JP2020068779A - Method for separating/selecting mesenchymal stem cell, culture method, and selection material used for the same - Google Patents

Abstract

To provide a method for separating/selecting a mesenchymal stem cell, a culture method, and selection material used for the same.SOLUTION: A method for separating/selecting mesenchymal stem cell in which a cell cluster containing a mesenchymal stem cell is disseminated onto a coating surface of a material for cell culture to which N-acetylglucosamine group-containing polymer having 2000 to 4000 of specific molecular weight is coated, culture medium is not replaced after charging culture medium, and culture medium is replaced in 2 to 10 days after dissemination. Further, it is preferable to further culture at least for 10 days after the culture medium replacement.SELECTED DRAWING: None

Description

  The present invention relates to a method for separating / selecting mesenchymal stem cells, a method for culturing, and a selecting material used therefor.

Mesenchymal stem cells are tissue stem cells that are present in tissues such as bone marrow and fat and have the ability to differentiate into bone, fat and cartilage. The mesenchymal stem cells have been reported to be differentiated into various tissues such as liver, nerve and vascular endothelial cells, and their clinical application is expected as a cell source for regenerative medicine.
Unlike iPS cells, mesenchymal stem cells do not have barriers such as canceration and the complexity of induction of differentiation, and they are attracting attention because of less ethical problems in isolation from living organisms and ease of induction of differentiation. In the clinical application of mesenchymal stem cells, cell transplantation therapy using mesenchymal stem cells is used to treat liver fibrosis, joint cartilage regeneration, bone tissue regeneration, periodontal tissue regeneration, and treatment during brain injury. Treatments for many diseases are expected. Furthermore, it has been shown that it is effective for repair and regeneration of cytokines, growth factors, and exosomes produced by mesenchymal stem cells themselves into injured tissues. Therefore, in addition to cell transplantation treatment, treatments have been attempted in which the culture supernatant of cultured mesenchymal cells is purified and administered to a living body.

  However, the differentiation ability and cytokine production ability of mesenchymal stem cells are greatly influenced by the establishment method from living tissues, the number of days, and the number of passages of culture. Therefore, it is currently difficult to obtain mesenchymal stem cells that are always in good condition, and it is difficult to realize a treatment that maintains a high therapeutic effect. Basically, mesenchymal stem cells, which have a low number of passages and have not been cultured for a long time, have high differentiation ability and cytokine production ability and are effective for treatment, but as the number of culture days increases, the effect decreases and the therapeutic effect Can not be expected. Therefore, in order to realize regenerative medicine using mesenchymal stem cells, it is important to isolate as many mesenchymal stem cells as possible from the living body, increase the number of cells in short-term culture, and use them for therapy. .. Mesenchymal stem cells are present in bone marrow and fat in an amount of about 0.001 to 0.01%, and it is important to efficiently separate and sort them.

  As a method for separating and selecting mesenchymal stem cells from bone marrow or fat, bone marrow cells are mainly seeded in a cell culture dish, and adherent cells are isolated as mesenchymal stem cells (Non-Patent Document 1). In this isolation method, cells other than mesenchymal stem cells are contaminated because they are performed on the basis of non-specific adhesion to cell culture dishes, and the contaminated cells are capable of differentiating and proliferating mesenchymal stem cells. Ability or migration ability is lost, or even if it is not lost, it causes a decline in mesenchymal stem cell performance, resulting in the problem that a sufficient amount of mesenchymal stem cells cannot be obtained. .. Therefore, although the dish surface coated with fibronectin is also used, since fibronectin is contained in the medium containing serum, the use of the fibronectin-coated dish has sufficient performance as compared with the tissue culture dish. Not obtained. Therefore, a method of isolating cells using a surface marker of mesenchymal stem cells has been proposed. Examples of mesenchymal stem cell markers at this time include CD10, CD13, CD73 (ecto-5 ′ nucleotidase, SH3, SH4), CD105 (endoglin, SH2), and CD166 (ALCAM) as positive markers such as CD34 and CD45 (negative markers). LCA: leukocyte common antigen) has been identified. Furthermore, a method of isolation using CD271 (LNGFR), CD140b (PDGFR-β), CD340 (HER-2 / erbB2), CD349 (frizzled-9), etc. has also been proposed (Non-patent document 2). Furthermore, a method of enriching human mesenchymal stem cells by selecting cells expressing the CD271 (LNGFR) and CD90 (Thy-1) from a cell population containing human mesenchymal stem cells (Patent Document 1), Separation of human mesenchymal stem cells in which only a passing cell population is selected by quantifying the abundance ratio of cells expressing Ror2 or Fzd5 from the cell population containing human mesenchymal stem cells to determine pass / fail of each cell population -A screening method (Patent Document 2) has been proposed.

JP, 2009-060840, A International Publication No. 2016/017795

Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Dougla s, R., Mosca, JD, Moorman, MA, Simonetti, DW, Craig, S., and Marshak, DR (1999) .Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147. Buhring Hans-Jorg, et al, Novel markers for the prospective isolation of human MSC, Annals of the New York Academy of Sciences, annals-1392-000, Haematopoietic Stem Cells VI, 10-Nov-2006.

  Although there are findings such as the above, the quality of mesenchymal stem cells is reduced due to contamination with cells other than mesenchymal stem cells, and the cost and effect on cells due to selection using multiple markers, passage The current situation is that there is no simple and efficient method for isolating and selecting mesenchymal stem cells from bone marrow or fat, such as an increase in the number of times. Further, when the number of mesenchymal stem cells is increased by post-selection culturing, the current situation is that there is no culturing method that does not affect the proliferative ability or the differentiation ability of the mesenchymal stem cells because the number of passages increases.

  Therefore, an object of the present invention is to provide a method for separating / selecting mesenchymal stem cells, a method for culturing, and a selection material used therefor.

  The present inventors have found that, for example, myeloid cells, erythrocytes, erythroblasts, eosinophils, hematopoietic stem cells, and stromal cells (mesenchymal stem cells) exist in the bone marrow cell population. Among them, vimentin-expressing cells are only stromal cells including mesenchymal stem cells, vimentin appears on the cell surface of cancer cells and mesenchymal cells despite the intracellular molecule, and It was discovered that vimentin, which appeared on the cell surface, binds to N-acetylglucosamine. This bond had a very strong bond by using a specific N-acetylglucosamine group-containing polymer (hereinafter, also referred to as AC-GlcNAc polymer). Therefore, the present inventors diligently studied from the findings, and when seeding a cell population containing mesenchymal stem cells such as bone marrow on a specific surface, only mesenchymal stem cells were selectively selected for N-acetylglucosamine on the specific surface. It was found that the mesenchymal stem cells can be separated and selected from other cells by binding to other cells, and the present invention has been completed.

That is, the present invention is the following [1] to [8] relating to a method for separating / selecting mesenchymal stem cells, a method for culturing, and a selecting material used therefor.
[1] Seeding a cell population containing mesenchymal stem cells on the coating surface of a selection material coated with a N-acetylglucosamine group-containing polymer having a weight average molecular weight of 2000 to 4000 and inserting a medium, and then performing no medium exchange A method for separating and selecting mesenchymal stem cells, which comprises performing medium exchange 2 to 10 days after seeding.
[2] The mesenchymal stem cells according to the above [1], wherein the cell population containing the mesenchymal stem cells is seeded, a medium is added, and then the medium is not replaced, and the medium is replaced 5 to 10 days after the seeding. Separation and selection method.
[3] A coating agent containing 0.01 to 1000 μg / mL of the N-acetylglucosamine group-containing polymer is coated so as to cover the entire surface of the cell culture substrate with the coating agent, and the temperature is from 4 ° C. to room temperature. The method for separating / selecting mesenchymal stem cells according to the above [1] or [2], which is characterized in that the solvent is dried without being discarded.
[4] Mesenchymal stem cells separated / sorted by the method for separating / sorting mesenchymal stem cells according to any one of [1] to [3] above are cultured for at least 10 days after the medium exchange. A method for culturing mesenchymal stem cells, which comprises:
[5] A N-acetylglucosamine group-containing polymer having a weight average molecular weight of 2000 to 4000, the polymer being a compound represented by the following chemical formula, and the polymer being coated on a culture substrate. A mesenchymal stem cell sorting material characterized by:

In the formula, n is an integer of 3 to 15.

[6] The N-acetylglucosamine group-containing polymer has a number average molecular weight in the range of 3000 to 5000, and a ratio of the weight average molecular weight to the number average molecular weight is 0.8 or more and less than 1.5. A material for selecting mesenchymal stem cells according to [5] above, which is characterized in that
[7] The mesenchymal stem cell selection material according to the above [5], wherein the culture substrate has a hydrophilic surface.
[8] A coating agent containing 0.01 to 1000 μg / mL of the N-acetylglucosamine group-containing polymer is coated so as to cover the entire surface of the cell culture substrate with the coating agent, and any temperature from 4 ° C. to room temperature is applied. The mesenchymal stem cell selection material according to any one of [5] to [7] above, wherein the material is dried without discarding the solvent.

  According to the present invention, it becomes possible to provide a method for separating and selecting mesenchymal stem cells, a method for culturing, and a selecting material used therefor.

The amount of RAFT agent used when polymerizing an AC-GlcNAc polymer, the weight average molecular weight (Mw), the number average molecular weight (Mn), the ratio of Mw and Mn, and the degree of polymerization of the polymer obtained by the polymerization are shown. The degree of polymerization (Number of carbohydrates per polymer) is calculated by the following formula. (Mw-364 [DTMPA]) / 346 [AC-GlcNAcmonomer] Using biotinylated succinylated WGA (wheat germ agglutinin), the coating state of each AC-GlcNAc polymer on the polystyrene dish was determined as the adsorption amount of the AC-GlcNAc on the dish, and the biotinylated succinylated WGA and AC-GlcNAc were treated with each other. It was evaluated based on the action. The amount of biotinylated succinylated WGA on the dish was developed with HRP-neutravidin and TMB solution, and the absorbance of each well at 450 nm was measured using a microplate reader (product name: iMark, Bio-Rad Laboratories). It was measured. It is a Western blot which shows the expression of cell surface vimentin and (beta) -actin in human immortalized UE7T-13 mesenchymal stem cell. The left side shows the expression of vimentin, and the right side shows the expression of β-actin. 1 is a cell surface protein and 2 is a Western blot of whole cell lysate, respectively. It is a graph which showed adhesion of human immortalized UE7T-13 mesenchymal stem cell to AC-GlcNAc polymer coat dish. The left side shows the adhesion at 37 ° C. and the right side shows the adhesion at 4 ° C., respectively. * P <0.01, n = 3. FIG. 8 is a graph showing inhibition of adhesion of human immortalized UE7T-13 mesenchymal stem cells to AC-GlcNAc polymer-coated dishes by vimentin, AC-GlcNAc5, and PV-MA. The left side shows the inhibition of adhesion at 37 ° C, and the right side shows the inhibition of adhesion at 4 ° C. n = 4. It is a Western blot which shows the expression of vimentin and (beta) -actin in vimentin knockdown UE7T-13 cell and UE7T-13 cell which is a negative control. 1 shows Western blots of control UE7T-13 cells and 2 shows vimentin knockdown UE7T-13 cells, respectively. It is a graph which showed the number of adhesion of a vimentin knockdown cell and a control cell to AC-GlcNAc5 coat dish at 4 ° C. 1 indicates the number of control cells and 2 indicates the number of adhered vimentin knockdown cells. * P <0.01, n = 5. It is the figure which showed the result of the flow cytometric analysis of the expression of the cell surface vimentin in rat bone marrow cells. The left side shows the results of flow cytometry using the anti-mouse IgG antibody, and the right side shows the results of flow cytometry using the cell surface vimentin monoclonal antibody. 1. On the 10th day of culture 2. AC-GlcNAc polymer coated dish (screening material) It is a figure showing a phase contrast image of a colony formed in a tissue culture dish. The left side is 3. Tissue culture dish, 1. The image of AC-GlcNAc polymer coat dish is shown. Each lower image is an enlarged view of the upper image. 1. Mesenchymal stem cells isolated and sorted from rat bone marrow 2. AC-GlcNAc polymer coated dish (sorting material) and 3. It is a figure which shows the colony forming property on the culture dish for tissues. Among the photographs in the upper row, the left side is a 35 mm tissue culture dish, and the right side is a 35 mm cell culture dish on which AC-GlcNAc polymer-coated dishes (selection material) are used to seed rat bone marrow, and 3 to 16 mL of 2 mL medium is used. An image of colonies on each dish after daily culture is shown. The lower graph shows the area of colonies for the entire dish area on the AC-GlcNAc polymer-coated dish (selection material) and the tissue culture dish after 3 to 16 days of culture ( %) Is a graph. * P <0.01, n = 3. 1. 2. AC-GlcNAc polymer coated dish (sorting material) and 3. It is a figure which shows the colony-forming property of the mesenchymal stem cell isolate | separated and selected from the rat bone marrow on the tissue culture dish. Among the photographs in the upper row, the left side is a 35 mm tissue culture dish, and the right side is a 35 mm cell culture dish on which an AC-GlcNAc polymer-coated dish (selection material) is used to inoculate rat bone marrow and cultured in 2 mL of medium for 17 days. Representative graphs of colonies on each of the subsequent dishes, the lower graph shows the area of the colonies (% of the total dish area on the AC-GlcNAc polymer-coated dish (selection material) and the tissue culture dish after 17 days of culture). ) Is a graph. * P <0.01, n = 8. 1. AC-GlcNAc polymer coated dish, 2. PV-MA coated dish and 3. FIG. 3 is a graph showing a representative image of colonies on each dish after culturing rat bone marrow using a tissue culture dish and culturing in 2 mL of medium for 10 days, and the area (%) of colonies of each dish with respect to the total dish area. .. * P <0.01, n = 3. 1. 2. AC-GlcNAc polymer coated dish and 3. For the colonies formed in each of the tissue culture dishes, the expression status of the mesenchymal stem cell marker colonized cells was determined by the positive markers (CD106 and CD90) and the mesenchymal stem cell negative marker (CD11b / CD11b / Observed using c). CD106, CD11b / c and CD90 were stained with PE-conjugated anti-rat CD106 antibody (red), FITC-conjugated anti-rat CD11b / c antibody (green), and FITC-conjugated anti-rat CD90 antibody (green). Vimentin was stained with mouse monoclonal anti-vimentin antibody and AlexaFluor647 conjugated anti-mouse IgG antibody (red). The results of nuclear staining using DAPI are shown. The scale bar is 500 μm. 1. 2. AC-GlcNAc polymer coated dish and 3. Expression of positive markers for mesenchymal stem cells (CD90, CD29, CD44, CD54, CD73, CD105 and CD106) and negative markers for mesenchymal stem cells (CD34, CD45 and CD11b / c) in cells grown in tissue culture dishes A representative chart of flow cytometric analysis is shown. 1. 2. AC-GlcNAc polymer coated dish and 3. Expresses positive markers for mesenchymal stem cells (CD90, CD29, CD44, CD54, CD73, CD105 and CD106) and negative markers for mesenchymal stem cells (CD34, CD45 and CD11b / c) in cells grown in tissue culture dishes It is the graph which showed the ratio of the cell number with respect to the total cell number of the living cell. Alizarin Red stain and Oil Red O stain were used to stain colonies with osteogenesis and adipogenic differentiation. The scale bar is 200 μm. Rat bone marrow cells (3 × 10 ⁶ cells) were seeded on 0.01 to 1000 μg / mL AC-GlcNAc polymer-coated dish (selection material of 1 and 4 to 8) and tissue culture dish (selection material of 3), It is the graph which showed the cell number of the cell cultured for 13 days. The upper chart shows rat bone marrow cells (3 × 10 ⁶ cells) containing 0.01 to 1000 μg / mL AC-GlcNAc polymer-coated dishes (selection materials 1 and 4 to 8) and tissue culture dishes (selection material 3). The results are obtained by analyzing the CD11b / c expression of the cells seeded in (4) and cultured for 13 days by flow cytometry. The lower graph shows the ratio (%) of the number of CD11b / c positive cells to the total number of cells. The upper chart shows rat bone marrow cells (3 × 10 ⁶ cells) containing 0.01 to 1000 μg / mL AC-GlcNAc polymer-coated dishes (selection materials 1 and 4 to 8) and tissue culture dishes (selection material 3). The results of flow cytometry analysis of CD90 expression in cells seeded in (4) and cultured for 13 days. The lower graph shows the ratio (%) of the number of CD90-positive cells to the total number of cells. ** P <0.05, n = 3. 1. 2. AC-GlcNAc polymer coated dish and 3. Figure 2 shows a representative chart of flow cytometric analysis of SA-β-galactosidase activity in cells grown in tissue culture dishes. 1. 2. AC-GlcNAc polymer coated dish and 3. It is a graph which showed SA- (beta) -galactosidase activity of the cell propagated in the tissue culture dish. * P <0.01, n = 11. 1. 2. AC-GlcNAc polymer coated dish and 3. The cells recovered from the tissue culture dish were cultured in a 35 mm tissue culture dish for 3 weeks using a bone differentiation induction medium, an adipose differentiation induction medium, and a cartilage differentiation induction medium, and alizarin red, oil red O, and al. An image stained with cyan blue is shown. The upper row shows a stained image of cells grown in the AC-GlcNAc polymer-coated dish and the lower row shows a tissue culture dish. Rat bone marrow cells (Rat21) were seeded on an AC-GlcNAc polymer-coated dish (selection materials of 1 and 4 to 8), wet100 and a dish for tissue culture (selection material of 3) at 297 × 10 ⁴ cells / 35 mm. .. MesenCult Expansion medium (manufactured by Veritas) was used as the medium, and the medium was exchanged 3 days after the cells were seeded, and then the medium was exchanged every 3 days. Then, after 13 days, cells were collected, the number of cells was counted, and a graph was drawn. Rat bone marrow cells (Rat21) were seeded on an AC-GlcNAc polymer-coated dish (selection material of 1 and 4 to 8), a tissue culture dish (selection material of 3), and wet100 at 297 × 10 ⁴ cells / 35 mm. .. MesenCult Expansion medium (manufactured by Veritas) was used as the medium, and the medium was exchanged 3 days after the cells were seeded, and then the medium was exchanged every 3 days. After 13 days, the cells were collected, stained with the CD11b / c antibody, and the stained cells were examined by flow cytometry. Rat bone marrow cells (Rat21) were seeded on an AC-GlcNAc polymer-coated dish (selection material of 1 and 4 to 8), a tissue culture dish (selection material of 3), and wet100 at 297 × 10 ⁴ cells / 35 mm. .. MesenCult Expansion medium (manufactured by Veritas) was used as the medium, and the medium was exchanged 3 days after the cells were seeded, and then the medium was exchanged every 3 days. After 13 days, the cells were collected and stained with the CD90 antibody, and the stained cells were examined by flow cytometry. Rat bone marrow cells (Rat21) were seeded on an AC-GlcNAc polymer-coated dish (selection material of 1 and 4 to 8), a tissue culture dish (selection material of 3), and wet100 at 297 × 10 ⁴ cells / 35 mm. .. MesenCult Expansion medium (manufactured by Veritas) was used as the medium, and the medium was exchanged 3 days after the cells were seeded, and then the medium was exchanged every 3 days. After 13 days, cells were collected and CD106 was examined by flow cytometry. Rat bone marrow cells (Rat21) cultured in each of AC-GlcNAc polymer-coated dishes (selection materials 1 and 4 to 8), tissue culture dishes (selection material 3) and wet100 were collected, and CD11b / c antibody was used. This is a graph obtained by staining, calculating the ratio of positive cells to the total number of cells (positive rate (%)). Rat bone marrow cells (Rat21) cultured in AC-GlcNAc polymer coated dishes (selection materials 1 and 4 to 8), tissue culture dishes (selection material 3) and wet100 were collected and stained with CD90 antibody. The ratio of positive cells to the total number of cells (positive rate (%)) was calculated and graphed. Rat bone marrow cells (Rat21) cultured in AC-GlcNAc polymer-coated dishes (selection materials 1 and 4 to 8), tissue culture dishes (selection material 3) and wet100 were collected and stained with anti-CD106 antibody. The average fluorescence intensity of each dish when the average fluorescence intensity of the tissue culture dish (selective material of 3) is set to 1 is a graph.

  The method for separating and selecting mesenchymal stem cells of the present invention is a mesenchymal stem cell such as bone marrow or adipose on a mesenchymal stem cell selecting material obtained by coating a specific N-acetylglucosamine group-containing polymer on a culture substrate. Efficiently separating and selecting only mesenchymal stem cells from the cell population by seeding a cell population containing the cells, adding a medium, culturing without exchanging the medium, and exchanging the medium 2 to 10 days after seeding. You can

  In the method for culturing mesenchymal stem cells of the present invention, after the medium is exchanged, the medium is appropriately exchanged and cultured to form a colony of mesenchymal stem cells, and the mesenchymal stem cells can be increased.

  Hereinafter, the method for separating / selecting mesenchymal stem cells of the present invention, the method for culturing, and the selection material used therefor will be described in detail.

[N-acetylglucosamine group-containing polymer]
In the present invention, a polymer containing an N-acetylglucosamine group, to which a compound such as acrylic acid, methacrylic acid, styrene, polyethyleneimine or Poly-L-lysine, biotin is bonded, and these N-acetylglucosamine group-containing polymers are A weight average molecular weight of 2,000 to 4,000 is used. When the weight average molecular weight of the N-acetylglucosamine group-containing polymer is less than 2,000 or more than 4,000, the coatability on polystyrene, cell culture dishes and plates is deteriorated, which is not preferable.
The N-acetylglucosamine group-containing polymer has a weight average molecular weight in the range of 3,000 to 4,000 from the viewpoint of the yield of the compound by polymerization, and 2,000 to 3,000 in terms of coatability on dishes and plates. The range is.

As a method for polymerizing the N-acetylglucosamine group-containing polymer, for example, one in which styrene, acrylic acid or the like is bound to the reducing end of the N-acetylglucosamine group, or polyethyleneimine or poly-L-lysine which is a cationic polymer is bound. There are things.
The method for producing the N-acetylglucosamine group-containing polymer is not particularly limited, and radical polymerization, living radical polymerization, reversible addition-cleavage chain transfer polymerization method using a chain transfer agent (RAFT agent) (RAFT polymerization method), etc. Can be used. For example, as a method for producing a polymer obtained by polymerizing the monomer having an N-acetylglucosamine group, a compound having a hydrophobic group such as 2-carboxyethyl acrylate is bonded to the reducing end of N-acetylglucosamine. Examples thereof include a method of polymerizing a monomer. As a more detailed production method, 2-carboxyethyl acrylate, which is a compound having a hydrophobic group, and an aminated N-acetylglucosamine are subjected to a condensation reaction with DMT-MM to produce an acryl having an N-acetylglucosamine group introduced. Examples include a method of producing a system monomer and then polymerizing the monomer to produce it. Further, as a method for producing a polymer in which N-acetylglucosamine is bound to the polymer compound, a hydrophobic group such as polyethyleneimine or poly-L-lysine which is a cationic polymer at the reducing end of the N-acetylglucosamine sugar chain is used. And a method of binding with a polymer compound having
Here, the method for binding the N-acetylglucosamine sugar chain to the compound is not particularly limited, but, for example, the amino group of the compound having an amino group and the reducing end of the N-acetylglucosamine sugar chain are subjected to a reductive amination method. Can be combined with. Further, by substituting the hydroxyl group of the N-acetylglucosamine sugar chain with a carboxyl group and by a 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) coupling method or the like, the amino group of the compound having an amino group is changed. You may combine.
Further, it is preferable to use the RAFT polymerization method for controlling the molecular weight because the molecular weight distribution can be narrowed. In controlling the molecular weight, the ratio (Mw / Mn) of the weight average molecular weight (Mw) and the number average molecular weight (Mn) is set to 0.8 or more and less than 1.5, whereby N- is added to the surface of the cell culture material. Since a large amount of acetylglucosamine can be present, more mesenchymal stem cells in the cell population can be captured.
As a specific example of the polymerization method, N-acetylglucosamine and 2-carboxyethyl acrylate as a compound having a hydrophobic group are mixed at a molar ratio of 1: 1 and then mixed in a solution such as DMT-MM, DMSO or water. And the AC-GlcNAc monomer is prepared by condensation reaction. Poly (N-ethylacrylic acid-O-2-acetamide-2-deoxy-β-D-glucopyranosylamine) (hereinafter referred to as AC-GlcNAc polymer) can be obtained by the RAFT polymerization method.

  Further, the N-acetylglucosamine group-containing polymer of the present invention preferably has 3 to 15 N-acetylglucosamine groups per molecule. When it is less than this number, the binding property to vimentin tends to be lowered, and when it exceeds this number, the amount of N-acetylglucosamine per unit area is small and the binding property to vimentin is also lowered. Therefore, the more preferable number is in the range of 3 to 14, the more preferable number is in the range of 5 to 14, and the particularly preferable number is in the range of 8 to 13.

<Sorting materials>
The sorting material of the present invention is a culture substrate, for example, a dish (also called a culture dish), a petri dish or a plate (6 holes, 24 holes, 48 holes, 96 holes, 384 holes, 9600 holes, etc., microtiter plate, microplate, Deep well plate, etc.), flask, chamber slide, tube, cell factory, roller bottle, spinner flask, phorofiber, microcarrier, beads, etc., whose surface is coated with the N-acetylglucosamine group-containing polymer. .. These culture substrates may be made of either an inorganic material such as glass or an organic material such as polystyrene, but a material such as polystyrene, polyethylene, polypropylene or the like having a high adsorbability for the N-acetylglucosamine group-containing polymer, Alternatively, it is preferable to use a material that has been subjected to a treatment for enhancing the adsorptivity, such as a hydrophilic treatment or a hydrophobic treatment. Further, it is preferably made of a material having heat resistance and water resistance that can be sterilized. As an example of a suitable device for that purpose, polystyrene dishes and / or plates (hereinafter referred to as untreated polystyrene plates) that are often used mainly for culturing Escherichia coli and the like, which are not particularly treated for cell culture, and A tissue culture dish and / or plate (hereinafter also referred to as a cell culture plate) whose surface has been hydrophilically treated can be mentioned. From the aspect of cell growth, a tissue culture dish having a hydrophilically treated surface can be used. It is preferably used. As the culture substrate, a commercially available one may be used, or one produced according to the characteristics may be used.

In the sorting material of the present invention, it is necessary to coat the surface of this culture substrate with the N-acetylglucosamine group-containing polymer. As a coating method, a physical method such as adsorption or a chemical method such as covalent bonding can be applied, but the adsorption method is preferable from the viewpoint of easy operation. For example, a solution of the compound is brought into contact with the surface of a culture substrate such as a plate, and the solvent is removed after a certain period of time (wet coating) to easily adsorb the compound or bring the solution of the compound into contact. After that, the compound can be adsorbed by a method of drying without removing the solvent (dry coating). However, since the N-acetylglucosamine group-containing polymer is overlaid on the solid surface of the culture substrate, the dry coating is overlaid. The binding property between the N-acetylglucosamine group-containing polymer and vimentin can be further improved. More specifically, for example, a solution of an N-acetylglucosamine group-containing polymer using distilled water, PBS or the like as a solvent is filtered and sterilized, and then contacted with a culture substrate such as a plate, for example, for several hours to one week. The screening material on which the polymer is fixed or coated is obtained, preferably by leaving it for one day and night.
At this time, the concentration of the N-acetylglucosamine group-containing polymer is preferably 0.01 to 1000 μg / mL, more preferably 10 to 200 μg / mL, and particularly preferably 10 to 100 μg / mL. It becomes easy to form a good surface.

<Mesenchymal stem cell separation / selection and culture method>
<Cell population>
The cell population used in the present invention is a cell population containing mesenchymal stem cells. Examples of such cell populations include bone marrow, peripheral blood, placental amnion, umbilical cord, and dental pulp. The shape of the cell population is not particularly limited, and a cell suspension, cell suspension, cell pellet, cell aggregate, or the like can be appropriately selected and used.
When using bone marrow of the spine, sternum, iliac bone, etc., the bone marrow is isolated from the bone by washing the bone with a non-serum medium, erythrocytes in the bone marrow are hemolyzed with a hemolytic agent, and further centrifuged to obtain mononuclear cells. By recovering the cells and using them as a cell population, it is possible to reduce the effect on the binding between blood-derived N-acetylglucosamine and vimentin and the effect of reducing the number of mesenchymal stem cells themselves.
<Mesenchymal stem cell separation / selection and culture method>
The method for separating and selecting mesenchymal stem cells of the present invention is to inoculate a cell population containing mesenchymal stem cells such as bone marrow and fat on the surface of the above-mentioned sorting material, add a medium for mesenchymal stem cell culture, and exchange the medium. After culturing for at least 2 days, preferably 2 to 10 days, more preferably 5 to 10 days, and further preferably 7 to 10 days, the medium is exchanged to remove cells that do not bind to N-acetylglucosamine. By doing so, only mesenchymal stem cells can be isolated and selected from the cell population. The number of cells to be seeded may be appropriately selected depending on the size of the sorting material used. For example, in the case of a 35 mm dish, the number of mesenchymal stem cells that can be collected at one time can be set to about 5 to 7 × 10 ⁶ cells. The number and the number obtained by the subsequent culture can be adjusted, and the number of passages can be reduced.
Further, after the medium is exchanged 2 to 10 days after seeding, the medium is appropriately exchanged while observing the state of the cells, and the mesenchymal stem cells are further cultured for at least 10 days to form a colony. The medium is exchanged usually every 3 to 4 days, and when colonies are formed on the entire surface of the selection material, the cells are passaged.
When the cells are subcultured, those normally used in cell culture such as EDTA and trypsin can be used for cell recovery.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the following, “%” is based on mass unless otherwise specified.

[Example 1]
<Polymerization of N-acetylglucosamine group-containing polymer>
In order to investigate the optimal N-acetylglucosamine group-containing polymer that can be coated on a polystyrene dish, various molecular weight AC-GlcNAc polymers were polymerized using a Reversible Addition Fragmentation chain Ttransfer (RAFT) agent. The specific polymerization method is as follows.
<Preparation of AC-GlcNAc Monomer>
5 g of GlcANc was dissolved in 50 mL of water and NH ₄ CO ₃ was added until saturated. The mixture was stirred in an eggplant-shaped flask and incubated at 30 to 35 ° C. for 4 to 5 days in an open system with an open lid, and when NH ₄ CO ₃ precipitation disappeared, it was added appropriately. Synthesis of GlcNAc-NH ₂ was confirmed by TLC. After 4 to 5 days, excess NH ₄ CO ₃ was removed by adding water to the reaction solution and performing an evaporator (30 ° C.). Note that this operation was repeated many times until the smell disappeared. After the evaporator was freeze-dried. Then GlcNAc-NH _2: The (4.5 mol approximately 1g) were dissolved in DMSO (10 mL), was added 2-carboxyethyl acrylate (4.5mmol). After dissolution, DMT-MM (6.8 mmol) was added and incubation was carried out at room temperature for 18 hours. After incubation, the reaction was added dropwise to 200-300 mL chloroform. Since a precipitate was obtained, it was collected with a Kiriyama funnel. The recovered precipitate was dissolved in methanol, the insoluble matter was removed with a Kiriyama funnel, and the product dissolved in methanol was recovered. Evaporation was performed to remove methanol. The solid substance from which methanol was removed was dissolved in water, and then purified by preparative HPLC (water / acetonitrile) to obtain an AC-GlcNAc monomer represented by the following chemical formula.
<Polymerization of AC-GlcNAc polymer>
50 mg of AC-GlcNAc monomer was dissolved in 250 μL of dimethyl sulfoxide (DMSO). About 5.26 mg of RAFT agent (2- (dodecylthiocarbamoylthioyl) -2-methylpropanoic acid (DTMPA: manufactured by Sigma)) and AIBN 1 mg (2% based on the monomer) were added, and after degassing (freezing, thawing, defrosting). The mixture was incubated for about 18 hours at 65 ° C. After the polymerization, the polymer was precipitated with isopropanol, collected by centrifugation, dissolved in water, and dialyzed (MW100-500) for about 1 day. After the dialysis, lyophilization was performed to obtain a polymer (AC-GlcNAc10).
Polymerization of other AC-GlcNAc polymers was carried out in the same manner as described above, with the mixing ratio of RAFT agent as shown in FIG. The molecular weight of the AC-GlcNAc polymer was controlled by changing the concentration of DTMPA. For each molecular weight, a Gel Permeation Chromatography (GPC) device (product name: LC-9110G NEXT, manufactured by Nippon Analytical Industry Co., Ltd.) was used to measure each average molecular weight of each material under the following conditions. The column was JAIGEL-GS510, and the eluent was 200 mM sodium nitrate / acetonitrile = 80/20. The flow rate was 1 mL / min, the detector was an RI detector, and the column temperature was 40 ° C. A molecular weight standard curve was run on pullulan. The physical properties of this product are shown in FIG.

  In addition, AC-GlcNAc described in Entry in FIG. 1 is an AC-GlcNAc polymer, and when the ratio of the AC-GlcNAc monomer to the RAFT agent is 5 monomer / RAFT agent, AC-GlcNAc 5 and 10 are AC. -GlcNAc10 and 15 were AC-GlcNAc15 and 20, AC-GlcNAc20 and 30 were AC-GlcNAc30 and 40, AC-GlcNAc40 and 50 were AC-GlcNAc50.

<Study of coating conditions of AC-GlcNAc polymer>
Each of the AC-GlcNAc polymers prepared above was dissolved in water at 100 μg / mL, and 50 μL of each AC-GlcNAc polymer solution was put into 1 well of a 96-well plate of polystyrene dish (Non Coating) for suspension culture, and the lid was left open. Coating was carried out by incubating at room temperature overnight and allowing the solution to dry.
After dry coating, blocking was performed with 0.002% BSA aqueous solution for 1 hour at 25 ° C., and then 100 μL of 0.25 μg / mL biotinylated succinylated WGA (wheat germ agglutinin) was added, and then for 1 hour at 25 ° C. Incubation was performed. Then, 100 μL of 0.05 μg / mL HRP-neutravidin was added and incubated at 25 ° C. for 2 hours. Then, 100 μL of TMB solution (manufactured by SeraCare Life Sciences) was added to each well for color development, and then 100 μL of hydrochloric acid solution (1M) was added to each well to stop the reaction. The coating conditions were examined by measuring the absorbance of each of these dishes at 450 nm using a microplate reader (product name: iMark, manufactured by Bio-Rad Laboratories). The result is shown in FIG.
From the result of FIG. 2, AC-GlcNAc5 having a weight average molecular weight of 2600 and AC-GlcNAc10 having a weight average molecular weight of 3500 showed high coating properties. This is because the main chain of the hydrophilic part of AC-GlcNAc5 and 10 is shorter than that of AC-GlcNAc15, and thus the terminal alkyl chain of the AC-GlcNAc polymer, which is the hydrophobic part, contributes to the interaction with the polystyrene dish. Presumed to be
Further, as a result of performing the same evaluation using the GlcNAc monomer of the above chemical formula, the absorbance was less than 0.1, and the coating property was worse than that of AC-GlcNAc50.
From these results, it can be seen that AC-GlcNAc5 and AC-GlcNAc10 have excellent coating properties.

<Expression of vimentin on cell surface and adhesion of mesenchymal cells to AC-GlcNAc polymer-coated dish>
To determine whether mesenchymal cells adhere to the AC-GlcNAc polymer-coated dish via cell surface vimentin, we examined cell surface vimentin expression on human immortalized UE7T-13 mesenchymal stem cells. .. UE7T-13 human immortalized mesenchymal stem cells (hereinafter, also referred to as UE7T-13 cells) were purchased from JCRB Cell Bank.
First, total cell surface proteins were biotinylated by incubating with 500 μg / mL of Biotin-AC5-Sulfo-Osu (manufactured by Dojindo Co., Ltd.) at 4 ° C. for 20 minutes. Next, total biotinylated proteins were collected using Manosphere (registered trademark) MS300 / Streptavidin (manufactured by Takara Bio Inc.), and cell surface proteins and whole cell lysates were analyzed by the following Western blotting.
The cells were lysed with 1% triton-X100, 20 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, and a protease / phosphatase inhibitor cocktail (manufactured by Nakarai Task Co.), and centrifuged at 20000 × g at 4 ° C. for 15 minutes. The soluble fraction was collected by. The obtained fraction was analyzed by SDS-PAGE, and the protein in the gel was transferred to Immobilon-P PVDF membrane (manufactured by EMD Millipore). The protein was detected using a mouse anti-vimentin monoclonal antibody (V9, manufactured by Sigma) and an anti-β-actin antibody (manufactured by Sigma). Detection was performed using C-DiGit (registered trademark) Blot Scanner (manufactured by LI-COR), and blotting data was analyzed using Image Studio Lite software (manufactured by LI-COR).
The result is shown in FIG. As shown in FIG. 3, a vimentin band was detected in the fraction of biotinylated surface protein from mesenchymal stem cells collected using streptavidin beads, but β-actin was not detected. From this result, vimentin was presumed to be a cell surface protein.

Next, it was confirmed whether UE7T-13 cells adhere to the AC-GlcNAc polymer-coated dish. Wells coated with AC-GlcNAc polymer were blocked with 0.002% BSA for 1 hour at 37 ° C. After blocking, 2 × 10 ⁴ cells / well in serum-free DMEM were seeded in each well of a 96-well plate and incubated at 37 ° C., 5% CO ₂ for 1 hour. After the incubation, non-adherent cells were removed by washing with PBS, and 10% methylthiazoletetrazolium (MTT) assay reagent (Dojindo Co., Ltd.) / 10% fetal bovine serum (FBS) was added to each well. Incubated at 37 ° C. for 2 hours in 5% CO ₂ . After the incubation, MTT formazan was lysed with 100 μL of dimethylsulfoxide. The absorbance of the obtained solution at 570 nm was measured using a microplate reader. The number of adherent cells was determined by a standard curve based on formazan production. The result is shown in FIG. As shown in FIG. 4, during incubation at 37 ° C., 80-90% of the seeded UE7T-13 cells adhered to the AC-GlcNAc5 and 10-coated dishes and the AC-GlcNAc5 and 10-coated dishes. To the dishes coated with AC-GlcNAc15, AC-GlcNAc20, or AC-GlcNAc30. Adhesion at 4 ° C was less than that at 37 ° C, but at 4 ° C and 37 ° C, adhesion to dishes coated with AC-GlcNAc5 and AC-GlcNAc10 produced longer polymers. Greater than adhesion to the coated dish.

Next, it was confirmed whether the adhesion of UE7T-13 cells to the AC-GlcNAc polymer-coated dish was caused by the interaction between cell surface vimentin and GlcNAc.
Cell suspension of recombinant vimentin, AC-GlcNAc5, or glucose-supporting polymer Poly [Np-vinylbenzyl-O-α-D-glucopyranosyl- (1 → 4) -D-gluconamide] (hereinafter referred to as PV-MA). It was investigated whether UE7T-13 cell adhesion to AC-GlcNAc5-coated dishes was inhibited after addition to the suspension. PV-MA is composed of polystyrene as a main chain and glucose as a side chain, and does not interact with vimentin. UE7T-13 cells were incubated with 20 μg / mL recombinant vimentin, 500 μg / mL AC-GlcNAc polymer, or 500 μg / mL PV-MA for 30 minutes at 37 ° C. After incubation, these cell suspensions were incubated for 1 hour at 37 or 4 ° C. on dishes coated with AC-GlcNAc5. Adhesion was assessed by MTT assay. The result is shown in FIG. As shown in FIG. 5, during incubation at 37 ° C. and 4 ° C., adhesion was reduced by about 40% by recombinant vimentin and by about 80% by AC-GlcNAc polymer, whereas by using PV-MA, AC was reduced. The reduction in adhesion was significantly lower than with the -GlcNAc polymer.

Vimentin knockdown UE7T-13 cells and negative control UE7T-13 cells were prepared to determine if vimentin is required for adhesion. The vimentin knockdown UE7T-13 cells were prepared by transfection using pcDNA6.2-GW / EmGFP-miR-human vimentin (vimentin-miRNA vector, manufactured by Thermo). Negative control UE7T-13 cells were prepared by transfecting pcDNA6.2-GW / EmGFP-MIR-negative control plasmid (negative miRNA vector, Thermo). The cells were transfected with the vector using Neon (registered trademark) Transfection System (manufactured by Thermo). Three days after the preparation, the expression of vimentin and β-actin in the vimentin knockdown UE7T-13 cells and the negative control UE7T-13 cells was analyzed by Western blotting in the same manner as above. The result is shown in FIG. As shown in FIG. 6, the expression of vimentin in the vimentin knockdown UE7T-13 cells was significantly lower than that of the negative control UE7T-13 cells.
To clarify the adhesion of vimentin knockdown UE7T-13 cells to AC-GlcNAc5-coated dishes, vimentin knockdown cells and negative control UE7T-13 cells (2 × 10 ⁴ cells) were placed in AC-GlcNAc5-coated dishes. Incubation was carried out at 4 ° C. for 1 hour, and the adhesion of each cell to the AC-GlcNAc5 coated dish was examined. The result is shown in FIG. 7. As shown in FIG. 7, the adhesion of vimentin knockdown UE7T-13 cells was about half that of the negative control UE7T-13 cells.

<Selection and culture>
<Adjustment of sorting material>
The following materials were prepared as sorting materials.
1. 1 mL of AC-GlcNAc10 dissolved in water at 100 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, Coating was performed by drying the solvent to obtain a 35 mm sorted material.
2. 1 mL of PV-MA dissolved in water at 100 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, A 35 mm PV-MA coated dish coated by drying the solvent was obtained.
3.35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific).
The PV-MA coated dish of 2 was used for comparison.

<Separation, selection and culture>
Bone marrow of female Wistar rats aged 6 to 9 weeks was hemolyzed with red blood cells from the bone marrow with a hemolytic agent (pH 7.55, 0.15 M ammonium chloride, 17 mM Tris-HCl, 0.25 mM EDTA2Na) at 200 xg for 3 minutes. After centrifugation, mononuclear cells were collected to obtain a cell population. The presence of cell surface vimentin-expressing cells in this cell population was examined by flow cytometry. Flow cytometry was carried out by suspending the cell population obtained above in 10% FBS / DMEM, and incubating with an anti-cell surface vimentin monoclonal antibody (clone 84-1; 1: 100; Abnova) at 4 ° C. for 1 hour. , Cells were incubated with a CF488-conjugated anti-mouse IgG secondary antibody (1: 500; manufactured by Biotium) at 4 ° C. for 1 hour. The result is shown in FIG. As shown in FIG. 8, cell surface vimentin-expressing cells were found at a frequency of 14 ± 2% (n = 7) in the cell population. From this result, it was estimated that mesenchymal stem cells were contained in this cell population.

Next, this cell population was seeded on the selection material or dish of 1 to 3 so that the cell number becomes 5 to 7 × 10 ⁶ cells, and 2 ml of a medium (product name: MesenCult proliferation medium with MesenPure, manufactured by Veritas Company). ) Was used for culturing for 17 days. On the way, the medium was exchanged 7 days after seeding to remove non-adherent cells, and then the medium was exchanged every 3 to 4 days. FIG. 9 shows a phase difference image of the formed colony after 10 days. As shown in FIG. 9, there were many cell-cell contacts in the colonies formed with the selection material (AC-GlcNAc polymer-coated dish) of 1 but not in the tissue culture dish of 3. On the 6th, 10th, 13th, and 16th days, colony formation in the selection material 1 (AC-GlcNAc polymer-coated dish) and the tissue cell dish 3 was confirmed by staining with crystal violet. As a confirmation method, the area of the formed stained colony was measured using image analysis software (Product name: Image J1.64r. National Institute of Health), and the ratio of the area of the colony to the entire area of the 35 mm dish was measured. Was calculated. The result is shown in FIG. The photograph in FIG. 10 is a photograph of the state of cells 3 to 16 days after seeding the bone marrow, and the graph below shows the result of calculating the colony state as the area. As shown in FIG. 10, on the 10th day, more colonies were detected in the 1 AC-GlcNAc polymer-coated dish than in the 3 tissue culture dishes, and on the 16th day, the AC-GlcNAc polymer-coated dish was detected. Formed larger colonies. The growth rate decreased on day 13-16 in 3 tissue culture dishes, but was maintained up to day 16 in 1 AC-GlcNAc polymer coated dishes.
The state of the colonies after 17 days was confirmed by staining with crystal violet. The result is shown in FIG.
The photograph in FIG. 11 is a photograph of the state of cells 17 days after seeding bone marrow, and the lower graph shows the result of calculating the colony state as the area. The left side of the photograph is a normal tissue culture dish (3), and the right side photograph is the selection material (1). In the tissue culture dish No. 3, the proportion of colonies was about 10%, but with the selection material No. 1 (AC-GlcNAc polymer coated dish), it was about 30 to 40%. Also, it can be seen that the number of formed colonies is larger and the number is larger in the selection material of 1 as compared with the normal tissue culture dish of 3. From this, it is considered that a large number of highly proliferative cells adhere to the sorting material coated with the AC-GlcNAc polymer. Furthermore, in order to examine whether mesenchymal stem cells are separated and sorted by the interaction between AC-GlcNAc and vimentin, PV-MA, which is a polymer that does not interact with vimentin, is coated on a cell culture dish. Used as. The results of culturing bone marrow cells on the above 2 by the same method as described above are shown in FIG.
From the results of FIG. 12, in the culture of bone marrow cells on PV-MA (center of the photo), adhesion of many cells was observed in the early stage of culture, but thereafter colony formation was not observed, and the number of formed colonies was 3. The tendency was similar to that of colony formation on the tissue culture dish. From this, it is shown that cells capable of forming colonies cannot adhere to the PV-MA-coated dish for cell culture, and that colony formation is promoted by vimentin-mediated interaction by AC-GlcNAc. It was revealed. From these results, it is clear that cells having a high proliferative colony-forming ability can be separated, sorted and cultured from bone marrow cells by using the sorting material coated with the AC-GlcNAc polymer.

<Confirmation of separated, sorted and cultured cells>
The colony-formed cells cultured using the selection material of 1 and the tissue culture dish of 3 were seeded in 24-well and cultured until the number of cells was about 70 to 80% of the well. The cells are collected and fixed with 4% paraformaldehyde / PBS, and methanol or 0.1% 4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (Product name: Triton-X100. Sigma. After permeabilizing with Aldrich) / PBS, the cells were immunostained with CD106 and CD90, which are positive markers for mesenchymal stem cells, and CD11b / c, which is a negative marker, and then subjected to a microscope (product name: FV3000. Olympus). (Made by the company). The fluorescence image at that time was processed using FV-31S-SW (manufactured by Olympus). The result is shown in FIG.
From the results of FIG. 13, staining with the positive markers CD106 and CD90 was confirmed in both the tissue culture dish of 3 and the colonies obtained from the AC-GlcNAc polymer-coated dish (selective material) of 1, and the negative marker was used. Certain CD11b / c staining was not confirmed. Vimentin expression was similarly observed in both cells. From the above, it can be seen that the colonies obtained from any of the dishes are mesenchymal stem cells.

Next, in order to determine whether or not the colonies formed by culturing using the selection material of 1 above and the tissue culture dish of 3 above have the characteristics of mesenchymal stem cells, mesenchymal cells were analyzed by flow cytometry. The expression of stem cell positive marker and mesenchymal stem cell negative marker was examined.
Bone marrow cells were cultured for about 3 weeks with the selection material of 1 above and the tissue culture dish of 3 above, and the proliferated cells were collected. The number of passages of these cells was 0 or 1. The collected rat bone marrow cells were suspended in 10% FBS / DMEM, and incubated with an anti-cell surface vimentin monoclonal antibody (clone 84-1; 1: 100; manufactured by Abnova) at 4 ° C. for 1 hour. After the incubation, the cells were incubated with a CF488-conjugated anti-mouse IgG secondary antibody (1: 500; Biotium) at 4 ° C. for 1 hour. Proliferated cells were collected by detaching them using Accutase (Nacalai Tesque). The cells were suspended in 10% FBS / DMEM, and FITC-conjugated anti-rat CD11b / c antibody (1: 100), FITC-conjugated anti-rat CD90 antibody (1: 100), FITC-conjugated anti-rat CD29 antibody (1: 100) FITC-conjugated Incubation was performed with anti-rat CD44 antibody (1: 100), PE-conjugated anti-rat CD54 antibody (1: 100), or PE-conjugated anti-rat CD106 antibody (1: 100) (all manufactured by BioLegend). For CD34 staining and CD45 staining, 10% FBS / DMEM cells were treated with anti-CD34 antibody (1: 100, Santa Cruz Biotechnology) or anti-CD45 antibody (1: 100; Abcam) at 4 ° C. for 1 hour. Then, it was incubated with a CF488-conjugated anti-rabbit IgG secondary antibody (1: 500, manufactured by Biotium) at 4 ° C. for 1 hour. For CD73 and CD105 staining, harvested cells were fixed with 4% paraformaldehyde (PFA) / PBS. After fixing, the cells were treated with anti-CD73 (1:50, Proteintech) or anti-CD105 (1:50, Bioss Antibodies) at 4 ° C. for 1 hour, and then with CF488-conjugated anti-rabbit IgG secondary antibody (1: 500). , Biotium) at 4 ° C. for 1 hour. Mouse IgG (Wako Pure Chemical Industries) and rabbit IgG (Thermo) were used as isotype controls.
The results are shown in FIGS. 14 and 15. As shown in FIG. 14, proliferating cells from the colonies of the dish coated with the selection material of 1 and the tissue culture dish of 3 were CD90, CD29, CD44, CD54, and CD73, which are mesenchymal stem cell positive markers. , CD105 and CD106, but not the mesenchymal stem cell negative markers CD34, CD45 and CD11b / c. In the cell populations of the dish coated with the selection material of 1 and the tissue culture dish of 3, the CD90-positive cells were 94 ± 5% and 81 ± 19%, and the CD34-positive cells were 0.65 ± 0.23%, respectively. 1.8 ± 0.46%, CD45 + cells 0.71 ± 0.09% and 1.6 ± 0.15%, CD11b / c positive cells 4.6 ± 3.7% and 3.1 It was ± 1.7%. In addition, as shown in FIG. 15, the proportions of CD90, CD29, CD44, CD54, and CD73 positive cells from the selection material of 1 were all about 80%, which was higher than the proportion from 3 tissue culture dishes. There were many.
The CD106-positive cells from the dish of the above selection material 1 were 35 ± 13% of the total cells, but 16 ± 11% in the tissue culture dish of 3. As shown in FIG. 15, the CD106 expression level in the dish coated with the selection material of 1 was significantly higher than that in the tissue culture dish of 3. Since CD106 is not expressed in fibroblasts and CD106-positive mesenchymal stem cells have high proliferative activity, it has been reported to be a reliable marker for mesenchymal stem cells. The above results indicate that the proliferating cells from the AC-GlcNAc polymer-coated dish, which is the sorting material of 1 above, have mesenchymal stem cell characteristics more than the proliferating cells from the uncoated tissue cell dish of 3. It shows a high percentage of cells.

<Confirmation of differentiation ability>
The colonized cells that were cultured using the selection material of 1 above and the tissue culture dish of 3 were dissociated using Acctase (manufactured by Nakarai Task Co.), and each cell was re-seeded in a separate 35 mm tissue culture dish. Then, the cells were cultured until the cell number reached 70 to 80% confluence. Then, after culturing for 21 days in a bone differentiation inducing medium and a fat differentiation inducing medium (manufactured by Veritas), each cell was stained with alizarin red (40 mM alizarin red, manufactured by Sigma) for bone differentiation, and for fat differentiation. Was subjected to oil red O dyeing (0.5% oil red O. manufactured by Wako Pure Chemical Industries, Ltd.). The result is shown in FIG.
From the results of FIG. 16, it was confirmed that the cells obtained from the colonies of both the tissue culture dish 3 and the selection material 1 were differentiated into bone and adipose. From the above, it can be seen that the cells obtained by the selection material 1 are mesenchymal stem cells having a differentiation ability.
From these results, by separating and selecting mesenchymal stem cells from a cell population containing mesenchymal stem cells using the selection material of 1 of the present invention, it is possible to reduce the amount of the mesenchymal stem cells to about 3 compared to the conventionally used tissue culture dish. It is possible to obtain a 4-fold amount of mesenchymal stem cells. Moreover, when sorting from a cell population, a large number of multiple markers (antibodies) are used in the conventional method, and flow cytometer, FACS, etc. Although it is not possible to sort a large amount at a time such as requiring a sorting means, and at a very high cost, a large amount of sorting processing can be performed at a time by using the method and material of the present invention. Moreover, the use of synthetic polymers and the fact that no special sorting equipment is required are excellent in terms of cost. Furthermore, since the cells can be cultured as they are after selection, they are not subjected to external stress due to cell detachment or various marker tests, so that the number of cells can be increased while maintaining the properties of the cells.
Further, the sorting material of the present invention strongly adheres by the interaction between N-acetylglucosamine and vimentin at the time of initial adhesion when cells are grown on the sorting material by using a culture substrate having a hydrophilic surface. Adhesion between the culture substrate and the AC-GlcNAc polymer becomes unstable due to long-term culture, AC-GlcNAc is released, and extracellular matrix (ECM) components such as fibronectin and collagen produced by mesenchymal stem cells in the space are selected. It is presumed that the mesenchymal stem cells will be promoted to proliferate.

[Example 2]
<Selection and culture>
<Adjustment of sorting material>
As the sorting material, in addition to the sorting materials 1 to 3 prepared in Example 1, the following sorting materials 4 to 8 were prepared.
4. 1 mL of AC-GlcNAc10 dissolved in water at 10 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, and the solvent was added. Coating was performed by drying to obtain a sorted material of 35 mm.
5. 1 mL of AC-GlcNAc10 dissolved in water at 1 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, and the solvent was added. Coating was performed by drying to obtain a sorted material of 35 mm.
6. 1 mL of AC-GlcNAc10 dissolved in water at 0.1 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, Coating was performed by drying the solvent to obtain a 35 mm sorted material.
7. 1 mL of AC-GlcNAc10 dissolved in water at 0.01 μg / mL was placed in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, Coating was performed by drying the solvent to obtain a 35 mm sorted material.
8. 1 mL of AC-GlcNAc10 dissolved in water at 1000 μg / mL was put in a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific) and incubated overnight at 4 ° C. with the lid open, and the solvent was added. Coating was performed by drying to obtain a sorted material of 35 mm.

<Separation, selection and culture>
Bone marrow cells were cultured in the same manner as in Example 1 except that the selection materials 1 to 3 described in Example 1 and the tissue culture dish described in Example 3 were used as the selection materials, and the selection materials 4 to 8 were used. The result is shown in FIG.
As shown in FIG. 17, the number of proliferating cells in the AC-GlcNAc polymer-coated dish (selection material of 1 and 4 to 8) of 0.01 to 1000 μg / mL was about 2 of the number of proliferating cells of the tissue culture dish of 3. Doubled. The percentage of CD11b / c positive cells from 10 and 100 μg / mL AC-GlcNAc polymer coated dishes (sorted material of 4 and 1) is shown in FIG. As shown in FIG. 18, the percentage of CD11b / c positive cells from the 10 and 100 μg / mL AC-GlcNAc polymer coated dishes was low, about 5%.
The percentage of CD90 positive cells from 0.01-100 μg / mL AC-GlcNAc polymer coated dishes (1 and 2-5 sorted material) is shown in FIG. As shown in FIG. 19, the percentage of CD90 positive cells from 0.01-100 μg / mL AC-GlcNAc polymer coated dish was about 90%. However, the proportion of CD90 positive cells from the 1000 μg / mL AC-GlcNAc polymer-coated dish (selective material of 8) and the tissue culture dish of 3 was about 80%. Contamination of other types of cells into mesenchymal stem cells was observed in 1000 μg / mL AC-GlcNAc polymer-coated dish (8 selection materials) and 3 tissue culture dishes, but 10 mesenchymal stem cells were found. Separation with high purity could be achieved by using ˜100 μg / mL AC-GlcNAc polymer. The above results suggest that the optimum concentration of the AC-GlcNAc polymer is 10 to 100 μg / mL.

<Confirmation of aging efficiency and differentiation>
Senescence of proliferated cells was analyzed by measuring β-galactosidase (hereinafter, also referred to as SA-βGal) activity related to senescence. In order to apply the cell senescence detection kit SPiDER-βGal (manufactured by Dojindo Kabushiki Kaisha) to the proliferated cells, the proliferating cells after culturing in the selection material of 1 and the tissue culture dish of 3 for about 3 weeks are each for 35 mm tissue culture. The cells were cultured in a dish for 1 day with 10% FBS / DMEM and stained with a cell senescence detection kit SPiDER-βGal (manufactured by Dojindo Co., Ltd.). The stained cells were analyzed using a Guava EasyCyte Flow Cytometry System (EMD Millipore). FIG. 20 shows the results of flow cytometry of the proliferating cells cultured in the selection material of 1 or the tissue culture dish of 3, and FIG. 21 shows the SA-β-galactosidase activity of each proliferating cell. As shown in FIG. 21, cells cultured in the tissue culture dish 3 which is not coated with the AC-GlcNAc polymer have SA-βGal activity higher than that of the cells in the AC-GlcNAc polymer-coated dish (selective material of 1). It was significantly higher. From this, it is considered that the mesenchymal stem cells separated on the AC-GlcNAc polymer-coated dish do not change even after long-term passage.

Mesenchymal stem cells have the potential to differentiate into osteogenesis, adipogenesis, and chondrogenesis. Therefore, the ability of mesenchymal stem cells to differentiate into bone, adipose, and cartilage was examined by staining the proliferating cells with alizarin red, oil red O, and alcian blue.
Cells collected from the AC-GlcNAc polymer-coated dish (selective material of 1) and the tissue culture dish of 3 were used in a 35 mm tissue culture dish using an osteodifferentiation induction medium, an adipose differentiation induction medium, and a cartilage differentiation induction medium, respectively. The cells were cultured for 3 weeks. The result is shown in FIG.
As shown in FIG. 22, no significant change was observed in long-term culture of mesenchymal stem cells separated with the AC-GlcNAc polymer-coated dish. The above results indicate that the mesenchymal stem cells obtained by the AC-GlcNAc polymer-coated dish have the same differentiation ability as the mesenchymal stem cells obtained by the tissue culture dish.

[Example 3]
<Selection and culture>
<Adjustment of sorting material>
As the sorting materials, 1 sorting material prepared in Example 1, 3 tissue culture dishes, 4 to 8 sorting materials prepared in Example 2 and wet100 (details will be described later) were prepared.

<Separation, selection and culture>
Bone marrow of female Wistar rats aged 6 to 9 weeks was hemolyzed with red blood cells from the bone marrow with a hemolytic agent (pH 7.55, 0.15 M ammonium chloride, 17 mM Tris-HCl, 0.25 mM EDTA2Na) at 200 xg for 3 minutes. After centrifugation, mononuclear cells were collected to obtain a cell population.

Next, from this cell population, the sorting material of 1 described in Example 1, the tissue culture dish of 3 and the sorting of 4 to 8 described in Example 2 so that the cell number becomes 5 to 7 × 10 ⁶ cells. 1 mL of the material and AC-GlcNAc10 dissolved in water at 100 μg / mL were put into a 35 mm tissue culture dish (product name: Nunc, manufactured by Thermo Scientific), and after incubation at 37 ° C. for 2 hours, AC-GlcNAc10 was contained. Water was removed and the material was further dried to prepare a coated selection material (wet100), which was then seeded and cultured for 13 days using 2 ml of a medium (product name: MesenCult proliferation medium with MesenPure, manufactured by Veritas). .. On the way, the medium was exchanged 3 days after seeding to remove non-adherent cells, and then the medium was exchanged every 3 days. Then, after 13 days, the cells were collected. The number of cells is shown in FIG. The collected cells were stained with each of CD11b / c antibody and CD90 antibody, and the stained cells were examined by flow cytometry. FIG. 24 shows the results of flow cytometry of the CD11b / c antibody, and FIG. 27 shows the CD11b / c positive rate. FIG. 25 shows the results of flow cytometry of the CD90 antibody, and FIG. 28 shows the CD90 positive rate.
From the result of FIG. 23, it was found that the number of cells was increased by about 2 times when the selection materials 1 and 4 to 8 were used as compared with the cell culture dish of 3. Further, many colonies appeared due to the AC-GlcNAc polymer. From the results of FIGS. 24 and 27, it was found that the expression level of the negative marker CD11b / c was different depending on the coating concentration of AC-GlcNAc. Since the CD11b / c positive cells are macrophages, the difference in the expression level indicates the difference in the mixing ratio of macrophages. The mixing ratio of CD11b / c tended to be low in the sorting materials of 1 and 4 to 5, and the mixing ratios of the other sorting materials and the cell culture dish of 3 tended to be high.

  From the results of FIGS. 25 and 28, CD90, which is an undifferentiated marker of mesenchymal stem cells, has a high positive rate in the selection materials of 1, 4 to 8 and wet100 and a low expression rate in the cell culture dish of 3. I found out. Considering CD90 as an index indicating the purity of mesenchymal stem cells, it was found that 4 to 6 sorting materials having a coating concentration of 0.01 to 10 μg / mL and 8 sorting materials having a coating concentration of 1000 μg / mL are particularly preferable. It was

  Further, from the results of FIGS. 27 and 28, it was found that the sorting materials having a low contamination rate of macrophages and satisfying both the purity of mesenchymal stem cells are the sorting materials of 4 to 5.

Then, rat bone marrow cells (Rat21) were seeded on the selection materials 1 and 4 to 8, tissue culture dishes 3 and wet100 at 297 × 10 ⁴ cells / 35 mm. MesenCult Expansion medium (manufactured by Veritas) was used as the medium, and the medium was exchanged 3 days after the cells were seeded, and then the medium was exchanged every 3 days. After 13 days, the cells were collected, and CD106, which is a positive marker for mesenchymal stem cells, was stained with an anti-CD106 antibody and examined by flow cytometry. The result is shown in FIG. From the results of FIG. 26, it was found that the cells obtained in the cell culture dish 3 were negative for CD106, and the cells obtained in the selection materials 1 and 4 to 8 and wet100 were positive for CD106. The CD106-positive mesenchymal stem cells can also be used as an index for cells having properties such as high proliferative activity and immunological tolerance.
FIG. 26 is a graph showing the average fluorescence intensity (relative fluorescence intensity) of each dish, where the average fluorescence intensity of the tissue culture dishes 3 that were negative (unstained) in the staining result of the CD106 antibody of FIG. 26 is 1. 29. As shown in FIG. 29, as compared with the tissue culture dish of 3, all the other sorting materials showed a fluorescence intensity of 1.3 times or more. Among them, the sorting materials of 5, 6 and 8 showed a fluorescence intensity of 2.7 times or more, and particularly the sorting material of 5 showed a fluorescence intensity of 3 times. Therefore, from the above results, the selection materials of 4 to 8 are preferable for the selection of mesenchymal stem cells, and the selection materials of 5, 6 and 8 are those that can obtain cells with a high expression level of CD106. Do you get it.
From the above-mentioned CD11b / c and CD90 positive rates and the expression results of CD106, it was found that mesenchymal stem cells having high purity and excellent properties were obtained by using the selection materials of 4 to 6 in particular.

Claims (8)

  After seeding a cell population containing mesenchymal stem cells on the coating surface of the sorting material coated with the N-acetylglucosamine group-containing polymer having a weight average molecular weight of 2000 to 4000 and inserting a medium, and without performing medium exchange, 2 after seeding A method for separating and selecting mesenchymal stem cells, characterized in that the medium is replaced after 10 to 10 days.   The method for separating and selecting mesenchymal stem cells according to claim 1, wherein the cell population containing the mesenchymal stem cells is seeded, a medium is added, and then the medium is not replaced, and the medium is replaced 5 to 10 days after the seeding. ..   A coating agent containing 0.01 to 1000 μg / mL of the N-acetylglucosamine group-containing polymer is coated to cover the entire surface of the cell culture substrate with the coating agent, and the solvent is added at any temperature from 4 ° C. to room temperature. The method for separating / selecting mesenchymal stem cells according to claim 1 or 2, wherein the mesenchymal stem cells are dried without being discarded.   Mesenchymal stem cells separated and sorted by the method for separating and sorting mesenchymal stem cells according to any one of claims 1 to 3 are further cultured for at least 10 days after the medium exchange. Method for culturing lineage stem cells. A N-acetylglucosamine group-containing polymer having a weight average molecular weight of 2000 to 4000, the polymer being a compound represented by the following chemical formula, wherein the polymer is coated on a culture substrate. Material for selecting mesenchymal stem cells.
In the formula, n is an integer of 3 to 15.   The N-acetylglucosamine group-containing polymer has a number average molecular weight in the range of 3000 to 5000, and a ratio of the weight average molecular weight to the number average molecular weight is 0.8 or more and less than 1.5. The material for selecting mesenchymal stem cells according to claim 5.   The mesenchymal stem cell selection material according to claim 5, wherein the culture substrate has a hydrophilic surface.   A coating agent containing 0.01 to 1000 μg / mL of the N-acetylglucosamine group-containing polymer is coated to cover the entire surface of the cell culture substrate with the coating agent, and the solvent is added at any temperature from 4 ° C. to room temperature. The mesenchymal stem cell selection material according to any one of claims 5 to 7, which is dried without being discarded.