JP2013201940A - Medium for inducing bone differentiation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medium capable of promoting osteocyte differentiation.SOLUTION: It has been found that osteocyte differentiation is remarkably promoted by culturing cells in a medium containing glycosaminoglycan in addition to known differentiation inducing agents such as ascorbic acid, β-glycerophosphate and dexamethasone. The osteocyte differentiation is further promoted by adding a bone growth factor in addition to the differentiation inducing agents.

Description

  The present invention relates to a medium for inducing bone differentiation and use thereof.

  In regenerative medicine, stem cell transplantation is generally carried out as a therapeutic method that has been researched and developed and is in practical use. Stem cells for cell transplantation include embryonic stem cells, mesenchymal stem cells collected from bone marrow and adipose tissue, hematopoietic stem cells, and various tissue stem cells (hepatic stem cells, neural stem cells, etc.). Among them, mesenchymal stem cells are relatively easy to obtain and culture. For this reason, in the field of cell transplantation, the research field of living tissue regeneration using mesenchymal stem cells is the most advanced.

  Examples of the target tissue for regeneration treatment using mesenchymal stem cells include bone, cartilage, fat, muscle, tendon and nerve. For example, bone regeneration uses a method in which mesenchymal stem cells cultured and expanded in vitro are seeded and expanded in a three-dimensional scaffold, then transplanted and treated, and there are therapeutic applications (for example, Patent Document 1).

Usually, in order to culture mesenchymal stem cells outside the body, various kinds of growth factors must be continuously added to the culture medium. As an approach for this, a method of introducing a growth factor gene into a cell, synthesizing the growth factor into a cell, and continuing to supply the growth factor in an autocrine manner, or using a viral vector incorporating a transcription factor gene, Methods have been developed that enable good bone / cartilage regeneration by continuously expressing transcription factor activity (for example, Patent Document 2).
However, the living body originally has a natural healing power, and in fact, simple fractures and the like are treated using the natural healing power. On the other hand, the natural healing power of the living body may not be sufficiently effective for large bone defects, etc., but for example, by increasing the natural healing power by administering a substance that promotes cell proliferation and differentiation Thus, it is possible to realize regeneration and repair of living tissue without performing stem cell transplantation or gene administration. In addition, when a substance having such an action is a substance present in the body, it is considered possible to evaluate the state of cell proliferation and differentiation by targeting this substance.

  From this point of view, development of a cell culture system that promotes cell proliferation and differentiation is expected.

JP 2008-29317 A International Publication No. 2005/035014

  This invention is made | formed in view of such a condition, The objective is to provide the culture medium which can accelerate | stimulate bone differentiation.

  As a result of intensive investigations to solve the above problems, the present inventors have found that cells are cultured in a medium containing glucosaminoglycan in addition to known differentiation inducers such as ascorbic acid, β-glycerophosphate, and dexamethasone. It was found that bone differentiation can be dramatically promoted by culturing. Further, the present inventors have found that bone differentiation can be further promoted by adding an osteogenic factor in addition to the differentiation inducer, and have completed the present invention.

  More specifically, the present invention provides the following inventions.

  (1) A medium for differentiating cells having the ability to differentiate into bone cells into bone cells, comprising glycosaminoglycan, ascorbic acid, β-glycerophosphate, and dexamethasone.

  (2) The medium according to (1), wherein the glycosaminoglycan is heparin, heparan sulfate, or a salt thereof.

  (3) The medium according to (1) or (2), further comprising an osteogenic factor.

  (4) The medium according to (3), wherein the bone morphogenetic factor is bone morphogenetic factor 2 (BMP-2).

  (5) A method for differentiating cells having the ability to differentiate into bone cells into bone cells, comprising culturing cells having the ability to differentiate into bone cells in the medium according to any one of (1) to (4) .

  By using the culture medium of the present invention, it has become possible to dramatically promote bone differentiation.

It is a graph which shows the result of having detected bone differentiation in the culture | cultivation using the differentiation induction culture medium which added heparan sulfate sodium salt. It is a graph which shows the result of having detected the bone differentiation in the culture | cultivation using the differentiation induction culture medium which added heparan sulfate sodium salt and rhBMP-2. It is a graph which shows the result of having detected bone differentiation in the culture | cultivation using the differentiation induction culture medium which added heparin sodium salt. It is a graph which shows the result of having detected bone differentiation in the culture | cultivation using the differentiation induction culture medium which added rhBMP-2 on the heparan sulfate sodium salt fixed plate.

  The present invention provides a medium for differentiating cells that have the ability to differentiate into bone cells, comprising glycosaminoglycan, ascorbic acid, β-glycerophosphate, and dexamethasone into bone cells.

  Examples of “cells having the ability to differentiate into bone cells” using the medium of the present invention include osteoblasts, osteoprogenitor cells, mesenchymal cells (including mesenchymal stem cells), and the like. Cell lines (for example, MC3T3-E1, C3H10T1 / 2), tissue stem cells collected from tissues other than bone, stem cells or progenitor cells having differentiation ability, and the like.

  “Glycosaminoglycan” in the culture medium of the present invention for culturing these cells is added to the medium together with known differentiation inducers ascorbic acid, β-glycerophosphate, and dexamethasone, to the bone cells. There is no particular limitation as long as it can promote differentiation. Examples of the glycosaminoglycan added to the medium include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronic acid, chondroitin, or a salt thereof, preferably heparin, heparan sulfate, or a salt thereof. It is. Regardless of its origin (animal isolation site or isolation method) and preparation method, various sulfated polysaccharides can be used in the present invention.

  The concentration of glucosaminoglycan in the medium can be appropriately set, but is usually 0.1 to 100 μg / mL, preferably 1 to 20 μg / mL. As shown in this example, glycosaminoglycan can be added to a medium to be a component in the medium. It can also be. As a method for binding glycosaminoglycan to an incubator, both a simple coating method and a chemical bonding method can be used. The chemical bonding method can be performed by performing chemical treatment and physical treatment on the surface of the incubator, but may be a method in which these treatments are not performed. As the chemical bonding method, a known chemical bonding method can be used.

The concentrations of ascorbic acid, β-glycerophosphate, and dexamethasone, which are known differentiation inducers used in combination with glucosaminoglycan, in the medium can be appropriately set. Ascorbic acid is usually 1 to 1000 μg / mL. Yes, preferably 10-100 μg / mL, β-glycerophosphate is usually 0.1-100 mM, preferably 1-20 mM, and dexamethasone is usually 5 × 10 ^{−9 to} 5 × 10 ⁻⁸ M And preferably 2 × 10 ⁻⁹ to 2 × 10 ⁻⁸ M.

  In the culture medium of the present invention, differentiation into bone cells can be further promoted by further adding an osteogenic factor. As the “bone forming factor” to be added to the medium, any substance that can promote differentiation into bone cells when added to the medium together with glycosaminoglycan, ascorbic acid, β-glycerophosphate, and dexamethasone is particularly useful. There is no limit. Examples of bone morphogenetic factors added to the medium include bone morphogenetic factor 2 (BMP-2), bone morphogenetic factor 3 (BMP-3), bone morphogenetic factor 4 (BMP-4) and bone morphogenetic factor 7 (BMP-7) Preferably, it is bone morphogenetic factor 2 (BMP-2).

  The concentration of the osteogenic factor in the medium can be appropriately set. For example, in the case of BMP-2, it is usually 1 to 500 ng / mL, preferably 5 to 200 ng / mL.

  In the medium of the present invention, the same components as those used in known basic media for mammalian cells can be used other than the above components. Well-known basal media that can be used for the medium of the present invention include, for example, Alpha Eagle basal medium (αMEM), Eagle basal medium (MEM), Dulbecco's modified Eagle medium (DMEM), Ham's F-12 medium and F- 10 medium, mesenchymal cell basic medium (MSCBM) and the like.

  The medium of the present invention can contain various amino acids, various vitamins, various inorganic salts and the like contained in these basic media, and can further contain other components.

  Examples of the amino acids contained in the medium of the present invention include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L -Histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine it can.

  Examples of inorganic salts include calcium chloride, copper sulfate, iron (III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Mention may be made of zinc sulfate.

  Examples of vitamins include biotin, choline, vitamin B12, folic acid, inositol, nicotinamide, pantothenic acid, vitamin B6, vitamin B2, and vitamin B1.

  The medium of the present invention may contain serum (for example, fetal calf serum, autoserum, synthetic serum), and may be serum-free or low serum.

  Other components added to the medium of the present invention include growth factors such as fibroblast growth factor (FGF) and epithelial cell growth factor (EGF), antibiotics such as penicillin, streptomycin, gentamicin and kanamycin, and carbon such as glucose. Sources, hypoxanthine, sodium pyruvate, thymidine, phenolsulfonephthalein, putrescine dihydrochloride, and the like.

  The present invention also provides a method for differentiating cells having the ability to differentiate into bone cells into bone cells, comprising culturing cells having the ability to differentiate into bone cells in the medium.

As for the culture conditions, normal conditions can be used except that the above medium is used. The culture can be performed, for example, in an environment of 37 ° C. and 5% CO ₂ . Further, it is preferable to change the medium once every 3 to 4 days. The culture period required for differentiation induction can be appropriately set according to the type of cells used for differentiation induction. Usually, it can be differentiated into bone cells in about 2 to 3 weeks.

Differentiation into bone cells can be detected using, for example, calcium deposition or alkaline phosphatase activity as an indicator. For these detections, commercially available kits such as a calcium measurement kit (Calcium C-Test Wako, Wako Pure Chemical Industries, Ltd.) and an alkaline phosphatase measurement kit (Lab Assay ^TM ALP, Wako Pure Chemical) can be used.

  In addition, it is preferable to seed | inoculate the cell which has the differentiation ability to a bone cell to the normal culture medium which does not contain said differentiation-inducing component, and to culture | cultivate for 2 to 4 days, before culturing with said differentiation-inducing medium.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

[Example 1] Differentiation induction medium supplemented with heparan sulfate sodium salt Mouse-derived osteoprogenitor cells MC3T3-E1 were placed in a 24-well plate (manufactured by Corning) containing medium supplemented with 10% fetal bovine serum in α-MEM. The cells were seeded at 1.25 × 10 ⁴ cells / well and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Ascorbic acid (Nacalai Tesque, Inc.) 50 [mu] g / mL, beta-glycerophosphate (SIGMA Co.) 10 mM, dexamethasone (Nacalai Tesque, Inc.) and 10 ^-8 M and heparan sulfate sodium salt (Seikagaku Corporation) 10 [mu] g / mL In this way, it was added to the culture medium to start induction of bone differentiation. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Example 2] Differentiation induction medium supplemented with heparan sulfate sodium salt and rhBMP-2 Mouse-derived osteoprogenitor cells MC3T3-E1 were added to a 24-well plate (Corning) containing medium supplemented with 10% fetal bovine serum in α-MEM. The cells were seeded at 1.25 × 10 ⁴ cells / well and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, Dexamethasone (Nacalai Tesque) 10 ^-8 M, Heparan sulfate sodium salt (Seikagaku Corporation) 10 μg / mL and Induction of bone differentiation was started by adding rhBMP-2 at 10 ng / mL to the medium. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Example 3] Differentiation-inducing medium supplemented with heparin sodium salt Mouse-derived bone progenitor cells MC3T3-E1 were added to a 24-well plate (manufactured by Corning) in a medium containing α-MEM plus 10% fetal bovine serum. The cells were seeded at 10 ⁴ cells / well and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Ascorbic acid (Nacalai Tesque, Inc.) 50μg / mL, β- glycerophosphate (SIGMA Co.) 10 mM, and dexamethasone (Nacalai Tesque, Inc.) 10 ^-8 M and heparin sodium salt (Wako Pure Chemical Industries, Ltd.) 10 [mu] g / mL In this way, it was added to the culture medium to start induction of bone differentiation. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Example 4] Culture in differentiation induction medium supplemented with rhBMP-2 in heparan sulfate sodium salt fixed plate
A 24-well plate (Sumitomo Bakelite Co., Ltd.) was treated with oxygen plasma for 10 minutes and then irradiated with UV. 100 μL / well of an aqueous solution of DMT-MM (10 equivalents / COOH, Wako Pure Chemical Industries, Ltd.) and ethylenedioxybisethylamine (50 equivalents / COOH, manufactured by SIGMA) was added and reacted at room temperature for 2 hours. After washing twice with 500 μL / well of sterilized water, add 100 μL / well of an aqueous solution of DMT-MM (3 eq / COOH) and heparan sulfate sodium salt (7 μg / well, Seikagaku Corporation), and react at room temperature for 2 hours. It was. 1 M NaCl in PBS (−) was added at 100 μL / well, left to stand for 10 min, removed, and then washed with 300 μL / well and washed twice with PBS (−) 500 μL / well.

Mouse-derived bone progenitor cells MC3T3-E1 were seeded at 1.25 × 10 ⁴ cells / well in the above-mentioned heparan sulfate-fixed 24-well plate containing medium containing 10% fetal bovine serum in α-MEM, The cells were cultured at 37 ° C. for 2 days in a% CO ₂ incubation. Ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, Dexamethasone (Nacalai Tesque) 10 ^-8 M and rhBMP-2 100 ng / mL bone added to the medium Differentiation induction was started. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 1 week in a 5% CO ₂ incubation.

[Comparative Example 1] Differentiation induction medium 1.25 × 10 ⁴ cells / well in 24-well plate (manufactured by Corning) containing mouse-derived bone progenitor cells MC3T3-E1 and 10% fetal bovine serum in α-MEM And cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Bone differentiation induction was started by adding to the medium so that ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, and dexamethasone (Nacalai Tesque) 10 ⁻⁸ M were added. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Comparative Example 2] Differentiation induction medium supplemented with rhBMP-2 Mouse-derived bone precursor cells MC3T3-E1 were added to a 24-well plate (manufactured by Corning) in a medium containing α-MEM plus 10% fetal bovine serum. The cells were seeded at 10 ⁴ cells / well and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, Dexamethasone (Nacalai Tesque) 10 ^-8 M and rhBMP-2 10 ng / mL are added to the medium. Differentiation induction was started. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Comparative Example 3] Differentiation induction medium 1.25 × 10 ⁴ cells / well in a 24-well plate (Corning) containing mouse-derived bone progenitor cells MC3T3-E1 and α-MEM plus 10% fetal bovine serum And cultured at 37 ° C. for 2 days in a 5% CO ₂ incubation. Bone differentiation induction was started by adding to the medium so that ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, and dexamethasone (Nacalai Tesque) 10 ⁻⁸ M were added. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 2 weeks in a 5% CO ₂ incubation.

[Comparative Example 4] Culturing in a differentiation induction medium supplemented with rhBMP-2 in a sterilized water-treated plate
A 24-well plate (Sumitomo Bakelite Co., Ltd.) was treated with oxygen plasma for 10 minutes and then irradiated with UV. Sterile water (100 μL / well) was added and allowed to stand at room temperature for 2 hours. After washing twice with 500 μL / well of sterilized water, 100 μL / well of sterilized water was further added, and the mixture was allowed to stand at room temperature for 2 hours. 1 M NaCl in PBS (−) was added at 100 μL / well, left to stand for 10 minutes, removed, and then washed with 300 μL / well and washed twice with PBS (−) 500 μL / well.

Mouse-derived bone progenitor cells MC3T3-E1 were seeded in the above-mentioned 24-well plate containing α-MEM plus 10% fetal bovine serum to 1.25 × 10 ⁴ cells / well and incubated with 5% CO ₂ Incubated for 2 days at 37 ° C. Ascorbic acid (Nacalai Tesque) 50 μg / mL, β-glycerophosphate (SIGMA) 10 mM, Dexamethasone (Nacalai Tesque) 10 ^-8 M and rhBMP-2 100 ng / mL bone added to the medium Differentiation induction was started. The medium was changed once every 3 to 4 days, and the cells and the culture supernatant were sampled by culturing at 37 ° C. for 1 week in a 5% CO ₂ incubation.

[Evaluation Method] Measurement of Calcium Deposition Amounts of sampled cells in solution (3M sodium chloride (Wako Pure Chemical Industries, Ltd.), 0.3M trisodium citrate (Nacalai Tesque), 200mg / L sodium dodecyl sulfate (Nacalai Tesque) Company)), add an equal volume of 1MHCL aqueous solution (Nacalai Tesque Co., Ltd.) and let stand at 4 ° C for 4 hours or longer, and then add a calcium measurement kit (Calcium C-Test Wako, Wako Pure Chemical Industries, Ltd.) Used to measure the amount of calcium deposition. Absorbance at 570 nm was measured with a microplate reader (VERSAmax, Molecular Devices), and the value obtained was divided by the number of cells to determine the amount of calcium deposition per cell.

  As a result, in addition to known differentiation inducers such as ascorbic acid, β-glycerophosphate, and dexamethasone, bone differentiation was dramatically promoted by culturing in a medium containing sodium heparan sulfate or heparin sodium salt ( 1 and 3). Further, the addition of rhBMP-2 to the medium further promotes bone differentiation (FIG. 2). Bone differentiation could also be promoted by using a heparan sulfate sodium salt-immobilized plate instead of addition of heparan sulfate sodium salt to the medium (FIG. 4).

  According to the culture medium of the present invention, it is possible to dramatically promote bone differentiation. Therefore, the present invention can greatly contribute to, for example, research on bone differentiation and medical treatment by bone cell transplantation. .

Claims (5)

  A medium for differentiating cells capable of differentiating into bone cells into bone cells, comprising glycosaminoglycan, ascorbic acid, β-glycerophosphate, and dexamethasone.   The medium according to claim 1, wherein the glycosaminoglycan is heparin, heparan sulfate, or a salt thereof.   Furthermore, the culture medium of Claim 1 or 2 containing an osteogenic factor.   The culture medium according to claim 3, wherein the bone morphogenetic factor is bone morphogenetic factor 2 (BMP-2).   A method for differentiating cells having the ability to differentiate into bone cells into bone cells, comprising culturing cells having the ability to differentiate into bone cells in the medium according to any one of claims 1 to 4.