WO2007055178A1 - Procédé de fractionnement de cellules et substrat à utiliser pour le procédé - Google Patents

Procédé de fractionnement de cellules et substrat à utiliser pour le procédé Download PDF

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Publication number
WO2007055178A1
WO2007055178A1 PCT/JP2006/322119 JP2006322119W WO2007055178A1 WO 2007055178 A1 WO2007055178 A1 WO 2007055178A1 JP 2006322119 W JP2006322119 W JP 2006322119W WO 2007055178 A1 WO2007055178 A1 WO 2007055178A1
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Prior art keywords
ligand
cells
antibody
cell
presenting
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PCT/JP2006/322119
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English (en)
Japanese (ja)
Inventor
Tetsuji Yamaoka
Atsushi Mahara
Soichiro Kitamura
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Japan Health Sciences Foundation
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Priority to JP2007544128A priority Critical patent/JPWO2007055178A1/ja
Publication of WO2007055178A1 publication Critical patent/WO2007055178A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • a cell suspension 101 containing hematopoietic stem cells is prepared.
  • CD34 of the hematopoietic stem cells is labeled with a fluorescent antibody 104.
  • FIG. 1 In FIG.
  • 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.
  • 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.
  • the sheath liquid containing cells is discharged from the nozzle 1 OOa.
  • 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.
  • 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).
  • FIG. 12 is a schematic diagram for explaining the principle of the MACS method.
  • a cell suspension 101 containing hematopoietic stem cells is prepared.
  • 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.
  • 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).
  • 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
  • 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.
  • a glass substrate coated with a selectin solution (2 ⁇ g / 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.
  • the average rolling rate of both cells is 2.2 times different, so the elution rate is calculated to be approximately 20 minutes different.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a ligand-presenting substrate By using a ligand-presenting substrate, cells having a receptor display density within a predetermined range are sorted.
  • 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.
  • 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.
  • the display density of the receptor can be adjusted.
  • it is characterized by sequentially sorting cells having a certain display density according to the level of the display density.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell is preferably a stem cell.
  • the ligand is a site force-in.
  • 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).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • SDF1 stromal cell-derived factor 1
  • PDGF platelet-derived growth factor
  • IGF Insulin-like growth factor
  • HGF hepatocyte growth factor
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell-derived neurotrophic factor
  • NT-3 Neurotrophin-3
  • NT-4 Eurotrophin-1
  • BMP Bone morphogenetic factor
  • IL Interleukin
  • the receptor may be an antigen
  • 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.
  • 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.
  • 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.
  • the ligand-presenting substrate according to the present invention is preferably a planar substrate having grooves.
  • the ligand-presenting substrate according to the present invention is preferably such that the ligand is formed in the groove via the graft chain! /.
  • 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.
  • 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.
  • 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.
  • 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) 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.
  • ligand-presenting substrate anti-CD34 antibody-immobilized tube substrate
  • 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.
  • 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.
  • a method for sorting cells having a receptor display density within a predetermined range is provided.
  • 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).
  • a protein having a ligand corresponding to this receptor hereinafter simply referred to as a ligand.
  • target cells cells that are desired to be sorted
  • 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.
  • 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.
  • cells that can differentiate into 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.
  • 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.
  • a stem cell is simply a cell.
  • 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.
  • 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.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • SDF1 stromal cell-derived factor 1
  • PDGF platelet-derived growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • GDNF derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4 -eurotrophin-4
  • BMP bone morphogenetic factor
  • IL interleukin
  • the target cell or group of cells is a Schwann cell or a neuron
  • nerve growth factor NNF
  • brain-derived neurotrophic factor BDNF
  • Ciliary neurotrophic factor CNTF
  • neurotrophin 3 NT-3
  • NT-4 NT-4
  • nerve growth factor NGF
  • brain-derived neurotrophic factor BDNF
  • CNTF ciliary neurotrophic factor
  • NT-3) and -Eurotrophin 1 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.
  • an antibody corresponding to a specific antigen on the surface of a target cell is used.
  • an antibody to a surface marker 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
  • a surface marker 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.
  • 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).
  • 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.
  • cyto force-in or antibody corresponding to any of them may be used, or two or more types may be used in combination.
  • two or more kinds of cytosines or antibodies are used in combination, higher selectivity can be realized in fractionation.
  • 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.
  • 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.
  • the substrate is not particularly limited, and a known inorganic material or organic material can be used.
  • a known inorganic material or organic material can be used.
  • 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.
  • the method for generating the polymerization start point is as follows: ⁇ ⁇ ⁇ ⁇ , plasma, corona, ultraviolet rays, and
  • 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.
  • 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.
  • 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.
  • polylactic acid or the like is used as the base material
  • 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.
  • EDC 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide
  • 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.
  • Collagen can also be used as the substrate.
  • 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.
  • 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. .
  • a tube-like material such as a tube
  • the protein having the above ligand is bound to the inner wall via a dial chain.
  • a film-like material such as a film
  • the protein having the ligand is bound to the membrane surface via a graft chain.
  • 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.
  • the protein may be bound to the entire base material, or may be bound to only a part thereof.
  • 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.
  • the ligand-presenting substrate preferably has a smooth surface formed by the ligand.
  • 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.
  • 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.
  • 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.
  • 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)
  • the separation device may include a collecting means for rolling the ligand surface to collect the sorted cells.
  • 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 uses the degree of.
  • 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”.
  • 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”.
  • binding force degree of binding
  • dissociating force force required to dissociate it
  • FIG. 1 is a schematic diagram for explaining the sorting method of this embodiment using this principle.
  • 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.
  • 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.
  • a dispersion liquid in which the cell group 1 is dispersed is supplied from upstream.
  • 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.
  • the moving speed is high, and the cells may contain cells that present the receptor.
  • 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.
  • 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.
  • the cell concentration in the dispersion 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.
  • each receptor presentation is made downstream in the movement direction.
  • cells having different receptor display densities can be sequentially sorted according to the receptor display density.
  • 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.
  • Non-Patent Document 3 the possibility of cell separation is suggested by controlling the cell rolling only by the flow of a single dispersion (share stress).
  • shared stress 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.
  • the method of Non-Patent Document 3 can realize cell rolling only within a very limited range.
  • 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.
  • the degree of shear stress Furthermore, cell separation can be achieved more efficiently by freely changing the inclination angle of the ligand-presenting substrate.
  • 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.
  • the ligand-presenting base material on the surface of the ligand is used via the graft chain.
  • 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.
  • 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.
  • the present inventors sorted cells having a desired receptor-presenting density.
  • a ligand-presenting substrate was prepared.
  • 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.
  • EDC 1-ethyl-3- (3 dimethylaminopropyl) force rubodiimide
  • 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.
  • FIG. 5 is a graph showing the results of quantification of the antibody amount of the anti-CD34 antibody.
  • a tube base material subjected to the following treatment is used as a control tube base material.
  • 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.
  • EDC 1-ethyl-3- (3 dimethylaminopropyl) carpositimide
  • 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 ⁇ g / m 2 and the ligand density is 1. 67 X 10 ” 9 mol / m 2 . It was.
  • ligand-presenting substrate anti-CD34 antibody-immobilized tube substrate
  • HL60 cells derived from acute leukemia which are CD34 negative cells
  • KG-la cells which are CD34 positive cells
  • 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.
  • 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.
  • 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.
  • Figs. 8 (a) and (b) 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)
  • Fig. 8 (b) shows the result collected in fraction no. 7 in Fig. 6 (a). It is the analysis result of the eluate.
  • 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.
  • 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 ⁇ fractions no. 11-15 in the fraction no. 1-10.
  • 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.
  • DMEM low glucose
  • dexamethasone 10_8 M dexamethasone 10_8 M
  • j8 glycephosphate phosphate 10 mM ascorbic acid 0.3 mM
  • Samples No. 2 and 3 show the results of inducing differentiation of cells with low and high CD34 expression by FACS method.
  • 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.
  • 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.
  • cells with a certain display density can be easily sorted and sequentially sorted according to the level of the display density.
  • 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.

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Abstract

L'invention concerne un procédé de fractionnement de cellules, lequel consiste, en utilisant un substrat présentant un ligand sur lequel est immobilisé via une chaîne de greffage un ligand capable de fixer temporairement un récepteur présent sur la surface d'une cellule qu'on souhaite fractionner, à transférer des cellules en rotations sur la surface du substrat présentant un ligand et à utiliser une différence de vitesse de transfert provoquée par le degré de fixation. Ceci permet de réaliser un procédé de fractionnement d'une cellule présentant un récepteur spécifique sur la surface de la cellule à partir d'un groupe de cellules comprenant la cellule, ledit procédé permettant de fractionner efficacement et de façon extrêmement précise la cellule ayant une densité des récepteurs comprise dans une plage déterminée et de fractionner en continu les cellules ayant des densités différentes à partir du groupe de cellules en fonction de la densité des récepteurs.
PCT/JP2006/322119 2005-11-08 2006-11-06 Procédé de fractionnement de cellules et substrat à utiliser pour le procédé WO2007055178A1 (fr)

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JP2010227016A (ja) * 2009-03-27 2010-10-14 Seiko Epson Corp 細胞分離装置および細胞分離方法
CN102337211A (zh) * 2011-08-25 2012-02-01 中国科学院深圳先进技术研究院 细胞培养装置
CN102686360A (zh) * 2009-11-12 2012-09-19 日立化成工业株式会社 Cmp研磨液、以及使用其的研磨方法和半导体基板的制造方法
JP2013215217A (ja) * 2013-07-31 2013-10-24 Seiko Epson Corp 細胞回収方法
JP2013230162A (ja) * 2013-07-24 2013-11-14 Seiko Epson Corp 細胞分離装置、医療機器
US9238791B2 (en) 2009-03-27 2016-01-19 Seiko Epson Corporation Cell separating apparatus and cell separating method

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009144928A1 (fr) * 2008-05-30 2009-12-03 国立大学法人 東京大学 Dispositif de séparation de cellules, système de séparation de cellules et procédé de séparation de cellules
JP2010227016A (ja) * 2009-03-27 2010-10-14 Seiko Epson Corp 細胞分離装置および細胞分離方法
US9238791B2 (en) 2009-03-27 2016-01-19 Seiko Epson Corporation Cell separating apparatus and cell separating method
CN102686360A (zh) * 2009-11-12 2012-09-19 日立化成工业株式会社 Cmp研磨液、以及使用其的研磨方法和半导体基板的制造方法
CN102337211A (zh) * 2011-08-25 2012-02-01 中国科学院深圳先进技术研究院 细胞培养装置
CN102337211B (zh) * 2011-08-25 2013-04-17 中国科学院深圳先进技术研究院 细胞培养装置
JP2013230162A (ja) * 2013-07-24 2013-11-14 Seiko Epson Corp 細胞分離装置、医療機器
JP2013215217A (ja) * 2013-07-31 2013-10-24 Seiko Epson Corp 細胞回収方法

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