Method for inducing differentiation into osteoblasts
The use of MCP-1 in the culture supernatant and optional purification enhances the efficiency of differentiating mesenchymal stem cells into osteoblasts, achieving rapid and stable calcification on substrates, addressing the inefficiency of long culture periods in existing methods.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- BIOFUTURE TECH LTD
- Filing Date
- 2025-02-26
- Publication Date
- 2026-07-23
AI Technical Summary
Existing methods for differentiating mesenchymal stem cells into osteoblasts require a long culture period in osteoblast differentiation induction medium, typically 7 to 14 days, which is inefficient and time-consuming.
A method involving the use of MCP-1 in the culture supernatant to induce early differentiation of mesenchymal stem cells into osteoblasts, with optional purification using organic solvents to enhance efficiency, and culturing on substrates like non-woven fabrics for adsorption and calcification.
Enables rapid differentiation of mesenchymal stem cells into osteoblasts and stable calcification on substrates, reducing the culture time to as little as 3 days and allowing for large-scale production with cost-effective and efficient differentiation.
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Abstract
Description
Methods for inducing differentiation into osteoblasts
[0001] The present invention relates to a method for inducing differentiation into osteoblasts.
[0002] Bone metabolism is maintained by a balance between osteoblasts, which build bone, and osteoclasts, which resorb bone. Osteoblasts are thought to originate from mesenchymal stem cells, while osteoclasts are thought to originate from morphogenetic monocytes / macrophages.
[0003] Conventionally, a known method for differentiating mesenchymal stem cells into osteoblasts involves culturing mesenchymal stem cells (MSCs) in a dish or plate containing cell proliferation medium, and then, once they reach a confluent state of 80% or more, replacing the medium with osteoblast differentiation induction medium and culturing for two weeks (for example, Patent Document 1). Furthermore, a known indicator for evaluating the induction of differentiation into osteoblasts is to assess the calcification that occurs as mesenchymal stem cells differentiate into osteoblasts by staining calcium with a dye such as alizarin red (for example, Patent Document 2).
[0004] Japanese Patent Publication No. 2012-037416 Japanese Patent Publication No. 2005-124460
[0005] However, the methods described above require culturing mesenchymal stem cells in osteoblast differentiation induction medium (OBM) for a long period of time, such as 7 to 14 days, in order to differentiate them into osteoblasts. Therefore, there is a need for a method that can induce early differentiation of mesenchymal stem cells into osteoblasts, but this problem remains unresolved.
[0006] In view of the above circumstances, the inventors conducted further investigations and made the novel discovery that MCP-1 is a differentiation-inducing protein for osteoblasts. Furthermore, they made the novel discovery that MCP-1 can induce early differentiation from mesenchymal stem cells to osteoblasts, leading to the present invention.
[0007] This invention has been made in view of the circumstances described above, and aims to induce early differentiation of mesenchymal stem cells into osteoblasts.
[0008] The present invention is based on the above findings and aims to advantageously solve the above problems. A first aspect of the present invention is a method for inducing differentiation into osteoblasts, wherein the culture supernatant contains MCP-1. According to this method for inducing differentiation into osteoblasts, mesenchymal stem cells for supernatant collection are cultured in a stem cell proliferation medium until they reach at least 80% confluence, then the mesenchymal stem cells for supernatant collection are transferred to an osteoblast differentiation induction medium and cultured for at least 3 days, the culture supernatant of the osteoblast differentiation induction medium used for the culture is collected, and the mesenchymal stem cells for differentiation induction are cultured for at least 3 days using the collected culture supernatant to induce differentiation into osteoblasts.
[0009] Furthermore, in the first embodiment described above, MCP-1 in a range of 150 to 250 ng / mL may be added to the culture supernatant. Doing so makes it possible to more reliably induce early differentiation from mesenchymal stem cells to osteoblasts.
[0010] Furthermore, in the first embodiment described above, a purified product obtained by purifying a portion of the collected culture supernatant with an organic solvent may be added to the culture supernatant for culturing the mesenchymal stem cells for differentiation induction. By doing so, it is possible to more efficiently induce early differentiation from mesenchymal stem cells to osteoblasts.
[0011] Furthermore, in the first embodiment described above, the process may further include the step of collecting the culture supernatant of the osteoblast differentiation induction medium used for the culture, adding a new osteoblast differentiation induction medium, culturing the mesenchymal stem cells for supernatant collection for at least three more days, and then collecting the culture supernatant of the new osteoblast differentiation induction medium used for the culture. In this way, a large amount of culture supernatant can be collected. Also, in the first embodiment described above, the mesenchymal stem cells for supernatant collection may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.
[0012] In the first embodiment described above, the mesenchymal stem cells for differentiation induction may be differentiated into osteoblasts on the substrate. In this way, the cells can be easily adsorbed onto the substrate and calcified on the substrate.
[0013] According to the present invention, mesenchymal stem cells can be rapidly differentiated into osteoblasts.
[0014] Figure 1 shows the results of Example 3. Figure 2 shows the results of Example 4. Figure 3 shows the results of Example 7. Figure 4 shows the results of Example 7. Figure 5 shows the results of Example 9. Figure 6 shows the results of Example 9. Figure 7 shows the results of Example 9. Figure 8 shows the results of Example 9. Figure 9 shows the results of Example 10. Figure 10 shows the results of Example 10. Figure 11 shows the results of Example 11. Figure 12 shows the results of Example 12. Figure 13 shows the results of Example 13.
[0015] Embodiments of the present invention will be described in detail below. A method for inducing differentiation into osteoblasts according to a first aspect of the present invention is a method for inducing differentiation into osteoblasts, wherein the culture supernatant contains MCP-1.
[0016] In the above method, the mesenchymal stem cells for supernatant collection are 1 × 10 5 cells / mL ~ 3 × 10 5 It is preferable to sow at a concentration of cells / mL, and more preferably at 2 × 10 5 cells / mL ~ 5 × 10 5The cell density is cells / mL. In the above method, it is preferable, and more preferably, that the mesenchymal stem cells for supernatant collection be proliferated to 80-100% confluence. In the above method, the culture period for the mesenchymal stem cells for supernatant collection is at least 3 days, but is preferably 3-6 days, and more preferably 3 days. In the above method, the mesenchymal stem cells for supernatant collection may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp, but are not limited to these.
[0017] In the above method, the mesenchymal stem cells for differentiation induction are 1 × 10⁶ 5 cells / mL ~ 3 × 10 5 It is preferable to sow at a concentration of cells / mL, and more preferably at 2 × 10 5 The cell density is cells / mL. In the above method, the culture period for mesenchymal stem cells for differentiation induction is required to be at least 3 days, but is preferably 3 to 6 days, and more preferably 3 days. In the above method, the mesenchymal stem cells for differentiation induction may be derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp, but are not limited to these.
[0018] In the above method, after collecting the culture supernatant of the osteoblast differentiation induction medium used for culture, a new osteoblast differentiation induction medium may be added, and the mesenchymal stem cells for supernatant collection may be cultured for at least three more days, and the culture supernatant of the new osteoblast differentiation induction medium used for the culture may be further included. In the above step, the culture period of the mesenchymal stem cells for differentiation induction is required to be at least three days, but it is preferably three to six days, and more preferably three days. The above step may also be repeated at least twice. In this way, a large amount of culture supernatant can be collected.
[0019] In the above method, the culture supernatant contains MCP-1, and its concentration is thought to be in the range of approximately 10 to 150 ng / mL, but it is thought that the value may vary depending on the cells and culture supernatant used. Therefore, in the above method, it is also possible to add MCP-1 in the range of 150 to 250 ng / mL to the culture supernatant. By doing so, it is possible to more reliably induce early differentiation from mesenchymal stem cells to osteoblasts. Preferably, 200 to 250 ng / mL of MCP-1 is added, and more preferably, 250 ng / mL of MCP-1 is added.
[0020] In the above method, a portion of the collected culture supernatant may be purified with an organic solvent to obtain a purified product, which may then be added to the culture supernatant for culturing the mesenchymal stem cells for differentiation induction. This allows for more efficient early differentiation induction from mesenchymal stem cells to osteoblasts. The collected culture supernatant can be purified, for example, by adding 4 to 8 times the amount of cooled organic solvent such as ethanol or acetone to 20 mL of the culture supernatant, letting it stand at room temperature for 5 minutes, centrifuging at 8,000 rpm for 10 minutes, aspirating the resulting precipitate under reduced pressure, suspending it in 20 mL of basal medium, and filtering it. The method is not limited to the above method, as long as it is a purification method that can produce a purified product. The organic solvent can be any organic solvent commonly used for purification, and organic solvents other than ethanol and acetone can also be used. Examples of basal media include IMDM medium, α-MEM medium, and DMEM medium, but the method is not limited to these, as it can be any medium that can suspend the purified product. The filter can be, for example, a 0.45 μm filter, but is not limited to any filter that can filter and sterilize the prepared purified product + culture medium.
[0021] In the above method, the mesenchymal stem cells for differentiation induction may be induced to differentiate into osteoblasts on a substrate. By doing so, cells can be easily adsorbed onto the substrate. For example, the substrate can be inserted into the target site, and the MSCs around the substrate can be differentiated into osteoblasts and calcified on the substrate. In the above method, non-woven fabrics, collagen sheets, biodegradable films, etc. can be used as the substrate.
[0022] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
[0023] (Example 1) Preparation of culture supernatant 5×10 5 cells / mL of adipose stem cells (ASC) (Lonza, product number: PT-5006) were cultured in a serum-free medium for mesenchymal stem cells (Fukoku Co., Ltd., product number: FKCM301T) with 2% addition of FBS (Cytiva, product number: SH30910) in a T-175 flask until 80-100% confluent. Then, it was replaced with 45 mL of (S) osteoblast differentiation induction medium (OBM) (BMK, product number: BMK-R008) and cultured for 3 days. The culture supernatant after the 3-day culture was collected. The culture supernatant collected after the 3-day culture was designated as (a) "OBM-CM1". Further, the above step (S) was repeated, and the culture supernatant of the 3-day culture was collected as (b) "OBM-CM2". Then, the medium was changed to a serum-free medium for mesenchymal stem cells (Fukoku Co., Ltd., product number: FKCM301T) and cultured for 3 days, and the culture supernatant after the 3-day culture was collected as (c) "301T-CM3".
[0024] (Example 2-1) Preparation of cultured cells The culture of adipose stem cells (ASC) (Lonza, product number: PT-5006) for "mesenchymal stem cells for differentiation induction" used after Example 3 was carried out using a KFCM301T medium containing 2% FBS for ASC, and 5× 5 cells / mL of ASC were grown to 90% confluence in a T-75 flask, detached with trypsin-EDTA (Nacalai Tesque, product number: 32777-44), and 5× 4Seed cells in 0.5 mL per well, then 2 × 10 × 10 × 12 well plates. 5 Seeds seeded at cells / mL (1mL) were used.
[0025] (Example 2-2) Preparation (Purification) of Protein Precipitate (Purified Product) 20 mL each of the culture supernatant (a) to (c) of the osteoblast differentiation induction medium prepared in Example 1 was mixed with 8 times the amount of cooled organic solvent ethanol (acetone can also be used), and after standing at room temperature for 5 minutes, the mixture was centrifuged at 8,000 rpm for 10 minutes to obtain a precipitate. The obtained precipitate was aspirated under reduced pressure, suspended in 20 mL of IMDM medium, and filtered through a 0.45 μm filter (Cytiva, product number: 6900-2504). The protein precipitates purified from the culture supernatant (a) to (c) in this manner are referred to as purified products (A) to (C), respectively.
[0026] (Example 3) Purification of OBM (control) Add 8 times the amount of cooled organic solvent, ethanol (acetone can be substituted), to 20 mL each of osteoblast differentiation induction medium (OBM) (BMK Corporation, product number: BMK-R008), and let stand at room temperature for 5 minutes. Then centrifuge at 8,000 rpm for 10 minutes to obtain a precipitate. After vacuum aspirate, dissolve in IMDM to obtain "purified OBM". As a control, untreated OBM was also diluted 2-fold with IMBM ("untreated OBM"). Adipose stem cells were placed in a 48-well plate in a 5 × 10⁶ 4 Cells were seeded in 0.5 mL wells, and the following day, the culture medium was changed between the "purified OBM" and "untreated OBM" described above. The culture medium was changed every 3 days, and 10 days after culturing, the cells were stained with the Alizarin Red S staining kit (Bio Mirai Kobo Co., Ltd., product number: BMK-R009). The results are shown in Figure 1 (microscope magnified image (×40)). ASCs cultured in "untreated OBM" calcified and stained red with Alizarin Red, but ASCs cultured in "purified OBM" did not calcify and did not stain red with Alizarin Red. From this, it is thought that the low molecular weight component osteoblast-inducing factor did not precipitate with organic solvents and was removed by purification.
[0027] (Example 4) Observation of calcification 1 As described in Example 2-1, on the day after seeding ASCs in a 48-well plate, the culture supernatants (a) to (c) prepared in Example 1 and the purified products (A) to (C) prepared in Example 2-2 were suspended in α-MEM medium and added to the wells, respectively, and cultured. The medium was changed at 3-day intervals, and staining with Alizarin Red was performed on the 1st, 3rd, and 7th days from the start of culture, as in Example 3. The results are shown in Figure 2 (microscope magnified photograph (×40)). High calcification was observed early on the 3rd day of culture in the culture supernatants (a) and (b), and in the purified products (A) and (B). Furthermore, calcification was observed from the 1st day in the culture supernatant (b). In contrast, it can be seen that 7 days were required for calcification in the control OBM. Also, slight calcification was observed in the purified product (C). Thus, it can be said that by using the culture supernatant according to the present invention, it is possible to induce high calcification at an early stage.
[0028] (Example 5) Osteoblast-related gene analysis 1 Cells cultured in Example 4 were sampled on days 1, 3, and 7, and mRNA was extracted using the Maxwell RSC simplyRNA Cells Kit (Promega Corporation, product number: AS1340). The extracted mRNA was used as a template, and qPCR (Thermo Fisher Scientific, product name: Step One PLUS Real-Time PCR System) was performed using the TaqMan primer / probe shown in Table 1.
[0029]
[0030] The results are shown in Tables 2 and 3. "cont." indicates the results of measurements for undifferentiated ASC mRNA. In all cases, OBM (control), culture supernatant (a) and (b), and purified products (A) and (B) showed high expression of osteocalcin mRNA after 3 to 7 days of culture, indicating differentiation into osteoblasts. Furthermore, as shown in Figure 2 (Example 4), purified product (C) showed slight calcification, but slight expression of osteocalcin was confirmed as shown in Table 3. In addition, as shown in Figure 2, calcification occurred in culture supernatant (b) from day 1 of culture, but high expression of osteocalcin mRNA was confirmed as shown in Table 2. Thus, it can be said that osteocalcin expression supports calcification.
[0031]
[0032]
[0033] (Example 6) Component analysis of culture supernatant For the culture supernatants (a) to (c) prepared in Example 1, the following were used for human cytokine quantification: - MCP-1 (R&D, product number: DY279-05) - TGF-β1 (BIOLEGEND, product number: 432907) - EGF (PROTEINTECH, product number: KE00138) - FGF-2 (BIOLEGEND, product number: 434309) - VEGF (BIOLEGEND, product number: 446507) - PDGF-BB (PROTEINTECH, product number: KE00161) - HGF (PROTEINTECH, product number: KE00168) - IGF-1 (R&D, product number: DG100B) - Type I collagen (manufactured by the company) The concentrations of cytokines and the like were measured by ELISA. As a control, the same ASC as in Example 1 was grown to 100% confluence in a T-75 flask using KFCM301T medium containing 2% FBS, and the culture supernatant collected was used. Further, it was grown to 100% confluence, and the culture supernatant collected after culturing for 3 days was used as CM0 (KFCM301T). The results are shown in Table 4. Those with higher expression levels than the control were MCP-1, TGF-β1, HGF, and Type I collagen. Incidentally, TGF-β1 and HGF have been reported in previous studies to be factors involved in the regulation of osteoblasts. Also, it was found that there is an inverse relationship between the state of induction of differentiation into osteoblasts and the MCP-1 concentration. As will be described later, MCP-1 is considered to be involved in promoting mineralization.
[0034]
[0035] (Example 7) Observation of mineralization 2 In a 12 well plate, 2×10 5 Adipose-derived stem cells (ASC) from a different Lot (Lonza Group Ltd., product number: PT-5006) seeded at cells / mL were cultured until 100% confluent, after which the medium was replaced with OBM and culturing was started on day 0. To slow down the differentiation rate, OBM was diluted 2 / 3-fold with IMDM. On day 0, day 3, day 6, day 9, and day 12, culture supernatants were collected from two wells each (301T-CM0, OBM-CM1 to OBM-CM4). Alizarin Red staining was performed on one well in the same manner as in Example 3, and cell lysate samples were taken from one well. During this period, the medium was changed every 3 days. The results of Alizarin Red staining are shown in Figure 3. Since the cells at stage 3 had a peak of calcification from the degree of Alizarin Red staining, stages 1 to 2 were considered to be the stage of osteoblast progenitor cells. Stage Confluence Medium Culture supernatant Stage 0 50 - 100% 2% FBS / FKCM301T 301T-CM0 Stage 1 100% OBM OBM-CM1 Stage 2 100% OBM OBM-CM2 Stage 3 100% OBM OBM-CM3 Stage 4 100% OBM OBM-CM⑷
[0036] Next, 0.5 mL of each of the above-mentioned collected culture supernatants (301T-CM0, OBM-CM1 to OBM-CM4) was added to each well of a 48-well plate seeded with ASC as described in Example 2-1, cultured for 7 days, and stained with Alizarin Red. The results of Alizarin Red staining are shown in Figure 4. The culture using OBM-CM2 at stage 2 showed the most calcification due to the culture supernatant.
[0037] (Example 8) Osteoblast-related gene analysis 2 RNA was extracted from the cell lysates at stages 0 to 4 collected in Example 7, and qPCR analysis was performed using the TaqMan primer probe in Table 1. The results are shown in Table 5.
[0038]
[0039] Regarding the expression of MCP-1 and its receptor CCR2, MCP-1 was expressed most highly in Stage 1, but the expression of CCR2, the receptor for MCP-1, was low in all stages. Therefore, no correlation was observed between CCR2 and calcification. Furthermore, regarding the expression of ALP, type I collagen, and osteocalcin, these were expressed most highly in Stage 1. This genetically demonstrated that ASCs differentiated into osteoblasts through culture with the culture supernatant according to the present invention. From these results, it is considered that MCP-1, which was most highly expressed in Stage 1, also contributes to promoting differentiation into osteoblasts. In Figure 2, (b) "OBM-CM2" showed high calcification ability, but in Table 4, the concentration of MCP-1 was low, which might suggest that MCP-1 suppresses calcification (differentiation into osteoblasts). However, the results in Table 4 show that the highest MCP-1 mRNA concentration is in Stage 1, which is the stage preceding Stage 2 with high calcification. This suggests that MCP-1 mRNA is translated and the MCP-1 protein is highly expressed in OBM-CM2 in Stage 2, indicating that MCP-1 promotes calcification.
[0040] (Example 9-1) Calcification using culture supernatant 3 To confirm whether MCP-1 promotes the differentiation (calcification) of osteoblasts, recombinant MCP-1 was added to 301T-CM0 and OBM-CM1 culture supernatants prepared in Example 7, and calcification was observed. Specifically, ASCs were placed in a 48-well plate in 5 × 10⁶ layers. 4 Cells were seeded in 0.5 mL wells, and the following day, culture was started by adding 124 ng / mL of recombinant MCP-1 (PEPROTECH, product number: AF-300-04) to 301T-CM0 and OBM-CM1 prepared in Example 7. The culture medium was changed every 4 days. In the second medium change, 170 ng / mL of MCP-1 was added to the culture supernatant, and in the third medium change, 250 ng / mL was added. In this way, the concentration of MCP-1 was gradually increased, and staining with Alizarin Red was performed 4, 8, and 12 days after culturing, as in Example 3. For comparison, the same experiment was performed on CM2 and OBM (control).
[0041] The results are shown in Figures 5 to 7. No calcification was observed in 301T-CM0 (culture supernatant cultured with FKCM301T), regardless of whether recombinant MCP-1 was added or not. On the other hand, in OBM-CM1 (culture supernatant of OBM), higher calcification was observed when recombinant MCP-1 was added (Figure 6, 16) compared to OBM-CM1 without MCP-1 (Figure 6, 15).
[0042] In addition to the visual observations described above, 200 μL of 5% formic acid aqueous solution was added to wells stained with Alizarin Red, and the dye was extracted for several minutes. 100 μL of this solution was then sampled into a 96-well plate, and the absorbance was measured at wavelengths of 450 nm and 630 nm. The results are shown in Table 6. Slight calcification was also observed in 301T-CM0 after the addition of MCP-1. There was a correlation between the numerical values and the degree of calcification observed visually, indicating that the addition of MCP-1 promoted calcification.
[0043]
[0044] (Example 9-2) Calcification using culture supernatant 4 The concentration of MCP-1 in the culture supernatant was measured 4 days after the third addition of MCP-1 (250 ng / mL) in Example 9-1. The results are shown in Figure 8. The recovery rate of MCP-1 in the culture supernatant 4 days after the third addition was approximately 14%. From this, it is considered that 86% of the added MCP-1 was consumed for calcification (differentiation).
[0045] As shown in Table 5 of Example 8, MCP-1 mRNA is highly expressed during the calcification process, and as shown in Table 4 of Example 6, it has been confirmed that the translated MCP-1 protein is consumed. As shown in Example 9-2, in OBM-CM1, OBM-CM2, and OBM in Figure 8, the MCP-1 concentration in the culture supernatant after induction of differentiation into osteoblasts (4 days after the third addition) is in the range of approximately 50-70 ng / mL (total addition amount 544 ng / mL). Therefore, it is considered that an MCP-1 concentration of approximately 400-500 ng / mL in the culture supernatant is required during the calcification (differentiation into osteoblasts) process.
[0046] (Example 10) Calcification of BM-MSCs, dental pulp cells, and periodontal ligament cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated from the provided bone marrow fluid, and dental pulp cells and periodontal ligament cells were isolated and cultured from human teeth according to the instructions on page 24 of the "Mesenchymal Stem Cell Handbook" from the Information Organization. Each cell was grown in a T-25 flask to 90% confluence using BSCM-PL1 medium (Bio Mirai Kobo, BMK-S001) supplemented with 2% FBS. The medium was then replaced with OBM and cultured for 3 days, after which the culture supernatant was collected and designated as "OBM-CM1". New OBM was added to the flask after the supernatant was collected, and the culture supernatant was collected and designated as "OBM-CM2". Adipose stem cells were placed in a 48-well plate in a 5×10⁶ 4 Cells were seeded in 0.5 mL / well, and the following day, "OBM-CM1" and "OBM-CM2" collected from the three types of cell cultures were added to each well and cultured. On the 7th day of culture, the cells were stained with Alizarin Red in the same manner as in Example 3. The results are shown in Figures 9 to 10. Even in the culture supernatant of OBM cultures using mesenchymal stem cells other than adipose-derived stem cells, calcification was observed much faster than in cultures using OBM (control).
[0047] (Example 11) Calcification on Substrate 1 A stem cell isolation substrate (Bio Mirai Kobo, BMK-R003) made of a nonwoven fabric with a PE-PP core-sheath structure coated with hydroxyapatite was placed in a 12-well plate. 1 mL of (i) "Purified OBM-CM (+)" obtained by collecting and purifying the culture supernatant from cell culture using FBS-containing OBM using the methods of Example 1 and Example 1, and Example 2-2, and 1 mL of (ii) "Purified OBM-CM (-)" obtained by collecting and purifying the culture supernatant from cell culture using FBS-free OBM were added to the substrate and allowed to stand in a refrigerator for 24 hours for thorough immersion. Similarly, purified culture supernatants ((iii) "Purified 301T-CM") and (iv) OBM (control) obtained by culturing in growth medium were added to the substrate and allowed to immerse thoroughly for 24 hours. After that, the substrate was washed twice with physiological saline. 1 × 10⁶ ASCs were placed in a 12-well plate. 5 Cells / well / mL were seeded in each well, and the following day, the substrate was placed in the wells where the ASC reached 95% confluence or higher and cultured for 24 hours. Then the substrate was transferred to another 12-well plate, and 2% FBS-containing α-MEM (Nacalai Tesque Co., Ltd., product number: 21445-95) was added and cultured for 7 days. After that, the substrate was immersed in 10% neutral buffered formaldehyde solution (Nacalai Tesque Co., Ltd., product number: 37152-51) for 10 minutes, washed with water, and stained with Alizarin Red as in Example 3 and observed. Furthermore, Alizarin Red was eluted from the stained substrate with a 5% formic acid aqueous solution, and the absorbance (OD) was measured. The results are shown in Figure 11. (i) Purified OBM-CM (+) (ii) Purified OBM-CM (-) (iii) Purified 301T-CM (iv) OBM Each magnified photograph is 120x. The results in Figure 11 show that ASCs that were adhered to the bottom of the well migrated to and adhered to the substrate, and then underwent calcification (differentiation into osteoblasts). Comparing (i) and (ii), the degree of calcification was similar with and without FBS, based on visual staining and OD. Magnified images of (i) and (ii) show that cells adhered to the fibers of the substrate are stained. On the other hand, in (iii) and (iv), there is almost no staining, indicating a low degree of calcification.
[0048] (Example 12) Calcification on the substrate 2 Similar to Example 11, the substrate was placed in a 12-well plate, and 1 mL of recombinant MCP-1 (10 μg / mL) used in Example 9-1 was added. The substrate was thoroughly immersed and left to stand in a refrigerator. After 24 hours, the substrate with adsorbed MCP-1 was washed twice with physiological saline. BM-MSCs were placed in a 12-well plate in a 2 × 10⁶ 5 Cells were seeded at a rate of cells / well / mL, and the following day, once at least 95% confluence was achieved, the substrate was placed in the well and cultured for 4 days. After that, the substrate was immersed in 10% neutral buffered formaldehyde solution (Nacalai Tesque Co., Ltd., product number: 37152-51) for 10 minutes, washed with water, and stained with Alizarin Red as in Example 3. The results are shown in Figure 12. (I) Substrate + BM-MSCs adhered to the substrate and calcified (160x magnification) (II) Adhered substrate only (no cells) (160x magnification) (III) Calcified BM-MSCs at the bottom of the well (64x magnification) As shown in Figure 12, in (I), calcification by BM-MSCs adhered to the substrate was observed. This indicates that BM-MSCs adhered to the fibers of the substrate to which recombinant MCP-1 was adsorbed and differentiated into osteoblasts. Fibers of the substrate to which cells were not adhered were not stained with Alizarin Red. Furthermore, since calcification was also observed at the bottom of the wells, it is thought that calcification was promoted by MCP-1 that detached from the substrate.
[0049] (Example 13) Calcification on a substrate 3 A nonwoven fabric made of norbornene polymer prepared from a COP substrate (Zeonor 1060R, Zeon Corporation) was cut into a rectangle so that it could be placed in a 12-well plate. 1 mL of purified OBM-CM(+) prepared in Example 11 was added, and the fabric was thoroughly immersed and left to stand in a refrigerator. After 24 hours, the substrate to which the purified OBM-CM(+) had been adsorbed was washed twice with physiological saline. Then, the COP substrate was placed in a 12-well plate and 1.3 × 10⁻⁶ 5 ASC was added at a concentration of cells / well / mL and cultured for 24 hours. The substrate was then transferred to another 12-well plate, and 2% FBS-containing α-MEM (Nacalai Tesque Co., Ltd., product number: 21445-95) was added and cultured for 7 days. Afterward, the substrate was immersed in 10% neutral buffered formaldehyde solution (Nacalai Tesque Co., Ltd., product number: 37152-51) for 10 minutes, washed with water, and stained with Alizarin Red as in Example 3. The results are shown in Figure 13. (X) COP substrate with purified OBM-CM(+) adsorbed. (Y) COP substrate with culture medium adsorbed. Magnified images of each are 64x. In (X), the entire COP substrate and its fibers were stained red, indicating calcification on the substrate. No calcification was observed in the control (Y).
[0050] The results from Examples 11-13 showed that by adsorbing OBM-CM or MCP-1 onto a substrate and bringing it into contact with MSCs, the MSCs adhered to the substrate and subsequently differentiated into osteoblasts. While it is difficult to retain multiple differentiation-inducing factors, such as low-molecular-weight compounds like OBM, in the necessary spaces and sites for osteoblast differentiation, purified OBM-CM and MCP-1 proteins can be easily adsorbed onto substrates such as nonwoven fabrics. By inserting this substrate into the target site, it becomes possible to differentiate the surrounding MSCs into osteoblasts and induce calcification. Furthermore, although BMP is known as a bone-forming protein, it is difficult to mass-produce, resulting in high costs, and may cause inflammation and pain as side effects. In contrast, OBM-CM can be mass-produced at low cost and can be adsorbed onto substrates such as nonwoven fabrics.
[0051] The method for inducing differentiation into osteoblasts according to the present invention enables early differentiation of mesenchymal stem cells into osteoblasts, and furthermore, allows for stable calcification on a substrate.
Claims
1. A method for inducing differentiation into osteoblasts, wherein the culture supernatant contains MCP-1, wherein mesenchymal stem cells for supernatant collection are cultured in a stem cell proliferation medium until they reach at least 80% confluence, then the mesenchymal stem cells for supernatant collection are transferred to an osteoblast differentiation induction medium and cultured for at least 3 days, the culture supernatant of the osteoblast differentiation induction medium used for the culture is collected, and the mesenchymal stem cells for differentiation induction are cultured for at least 3 days using the collected culture supernatant to induce differentiation into osteoblasts.
2. The method for inducing differentiation into osteoblasts according to claim 1, wherein MCP-1 in an amount in the range of 150 to 250 ng / mL is added to the culture supernatant.
3. A method for inducing differentiation into osteoblasts according to claim 1, wherein a purified product obtained by purifying a portion of the collected culture supernatant with an organic solvent is added to the culture supernatant for culturing the mesenchymal stem cells for differentiation induction.
4. The method for inducing differentiation into osteoblasts according to claim 1, further comprising the step of collecting the culture supernatant of the osteoblast differentiation induction medium used in the culture, adding a new osteoblast differentiation induction medium, culturing mesenchymal stem cells for supernatant collection for at least 3 days, and collecting the culture supernatant of the new osteoblast differentiation induction medium used in the culture.
5. The method for inducing differentiation into osteoblasts according to claim 1, wherein the mesenchymal stem cells for supernatant collection are derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.
6. A method for inducing differentiation into osteoblasts according to any one of claims 1 to 5, wherein the mesenchymal stem cells for differentiation induction are differentiated into osteoblasts on a substrate.