WO2007055178A1

WO2007055178A1 - Method of cell fractionation and substrate to be used for the method

Info

Publication number: WO2007055178A1
Application number: PCT/JP2006/322119
Authority: WO
Inventors: Tetsuji Yamaoka; Atsushi Mahara; Soichiro Kitamura
Original assignee: Japan Health Sciences Foundation
Priority date: 2005-11-08
Filing date: 2006-11-06
Publication date: 2007-05-18
Also published as: JPWO2007055178A1

Abstract

In a method of cell fractionation according to the invention, by using a ligand-presenting substrate in which a ligand capable of temporarily binding to a receptor present on the surface of a cell desired to be fractionated is immobilized via a graft chain, cells are rotationally transferred on the surface of the ligand-presenting substrate and a difference in the transfer rate caused by the binding degree is utilized. This achieves a method of fractionating a cell presenting a specific receptor on the cell surface from a group of cells including the cell, in which the cell having a receptor density within a predetermined range can be fractionated efficiently and highly accurately and the cells having respective densities can be continuously fractionated from the group of cells depending on the receptor density.

Description

Specification

Cell sorting method and substrate used in the method

Technical field

[0001] The present invention relates to a cell sorting method and a base material used in the method, and more specifically, temporarily binds to the receptor from a cell group including cells presenting a specific receptor on the cell surface. By using a ligand-presenting substrate on which the ligand to be displayed is displayed, a method for sorting cells efficiently and with high accuracy, and a high-density ligand immobilization for use in the method The present invention relates to a ligand-presenting substrate.

Background art

[0002] In the field of regenerative medicine, aiming for the realization of the treatment method proposed by Langer et al. In 1993 in the United States, that is, the realization of the treatment method by tissue regeneration using cells and the substrate (shearhold) that supports them Various research * development is being developed. In order to establish this method as a highly safe and therapeutic method, it is necessary to collect stem cells and the like used for tissue regeneration efficiently with the use of self-tissue.

[0003] Currently, as a method of collecting stem cells and the like from autologous tissue, a physical method represented by the Ficoll method, a fluorescence activated cell sorting (FACS) method, a magnetic bead (Magnetic eel 1 sorting) method (hereinafter referred to as MACS) Biological methods). Among them, the FACS method, whose basic principle was established by Fulwyler in 1965 based on the principle of a flow cytometer developed in 1947 by Coulter et al. It is adopted as a method for separating stem cells used in the above. Hereinafter, the principles of the FACS method and the MACS method will be described with reference to FIG. 11 and FIG.

FIG. 11 is a schematic diagram illustrating the principle of the FACS method. The FACS method can be performed using an apparatus 100 shown in FIG. The sorting mechanism includes a droplet charging method and a cell capture method. FIG. 11 shows the principle of the droplet charging method. The apparatus 100 is provided with a laser transmitter 102 and a detector 103 as shown in FIG. This device 100 determines the amount of antigen on the cell surface by allowing cells labeled with a fluorescent antibody to flow in a liquid flow, passing through the focal point of the laser beam, and measuring the fluorescence emitted by individual cells. It can be measured quantitatively and specific cells can be sorted.

[0005] The following is a detailed description of the case where hematopoietic stem cells contained in the bone marrow are sorted by the FACS method. CD34 is presented on the surface of hematopoietic stem cells as a receptor specific for hematopoietic stem cells, and this CD34 can be used as a marker for identifying hematopoietic stem cells. First, a cell suspension 101 containing hematopoietic stem cells is prepared. At the time of preparation, CD34 of the hematopoietic stem cells is labeled with a fluorescent antibody 104. In FIG. 11, CD34 (+) cells (hematopoietic stem cells) fluorescently labeled with fluorescent antibody 104 are shown in black, and CD34 () cells (cells other than hematopoietic stem cells) are shown in white. Next, the prepared cell suspension 101 is also passed through the apparatus 100 with the directional force indicated by the arrow a in FIG. 11, and the sheath liquid is also flowed with the directional force indicated by the arrow b. Then, the sheath liquid containing cells is discharged from the nozzle 1 OOa. Here, the discharged sheath liquid is allowed to pass through the focal point of the laser beam emitted from the laser transmitter 102, whereby the fluorescence emitted by each cell is detected by the detector 103. At this time, since the nozzle 100a of the apparatus 100 is ultrasonically vibrated, the sheath flow that flows out of the nozzle 100a also forms a droplet in the middle force. Therefore, the sheath where hematopoietic stem cells in which fluorescence is detected by the detector 103 is present A charged hematopoietic stem cell-containing droplet is formed by charging immediately before the liquid is about to form a droplet. Therefore, as shown in FIG. 11, if the deflector plate 105 is provided in the apparatus 100, the charged hematopoietic stem cell-containing droplets can be drawn to the deflector plate 105 and separated into the collection container 106 (non-patent document). (Ref. 1).

Next, FIG. 12 is a schematic diagram for explaining the principle of the MACS method. Here, the case where hematopoietic stem cells are sorted will be described. First, a cell suspension 101 containing hematopoietic stem cells is prepared. In the MACS method, magnetic beads 107 to which antibodies are attached are used to bind the antibodies to CD34 (antigen) to display magnetic beads 107 on the surface of hematopoietic stem cells. Then, the cell suspension 101 containing hematopoietic stem cells on which the magnetic beads 107 are presented is placed in the column 109 provided in the magnetic device 108 shown in FIG. 12, and the magnetic beads 107 are adsorbed on the magnetic device 108 to cause hematopoiesis. Stem cells can be sorted (see Non-Patent Document 2).

Special Reference 1: Visser J.W.M. (1984) Isolation of murine plunpotent hemopoietic stem cells. J. Exp. Med., 59, 1576—1590

Non-Patent Document 2: Miltenyi S. (1990) High Gradient Magnetic Cell Separation With MAC S, Cytometry, 11, 231-238

Non-Patent Document 3: Greenberg and Hammer: Cell Separation Mediated by Differential Rolling Adhesion, BIOTECHNOLOGY AND BIOENGINEERING, VOL.73, NO.2, APRI L 20, 2001, 111-124

Disclosure of the invention

Problems to be solved by the invention

[0007] However, the isolation of cells by the conventional method described above is complicated and requires a large-scale apparatus, and further, since the number of cells that can be isolated is limited, it has become a clinical practice. Is difficult to deploy.

[0008] In addition, in the isolation of cells by the conventional method described above, it is not possible to continuously isolate droplets or cells with magnetic beads at each stage according to the gradient of magnitude of charge or strength of magnetic force. It is impossible. In other words, if the charge amount of the droplet or the magnetic force of the cell with magnetic beads is proportional to the number of receptors on the cell surface (receptor density), the FACS method and the MAC S method allow the cell to be It can be said that it is difficult or a complicated process is necessary to continuously isolate cells having the density of each receptor depending on the density of receptors present in the cells.

[0009] By the way, Non-Patent Document 3 discloses a technique for separating cells based on the presence or absence of a receptor by using a ligand that binds to a receptor on the cell surface and using a base material on which the ligand is immobilized. Has been. Specifically, in Non-Patent Document 3, a glass substrate coated with a selectin solution (2 μg / mL) as a ligand is adopted as a ligand-presenting substrate, and CD34-negative cells (without receptors and cells) and CD34-positive cells are used. By rolling cells (cells with receptors) on the surface of the ligand-presenting substrate, the average rolling rate of both cells is 2.2 times different, so the elution rate is calculated to be approximately 20 minutes different. In addition, when 5. or 10.9 m latex particles coated with sialyl Lewis antigen are rolled in a chamber (width 15 mm, height 55 mm) composed of a ligand-presenting substrate surface, latex with a small particle size is used. For the particles, the rolling speed is slower than the larger one, indicating that the elution time is delayed.

However, with the technique disclosed in Non-Patent Document 3, the ligand is applied to a smooth surface. A non-covalently bound ligand-presenting substrate is used. For this reason, it is not possible to expect an efficient contact frequency between the cell surface and the ligand-presenting substrate surface, which is difficult to modify with a high density of ligand, and therefore, cells having a receptor density within a predetermined range can be accurately separated. There is a risk of not.

[0011] That is, a method for efficiently and highly accurately sorting cells having a receptor density within a predetermined range is still desired.

Means for solving the problem

[0012] The present invention has been made in view of the above problems, and an object of the present invention is a method of sorting cells from a group of cells including cells presenting a specific receptor on the cell surface. (1) a method for efficiently and accurately sorting the cells having a receptor density within a predetermined range; and (2) the cells having each density according to the receptor density from the cell group. It is an object to provide a method for efficiently and continuously fractionating the water.

[0013] As a result of intensive studies in view of the above problems, the inventors of the present application have fixed a ligand capable of temporarily binding to a receptor present on the surface of a cell desired to be sorted via a graft chain. It has been found that cells having a display density of a receptor that is desired to be sorted can be sorted efficiently and with high accuracy by using a trapped substrate, and the present invention has been completed. In addition, the present inventors have found that by using the above-mentioned base material, cells having a certain display density can be sequentially sorted according to the display density of the receptor according to the receptor display density, and the present invention has been completed. I came to let you.

[0014] That is, in the sorting method according to the present invention, a ligand that temporarily binds to the receptor is presented on the surface via a graft chain from a cell group including cells in which a specific receptor is presented on the cell surface. By using a ligand-presenting substrate, cells having a receptor display density within a predetermined range are sorted.

It should be noted that the cells whose receptor display density is within a predetermined range are cells whose receptor display density is within the range of the display density desired to be sorted.

[0016] In another sorting method according to the present invention, a ligand that temporarily binds to the receptor is displayed on the surface via a graft chain from a group of cells including cells on which the specific receptor is presented on the cell surface. By using an improved ligand-presenting substrate, the display density of the receptor can be adjusted. Next, it is characterized by sequentially sorting cells having a certain display density according to the level of the display density.

[0017] In these sorting methods according to the present invention, a smooth surface is formed by the ligand-presenting base material and the ligand, and the sorting is performed by moving a group of cells along the surface. The cell is rotated by moving on the smooth surface while binding with the ligand, and the cell moving speed is preferably reduced by the degree of binding with the ligand according to the receptor display density. Better ,.

[0018] Note that the smooth surface refers to a surface structure other than a surface having a structure that restricts the movement of cells when a cell group moves along the surface. This excludes the surface having a structure in which the cell just fits in the recess about the size of the cell.

[0019] Further, in the sorting method according to the present invention, a dispersion liquid in which the cells are dispersed is allowed to flow on the smooth surface, and the bonds are dissociated by shear stress that the cells receive a dispersion liquid force. Is preferred.

[0020] Further, the sorting method according to the present invention uses a force that rolls on the inclined surface generated in the cells by flowing a dispersion liquid in which the cell group is dispersed on the inclined smooth surface. It is preferable to dissociate the bonds.

[0021] In the sorting method according to the present invention, the cell is preferably a stem cell.

[0022] In the fractionation method according to the present invention, it is preferable that the ligand is a site force-in.

More specifically, the above cyto force-in levels are vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor 1 (SDF1), and platelet-derived growth factor (PDGF). , Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), Neurotrophin-3 (NT-3), Eurotrophin-1 (NT-4), Bone morphogenetic factor (BMP), or Interleukin (IL).

[0023] In the fractionation method according to the present invention, the receptor may be an antigen, and the ligand may be an antibody that causes an antigen-antibody reaction against the antigen. More specifically, on The antigen is a specific antigen on the surface of vascular endothelial progenitor cells, and the above-mentioned antibodies are anti-CD31 antibody, anti-CD34 antibody, anti-CD133 antibody, anti-CD144 antibody, anti-Flk-l antibody, anti-Flk-2 antibody, anti-antibody Flk-3 antibody, anti-Flk-4 antibody, anti-Fit-1 antibody, anti-Fit-2 antibody, anti-Fit-3 antibody, anti-Fit-4 antibody, anti-tie-2 antibody, anti-PECAM antibody, anti-VE cadherin antibody And at least one selected from the group consisting of anti-VEGF receptor antibody strength.

In addition, the ligand-presenting substrate according to the present invention is characterized in that the ligand is presented on the surface via a graft chain for use in the above-described sorting method.

[0025] Further, the ligand-presenting substrate according to the present invention is preferably a tubular substrate, and the ligand forms a smooth surface on the inner wall of the tube via the graft chain.

[0026] Further, the ligand-presenting substrate according to the present invention is preferably a planar substrate having grooves.

[0027] The ligand-presenting substrate according to the present invention is preferably such that the ligand is formed in the groove via the graft chain! /.

[0028] Further, in the method for producing a ligand-presenting substrate according to the present invention, a polymerization step of polymerizing a graft chain on the surface of the substrate before presenting the ligand, and the graft chain polymerized by the polymerization step, And a fixing step of fixing the ligand.

[0029] Further, the separation apparatus including the ligand-presenting base material used in the sorting method includes the above-described ligand-presenting base material and a tilting means for tilting the ligand surface of the ligand-presenting base material. It is characterized by that.

The invention's effect

[0030] According to the display device of the present invention, cells having a receptor density within a predetermined range can be efficiently and accurately sorted from a cell group containing the cells, and the cell group described above. From the above, according to the receptor density, the cells having each density can be continuously collected. Specifically, in the present invention, since a ligand-presenting substrate in which a ligand is presented on the surface via a graft chain is used, a conventional ligand-presenting substrate in which the ligand is non-covalently bonded to the substrate surface. Compared to the above, in order to achieve high-density ligand modification, it provides efficient contact frequency between the cell surface and the ligand-presenting substrate surface, so that cells having a receptor density within a predetermined range can be accurately detected. It becomes possible to separate. [0031] Still other objects, features, and advantages of the present invention will be sufficiently enhanced by the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

Brief Description of Drawings

FIG. 1 is a diagram for explaining the principle of a sorting method according to an embodiment of the present invention.

FIG. 2 is a diagram schematically showing a ligand-presenting substrate that can be used in a sorting method according to an embodiment of the present invention and a state of cells that rotate and move on the surface thereof.

FIG. 3 (a) is a diagram showing a configuration of a separation apparatus provided with a ligand-presenting substrate that can be used in a sorting method according to an embodiment of the present invention.

[FIG. 3 (b)] FIG. 3 (b) is a perspective view showing the shape of a ligand-presenting base material provided in the separation apparatus shown in FIG. 3 (a).

FIG. 4 is a view showing a ligand-presenting base material that can be used in a sorting method according to an embodiment of the present invention.

FIG. 5 is a graph showing the results of quantifying the amount of ligand immobilized on the ligand-presenting substrate used in one example of the present invention.

[FIG. 6 (a)] A dispersion in which a cell group containing CD34-positive cells is dispersed in the ligand-presenting substrate (anti-CD34 antibody-immobilized tube substrate) used in one example of the present invention is passed through. It is a graph which shows the result of having quantified the number of cells in the fraction collected.

FIG. 6 (b) is a graph showing the results of quantifying the number of cells in the fraction recovered by passing the dispersion liquid of FIG. 6 (a) through a substrate with a ligand immobilized as a comparative example. is there.

[Fig. 7 (a)] The ligand-presenting substrate (anti-CD34 antibody-immobilized tube substrate) used in one example of the present invention was collected by passing a dispersion in which CD34-negative cells were dispersed. It is a graph which shows the result of having quantified the number of cells in a fraction.

[FIG. 7 (b)] As a comparative example, a graph showing the results of quantifying the number of cells in a fraction collected by passing a dispersion in which CD34 negative cells were dispersed in a base material, with a ligand immobilized. It is.

[Fig. 8 (a)] Analysis result by flow cytometer, analysis result of the dispersion before passing through the ligand-presenting substrate. [Fig. 8 (b)] Analysis results by flow cytometer, and analysis results of the eluate collected in fraction no. 7 in Fig. 6 (a).

FIG. 9 is a graph showing the results of plotting the percentage of CD34 high-density cells against fraction no.

FIG. 10 is a graph showing the results of quantifying the expression level of collagen type 1 in mouse bone marrow-derived mesenchymal stem cells separated by a ligand-presenting substrate (anti-CD34 antibody-fixed tube substrate).

FIG. 11 is a diagram showing a conventional technique and explaining the principle of the FACS method.

FIG. 12 is a diagram showing a conventional technique and explaining the principle of the MACS method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0033] The present invention uses a ligand-presenting substrate in which a ligand that temporarily binds to the receptor is presented on the surface via a graft chain from a group of cells containing cells on which the specific receptor is presented on the cell surface. Thus, a method for sorting cells having a receptor display density within a predetermined range is provided.

[0034] The present inventors have focused on a specific receptor that is present on the surface of a cell and serves as a marker for identifying the cell, and a protein having a ligand corresponding to this receptor (hereinafter simply referred to as a ligand). In other words, cells that are desired to be sorted (hereinafter also referred to as target cells) can be efficiently and accurately sorted from a group of cells by using a base material that is bound to the surface via a graft chain. As a result, they have found that they can do it and have completed the method according to the present invention.

Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0036] Examples of target cells to be sorted include stem cells. Stem cells are cells that constitute the target tissue or organ, cells that can differentiate into cells that constitute the target tissue or organ, and the like. In addition to such cells, there are cells that can differentiate into cells constituting the target tissue or organ or cells constituting the target tissue or organ by secreting a specific site force-in or the like. This also includes cells that encourage entry into the support body or induce differentiation of these cells. [0037] The cells that can differentiate into cells that constitute the tissue or organ, cells that constitute the tissue or organ are not particularly limited, and for example, human or animal-derived mesenchymal stem cells, ES cells, keratinocytes, Examples include fibroblasts, bone marrow cells, endothelial cells, smooth muscle cells, Schwann cells, chondrocytes, fat cells, osteoblasts, vascular endothelial progenitor cells, and the like.

[0038] The cell that secretes the site force-in is not particularly limited, and examples thereof include a cell constituting the tissue or organ, a cell capable of differentiating into a cell constituting the tissue or organ, and the like. In the following explanation, a stem cell is simply a cell.

[0039] The ligand that temporarily binds to a specific receptor displayed on the cell surface of the cell to be sorted is not particularly limited, and can bind to a receptor on the cell surface and has the conditions described below. Any ligand can be used as long as it can be dissociated. Specific examples include site force-in and antibodies.

[0040] The site force-in is not particularly limited, and examples thereof include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor 1 (SDF1), and platelet-derived growth factor. (PDGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell Examples include derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), -eurotrophin-4 (NT-4), bone morphogenetic factor (BMP), or interleukin (IL).

[0041] For example, when the target cell or group of cells is a Schwann cell or a neuron, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) for receptors specific to these cells , Ciliary neurotrophic factor (CNTF), neurotrophin 3 (NT-3) and -eurotrophin-4 (NT-4) can be specifically bound. Therefore, when Schwann cells or nerve cells are separated by the method of the present invention, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), di-eurotrophin. 1 (NT-3) and -Eurotrophin 1 (NT-4) At least one site force selected from the group consisting of forces is bonded to the surface via a graft chain. (Ligand-presenting substrate) is used.

[0042] As the antibody, an antibody corresponding to a specific antigen on the surface of a target cell is used. The

[0043] For example, an antibody to a surface marker (receptor) specifically present on the surface of a vascular endothelial cell or vascular endothelial progenitor cell, Schwann cell or nerve cell (anti-cell surface marker antibody), vascular endothelial cell or vascular endothelium Examples thereof include antibodies against a progenitor cell, a cytoforce-in receptor that specifically acts on Schwann cells or neurons (anti-site force-in receptor antibody), and the like.

[0044] Specifically, for example, CD34, CD31, CD133, CD144, Flk-1, Flk-2, Flk-3, on the surface of vascular endothelial progenitor cells that can differentiate into cells constituting vascular endothelium. Specific antigens such as Flk—4, Fit—1, Fit—2, Fit—3, Fit—4, tie—2, VE cadherin, PECAM, and VEGF receptor are present to identify vascular endothelial progenitor cells. It can be used as a marker (receptor). Therefore, when vascular endothelial progenitor cells are collected by the method of the present invention, anti-CD31 antibody, anti-CD34 antibody, anti-CD133 antibody, anti-CD144 antibody, anti-Flk-1 antibody, anti-Flk-2 antibody, anti-Flk— 3 antibodies, anti-Flk—4 antibodies, anti-Fit—1 antibody, anti-Fit—2 antibodies, anti-Fit—3 antibodies, anti-Fit—4 antibodies, anti-tie—2 antibodies, anti-PECAM antibodies, anti-VE cadherin antibodies or anti-VEGF Use a base material in which at least one kind of antibody selected from the group consisting of receptor antibodies is bound (presented) to the surface via a graft chain.

[0045] When a plurality of types of receptors are present on the cell surface, only one type of cyto force-in or antibody corresponding to any of them may be used, or two or more types may be used in combination. When two or more kinds of cytosines or antibodies are used in combination, higher selectivity can be realized in fractionation.

[0046] A method for preparing the above-mentioned site force-in or antibody is not particularly limited, and a conventionally known method can be used. In recent years, antibodies corresponding to various types of site force-in and main cell surface antigens have been marketed, so use them.

[0047] Next, the ligand-presenting base material on which the ligand is presented on the surface used in the sorting method of the present invention will be described.

[0048] The ligand-presenting substrate of the present invention is obtained by binding a protein having a ligand to the surface of the substrate via a graft chain.

[0049] The substrate is not particularly limited, and a known inorganic material or organic material can be used. . Thus, for example, polylactic acid, polydaricholic acid, poly ε-strength prolatatone, lactic acid-darlicholic acid copolymer, glycolic acid ε-strength prolatatone copolymer, lactic acid ε-strength prolatatone copolymer, polykenic acid, polymalic acid , Poly (X-cyanoacrylate, poly 13-hydroxy acid, polytrimethylene oxalate, polytetramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylene carbonate, poly γ-benzyl lu glutamate, poly γ —Synthetic polymers such as methyl-L-glutamate and poly-L-alanine; natural polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectinic acid and derivatives thereof, and proteins such as gelatin, collagen, albumin and fibrin It is also possible to use a polymer. , Even those that have been constructed by combining the Yogu multiple types even constitute using one type from among these! /,.

[0050] Substances that can be used as a graft chain to be polymerized on the above-mentioned substrate include, for example, bur monomers such as acrylic acid, methacrylic acid, and butyl acetate, and derivatives thereof, and cyclic amines such as ethylene imide lactide. Examples include compounds.

[0051] Further, the method for generating the polymerization start point is as follows: プラズマ, plasma, corona, ultraviolet rays, and

Three

This includes the introduction of response starting points and is not limited to a specific method. In addition, the polymerization method of the graft chain is not limited to the radical polymerization by the vinyl monomer shown in the examples, but includes coordination anion polymerization, ring-opening polymerization, hydrogen transfer polymerization, polycondensation, addition condensation, and the like. The combination is also included.

[0052] Further, the method for immobilizing an antibody to the generated graft chain is not limited to the method for the activation of an amino group with carpositimide, but is immobilized with a compound having an amino group and an isothiocyanate group or a succinimidyl ester. And various methods for hydroxyl groups, thiol groups, and carboxyl groups.

[0053] In the present invention, the density of the ligand and the contact frequency with the receptor-presenting cell are improved by immobilizing the antibody on the base material via a graft chain. It is not limited. That is, as long as the density of the ligand and the frequency of contact with the receptor-presenting cell can be improved, for example, immobilization of Fab, protein having a multi-binding site, and introduction of a high-density active site may be employed. It is possible. The following can be illustrated as such a form. [0054] When polylactic acid or the like is used as the base material, for example, the surface of polylactic acid is hydrolyzed with an alkali or the like to expose a carboxyl group, and then a kind of water-soluble carbodiimide to the carboxyl group. A protein can be bound by reacting a certain 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide (EDC) and using this EDC active group to covalently bond the amino group of the protein. The unreacted EDC active group remaining on the surface of the base material inhibits the binding between the receptor and the ligand, and there is a possibility that accurate fractionation cannot be realized. It is preferable to perform a cabbing treatment such as reacting with.

[0055] Collagen can also be used as the substrate. In this case, for example, the protein is shared via various divalent crosslinkers such as dialdehydes, epoxy compounds, and acid anhydrides by using functional groups of amino acids constituting collagen. Can be combined.

[0056] Even in the above-described form, it is expected that the density of the ligand is increased and the frequency of contact with the receptor-presenting cell is improved.

[0057] The shape of the substrate is not particularly limited, and for example, a tube or a film such as a tube or a film may be used. . In the case of a tube-like material such as a tube, it is preferable that the protein having the above ligand is bound to the inner wall via a dial chain. In the case of a film-like material such as a film, it is preferable that the protein having the ligand is bound to the membrane surface via a graft chain.

[0058] The binding amount of the protein (ligand) is not particularly limited, but according to the configuration of the present invention, since the ligand is bound via the graft chain, the ligand is bound at a high density. Can do. In addition, the protein may be bound to the entire base material, or may be bound to only a part thereof.

[0059] Although the details of the present invention will be described later, the target cells are sorted by the cell group containing the target cells moving while rotating on the ligand surface of the ligand-presenting substrate. Accordingly, the ligand-presenting substrate preferably has a smooth surface formed by the ligand. In the present specification, the term “smooth surface” means that a cell group is on the surface. A surface that does not have a structure that restricts the movement of cells when moving along. For example, if a recess corresponding to the size of the cell is formed so that the cell completely fits in, the cell cannot be moved due to being fitted into the recess during movement. It becomes impossible to carry out taking method. Therefore, a surface that excludes the surface on which such a structure is formed is called a “smooth surface”. For example, a porous body having a large number of fine pores is suitable because it may cause the above problems.

[0060] However, if there is no risk of causing the above-described problems, the ligand surface of the ligand-presenting substrate has a planar surface with grooves that do not have to be a perfect plane. It may be a thing. In this case, it is also preferable that the protein having the ligand is bound to the groove via a graft chain.

[0061] By using the base material having the groove to which the ligand is bound in this way, the surface area of the ligand that interacts with the cell group can be widened, and sorting can be efficiently performed. For the same reason, the ligand-presenting substrate has a structure in which a plurality of tubes are formed with the flow channel directions parallel to each other, and the protein having the ligand is bound to the inner wall of each tube via a graft chain. It may be a thing.

FIG. 3 (a) shows an embodiment of a separation apparatus that can be used in the separation method of the present invention.

As shown in FIG. 3 (a), the separation device is provided with a ligand-presenting base material 2, a tilting means 6 for tilting the ligand-presenting base material 2, and a syringe pump 7. FIG. 3 (b) shows the shape of the ligand-presenting substrate 2. The separation device according to the present embodiment is configured such that a dispersion liquid in which a cell group is dispersed is supplied from a syringe pump 7 to the ligand surface, and the cell group rotates and moves on the ligand surface inclined by the inclination means 6. (Rolling movement)

[0063] Further, the separation device may include a collecting means for rolling the ligand surface to collect the sorted cells.

[0064] Next, a sorting method according to the present invention will be described in detail.

[0065] In the sorting method according to the present invention, the interaction between a receptor on the surface of a cell desired to be sorted and a ligand provided on the ligand-presenting substrate via a graft chain (temporary binding). Use the degree of. [0066] The degree of interaction (hereinafter referred to as binding) is proportional to the density of receptors presented on the cell surface (hereinafter referred to as receptor presentation density). That is, the degree of binding to the ligand immobilized on the substrate is high, and the cell has a receptor display density of “high”. On the other hand, a cell with a low degree of binding to the ligand of the substrate can be said to have a receptor presentation density force S “low”. Also, from here, when the degree of binding (hereinafter referred to as binding force) is high, the force required to dissociate it (hereinafter referred to as dissociating force) is large, and when the binding force is low, the dissociating force may be small. It will be.

[0067] The present invention sorts target cells using such a principle. FIG. 1 is a schematic diagram for explaining the sorting method of this embodiment using this principle.

[0068] In the sorting method of the present embodiment, as shown in FIG. 1, the cell group 1 is rolled and moved on the ligand surface 3 fixed to the draft chain 5 on the ligand-presenting substrate 2.

In this embodiment, as shown in FIG. 2, the ligand-presenting base material 2 is tilted using the tilting means 6 (FIG. 3 (a)), and a dispersion liquid in which the cell group 1 is dispersed is supplied from upstream. To do. As a result, the cell 4 rolls due to the force of the dispersion (shear stress) and the force that rolls on the slope generated in the cell 4 itself. That is, in the present embodiment, the shear stress and the force rolling on the inclined surface are the dissociating force.

[0070] Therefore, by applying this dissociation force to a plurality of cells (cell groups) having different binding forces, the binding force is small, and the binding force of cells 4b is easily dissociated. The slowing of the moving speed is small, but the binding force is high, and the binding force of the cell 4a is difficult to dissociate. Therefore, the rolling moving speed caused by the binding force is slow compared to the cell 4b having a small binding force. In other words, the movement speed varies depending on the difference in receptor presentation density. In other words, a cell 4b with a high migration speed can be said to have a "low" receptor presentation density, and a cell 4a with a low migration speed can be said to have a "high" receptor presentation density.

[0071] It should be noted that the moving speed is high, and the cells may contain cells that present the receptor.

[0072] Thus, according to the method of the present embodiment, the receptor presentation density in each cell can be classified as a difference in migration speed. [0073] Therefore, for example, by using a cell in which the receptor presentation density is pre-divided, the cell can be rolled on the ligand presentation substrate, and the time during which the downstream force of the ligand presentation substrate is eluted according to the receptor presentation density can be set. By measuring, cells having a desired receptor display density can be sorted using the difference in migration speed, that is, using the difference in elution time.

[0074] The cell concentration (number) of the dispersion in which the cell group 1 is dispersed is not particularly limited as long as the above dissociation force is applied to individual cells that move rolling on the ligand surface. When using a ligand-presenting substrate of μg / m ² , the cell concentration in the dispersion (including cells without receptors) is 1 X 10 ° to 1 X 10 ⁷ cells / mL, and the number of cells is 1 X A dispersion of 10 ° to 1 × 10 ^1G Zm ² can be used.

[0075] Further, according to the sorting method according to the present invention, as shown in FIG. 1, since the moving speed can be differentiated by the difference in receptor presentation density, each receptor presentation is made downstream in the movement direction. By providing a means for collecting cells having a different density, cells having different receptor display densities can be sequentially sorted according to the receptor display density.

[0076] That is, by using a ligand-presenting substrate in which a ligand that temporarily binds to the receptor is presented on the surface via a graft chain from a group of cells containing cells on which the specific receptor is presented on the cell surface. A method of sequentially sorting cells having a certain display density according to the display density of the receptor according to the level of the display density is also one of the sorting methods of the present invention.

In the above description, as shown in FIG. 2, the ligand-presenting base material 2 is tilted, and the shear stress received from the dispersion and the force that rolls on the slope generated in the cells themselves are used as the dissociation force. Here, in the technique of Non-Patent Document 3 described above, the possibility of cell separation is suggested by controlling the cell rolling only by the flow of a single dispersion (share stress). However, an increase in shear stress generates a phase flow state between the ligand substrate surface and the liquid flow, and decreases the contact frequency between the cells and the substrate surface. For this reason, the method of Non-Patent Document 3 can realize cell rolling only within a very limited range. Further, as described above, the technique of Non-Patent Document 3 has a configuration in which a ligand is non-covalently bonded to the surface of a substrate. For this reason, the cell surface and the ligand substrate surface, on which high-density ligand modification is difficult, are more effective. Since it is not possible to expect an efficient contact frequency, there is a limit to the applicable share stress, and when the binding force between the ligand and the receptor is strong, the dissociation can be promoted only with a single share stress. Is difficult. On the other hand, the present invention utilizes the force of rolling on the slope generated in the cell itself together with the shear stress. This method increases the frequency of contact between the ligand-presenting substrate and the cell surface receptor, enabling more efficient separation of the cells. As described in the examples described later, the degree of shear stress Furthermore, cell separation can be achieved more efficiently by freely changing the inclination angle of the ligand-presenting substrate.

[0078] As described above, according to the fractionation method of the present invention, the ligand is noncovalently bound to the substrate surface because the ligand-presenting base material on the surface of the ligand is used via the graft chain. Thus, compared with the conventional ligand-presenting substrate, in order to realize a high-density ligand modification, it provides an efficient contact frequency between the cell surface and the ligand-presenting substrate surface, and thus within a predetermined range. Cells having a receptor density can be accurately separated, and the cells having each density can be continuously collected according to the receptor density.

[0079] In the above description, the cells that have migrated on the ligand surface 3 of the ligand-presenting substrate 2 are collected downstream using the collecting means and sorted. However, the present invention is not limited to this. That is, in the fractionation method according to the present invention, a dissociation force is released and a plurality of mobility different in the mobility is obtained in a state in which the movement speed is made different on the ligand surface 3 of the ligand-presenting substrate 2 with a difference in movement speed. The cells may be placed (bound) on the ligand surface 3 as they are.

[0080] Thereby, it is possible to provide a base material in which cells are unevenly distributed on the ligand surface 3 in accordance with the receptor display density on the cell surface.

[0081] The present invention is not limited to the force described in more detail by the following examples.

Example

[0082] Using the ligand-presenting base material having the above-described configuration, the present inventors sorted cells having a desired receptor-presenting density. First, a ligand-presenting substrate was prepared.

[0083] (1) Creation of ligand-presenting substrate As shown in FIG. 4, a polyethylene tube substrate having an inner diameter of 1 mm, an outer diameter of 2 mm, and a length of 100 mm was used as the substrate. First, the polyethylene tube substrate shown in FIG. 4 was treated with ozone gas at room temperature for 4 hours. Thereafter, the grafted chain of polyacrylic acid was polymerized on the polyethylene surface by heating at 60 ° C. for 4 hours in a 20% acrylic acid Z methanol solution. The grafted tube substrate was cleaned by sonication at 37 ° C for 30 minutes, and then 0.1M 1-ethyl-3- (3 dimethylaminopropyl) force rubodiimide (EDC) solution at 4 ° C. Soaked for 2 hours to activate the carboxyl groups on the surface.

[0084] Next, the EDC activity tube substrate was immersed in 0.04 mgZmL anti-CD34 antibody-phosphate buffer solution at 37 ° C for 4 hours to bind the anti-CD34 antibody to the inner wall.

[0085] Finally, it was immersed in a 10 mM 2-aminoethanol-phosphate buffer solution at 4 ° C for 2 hours to cap the unreacted active carboxyl group to obtain a ligand-presenting substrate.

[0086] Next, the amount of the immobilized anti-CD34 antibody was quantified. Quantification was performed using peroxidase-modified anti-mouse IgG goat IgG. Figure 5 shows the quantitative results.

FIG. 5 is a graph showing the results of quantification of the antibody amount of the anti-CD34 antibody. In FIG. 5, a tube base material subjected to the following treatment is used as a control tube base material.

[0088] As the control tube substrate, for example, a polyethylene tube substrate having an inner diameter of 1 mm, an outer diameter of 2 mm, and a length of 100 mm was used in the same manner as the ligand-presenting substrate. The tube substrate was cleaned by sonication at 37 ° C for 30 minutes, and then added to 0.1M 1-ethyl-3- (3 dimethylaminopropyl) carpositimide (EDC) solution at 4 ° C for 2 hours. The surface carboxyl group was activated by immersion. The EDC active tube substrate was then immersed in phosphate buffer solution at 37 ° C for 4 hours, and finally immersed in 1 OmM 2-aminoethanol phosphate buffer solution at 4 ° C for 2 hours. The reaction active carboxyl group was capped to obtain a control tube base.

FIG. 5 shows that the amount of antibody immobilized on the inner wall of the tube base material (ligand-presenting base material) increases as the EDC activity time elapses. That is, this result showed that the anti-CD34 antibody was immobilized on the inner wall of the tube base material. The ligand amount of the substrate is 200 μg / m ² and the ligand density is 1. 67 X 10 ” ⁹ mol / m ² . It was.

[0090] (2) Performance of ligand-presenting substrate

[Examination of separation by displayed marker density]

The performance of the ligand-presenting substrate (anti-CD34 antibody-immobilized tube substrate) prepared in (1) above was verified. First, a dispersion in which cell groups were dispersed was prepared. HL60 cells derived from acute leukemia, which are CD34 negative cells, and KG-la cells, which are CD34 positive cells, were suspended in phosphate buffer to prepare cell dispersions.

[0091] The prepared dispersion (cell concentration: 2 X 10 ⁶ ZmL) was placed in a cell injection tube connected to the ligand-presenting substrate shown in Fig. 3 (a) using a syringe with 10 L (cell number: 2 X 10 ⁴ pieces) Then, using a syringe pump 7 (KD Scientific Inc., Inlusion Pomp, Model: KDS100) shown in FIG. 3 (a), the dispersion liquid was flowed for 50 LZ (fraction no. 1 to 5), flow rate 60 O / z LZ fraction (fraction no. 6 to 15) was passed through the hollow part of the ligand-presenting substrate. The eluted cells were collected for each fraction. The recovered amount is 50 / z L in fractions no. 1-10 and 100 in fractions no. 11-15.

FIG. 6 (a) shows the results of measuring the number of cells in the eluate (fractions no. 1 to 8) collected every minute.

[0093] In FIG. 6 (b), as a comparative example, phosphoric acid was added to the control tube base material (tube base material not presenting the ligand) prepared in FIG. The results of collecting cells under the same conditions as in Fig. 6 (a) after passing a buffer solution are shown.

[0094] From the results in Fig. 6 (a) · (b), almost no cell elution was observed in fractions no. 6 'to 8' in Fig. 6 (b), whereas Fig. 6 Cell elution was observed in fractions no. 6 to 8 in (a). That is, a cell group with a delayed cell elution time was confirmed only when a ligand-presenting substrate on which an antibody was presented was used.

[0095] As a control experiment, HL60 cells (CD34 negative cells) were allowed to flow through the ligand-presenting substrate. As a result, no delayed fraction was observed (Fig. 7 (a)). In addition, even when HL60 cells were allowed to flow through the control tube substrate, delayed fraction was not confirmed (Fig. 7 (b)). From the above results, it was confirmed that the cells of fractions no. 6 to 8 observed in FIG. 6 (a) were delayed in elution time by the action of CD34 expression. [0096] Next, using a flow cytometer, the dispersion containing HL60 cells and KG-la cells before passing through the ligand-presenting substrate and the eluate collected in fraction no. 7 in Fig. 6 (a) are used. The amount of fluorescence labeled on CD34 positive cells was quantified. The fluorescent labeling reagent used was FITC anti-human CD34 (Cat No: 558221) manufactured by BD Biosciences Pharmingen. 2 L of fluorescent labeling reagent was added to the collected 100 L fraction and allowed to stand at 4 ° C for 30 minutes. Thereafter, FACS measurement tube (Becton Dickinson Labware, FALCON352058 Polystyrene tube, 5 mL) and PBS were added to prepare a final volume of 1 mL.

[0097] The results of analysis using a flow cytometer are shown in Figs. 8 (a) and (b). Fig. 8 (a) shows the analysis results of the eluate collected in fraction no. 2 in Fig. 6 (a), and Fig. 8 (b) shows the result collected in fraction no. 7 in Fig. 6 (a). It is the analysis result of the eluate.

[0098] From the results in Figs. 8 (a) and (b), it is clear that the fluorescence intensity included in the circled region in the analysis results in Fig. 8 (a) increases in Fig. 8 (b). Recognize. The cells detected in this circled area are cells with a higher cell surface receptor density than the cells detected in other areas. From this, it was shown that the cells within a certain fluorescence intensity range are separated and concentrated by the fractionation method according to the present invention. As a result of plotting the high cell surface receptor density and the percentage of cells against the fraction no., It was shown that the density of CD34 high density cells increased with the fraction no. . This indicates that this column can separate high-density CD34 cells. That is, it shows that cells can be separated by the surface receptor density of the cells.

[0099] [Induction of differentiation of mesenchymal stem cells separated by ligand-presenting base material into bone cells]

O Conducted an experiment to induce differentiation into bone cells using mesenchymal stem cells separated by a ligand-presenting substrate o

[0100] Using a ligand-presenting base material (a silicon tube base material having an inner diameter of 0.5 mm, an outer diameter of 1.5 mm, and a length of 100 mm, anti-mouse CD34 antibody immobilization agent) prepared in the same manner as in (1) above. Then, mouse bone marrow-derived mesenchymal stem cells that are CD34 positive cells were isolated. The amount of cell dispersion injected into the ligand-presenting substrate, the number of cells, and the flow rate of the dispersion are the same as in the above method (2). The recovered cell volume is 25 L in the 12.5 μ fractions no. 11-15 in the fraction no. 1-10. The collected fractions no. 2 to 7 The cells were seeded in a 24-well culture dish and allowed to stand for 24 hours at a temperature of 37 ° C and a carbon dioxide concentration of 5%. Then, the bone cell fraction induction medium (DMEM—low glucose (manufactured by Aldrich), dexamethasone ^10_8 M, j8 glycephosphate phosphate 10 mM, ascorbic acid 0.3 mM) was added, and the temperature was 37 °. C, left at 5% carbon dioxide concentration.

[0101] Four days after the start of the addition of the bone cell differentiation induction medium, mRNA was recovered from the cells by a conventionally known method, and the gene expression level of collagen type 1 was quantified by the PCR method (forward primer sequence: 5'—GAAGTCAGCTGCATACAC—3, reverse primer Sequence: 5′-AGG AAGTCC AGGCTGTCC 3, PCR conditions: 50 ° C (2 minutes) → 95 ° C (1 minute) → [95 ° C (1 minute) → 60 ° C (15 seconds) → 74 ° C (1 minute)]) X 40 cycles → end). Figure 10 shows the results. Sample no. 1 in Fig. 10 shows the results of similarly inducing differentiation of cells (mouse bone marrow-derived mesenchymal stem cells) as a control. Samples No. 2 and 3 show the results of inducing differentiation of cells with low and high CD34 expression by FACS method.

[0102] From this result, in samples no. 5 to 8 separated with the ligand-presenting substrate, the gene was 5 times more potent and 7 times higher than unseparated V and cells or cells separated by the FACS method. The expression level was shown.

[0103] This indicates that the mesenchymal stem cells separated by the ligand-presenting substrate can isolate the stem cells that can be more efficiently induced.

[0104] From the above, it was shown that by using a ligand-presenting substrate, cells having a predetermined receptor-presenting density can be easily and quickly sorted by a cell group dispersed in a dispersion. In addition, mesenchymal stem cells isolated by this ligand-presenting substrate showed significantly higher differentiation-inducing efficiency than mesenchymal stem cells isolated by the conventional FACS method. It is considered to be a very useful aid for elucidating the correlation between cell surface receptors and separations. In addition, if cells that can be sorted into specific cells or sorted into specific cells can be realized based on cell surface receptors, it will greatly contribute to the research and development of cell transplantation and regenerative medicine. Can be expected.

It should be noted that the present invention is not limited to the above-described embodiment, but includes the scope shown in the claims. Various changes can be made in the box.

Industrial applicability

The sorting method according to the present invention uses a ligand-presenting substrate in which a ligand that temporarily binds to the receptor is presented on the surface from a group of cells containing cells on which the specific receptor is presented. Depending on the receptor display density, cells with a certain display density can be easily sorted and sequentially sorted according to the level of the display density. For example, cells in stem cells that are divided into specific cells It can be widely applied to the field of regenerative medicine where various research and development are being carried out for the realization of treatment methods by tissue regeneration, such as elucidating the correlation between surface receptors and molecules.

Claims

The scope of the claims

[1] By using a ligand-presenting substrate in which a ligand that temporarily binds to the above receptor is presented on the surface via a graft chain from a group of cells containing cells on which the specific receptor is presented on the cell surface, A sorting method comprising sorting cells having a display density within a predetermined range.

[2] By using a ligand-presenting substrate in which a ligand that temporarily binds to the above receptor is presented on the surface via a graft chain from a group of cells including cells on which the specific receptor is presented on the cell surface, A sorting method comprising sequentially sorting cells having a certain display density according to the display density according to the level of the display density.

[3] The ligand-presenting substrate has a smooth surface formed by the ligand, and the sorting is performed by moving a group of cells along the surface and binding the cells to the ligand. Done by rotating the smooth surface,

3. The sorting method according to claim 1, wherein the moving speed of the cells is reduced depending on the degree of binding with the ligand according to the display density.

[4] The fractionation according to [3], wherein a dispersion in which the cell group is dispersed is allowed to flow on the smooth surface, and the binding is dissociated by a shear stress that the cells receive from the dispersion. Method.

[5] The dissociation of the bond is performed by using a force that rolls on the inclined surface generated in the cell by flowing a dispersion liquid in which the cell group is dispersed on the inclined smooth surface. The preparative method according to claim 3 or 4.

[6] The sorting method according to any one of [1] to [5], wherein the cells are stem cells.

[7] The fractionation method according to any one of [1] to [6], wherein the ligand is site force-in.

[8] The above-mentioned cyto force in is vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor 1 (SDF1), platelet-derived growth factor (PDGF), insulin-like Growth factor (IGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic It is a factor (GDNF), neurotrophin 1 (NT-3), neurotrophin 4 (NT-4), bone morphogenetic factor (BMP), or interleukin (IL). The preparative method described in 7.

[9] The receptor is an antigen,

The sorting method according to any one of claims 1 to 6, wherein the ligand is an antibody that causes an antigen-antibody reaction against the antigen.

[10] The antigen is a specific antigen on the surface of vascular endothelial progenitor cells,

Anti-CD31 antibody, anti-CD34 antibody, anti-CD133 antibody, anti-CD144 antibody, anti-CD31 antibody

Flk-1 antibody, anti-Flk-2 antibody, anti-Flk-3 antibody, anti-Flk-4 antibody, anti-Fit-1 antibody, anti-F

It 2 antibody, anti-Fit—3 antibody, anti-Fit—4 antibody, anti-tie—2 antibody, anti-PECAM antibody, anti-V

10. The sorting method according to claim 9, wherein the sorting method is at least one selected from the group consisting of an E-cadherin antibody and an anti-VEGF receptor antibody.

[11] A ligand-presenting substrate for use in the sorting method according to any one of claims 1 to 10, wherein the ligand is presented on the surface via a graft chain.

[12] The ligand-presenting substrate is a tubular substrate, and the ligand forms a smooth surface on the inner wall of the tube via the graft chain. The base material for presenting.

[13] The ligand-presenting substrate according to claim 11, wherein the ligand-presenting substrate is a planar substrate having a groove.

[14] The ligand-presenting base material according to claim 13, wherein the ligand is formed in the groove via the graft chain.

[15] A method for producing a ligand-presenting substrate for use in the fractionation method according to any one of claims 1 to 10,

A ligand presenting substrate comprising: a polymerization step of polymerizing a graft chain on the surface of the substrate before presenting the ligand; and a fixing step of immobilizing the ligand on the graft chain polymerized by the polymerization step. Manufacturing method.

[16] A separation apparatus comprising a ligand-presenting base material used in the sorting method according to any one of claims 1 to 10, The ligand-presenting substrate;

A separation apparatus comprising a tilting means for tilting the ligand surface of the ligand-presenting substrate.