Cell calcification method and screening method using same

Abstract

This cell calcification method involves suspending mesenchymal stem cells in 85-100% serum, allowing the suspended mesenchymal stem cells to stand at 4-10°C for at least 4 days, seeding, in a culture container, the mesenchymal stem cells after being left to stand, and culturing the cells in a serum-free basal medium to calcify the cells.

Description

Method for cellular calcification and screening method using the same

[0001] The present invention relates to a method for calcifying cells, and a screening method using the said method for calcifying cells.

[0002] In recent years, there has been a growing demand for the development of drugs related to calcification, such as calcification accelerators that have therapeutic effects on osteoporosis, and calcification inhibitors that suppress cartilage calcification and stone formation in the body.

[0003] For example, Patent Document 1 provides a calcium calcification inhibitor that suppresses cartilage calcification and stone formation in the body, based on the mechanism of calcium calcification in the human body. Patent Document 2 also provides an osteoblast calcification promoter that can be used as a treatment for osteoporosis.

[0004] However, in order to test (screen) the effectiveness of the developed calcification promoters and inhibitors, for example, preparing a calcification test environment using stem cells requires time to culture the stem cells, induce bone differentiation, and induce calcification, resulting in a complex and time-consuming process.

[0005] Japanese Patent Application Publication No. 2006-104152 Publication No. 05-000965

[0006] Osteoblasts are thought to differentiate from mesenchymal stem cells, and in bone and tooth tissue, osteoblasts differentiate into osteocytes, causing calcification. Normally, calcification by osteocytes is controlled to occur only where needed in the body. However, due to some abnormality, calcification occurs in various parts of the body (other than bone and teeth), causing problems with movement and physiological functions. This phenomenon is called ectopic calcification (ossification), and it is known to occur when hydroxyapatite crystals are deposited on collagen fibers in soft tissue, but the mechanism was unknown.

[0007] In light of the above circumstances, the inventors conducted further investigations and, as a result, discovered a novel method for inducing calcification by a mechanism different from that which occurs when mesenchymal stem cells (MSCs) are differentiated into osteoblasts, by using 85-100% serum in cell culture. This led to the present invention. Specifically, it was newly discovered that calcification occurs when cells are cultured in 85-100% serum for a certain period of time. Depending on the type of cell, fetal bovine serum (FBS), adult bovine serum (BS), or adult human serum can be used. It was also discovered that a calcium concentration of 100 mg / L or higher is important for efficiently inducing calcification.

[0008] This invention has been made in view of the above-described circumstances, and aims to provide a method for inducing cell calcification that enables early calcification through cell culture without the use of differentiation induction media, etc.

[0009] The present invention is based on the above findings and aims to advantageously solve the above problems. The first aspect of the present invention is a method for calcifying cells, comprising: suspending mesenchymal stem cells in 85-100% serum; allowing the suspended mesenchymal stem cells to stand at 4-10°C for at least 4 days; seeding the mesenchymal stem cells after standing in a culture vessel; and culturing them in serum-free basal medium to induce calcification. With such a method for calcifying cells, it is possible to induce calcification early in cell culture without using differentiation induction medium or the like.

[0010] Furthermore, a second aspect of the present invention is a method for calcifying cells, comprising a culture step of culturing the cells in 85-100% serum for at least four days. With such a method for calcifying cells, it is possible to induce calcification early in cell culture without using differentiation induction media or the like.

[0011] In the second embodiment described above, the serum is bovine-derived, the cells are selected from the group consisting of mesenchymal stem cells, dental pulp cells, periodontal ligament cells, vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, dermal papilla cells, cell lines, and cancer cells, the cell lines may be HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cells, and the cancer cells may be A549, THP-1, BRL-2H3, or P3U1. Such specific serum and cell combinations can more effectively induce calcification.

[0012] In the second embodiment described above, the serum may be of human origin, and the cells may be mesenchymal stem cells or epidermal keratinocytes. Such specific serum and cell combinations can more effectively induce calcification.

[0013] In the second embodiment described above, calcium may be added to the serum to a concentration of 100 mg / L or more. This allows for more efficient calcification.

[0014] Furthermore, a third aspect of the present invention is a screening method for screening calcification inhibitors using cells calcified by the cell calcification method described in the first or second aspect. With such a screening method, candidate drugs can be screened using the calcification with extremely high efficiency.

[0015] According to the present invention, calcification can be induced early by cell culture without using differentiation induction media, etc.

[0016] Figure 1 shows the results of Example 1. Figure 2 shows the results of Example 2. Figure 3 shows the results of Example 4. Figure 4 is a table showing the results of Example 5. Figure 5 is a table showing the results of Example 6. Figure 6 is a table showing the results of Example 7. Figure 7 shows the results of Example 8. Figure 8 shows the results of Example 9. Figure 9 shows the results of Example 10.

[0017] Embodiments of the present invention will be described in detail below. A first embodiment of the present invention is a method for calcifying cells, characterized by suspending mesenchymal stem cells in 85-100% serum, allowing the suspended mesenchymal stem cells to stand at 4-10°C for at least 4 days, seeding the standing mesenchymal stem cells in a culture vessel, and culturing them in serum-free basal medium to induce calcification. A second embodiment of the present invention is a method for calcifying cells, characterized by a culture step in which cells are cultured in 85-100% serum for at least 4 days. A third embodiment of the present invention is a screening method that screens for calcification inhibitors using cells calcified by the first or second embodiment of the present invention.

[0018] The inventors have newly discovered that calcification occurs when MSCs treated with low-temperature therapy are cultured in 85-100% FBS for a predetermined period. Furthermore, they have newly discovered that calcification occurs even without low-temperature therapy by culturing cells in 85-100% FBS under predetermined conditions.

[0019] In the first aspect of the present invention, the mesenchymal stem cells that can be used are, for example, bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (ASCs), peripheral blood-derived mesenchymal stem cells, umbilical cord Wharton's Jelly-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and the like.

[0020] In the first embodiment described above, the serum used for cell suspension can be fetal bovine serum (FBS), adult bovine serum (BS), human adult serum, etc.

[0021] In the first embodiment described above, IMDM, DMEM, and α-MEM can be used as the basal culture medium, but are not limited to these, and any commercially available basal culture medium suitable for culturing mesenchymal stem cells can be used.

[0022] In the first embodiment described above, the low-temperature treatment, in which the material is left to stand at 4 to 10°C, is carried out for at least 4 days, preferably 4 to 5 days, and more preferably 4 days.

[0023] In the first aspect described above, the concentration of cells suspended in 85 to 100% serum during the low-temperature treatment is 1×10 5 cells / ml to 1×10 7 cells / ml is preferred. Most preferably, 1.5×10 6 cells are suspended in 1.5 ml of 85 to 100% serum. Also, in the first aspect described above, the concentration of cells when seeding into a culture vessel such as a plate during the culture after the low-temperature treatment is 2.5×10 4 cells / ml to 10×10 4 cells / ml is preferred. Most preferably, it is 5×10 4 cells / well. When using serum at a concentration other than 100%, it is appropriately diluted with a basal medium or the like.

[0024] In the method for calcifying cells according to the second aspect of the present invention, culturing is performed in a serum of 85 to 100% for at least 4 days, but it can be cultured up to a maximum of 14 days.

[0025] In the second aspect described above, the concentration of cells in the culturing step of culturing in a serum of 85 to 100% for at least 4 days is 2.5×10 4 [[ID=二十一]] cells / ml to 10×10[[ID=二十二]] 4 [[ID=二十三]] cells / ml is preferred. Most preferably, cells at a concentration of 5×10[[ID=二十四]] 4 [[ID=二十五]] cells / well are cultured in a serum of 85 to 100%. When using serum at a concentration other than 100%, it is appropriately diluted with a basal medium or the like. [[ID=二十六]] [[ID=二十七]]

[0026] [[ID=二十八]] In the second aspect described above, the serum used in the culturing step can be fetal bovine serum (FBS), adult bovine serum (BS), human adult serum, etc., depending on the type of cells to be cultured, but is not limited thereto. [[ID=二十九]] [[ID=三十]]

[0027] For example, if the serum is of bovine origin, suitable cells include mesenchymal stem cells, dental pulp cells, periodontal ligament cells, vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, dermal papilla cells, cell lines, and cancer cells. Examples of cell lines include HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cells, and examples of cancer cells include HA549, THP-1, BRL-2H3, or P3U1, but are not limited to these.

[0028] For example, if the serum is of human origin, the cells are suitable, but not limited to, mesenchymal stem cells or epidermal keratinocytes.

[0029] In the second embodiment described above, calcification can be more efficiently induced by adding calcium to the serum to a concentration of 100 mg / L or more. Up to 250 mg / L of calcium can be added. Preferably, the concentration is 100 to 150 mg / L.

[0030] In the screening method according to the third aspect of the present invention described above, candidate drugs for calcification inhibitors and calcification promoters can be screened using the calcification of cells produced by the calcification method according to the first or second aspect described above. However, the candidate drugs are not limited to calcification inhibitors and calcification promoters, and can be used for any candidate drugs related to calcification.

[0031] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0032] (Example 1) Calcification of adipose-derived stem cells (ASCs) by serum culture ASCs (Lonza Corporation, product number: PT-5006) were cultured in a T-75 flask in BSCM-PL1 medium (Bio Mirai Kobo Co., Ltd., product number: BMK-S002) supplemented with 2% FBS (Cytiva, product number: SH30910) until 80-90% confluence was reached. Then, they were detached with trypsin-EDTA (Nacalai Tesque, product number: 32777-44), suspended in the above BSCM-PL1 medium, and 0.5 mL (5 × 10) was placed in a 48-well plate. 4 Cells were seeded (cells / well), and the following day, the culture medium of cells that had reached 100% confluence was replaced with 100% serum (fetal bovine serum (FBS) / adult bovine serum (BS) / human adult serum) and cultured for 10 days. The three types of serum used were FBS (Cytiva, product number: SH30910), BS (Kojin Bio, product number: 12183610), and human adult serum (SigmaAldrich, product number: B9433). On days 3, 7, and 10, the cells were stained with an Alizarin Red S staining kit (Bio Mirai Kobo Co., Ltd., product number: BMK-R009) and observed under a microscope for the presence or absence of calcification. The results are shown in Figure 1 (microscopic magnified image (×64)). As shown in Figure 1, FBS and BS calcified from day 3 of culture. In contrast, no calcification was observed in human adult serum even on day 10 (photo omitted).

[0033] (Example 2) Calcification of various cells by serum culture Next, calcification was observed in the following cells using the three types of serum mentioned above: bone marrow-derived MSCs, Wharton's Jelly-derived mesenchymal stem cells (isolated and cultured from provided human umbilical cord using the method described in the "Mesenchymal Stem Cell Handbook" (Information Agency)), dental pulp cells, periodontal ligament cells (isolated and cultured from provided human teeth using the method described in the "Mesenchymal Stem Cell Handbook" (Information Agency)), vascular endothelial cells (HUVEC) (Takara Bio Inc., product number: C-12200), epidermal keratinocytes (Takara Bio Inc., product number: C-12006), chondrocytes (Takara Bio Inc., product number: C-12710), dermal papilla cells (Takara Bio Inc., product number: C-12071), cell lines: HEC293, L929, Balb / 3T3, MC3T3-E1, Vero cells, corneal cells (SIRC) (JCRB Cell Bank / RIKEN) Cancer cells: A549, THP-1, BRL-2H3, P3U1 (obtained from JCRB Cell Bank / RIKEN BRC Cell Bank).

[0034] For the above cells, except for culturing them in a commercially available medium suitable for each cell until they reached confluence, culturing was performed in the same manner as in Example 1. On days 7 to 11, staining was performed with an Alizarin Red S staining kit, and the presence or absence of calcification was observed under a microscope. The results are shown in Table 1 and Figure 2 (microscopic magnified photograph (×64)). As shown in Table 1 and Figure 2, calcification was observed in all the cells used in the test with FBS and BS, but with human adult serum, calcification was observed only in Wharton's Jelly-derived mesenchymal stem cells and epidermal keratinocytes. In Table 1, "〇" indicates that 50% or more of the well bottom area was stained, "△" indicates that 1% or more and less than 50% of the well bottom area was stained, "×" indicates that there was no staining at all, and "-" indicates no data.

[0035]

[0036] (Example 3) Measurement of calcium concentration in serum Since calcium concentration is considered to be one of the important conditions in calcification, the calcium concentrations of the following FBS, BS, and human adult serum, which included the serum used in Examples 1 to 2, were measured using a Metallometric Calcium Measurement LS kit (Metallogenics Co., Ltd., product number: CA30M). ・FBS (Cytiva, product numbers: Lot. AG29485626 and Lot. AE24573269 of SH30910) ・BS (Cosmo Bio, product number: 12183610; Sigma Aldrich, product number: B9433) ・Human adult serum (Sigma Aldrich, product number: B9433; Cosmo Bio, product numbers: Lot. 20952040 and Lot. 23080478-1 of 12181201) The average values of the measurement results for FBS, BS, and human adult serum are shown in Table 2.

[0037] <000009​Regarding the results in Table 2, since the datasheet of FBS (Cytiva, product number: SH30910) indicated 139 mg / L, it is considered that accurate calcium concentrations can also be measured for BS and human adult serum by the above method. Also, from the results in Table 2, it was found that the calcium concentration in human adult serum is lower compared to FBS and BS.

[0039] (Example 4) Mineralization by Culturing Human Adult Serum with Added Calcium Chloride Thus, mineralization by culturing human adult serum with increased calcium concentration by addition was observed. Specifically, for the ASC and periodontal ligament cells used in Examples 1 and 2, they were cultured for 11 days in human adult serum (the same serum as in Example 1) with calcium chloride (Nacalai Tesque, product number: 06900-14) added to a concentration of 100 mg / L, and similar to Example 1, stained with an Alizarin Red S staining kit and the presence or absence of mineralization was observed under a microscope. The results are shown in Fig. 3 (microscopic magnified photograph (×64)). In the culture with human adult serum with calcium chloride added to a concentration of 100 mg / L, mineralization was observed for both ASC and periodontal ligament cells. From the results of Example 4, it was found that mineralization is effectively promoted by setting the calcium concentration in the serum to 100 mg / L or more.

[0040] (Example 5) mRNA Gene Expression of ASC (1) From the Lysate of ASC on the 3rd, 5th, and 7th days of culture in Example 1, RNA was extracted using the Maxwell (registered trademark) RSC simplyRNA Cells Kit (Promega Corporation, product number: AS1340). Using the extracted RNA as a template and the TaqMan primer / probe described in Table 3, qPCR (ThermoFisher Scientific, Step One PLUS PCR system) was performed.

[0041]

[0042] The results are shown in Figure 4. In the control osteoblast differentiation induction medium (OBM), the expression of osteoblast-related genes ALP, type I collagen, osteocalcin, Runx2, and Sp7 mRNA was elevated. On the other hand, in 100% FBS, 100% BS, and 100% human adult serum, the elevation of expression of all of the above genes was not observed as in OBM, but the expression of some genes was elevated.

[0043] (Example 6) mRNA gene expression in Wharton's Jelly-derived mesenchymal stem cells RNA was extracted from the lysate of Wharton's Jelly-derived mesenchymal stem cells on days 3, 5, and 7 of culture in Example 2 using the same method as in Example 5, with the Maxwell® RSC simplyRNA Cells Kit. The results are shown in Figure 5. In the control OBM, the expression of osteocalcin and Runx 2 mRNA was elevated. On the other hand, no elevation in gene expression was observed in 100% FBS, 100% BS, and 100% human adult serum.

[0044] (Example 7) mRNA gene expression of ASC (2) Except for using ASCs from a different lot than those used in Example 4, ASCs were cultured in 100% human adult serum and human adult serum with increased calcium concentration, similar to Example 4. RNA was extracted from lysates on days 1, 3, and 7 of culture using the Maxwell® RSC simplyRNA Cells Kit in the same manner as in Example 5. The results are shown in Figure 6. In the control OBM, the expression of osteocalcin and ALP mRNA was significantly increased. On the other hand, no increase in gene expression over time was observed in either 100% human adult serum or calcium-supplemented human adult serum. From the results of Examples 5 to 7, it is considered that calcification by 100% serum has a different mechanism of action than calcification by induction of osteogenic differentiation using OBM.

[0045] (Example 8) Calcification by culture after low-temperature treatment 1.5 × 10⁶ 6 Cell ASCs were suspended in 1.5 ml of 100% FBS and subjected to a low-temperature treatment in a refrigerator (4-10°C) for 2-4 days. Afterwards, 5 × 10⁶ cells were placed in a 48-well plate. 4 Cells were seeded in cells / well and cultured for 7 days in serum-free α-MEM after 2 hours. The culture medium was changed every 3 days during the 7-day culture period. Alizarin Red staining was performed on days 1, 3, and 7 of culture in α-MEM. The results are shown in Figure 7. Cells treated with 4 days of cryogenic treatment calcified from day 1 of culture, but cells treated with 2 days of cryogenic treatment (photo omitted) and 3 days of cryogenic treatment did not calcify even on day 7 of culture. In addition, even when cells were suspended in a medium other than serum (such as osteoblast differentiation induction medium) and subjected to cryogenic treatment, no calcification occurred in subsequent culture in serum-free basal medium (data not shown).

[0046] (Example 9) mRNA was extracted from lysates of cells sampled on days 1, 3, and 7 of culture using α-MEM in Example 8, in the same manner as in Example 5, and qPCR was performed. The results are shown in Figure 8. The control was measured for expression in cells cultured only in growth medium (α-MEM). As shown in Figure 8, no expression trend for specific osteoblast-related genes was observed. From this, it is considered that calcification by 100% serum has a different mechanism of action than calcification by induction of osteogenic differentiation using OBM.

[0047] (Example 10) Differences in calcification depending on FBS concentration ASC was suspended in serum-free medium for mesenchymal stem cells containing 2% FBS (Fukoku Co., Ltd., product number: FKCM301T) and measured at 1 × 10 5The cells were adjusted to cells / mL and seeded at 500 μL / well in a 48-well plate. The cells were cultured until 100% confluence. During the medium change, 300 μL / well each of 100% FBS, 90% FBS, 85% FBS, 75% FBS, and 50% FBS was used. Cytiva FBS (product number: SH30910) was used for dilution with physiological saline (Otsuka Pharmaceutical, product number: 1326). On day 4 of culture, the cells were stained with Alizarin Red using the same method as in Example 1, then extracted with 5% formic acid, and the OD at 450 nm / 630 nm was measured using a Nivo multimode plate reader (Revvity). The results are shown in Figure 9. Calcification was highest with 100% FBS, high calcification was observed up to 85% FBS, the degree of calcification decreased with 75% FBS, and no calcification occurred with 50% FBS. This indicates that a high concentration of FBS is important for calcification.

[0048] (Example 11) Screening of calcification inhibitors 1 To investigate whether it is possible to screen for calcification inhibitors using cell calcification by 100% serum culture, the calcification inhibitors listed in Table 4 No. 1 to 3 were prepared at 10 mM, 1 mM, and 100 μM in 2% FBS-containing α-MEM medium (Nacalai Tesque Co., Ltd., product number: 21445).

[0049]

[0050] Next, the ASCs are suspended in serum-free medium for mesenchymal stem cells containing 2% FBS (Fukoku Corporation, product number: FKCM301T) and 1 × 10⁻⁶ 5 The cells were adjusted to cells / mL and seeded at 120 μL / well in a 96-well plate. Once 100% confluence was reached, the culture medium was replaced with 10% FBS (Cytiva, product number: SH30910) at three different concentrations (No. 1-3 in Table 4) and with physiological saline (No. 4) containing 90% FBS as a control (10 types in total), and cultured for 7 days (the medium was changed on the 4th day of culture).

[0051] The substances listed as No. 1-3 in Table 4 were diluted in three stages using a 10-fold dilution series, with a sample size of 3. After the 7-day culture period, SF reagent (Nacalai tesque, product number: 07553-44) was added, and the wavelength was measured using a Nivo multimode plate reader (Revvity) to evaluate the viability of the cells. Next, the reagent was removed, and calcium deposition was stained using Alizarin Red in the same manner as in Example 1. Subsequently, the cells were extracted with 5% formic acid in the same manner as in Example 10, and calcification was evaluated by measuring the OD at 450nm / 630nm using a Nivo multimode plate reader.

[0052] The results are shown in Table 5. As described above, the OD of the formic acid extract was used as an indicator of calcification, and the OD of the SF reagent was used as an indicator of cell viability, and the calcification / SF results were compared. Based on the "control" calcification / SF value for control No. 4 (physiological saline containing 90% FBS), the "evaluation value (%)" for each substance No. 1 to 3 was calculated as ("substance" calcification / SF) / ("control" calcification / SF) × 100. Compared to the evaluation value of the control (100%), the evaluation values ​​of substances No. 1 to 3 were lower than 100%. From this, it can be seen that the calcification inhibition by substances No. 1 to 3 can be measured using this method. Thus, it can be said that it is possible to screen for calcification inhibitors using the calcification of cells cultured in 100% serum.

[0053]

[0054] (Example 12) Screening of calcification inhibitors 2 To investigate whether it is possible to screen for calcification inhibitors using calcification of cells treated with 100% serum at low temperature, 4.2 × 10⁶ ASCs cultured to 80-90% confluence, similar to Example 8, were used. 5 Cells were suspended in 100% FBS at a concentration of cells / mL. 360 μL of the cell suspension and 40 μL of substances No. 1-3 from Table 4 were added to a 2 mL tube, and the tube was left to stand in a refrigerator (4-10°C) for 4 days. After that, the cells were seeded into a 96-well plate at 130 μL / well and cultured for 7 days (the culture medium was changed on the 4th day of culture). After the above 7 days of culture, the evaluation values ​​(%) for substances No. 1-3 were calculated using the same method as in Example 11. The results are shown in Table 6.

[0055]

[0056] Since the evaluation values ​​for substances No. 1 to 3 are lower than 100% compared to the control evaluation value (100%), it can be seen that the calcification inhibition by substances No. 1 to 3 can be measured using this method. This suggests that it is possible to screen for calcification inhibitors using calcification in cells treated with 100% serum at low temperatures.

[0057] These results show that it is possible to induce calcification in specific cells by low-temperature treatment / cell culture using 85-100% serum, without using differentiation induction media or the like. Furthermore, according to this calcification method of the present invention, it is possible to induce calcification from cells with extremely high efficiency using components derived from the cells themselves, without using differentiation induction media or the like. In addition, candidate drugs for calcification inhibitors and calcification promoters can be screened with extremely high efficiency using this calcification.

Claims

1. A method for calcifying cells, in which mesenchymal stem cells are suspended in 85-100% serum, the suspended mesenchymal stem cells are left to stand at 4-10°C for at least 4 days, the mesenchymal stem cells are seeded in a culture vessel after standing, and then cultured in serum-free basal medium to induce calcification.

2. A method for calcifying cells, comprising a culture step of culturing the cells in 85-100% serum for at least four days to calcify the cells.

3. The method for calcifying cells according to claim 2, wherein the serum is derived from a bovine, the cells are selected from the group consisting of mesenchymal stem cells, dental pulp cells, periodontal ligament cells, vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, dermal papilla cells, cell lines, and cancer cells, the cell lines are HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cells, and the cancer cells are A549, THP-1, BRL-2H3, or P3U1.

4. The method for calcifying cells according to claim 2, wherein the serum is of human origin and the cells are mesenchymal stem cells or epidermal keratinocytes.

5. The method for calcifying cells according to claim 4, wherein calcium is added to the serum to a concentration of 100 mg / L or more.

6. A screening method for screening for calcification inhibitors using cells calcified by the cell calcification method described in any one of claims 1 to 5.

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