and skin ulcers.

[0026] The pharmaceutical composition according to this embodiment contains, as a major component, a culture supernatant obtained by culturing immortalized dental pulp stem cells. The culture supernatant of immortalized dental pulp stem cells contained in the pharmaceutical composition according to this embodiment is preferably a culture supernatant of immortalized dental pulp stem cells that have been passaged five or more times, and more preferably a culture supernatant of immortalized dental pulp cells that have been passaged approximately 40 to 80 times (a culture supernatant containing approximately 200 pg / mL to approximately 600 pg / mL of bFGF). The pharmaceutical composition according to this embodiment can be used to treat wounds, particularly skin wounds, such as pressure sores and skin ulcers.

[0027] Those skilled in the art can appropriately select the type of formulation additive used in the production of the pharmaceutical composition of this embodiment, the ratio of the formulation additive to the culture supernatant or its lyophilized product as the active ingredient, the production method, etc. The formulation additive can be an inorganic or organic substance, or a solid or liquid substance, and can generally be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0028] When the pharmaceutical composition of this embodiment is produced as an injection, the culture supernatant or a freeze-dried product thereof, which is the active ingredient, is mixed with distilled water for injection, optionally together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, or the like, and an isotonicity adjuster such as sodium chloride or glucose, and the mixture is sterile filtered and filled into ampoules; alternatively, mannitol, dextrin, cyclodextrin, gelatin, or the like may be further added, followed by vacuum freeze-drying to produce an injection that is dissolved immediately before use.

[0029] When the pharmaceutical composition according to this embodiment is prepared as an ointment or cream, it can be produced by kneading and mixing the culture supernatant or its lyophilized product, which is the active ingredient, with a base and additives. An oleaginous ointment can be produced, for example, by warming and melting an oleaginous base such as oils, waxes, or hydrocarbons such as paraffin, adding the active ingredient, mixing to dissolve or disperse the active ingredient, and kneading the mixture until homogeneous. A water-soluble ointment can be produced, for example, by warming and melting a water-soluble base such as macrogol, adding the active ingredient, and kneading the mixture until homogeneous.

[0030] The pharmaceutical composition according to this embodiment can be administered by known methods such as injection or application to a wound such as a bedsore or skin ulcer. The dosage of the pharmaceutical composition can be easily determined by a specialist such as a physician, but preferably, for example, the amount of culture supernatant administered per administration is in the range of approximately 0.1 mL to 5.0 mL.

[0031] A fourth embodiment is a method for preventing or treating wounds, particularly skin wounds (such as bedsores and skin ulcers), comprising administering the pharmaceutical composition according to the third embodiment to a subject.

[0032] Here, "treatment" means preventing or alleviating the progression or worsening of the pathological condition of a wound, and "prevention" means treatment aimed at preventing the onset of a wound in advance.

[0033] The subjects of the treatment and prevention methods according to the present embodiment are not particularly limited, and may be any animal classified as a mammal, including, in addition to humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses. Particularly preferred "mammals" are humans and dogs.

[0034] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include the plural as well as the singular unless the context clearly indicates otherwise. Also, in this specification, "about" or "to the extent of" means a numerical range of ±10%.

[0035] The present invention will be further explained below by showing examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention.

[0036] I. Materials, Reagents, and Experimental Methods I-1. Materials and Reagents: NIH3T3 cells were purchased from the JCRB Cell Bank. Dulbecco's Modified Eagle Medium (DMEM) was purchased from GIBCO. Bovine Calf Serum (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) 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 membranes, 30,000, 50,000, and 100,000 MWCO) were purchased from SARTORIUS. Millex-GV low protein binding durapore (PVDF) membrane 0.22 μm was purchased from Merck Millipore. PD-10 columns were purchased from Cytiva. Micro BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. 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-mouse IgG-HRP antibody (#62-6520) and anti-rabbit IgG-HRP antibody (#65-6120) were purchased from Invitrogen. Anti-FGF2 antibody (#ab208687) was purchased from Abcam. Blocking One (#03953-95) was purchased from Nacalai Tesque. Recombinant Human FGF basic / FGF2 (#3718-GMP) was purchased from R&D Systems.Human VEGF (#DVE00), Human HGF (#DHG00B), Human FGF basic / FGF2 / bFGF (#DFB50), Human CCL2 / MCP-1 (#DCP00), Human IL-6 (#6050), and Human PDGF-CC (#DCC00) Quantikine ELISA Kits were purchased from R&D Systems. Human beta NGF (#ARG80133) ELISA Kit was purchased from Arigo Biolaborators Corporation.

[0037] I-2. Preparation of deciduous tooth pulp-derived stem cell culture supernatant and its fraction I-2-1. Preparation of human deciduous tooth pulp-derived stem cells 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% FBS by volume, and 2 mg / mL collagenase (Fujifilm Wako Pure Chemical Industries, Ltd.) and dispase (Fujifilm Wako Pure Chemical Industries, Ltd.) were added and incubated at 37°C for 1 hour. The mixture was centrifuged at 777 × g for 5 minutes, the supernatant was removed, and dental pulp cells were collected. The culture was then sterilized with 10% FBS by volume and 1% antibiotic-antimycotic agent (Gibco). TM Dental pulp cells were suspended in DMEM (4 mL) supplemented with an antibiotic-antimycotic and seeded onto a 6-well plate. The cells were cultured at 37°C under 5% CO2 conditions until they became subconfluent. 0.05% trypsin / EDTA solution was added and incubated at 37°C for 5 minutes, after which the cells were harvested 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 7 The cells were grown until they became cells. A 0.05% trypsin / EDTA solution was added and incubated at 37°C for 5 minutes. The collected cells were named "SHED (Stem cells from Human Exfoliated Deciduous teeth)."

[0038] I-2-2. Preparation of immortalized human deciduous dental pulp-derived stem cells. Immortalized SHED (IM-SHED) was prepared by introducing the SV40 gene into the human deciduous dental pulp-derived stem cells prepared in I-2-1 using a viral vector. The actual preparation of IM-SHED was outsourced to Applied Biological Materials.

[0039] I-2-3. Preparation of culture supernatant. SHED or IM-SHED prepared in I-2-1 and I-2-2 were subcultured in DMEM containing 10% FBS by volume. Culture supernatants were prepared using SHED cultures that had been subcultured up to 8 times, and IM-SHED cultures that had been subcultured 50–55 times. The medium was replaced with serum-free DMEM and cultured for 48 hours. The medium was then collected and filtered through a separation membrane (Stericup Quick Release-GP, PVDF (0.22 μm); Merck Millipore) to obtain the culture supernatant (conditioned medium: CM). Hereafter, 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] I-2-4. Fractionation and Concentration of Culture Supernatant. The CM was centrifuged at 180 × g for 3 minutes at room temperature, 500 × g for 30 minutes at 4°C, and 2,000 × g for 30 minutes at 4°C. Cells and other impurities were removed by filtration through a Millex-GV low protein binding durapore (PVDF) membrane (0.22 μm). 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 (hereafter 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 passed through a 30,000 MWCO ultrafiltration unit and centrifuged at 2,000 × g for 20 minutes at 4°C.

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

[0042] I-3. ELISA Quantification of VEGF, HGF, bFGF, MCP-1, IL-6, NGF, and PDGF The following describes the quantification of human VEGF. Reagent Preparation: The reagents included with the Human VEGF Quantikine ELISA Kit were used. Wash buffer was prepared by adding 480 mL of distilled water to 20 mL of wash buffer concentrate. The substrate solution (TMB) was prepared by mixing equal volumes of Color Reagent A and B 15 minutes before use. The mixture was protected from light. The human VEGF standard was prepared by reconstituting the standard vial with 1 mL of Calibrator Diluent RD5K and gently stirring for 15 minutes before dilution. 500 μL of Calibrator Diluent RD5K was dispensed into each tube in advance, and the VEGF standard was diluted as shown in Figure 1.

[0043] Assay: 50 μL of Assay Diluent RD1W was added to each well. Then, 200 μL of standard, control, and supernatant samples were added to each well, sealed with a plate sealer, and incubated at room temperature for 2 hours. The plate was washed three times with wash buffer, and 200 μL of VEGF conjugate (secondary antibody) was added to each well and incubated at room temperature for 2 hours. After washing the plate three times with wash buffer, 200 μL of substrate solution (TMB) was prepared (see Reagent Preparation) and added to each well. The plate was incubated at room temperature for 20 minutes. After adding 50 μL of stop solution to each well, the absorbance at 450 nm (corrected to 570 nm) was measured using an Infinite F Plex multimode microplate reader.

[0044] Assays using the Human HGF, bFGF, MCP-1, IL-6, NGF, and PDGF ELISA Kits were performed according to the VEGF quantification method described above, with reference to the respective kit instructions for standard and sample dilution. For HGF and NGF assays, 200 μL of peroxidase (1× HRP-Streptavidin), an enzyme that catalyzes the oxidation of hydrogen peroxide as a substrate, was added to each well after incubation at room temperature for 30 minutes. After washing the plate four times with wash buffer, 200 μL of substrate solution (TMB) was added, as in the VEGF assay. After incubation at room temperature for 30 minutes (HGF) or 15 minutes (NGF), 50 μL of stop solution was added to each well, and the absorbance at 450 nm (corrected at 570 nm) was measured.

[0045] I-4. Western blotting of bFGF. Desalted culture supernatant or fraction samples were added to SDS sample buffer and heated at 95°C for 5 minutes, followed by electrophoresis on a 12.5% ​​polyacrylamide gel. The electrophoresed proteins were transferred to a nitrocellulose membrane and blocked with Blocking One (room temperature, 1 hour). The nitrocellulose membrane was then incubated overnight at 4°C in an anti-FGF2 antibody (1:2000) solution. After washing three times with 0.05% Tween-TBS, the membrane was incubated in an anti-rabbit IgG-HRP antibody (1:20,000) solution at room temperature for 1 hour. After washing three times with 0.05% Tween-TBS, the membrane was detected using Clarity Western ECL Substrate.

[0046] I-5. Activation of cell migration by IM-SHED-CM fraction (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 linear scratch was created on the cell monolayer using a 200 μL pipette tip. After two washes with DMEM, cells were added to 0.4% CH-CS / DMEM with CM (1 / 2 dilution); recombinant human FGF-2 (10 ng / mL); >3.5 kDa (1x concentrated, 42.7 μg / mL), >100 kDa (1x concentrated, protein concentration undetermined), or 50-100 kDa (2x concentrated, 48.2 μg / mL) for 24 hours.

[0047] II. Results II-1. Comparison of cytokines contained in IM-SHED-CM and SHED-CM Cytokines (VEGF, HGF, bFGF, MCP-1, IL-6, NGF, PDGF) contained in IM-SHED-CM and SHED-CM were quantified by ELISA. Compared to SHED-CM, the secretion levels of most cytokines in IM-SHED-CM were increased. In particular, the secretion level of bFGF increased approximately 190-fold compared to the level in SHED-CM, and this increase was more significant than that of other cytokines (Figure 2).

[0048] II-2. Effect of IM-SHED passage on the amount of bFGF in IM-SHED-CM IM-SHED were cultured for a long period of time, and the amount of bFGF secreted was measured as a function of the passage number. It was found that the secretion amount was maximized at 65-70 passages, indicating that there is an optimal passage number for secretion (Figure 3).

[0049] Furthermore, IM-SHED cells at passage 50 were cultured for an extended period in an automated culture system, with the supernatant collected twice a week for a total of 53 collections. The amount of bFGF secreted from each supernatant was measured, and the results are shown in Figure 4. The amount of bFGF secreted decreased as the culture time increased. It was found that there was an optimal number of passages for secretion, even within the culture time using the automated culture system (Figure 4).

[0050] II-3. Western Blotting Detection of bFGF in IM-SHED-CM. bFGF was detected by Western blotting using an anti-bFGF antibody in IM-SHED-CM, SHED-CM, culture supernatants of human adipose-derived mesenchymal stem cells (MSCs), human bone marrow-derived mesenchymal stem cells, and human umbilical cord-derived stem cells. Recombinant human bFGF (17.2 kDa, rhbFGF) was also detected as a control. Western blotting revealed a band for rhbFGF near the 15 kDa marker. It was also detected near the 15 kDa marker in the culture supernatants of IM-SHED-CM and human umbilical cord-derived stem cells (Figure 5). Four additional high-molecular-weight bands (18 kDa, 20 kDa, 37 kDa, and 40 kDa) were detected in IM-SHED-CM, indicating the presence of variants and multimers.

[0051] II-4. Separation of bFGF with different apparent molecular weights in IM-SHED-CM bFGF was detected by Western blotting for IM-SHED-CM (unfractionated), the >3.5 kDa fraction, the 50-100 kDa fraction, and the >100 kDa fraction. The results are shown in Figure 6. A bFGF band was detected in each fraction except for the >100 kDa fraction.

[0052] II-5. Cell migration activity of bFGF in IM-SHED-CM. Scratch assays using NIH3T3 cells were performed using IM-SHED-CM (unfractionated), the >3.5 kDa fraction, the 50-100 kDa fraction, and the >100 kDa fraction. Strong cell migration was observed in the groups treated with IM-SHED-CM (unfractionated), the >3.5 kDa fraction, and the 50-100 kDa fraction, and after 24 hours, the scratched area was confirmed to be covered with cells (Figure 7). On the other hand, almost no cell migration was observed with the >100 kDa fraction. Numerical values ​​of the scratch area for the results after 24 hours in Figure 7 are shown in Figure 8. In the groups containing IM-SHED-CM (unfractionated), the >3.5 kDa fraction, and the 50-100 kDa fraction, the scratch area was reduced to approximately 10-20% (Fig. 8). As shown in Fig. 4, this result indicates that fractions containing bFGF and its variants and multimers have high cell migration activity, and that bFGF activity (cell migration activity) is maintained regardless of whether it is bFGF, its variants, or multimers.

[0053] Therefore, it is believed that bFGF produced by the method of the present invention has cell migration activity and the ability to induce angiogenesis and benign sarcoma formation.

[0054] The present invention provides a method for producing bFGF, which is known to be effective in wound healing, etc. Therefore, the present invention is expected to be utilized in the medical and healthcare fields.

Claims

1. A method for producing basic fibroblast growth factor (bFGF), comprising the steps of culturing immortalized dental pulp stem cells to prepare a culture supernatant, and purifying bFGF from the culture supernatant.

2. The production method according to claim 1, wherein the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells.

3. The method according to claim 1 or 2, wherein the dental pulp is deciduous dental pulp.

4. A method for producing a culture supernatant containing 100 pg / mL or more of bFGF, comprising the steps of culturing immortalized dental pulp stem cells to prepare a culture supernatant, and recovering the culture supernatant.

5. The production method according to claim 4, wherein the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells.

6. The method according to claim 4 or 5, wherein the dental pulp is deciduous dental pulp.

7. A pharmaceutical composition for treating or preventing wounds, comprising a culture supernatant of immortalized dental pulp stem cells containing at least 100 pg / mL or more of bFGF.

8. The pharmaceutical composition according to claim 7, wherein the immortalized dental pulp stem cells are immortalized by introducing the SV40 gene into dental pulp stem cells.

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