Carriers for cell adsorption materials

Abstract

To provide a water-insoluble carrier that is optimal as a substrate of a cell adsorbent, wherein the carrier can adsorb selectively a target cell, does not have adverse effects on cell viability or bioactivity, prevents cells from being trapped between gaps in the substrate to cause clogging, and can reduce nonspecific adsorption of nontarget cells.SOLUTION: When polymerizing a methacrylate polymer to prepare water-insoluble particles, their particle size and particle size distribution are controlled in a specific range, the polymer is optimally modified with a functional group for hydrophilicization, and an end of the polymer is fixed with a ligand for specifically recognizing a target animal cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a carrier for specifically adsorbing animal cells onto its surface. Specifically, the surface of a water-insoluble base material is modified to prevent non-specific adsorption of non-target cells, and a physiologically active substance that recognizes target cells is chemically bound to the surface of the base material, thereby enabling the carrier to exhibit selective adsorption of target cells. [Background technology]

[0002] Well-known carriers used for cell adsorption, separation, and purification are magnetic particles whose surface is coated with carboxydextran or cross-linked agarose, etc., on magnetizable materials such as γFe2O3 or Fe3O4. Functional groups such as amino, carboxyl, hydroxyl, and epoxy groups are introduced into these coating molecules, and physiologically active substances such as antibodies that recognize cells are bound via these functional groups, thereby exhibiting adsorption properties to surface antigens on target cells.

[0003] A so-called immunomagnetic separation method is known, in which a cell suspension containing target cells is mixed with antibody-bound magnetic particles, and the mixture is brought close to a magnetic field to separate cells that bind to the antibody from those that do not (Patent Document 1). In this case, if the target cells have the ability to bind to the antibody, this is defined as positive selection, and if the target cells do not have the ability to bind to the antibody, this is defined as negative selection.

[0004] The amount of antibody that can be introduced is proportional to the specific surface area of ​​the magnetic particles, and the smaller the particle diameter of the magnetic particles, the higher the antibody binding rate and the better the sensitivity and accuracy for target cells.However, since the magnetization per particle is weaker, there is a higher risk of missing particles during the magnetic field separation process.

[0005] Furthermore, magnetic particles have a high specific gravity, so they settle immediately when they come into contact with a cell suspension, making them unsuitable for mass processing. In the case of positive selection, it is difficult to dissociate the cells from the antibodies bound to them. Furthermore, the molecules that coat the particle surface have low mechanical strength, so some of them break off and fall off, remaining as foreign bodies in the separated cell population.

[0006] Furthermore, when separating antibody-bound cells from non-bound cells, drawbacks have been pointed out, such as the risk of bound cells being crushed between the magnetic particles and the container wall, the difficulty of applying a high-strength external magnetic field, and the effects of a high-strength magnetic field on cells (Patent Document 2).

[0007] In addition, cell culture carriers called microcarriers are known, which are used as scaffolds when culturing animal cells in bioreactors and the like.

[0008] Known cell culture carriers include a method using collagen gel (Non-Patent Document 1), cross-linked poly(meth)acrylic acid particles having cell adhesion factors (Patent Document 3), dextran beads having animal-derived collagen (Non-Patent Document 2), and polystyrene beads having a polypeptide having a minimal amino acid sequence that expresses a cell adhesion signal (Patent Document 4).

[0009] However, these cell culture carriers do not have the function of specifically binding specific cells, and are therefore essentially difficult to use for cell adsorption, separation, and purification.

[0010] Since cell surfaces are generally negatively charged, it has been shown that using a carrier whose surface is modified with, for example, DEAE (diethylaminoethyl) groups improves the adhesion of the cells to be cultured to the carrier and improves the culture efficiency (Non-Patent Document 3), but there is no selectivity for the type of adherent cells.

[0011] Furthermore, the dextran beads described above have another problem in that they contain animal-derived components and therefore may be contaminated with infectious agents such as viruses.

[0012] Meanwhile, in previous studies, the present inventors have previously proposed a cell adsorbent capable of binding to undifferentiated cells of human pluripotent stem cells (human iPS cells, human ES cells, etc.), which is made by immobilizing BC2LCN lectin, a protein with binding affinity to glycans containing fucose residues and derived from the N-terminal domain of BC2L-C lectin produced by Gram-negative bacteria (Burkholderia cenocepacia), as a ligand on a water-insoluble carrier (Patent Document 5).

[0013] BC2LCN lectin is a protein with binding affinity to glycans containing fucose residues, and is known to have high binding affinity to multiple types of glycans containing fucose residues, such as H-type 1 glycans (Fucα1-2Galβ1-3GlcNAc) and H-type 3 glycans (Fucα1-2Galβ1-3GalNAc), which are known as glycan markers on the surface of undifferentiated human iPS cells, as well as Lewis Y glycans (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and Lewis X glycans (Galβ1-4(Fucα1-3)GlcNAc) (Non-Patent Document 4). BC2LCN is also known to bind to undifferentiated human iPS cells and human ES cells, which highly express H-type 1 and H-type 3 glycans, but not to human somatic cells (Non-Patent Document 5). Furthermore, because BC2LCN has the ability to bind to the undifferentiated glycan markers, it is used, for example, to detect glycoconjugates containing undifferentiated glycan markers and to detect undifferentiated cells such as human iPS cells and human ES cells (Patent Documents 6 and 7). In addition, it is known that H-type 1 glycans are highly expressed in certain cancer cells as SSEA-5 (Non-Patent Document 6). [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Application Publication No. 3-155778 [Patent Document 2] Special Publication No. 2006-516890 [Patent Document 3] Japanese Patent Application Laid-Open No. 2007-275056 [Patent Document 4] Japanese Patent Application Laid-Open No. 2003-189848 [Patent Document 5] Japanese Patent Publication No. 2020-25535 [Patent Document 6] WO2013 / 065302 issue [Patent Document 7] WO2013 / 128914 [Patent Document 8] Japanese Patent Application Laid-Open No. 2011-206046 [Non-patent literature]

[0015] [Non-Patent Document 1] Toshio Nishiyama et al., Tissue Culture Research (1991), 9:87 [Non-patent document 2] Microcarrier cell culture principles & methods (Pharmacia Biotech Co., Ltd., October 10, 1996) pp. 27-31 [Non-patent document 3] Polymer, 1980, Vol. 29, February, 102 [Non-patent document 4] Sulak, O et al., Structure.2010, 18(1):59-72. [Non-patent document 5] Tateno, H et al., J BiolChem.2011, 286(23):20345-20353. [Non-patent document 6] Tang, C et al., Nat Biotechnol. 2011, 29(9):829-835. Summary of the Invention [Problem to be solved by the invention]

[0016] The object of the present invention is to provide a water-insoluble carrier that is optimal as a substrate for a cell adsorbent, capable of selectively adsorbing target cells without adversely affecting cell viability or physiological activity, preventing cells from becoming trapped in gaps in the substrate and causing clogging, and suppressing non-specific adsorption of non-target cells. [Means for solving the problem]

[0017] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that, when preparing water-insoluble particles by polymerizing a (meth)acrylic acid ester polymer, it is possible to obtain an adsorbent that can selectively adsorb target animal cells while suppressing non-specific adsorption of non-target cells by controlling the particle size and particle size distribution within a specific range, and further by optimally modifying the polymer with functional groups and immobilizing a ligand at the end of the polymer for specifically recognizing target animal cells. Based on these findings, the present inventors have completed the present invention.

[0018] That is, the present invention has the following gist. 1. A water-insoluble carrier for cell adsorption material, characterized in that the carrier is a polymer particle obtained by polymerizing a vinyl monomer, and when wetted with water, the proportion of particles with a diameter of less than 105 μm is 10% by weight or less of the total, the pore size of the polymer particles is 100 nm or less, and the polymer has hydroxyl groups and / or amino groups and / or epoxy groups as functional groups. 2. The water-insoluble carrier for a cell adsorbent according to item 1 above, characterized in that the water-insoluble carrier has a volume average particle diameter of 130 μm or more in a water-wet state. 3. A water-insoluble carrier for a cell adsorbent according to 1 or 2 above, characterized in that the particle size distribution width ε of the water-insoluble carrier in a water-wet state is less than 0.7. ε = (cumulative 90% particle size - cumulative 10% particle size) ÷ cumulative 50% particle size 4. The water-insoluble carrier for a cell adsorbent according to any one of the above items 1 to 3, wherein the polymer particles are a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer. 5. A water-insoluble carrier for a cell adsorbent according to any one of 1 to 5 above, characterized in that a hydrophilic polymer is covalently bonded via a functional group of the polymer, and the polymer particles are hydrophilically modified. 6. A water-insoluble carrier for a cell adsorbent according to item 5 above, characterized in that the hydrophilic polymer is a neutral polysaccharide having a number-average molecular weight of 10,000 or more and less than 1,000,000. 7. The water-insoluble carrier for a cell adsorbent according to item 5 above, characterized in that the hydrophilic polymer is polyethylene glycol polyglycidyl ether having a number average molecular weight of 100 or more. 8. A water-insoluble carrier for a cell adsorbent described in 1 to 7 above, characterized in that a maleimide group and / or a haloacetyl group is introduced into the polymer in order to immobilize a ligand that specifically recognizes target cells. 9. The water-insoluble carrier for a cell adsorbent according to any one of 1 to 8 above is provided with a ligand that specifically recognizes target cells and has binding affinity for a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc, A cell adsorbent on which a fucose-binding protein is immobilized, the fucose-binding protein having the amino acid sequence shown in SEQ ID NO: 1, and comprising any of the amino acid sequences (a) to (d) below: (a) an amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, where X is an integer of 110 or more and 155 or less; (b) an amino acid sequence comprising one or more deletions, substitutions, insertions, and / or additions of amino acid residues in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 110 or more and 155 or less; (c) an amino acid sequence having 90% or more homology to the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 110 or more and 155 or less; (d) An amino acid sequence comprising specific amino acid substitutions in the amino acid sequence of any one of (a) to (c), wherein the specific amino acid substitutions are one or more amino acid substitutions selected from the amino acid substitutions of (1) to (5) below: (1) substitution of an amino acid residue corresponding to the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glutamine; (2) Substitution of the amino acid residue corresponding to the 72nd cysteine ​​residue in the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than a cysteine ​​residue; (3) substitution of an amino acid residue corresponding to the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glutamine; (4) substitution of the amino acid residue corresponding to the glutamic acid residue at position 81 of the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glutamic acid; (5) Substitution of the amino acid residue corresponding to the 36th glycine residue in the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glycine. 10. The fucose-binding protein according to claim 1, wherein the amino acid substitutions described in (1) to (5) are the amino acid substitutions described in the following (6) to (10), respectively: (6) Substitution of the amino acid residue corresponding to the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue or a methionine residue; (7) Substitution of the amino acid residue corresponding to the 72nd cysteine ​​residue in the amino acid sequence shown in SEQ ID NO: 1 with a glycine residue or an alanine residue; (8) substitution of the amino acid residue corresponding to the glutamine residue at position 65 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue; (9) Substitution of the amino acid residue corresponding to the glutamic acid residue at position 81 of the amino acid sequence shown in SEQ ID NO: 1 with a cysteine ​​residue, glutamine residue, histidine residue, methionine residue, valine residue, lysine residue, serine residue, isoleucine residue, tyrosine residue, glycine residue, proline residue, leucine residue, or asparagine residue; (10) Substitution of the amino acid residue corresponding to the 36th glycine residue in the amino acid sequence shown in SEQ ID NO: 1 with a cysteine ​​residue.

[0019] The present invention will be described in detail below.

[0020] The water-insoluble carriers for the cell adsorbent of the present invention are polymer particles obtained by polymerizing vinyl monomers. The particle size and particle size distribution in a water-wet state are important indicators. If three spherical carriers of the same diameter were packed closely together, the diameter of the spherical particle that could pass through the narrowest gap would be calculated to be 15.5% of the diameter of the carrier. If smaller-diameter carriers were mixed in, the gaps between the carriers would be correspondingly narrower. Typical animal cells are said to have a diameter of 10 μm to 30 μm when suspended as single cells. If the diameter of non-target cells that do not adsorb to the cell adsorbent is assumed to be 20 μm, the diameter of the carrier must be greater than 129 μm in order for these cells to smoothly flow through the gaps in the cell adsorbent in a spherical form. Therefore, in the present invention, in consideration of cell clogging between the carriers, the proportion (volume percentage) of particles with a diameter less than 105 μm is preferably 10% or less of the total carrier. This is more preferably 5% or less, and most preferably 3% or less.

[0021] For the same reason, the average particle size of the polymer particles is preferably 130 μm or more in terms of volume average particle size in a water-wet state. However, since the larger the particle size of the carrier, the fewer the number of carriers per volume, and the smaller the area for adsorption of target cells, the volume average particle size is preferably 130 μm to 1,000 μm, more preferably 140 μm to 800 μm, and most preferably 150 μm to 600 μm.

[0022] Furthermore, the more uniform the particle size, the more uniform the gaps between the carriers, improving the outflow of non-target cells. Therefore, the value of ε, an index of particle size distribution width, is preferably less than 0.7, particularly preferably less than 0.6, and most preferably less than 0.5. Here, ε is defined as {(cumulative 90% particle size - cumulative 10% particle size) ÷ cumulative 50% particle size}. If the carrier is a dispersion of completely single particle size, ε = 0. In other words, the closer ε is to zero, the narrower the particle size distribution width of the entire carrier. Therefore, the smaller the ε of the water-insoluble carrier of the present invention, the better. Note that the "cumulative 10% particle size," "cumulative 50% particle size," and "cumulative 90% particle size" herein refer to the particle sizes (μm) at which the cumulative curve is 10%, 50%, and 90%, respectively, when the particle size distribution of a single powder aggregate is determined and the total volume of the powder aggregate is taken as 100%. In particular, the cumulative 50% particle size is known as the cumulative median size and is generally used as one of the parameters for evaluating particle size distribution.

[0023] Commonly known particle size measurement methods include microscopy, sieving, digital Coulter (pore electrical resistance), laser diffraction / scattering, centrifugal sedimentation, flow-based image analysis, and dynamic light scattering. Each method has its own advantages and disadvantages depending on the type, refractive index, specific gravity, and particle size of the particles being measured. Laser diffraction / scattering is the most preferred method for measuring the particle size of the water-insoluble carrier for the cell adsorbent of the present invention. Unless otherwise noted, the particle size measurement results used in the present invention are measurements taken when the carrier is dispersed in pure water using a Microtrac Bell MT3200II particle size distribution analyzer (laser diffraction / scattering method), with the vertical axis representing the volume, i.e., the mass ratio, calculated. It is known that calculation results for particle size measuring instruments from different manufacturers vary depending on the control software; in this invention, Microtrac Version 11.1 is used, and the values ​​obtained by inputting the following measurement parameters (calculation mode: MT3000, particle transmittance: transmittance, particle refractive index: 1.49, particle shape: spherical, solvent (pure water) refractive index: 1.333, distribution display: volume) are used.

[0024] The water-insoluble carrier of the present invention is a polymer particle obtained by polymerizing a vinyl monomer, specifically a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer. A monofunctional vinyl monomer is a monomer having one reactive vinyl group in the molecule, while a polyfunctional vinyl monomer has multiple reactive vinyl groups. When these react in the presence of a catalyst, a linear polymer is formed when the monofunctional vinyl monomer is continuous, and when a polyfunctional vinyl monomer is mixed therewith, a three-dimensional crosslinked structure is formed, improving the polymer's properties such as heat resistance and solvent resistance.

[0025] The polymer particles themselves can be produced by, for example, a method of suspension polymerization of a mixture of a monofunctional vinyl monomer (such as glycidyl monovinyl ester or glycidyl monovinyl ether) and a polyfunctional vinyl monomer (such as alkylene glycol divinyl ester), as disclosed in Japanese Patent Publication No. 58-58026 and Japanese Patent Application Laid-Open No. 53-90991, or a method of seed polymerization of a combination of the above monomers, as described in Japanese Patent Application Laid-Open No. 2001-2716. Other monofunctional vinyl monomers include glycidyl esters of monovinylcarboxylic acids having 3 to 12 carbon atoms and glycidyl ethers of vinyl alcohols having 3 to 12 carbon atoms. Among these, those with fewer carbon atoms are particularly preferred, and examples include glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, vinylbenzyl glycidyl ether, and the like.

[0026] As the polyfunctional vinyl monomer, alkylene glycols having 2 to 3 carbon atoms or esters of polyalkylene glycols thereof with acrylic acid or methacrylic acid are preferably used. Examples include alkylene glycol divinyl esters such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, and glycerin polymethacrylate, polyalkylene glycol divinyl esters such as polyethylene glycol dimethacrylate and polypropylene glycol dimethacrylate, and divinylbenzene. Among these, a combination of a (meth)acrylic acid ester and a polyalkylene glycol di(meth)acrylic acid ester is preferred. In addition to these monomers, lower vinyl esters such as methyl methacrylate and methyl acrylate may be used as comonomers in order to adjust the hydrophilicity of the particles.

[0027] The preferred ratio of monofunctional vinyl monomer to polyfunctional vinyl monomer is 60-98 mol% of the former and 2-40 mol% of the latter. More preferably, it is 70-97 mol% of the former and 3-30 mol% of the latter. The higher the ratio of polyfunctional vinyl monomer, the higher the degree of crosslinking and the harder the polymer. Therefore, the network structure becomes denser, improving heat resistance, organic solvent resistance, acid / alkali resistance, and other properties. On the other hand, although the degree of crosslinking increases, the polymer also becomes brittle, making the particles more susceptible to breakage during polymerization or post-processing. The presence of these fragments as foreign matter can disrupt the uniformity of the particle size distribution and, when used as a water-insoluble carrier for a cell adsorbent, can lead to the aforementioned clogging of the non-target cells. When heat resistance and organic solvent resistance are not as important, as in the case of water-insoluble carriers for cell adsorbents, a higher ratio of monofunctional vinyl monomer to polyfunctional vinyl monomer is preferred. In other words, the most preferred ratio is 90-96 mol% of the former and 4-10 mol% of the latter.

[0028] When synthesizing the water-insoluble polymer particles of the cell adsorbent using aqueous suspension polymerization, seed polymerization, or other so-called oil-in-water polymerization methods, known dispersion stabilizers for emulsion polymerization or suspension polymerization can be used in combination. Among these, polyvinyl alcohol (hereinafter abbreviated as PVA), especially partially saponified PVA, is preferred in terms of particle shape stability and polymerization reaction stability. The average degree of polymerization (measured according to JIS K6726) of PVA used as a dispersion stabilizer is typically 50 to 4,000, with those with a degree of polymerization of 100 to 3,800 and even 200 to 3,600 being preferred. If the average degree of polymerization is too low, protective colloidal properties cannot be obtained. Conversely, if the average degree of polymerization is too high, the resulting emulsion becomes too viscous, making it difficult to stir during polymerization and resulting in polymerization difficulties. The saponification degree of the PVA resin (measured in accordance with JIS K6726) is preferably 70 to 100 mol%, particularly 75 to 99 mol%, and even more preferably 80 to 95 mol%. 85 to 93 mol% is most preferred. If the saponification degree is too low, insoluble matter in the PVA resin may precipitate during polymerization due to the cloud point phenomenon, inhibiting the polymerization. Conversely, if the saponification degree is too high, the emulsifying power may be insufficient, making it difficult to obtain a stable aqueous emulsion.

[0029] When polymer particles, which serve as water-insoluble carriers for cell adsorbents, are synthesized using aqueous suspension polymerization, seed polymerization, or other so-called oil-in-water polymerization methods, an organic solvent that is inert to the polymerization reaction and insoluble or poorly soluble in water but capable of dissolving the raw monomers can be mixed into the monomer droplets. Examples of such organic solvents include cyclohexanone, monochlorobenzene, benzene, toluene, n-propyl acetate, n-butyl acetate, n-octane, and 1,2-dichloroethane. The use of these organic solvents in combination allows for the production of polymer particles with pores, and the pore size and pore volume can be adjusted by adjusting the type, amount, and combination of the organic solvent with the monomer. The amount of organic solvent used is preferably 90% by volume or less, more preferably 85% by volume or less, and most preferably 80% by volume or less, based on the total amount of the monomer components and organic solvent.

[0030] In the case of general liquid chromatography packing materials, the presence of pores is necessary for the formation of a separation field, but in the case of the cell adsorbent of the present invention, it is difficult to create pores in the polymer particles large enough for cells to enter, and the presence of pores may actually induce nonspecific adsorption of non-target cells to the cell adsorbent.Therefore, the pore diameter of the polymer particles is preferably 100 nm or less, more preferably 50 nm or less, and most preferably 10 nm or less.

[0031] When a glycidyl ether or glycidyl ester monomer is used as the vinyl monomer, if the polymerization is carried out without ring-opening the epoxy group, the polymer particles will contain epoxy groups. By ring-opening these epoxy groups to convert them into hydroxyl groups after polymer particle formation, it is possible to increase the hydrophilicity of the polymer particles. A commonly known modification method is to heat the epoxy group with water under acidic or basic conditions, converting it into a 1,2-dihydroxyethyl group (i.e., two moles of hydroxyl groups are generated per mole of epoxy resin). Epoxy groups can be introduced into the polymer particles by reacting these hydroxyl groups under basic conditions with compounds containing multiple epoxy groups in the molecule, such as halohydrins containing one epoxy group in the molecule, such as epichlorohydrin or epibromohydrin; diglycidyl ethers such as ethylene glycol diglycidyl ether, glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, diethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, and resorcinol diglycidyl ether; triglycidyl ethers such as glycerol triglycidyl ether, erythritol triglycidyl ether, and diglycerol triglycidyl ether; and tetraglycidyl ethers such as erythritol tetraglycidyl ether and pentaerythritol tetraglycidyl ether. In the present invention, when the polymer particles, which are the water-insoluble carrier of the cell adsorbent, are dispersed in pure water and allowed to settle, it is preferable that 100 mmol or more of epoxy groups are bound to the polymer particles, and it is even more preferable that 200 mmol or more of epoxy groups are bound to the polymer particles per 1 L of volume.

[0032] In the present invention, it is preferable that the hydrophilic polymer is covalently immobilized to the water-insoluble carrier polymer particle via this epoxy group. Modification with a hydrophilic polymer significantly suppresses nonspecific cell adsorption, making the cell-selective substrate more suitable for cell adsorption. Examples of hydrophilic polymers that modify the surface of polymer particles include neutral polysaccharides such as agarose, cellulose, dextran, pullulan, and starch, and synthetic polymers with hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan, and starch are preferred, with dextran and pullulan being more preferred, due to their high hydrophilicity and ease of immobilization to polymer particles via covalent bonds. While there are no particular limitations on the molecular weight of dextran or pullulan, a number-average molecular weight of 10,000 to 1,000,000 is preferred, as this allows for sufficient hydrophilic modification of the polymer particles. If the molecular weight is too small, the hydrophilization effect will be reduced, while if the molecular weight is too large, the liquid viscosity will increase during solution formation, making handling difficult and potentially leading to problems such as an uneven reaction. In the present invention, when the polymer particles, which are the water-insoluble carrier of the cell adsorbent, are dispersed and precipitated in pure water, it is preferred that 200 mg or more of hydrophilic polymer be bound to 1 L of the volume, and more preferably 400 mg or more of hydrophilic polymer be bound to 1 L of the volume.

[0033] Further epoxy groups can be introduced into the polymer particles by further reacting the hydroxyl groups of the covalently immobilized hydrophilic polymer with the aforementioned compound containing multiple epoxy groups in the molecule as a linker under basic conditions. Repeating this reaction can also form so-called multifunctional hydrophilic dendrimers. In the present invention, it is preferable to immobilize high-molecular-weight dextran on the polymer particles and then add polyethylene glycol diglycidyl ether as a linker, thereby forming tentacle-like hydrophilic linkers extending from the hydrophilized polymer particle surface. The number-average molecular weight of the polyethylene glycol portion of the polyethylene glycol diglycidyl ether molecule is preferably 90 to 5,000, more preferably 100 to 3,000, and most preferably 105 to 2,000. A copolymer in which the polyethylene glycol portion also contains a structure formed by ring-opening polymerization of propylene oxide, tetrahydrofuran, or the like is also acceptable. In the present invention, when a compound containing multiple epoxy groups is reacted with the hydroxyl groups of a hydrophilic polymer immobilized on a water-insoluble carrier of a cell adsorbent under basic conditions, it is preferable to adjust the base concentration and reaction temperature so that an appropriate amount of epoxy groups remain after the reaction without ring-opening. More specifically, when the polymer particles that are the water-insoluble carrier of the cell adsorbent are dispersed in pure water and allowed to settle, it is preferable that 10 mmol or more, and even more preferably 30 mmol or more, of epoxy groups remain per 1 L of volume.

[0034] Furthermore, amino groups can be introduced into the water-insoluble polymer particles by reacting the aforementioned epoxy groups with a compound having at least two amino groups per molecule, such as ethylenediamine, diethylenetriamine, or tris(2-aminoethyl)amine. In the present invention, it is preferable to use an amino group-containing compound in the reaction so that the ratio of amino groups per 1 mole of epoxy groups is 3 times or more, and 5 times or more is even more preferable. If the amino group ratio is less than 3 times, additional epoxy groups will be bonded to the amino group-containing compound already bonded to the epoxy groups on the polymer particles, thereby failing to achieve the original purpose of introducing amino groups. On the other hand, if the proportion of amino group-containing compound is too high, it will be time-consuming to wash off the remaining unreacted amino group-containing compound, so it is desirable to keep the ratio at most 30 times. In the present invention, it is desirable that at least 60% of the moles of epoxy groups contained in the polymer particles before the amino group reaction are bonded to amino groups, and more desirable that at least 80% of the moles of epoxy groups are bonded to amino groups.

[0035] An example of a method for introducing maleimide groups into water-insoluble polymer particles is to react polymer particles having hydroxyl and / or amino groups with maleimide-containing carboxylic acids, such as 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, and 4-(N-maleimidomethyl)cyclohexanecarboxylic acid, in the presence of a condensing agent such as EDC. Another example is to react the aforementioned maleimide-containing carboxylic acids with N-hydroxysuccinimide esters or N-hydroxysulfosuccinimide esters. In the present invention, it is preferable to react the aforementioned polymer particles having primary amino groups with an NHS ester crosslinker, such as BMPS (N-β-maleimidopropionyloxysuccinimide ester), under weakly basic conditions (pH 7.2 to 8.5). In addition to BMPS, NHS ester crosslinkers with different spacer atom lengths and NHS ester crosslinkers with polyethylene glycol chains inserted can also be used. The N-hydroxysuccinimide liberated by the reaction of the amino group with the NHS ester can be easily removed by dialysis or desalting. Specifically, it can be removed by washing the polymer particles after the reaction with pure water and filtering them. The amount of maleimide groups introduced into the water-insoluble polymer particles in the present invention is preferably 5 mmol or more, and more preferably 10 mmol or more, per 1 L of volume when the polymer particles are dispersed and precipitated in pure water.

[0036] Examples of methods for introducing formyl groups into polymer particles as other functional groups include reacting the hydroxyl groups of the polymer particles with bifunctional aldehydes such as glutaraldehyde, and reacting the polymer particles with an oxidizing agent such as sodium periodate. Another example is reacting polymer particles into which epoxy groups have been introduced by the above-mentioned method with compounds such as D-glucamine, N-methyl-D-glucamine, and α-thioglycerol to introduce adjacent hydroxyl groups, and then reacting the resulting polymer particles with an oxidizing agent such as sodium periodate.

[0037] In addition, examples of methods for introducing carboxyl groups include reacting the hydroxyl groups of polymer particles with haloacetic acids such as monochloroacetic acid and monobromoacetic acid under basic conditions, and reacting polymer particles into which epoxy groups have been introduced by the above-mentioned method with amino acids such as glycine, alanine, aspartic acid, and glutamic acid, amino group-containing carboxylic acids such as β-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid, and sulfur-containing carboxylic acids such as thioglycolic acid and thiomalic acid under basic conditions.Further examples include a method in which the carboxyl groups introduced into the polymer particles are converted to N-hydroxysuccinimide esters, which are active ester groups, by reacting them with N-hydroxysuccinimide in the presence of a condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDC).

[0038] Other methods for introducing haloacetyl groups into polymer particles include, for example, reacting polymer particles having hydroxyl groups or polymer particles into which amino groups have been introduced by the above-mentioned method with acid halides such as chloroacetic acid chloride, bromoacetic acid chloride, and bromoacetic acid bromide, or reacting halogenated acetic acids such as chloroacetic acid, bromoacetic acid, and iodoacetic acid in the presence of a condensing agent such as EDC.Furthermore, a method for reacting N-hydroxysuccinimide esters or N-hydroxysulfosuccinimide esters of the halogenated acetic acids described above can be mentioned.

[0039] The polymer particles, which are the water-insoluble carriers of the cell adsorbent of the present invention, can be immobilized with a ligand that specifically recognizes target cells. Proteins such as antibodies and lectins are used as ligands. Examples of ligand immobilization methods include reacting a formyl group or active ester group introduced into the insoluble carrier with an amino group on the protein, or reacting a maleimide group or haloacetyl group introduced into the insoluble carrier with a mercapto group on the protein. Furthermore, a more preferred immobilization method, which can be performed at a near-neutral pH and can suppress protein denaturation, is reacting a maleimide group or haloacetyl group introduced into the insoluble carrier with a mercapto group on the protein. Furthermore, a more preferred immobilization method, which provides high stability of the functional group, is reacting a maleimide group introduced into the insoluble carrier with a mercapto group on the protein. In this case, the protein is preferably used in a buffer solution.

[0040] One example of target cells for the cell adsorbent of the present invention is human iPS cells (hereinafter abbreviated as hiPS cells), and an example of a protein that specifically recognizes hiPS cells is the fucose-binding protein described in JP 2020-25535 A (Patent Document 5). As mentioned above, BC2LCN, a fucose-binding protein derived from the N-terminal domain of BC2L-C lectin produced by the gram-negative bacterium Burkholderia cenocepacia, is a protein with binding affinity for glycans containing fucose residues. It is known to have high binding affinity for multiple types of glycans containing fucose residues, which are known as glycan markers on the surface of undifferentiated hiPS cells, and is known to bind to undifferentiated human iPS cells and human ES cells, but not to human somatic cells. In other words, in this invention, by reacting and immobilizing maleimide or haloacetyl groups introduced into a hydrophilized water-insoluble carrier with the mercapto group of BC2LCN, it is possible to obtain a cell adsorbent that specifically adsorbs undifferentiated hiPS cells (target cells) and suppresses non-specific adsorption of other cells. Furthermore, non-target cells can easily pass through the gaps in the water-insoluble carrier and be expelled from the system, allowing for easy separation of target cells from non-target cells.

[0041] There are no particular limitations on the buffer solution used to dissolve the fucose-binding protein when reacting it with the water-insoluble carrier of the cell adsorbent of the present invention, and examples include commercially available buffer solutions such as acetate buffer, phosphate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and D-PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Furthermore, to increase the efficiency of the immobilization reaction, inorganic salts such as sodium chloride or surfactants such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added to the buffer solution. The reaction temperature and pH when immobilizing a fucose-binding protein on a water-insoluble carrier may be appropriately set within the ranges of 0°C or higher and 50°C or lower, and pH or higher and 10 or lower, taking into consideration the reactivity of the active functional group and the stability of the fucose-binding protein of the present invention. From the viewpoint of preventing inactivation of the fucose-binding protein, the reaction temperature is preferably set within the ranges of 15°C or higher and 40°C or lower, and pH is preferably set within the ranges of pH 5 or higher and 9 or lower.

[0042] The amount of fucose-binding protein immobilized on a water-insoluble carrier can be appropriately determined taking into consideration the binding affinity between the target hiPS cells and the fucose-binding protein, and is preferably 10 mg to 50 g, more preferably 50 mg to 30 g, per liter of water-insoluble carrier. The amount of fucose-binding protein immobilized on a water-insoluble carrier can be adjusted by adjusting the amount of protein used in the immobilization reaction or the amount of active functional groups introduced into the water-insoluble carrier. The amount of fucose-binding protein immobilized on a water-insoluble carrier can be calculated by recovering the immobilization reaction solution and the washing solution after the reaction, determining the amount of unreacted fucose-binding protein, and then subtracting the amount of unreacted fucose-binding protein of the present invention from the amount of fucose-binding protein used in the immobilization reaction.

[0043] Next, the fucose-binding protein of the present invention will be described in detail. The fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention is a protein that has the ability to bind to fucose-containing sugar chains such as H-type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc), H-type 3 sugar chain (Fucα1-2Galβ1-3GalNAc), Lewis Y sugar chain (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), and Lewis b sugar chain (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), and the aforementioned recombinant BC2LCN lectin is also included in the fucose-binding proteins immobilized on the water-insoluble carrier of the cell adsorbent of the present invention.

[0044] Specifically, the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention is (a) a protein comprising the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence of the recombinant BC2LCN lectin shown in SEQ ID NO: 1 (which corresponds to the amino acid sequence from the second to the 156th amino acid in the amino acid sequence registered with GenPept under registration number WP_006490828), where X is an integer of 120 or greater, or (b) a protein consisting of an amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acids have been deleted, substituted, or added, and which has the ability to bind to H-type 1 glycans and / or H-type 3 glycans, where X is an integer of 120 or greater, expressed as a recombinant protein in an Escherichia coli transformant.

[0045] As long as the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention has the ability to bind to the fucose-containing glycans, particularly glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acids may be deleted, substituted, or added in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1. For example, 15 or fewer, preferably 10 or fewer, amino acids may be deleted, substituted, or added. Furthermore, X may be 120 to 155, or 125 to 155. As disclosed in JP 2020-25535 A, the productivity (expression level) of the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention can be improved by deleting multiple amino acid residues on the C-terminus of the amino acid sequence shown in SEQ ID NO: 1, compared to when these amino acid residues are not deleted.

[0046] Furthermore, the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention may contain one or more of the following to improve heat stability: (i) substitution of the glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue; (ii) substitution of the cysteine ​​residue at position 72 in the amino acid sequence shown in SEQ ID NO: 1 with one amino acid residue selected from glycine and / or alanine; or (iii) substitution of the glutamine residue at position 65 in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue. As disclosed in JP 2020-25535 A, the heat stability of the fucose-binding protein can be improved by making the amino acid substitutions described in (i) to (iii) above. While the amino acid substitutions described in (i) to (iii) above, either singly or in combination, are effective in improving heat stability, combining multiple amino acid substitutions described in (i) to (iii) above is preferable in terms of further improving heat stability.

[0047] Furthermore, as long as the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention has the ability to bind to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, one or more amino acid residues may be deleted, substituted, or inserted in a region other than the positions substituted by the substitutions (1) to (3) in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown in SEQ ID NO: 1; for example, 15 or fewer, preferably 10 or fewer amino acid residues may be deleted, substituted, or inserted.

[0048] The fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention may have an additional amino acid sequence at its N-terminus and / or C-terminus that is useful for detecting the fucose-binding protein, as long as it has the ability to bind to a glycan containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. Examples of the additional amino acid sequence include an oligopeptide containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose-binding protein, cellulose-binding domain, myc tag, FLAG tag, etc. Among these additional amino acid sequences, an oligopeptide containing a polyhistidine sequence or GST is preferred, and an oligopeptide containing a polyhistidine sequence is more preferred, because they have high productivity when produced using Escherichia coli and allow fucose-binding proteins to be easily detected using fluorescently labeled anti-polyhistidine antibodies or anti-GST antibodies. There are no particular restrictions on the number of histidine repeats in an oligopeptide containing a polyhistidine sequence, but if the histidine repeats are short, detection with an anti-polyhistidine antibody becomes difficult, and if they are long, the binding ability of the fucose-binding protein to the sugar chain may be impaired. Therefore, the length of the histidine repeat sequence in an oligopeptide containing a polyhistidine sequence is preferably a repeat sequence consisting of 5 to 15 histidines, and more preferably a repeat sequence consisting of 5 to 10. The position at which the oligopeptide containing a polyhistidine sequence is added to a fucose-binding protein is not particularly limited, and it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus, but from the viewpoint of efficient detection with an anti-polyhistidine antibody, it is preferable that the oligopeptide containing a polyhistidine sequence be added to the N-terminus of the fucose-binding protein.

[0049] Furthermore, the fucose-binding protein immobilized on the water-insoluble carrier of the cell adsorbent of the present invention may have an additional amino acid sequence (hereinafter referred to as a carrier immobilization tag) at its N-terminus and / or C-terminus, which is composed of an oligopeptide containing a cysteine ​​or lysine residue, useful for immobilizing the fucose-binding protein on a water-insoluble carrier. By immobilizing the fucose-binding protein on a carrier, it is possible to prepare an undifferentiated cell adsorbent for removing undifferentiated cells, such as human iPS cells, as described in JP 2020-25535 A. There are no particular limitations on the length of the carrier immobilization tag, as long as the fucose-binding protein has the ability to bind to glycans containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. As a tag for immobilization on a support, an oligopeptide consisting of 2 to 10 amino acid residues containing one or more cysteine ​​residues is preferred, as it allows for highly selective and efficient immobilization on a water-insoluble support. Specific examples include an oligopeptide consisting of the three amino acid residues "Gly-Gly-Cys," an oligopeptide consisting of the five amino acid residues "Ala-Ser-Gly-Gly-Cys," and an oligopeptide consisting of the seven amino acid residues "Gly-Gly-Gly-Ser-Gly-Gly-Cys." There are no particular limitations on the position at which the oligopeptide containing one or more cysteines is added to the fucose-binding protein, and it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus. However, it is preferable that the oligopeptide containing one or more cysteine ​​residues is added to the C-terminus of the fucose-binding protein, because this allows for efficient immobilization of the fucose-binding protein to the carrier and, further, because it is away from the active center of the fucose-binding protein and therefore is less likely to inhibit the binding activity.

[0050] Specific examples of fucose-binding proteins in the cell separation method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (the amino acid sequence from position 1 to position 127 of the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 (the amino acid sequence in which the 72nd cysteine ​​residue of SEQ ID NO: 2 is substituted with a glycine residue), SEQ ID NO: 4 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), SEQ ID NO: 5 (the amino acid sequence in which the 39th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue, the 65th glutamine residue of SEQ ID NO: 2 is substituted with a leucine residue and the 72nd cysteine ​​residue is substituted with a glycine residue), SEQ ID NO: 6 (the amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus ), SEQ ID NO: 7 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 8 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), SEQ ID NO: 9 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 4 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus), and SEQ ID NO: 10 (an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 5 and an oligopeptide containing a cysteine ​​residue is added to the C-terminus).

[0051] A signal peptide may be added to the N-terminus of the fucose-binding protein in the cell separation method of the present invention to promote efficient expression in the host. When the host is Escherichia coli, examples of the signal peptide include signal peptides that cause protein secretion into the periplasm, such as PelB, DsbA, MalE, and TorT. DNA encoding the fucose-binding protein in the cell separation method of the present invention can be prepared by known methods. Examples of methods for preparing the DNA include a method in which the amino acid sequence of the fucose-binding protein in the cell separation method of the present invention is converted into a nucleotide sequence and then artificially synthesized DNA containing the nucleotide sequence; a method in which DNA encoding the fucose-binding protein in the cell separation method of the present invention is directly and artificially prepared; and a method in which DNA is prepared from genomic DNA of Burkholderia cenocepacia using a DNA amplification method such as PCR. In this preparation method, when designing the base sequence, it is preferable to consider the codon usage frequency in the E. coli to be transformed. For example, AGA, AGG, CGG, or CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are all rarely used codons (rare codons), so it is preferable to select and convert codons other than these codons. Analysis of codon usage frequency can also be done using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website, http: / / www.kazusa.or.jp / codon / , accessed May 7, 2020).

[0052] To transform Escherichia coli with DNA encoding a fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. However, it is preferable to use an expression vector prepared by inserting the DNA into an appropriate position in a vector based on a bacteriophage, cosmid, or plasmid, which is commonly used for transforming prokaryotic and eukaryotic cells, to achieve stable transformation. Here, "appropriate position" refers to a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. Furthermore, when inserting the DNA into a vector, it is preferably inserted into the vector in a state linked to functional DNA, such as a promoter required for expression. There are no particular limitations on the vector used as the expression vector, as long as it is stable and replicable in the host. Examples of such vectors include pET vectors, pUC vectors, pTrc vectors, pCDF vectors, and pBBR vectors. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter, as well as the λPL promoter and λPR promoter of λ phage. Transformation of the host Escherichia coli with the expression vector may be carried out using a method commonly used by those skilled in the art. For example, when selecting Escherichia coli JM109 strain, Escherichia coli BL21(DE3) strain, Escherichia coli NiCo21(DE3) strain, Escherichia coli W3110 strain, or the like as the host, methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

[0053] The fucose-binding protein of the present invention can be produced by culturing the transformant and recovering and purifying the fucose-binding protein using the method disclosed in Japanese Patent Application Laid-Open No. 2020-25535. The purity and molecular weight of the fucose-binding protein of the present invention can be determined using methods known in the art, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography.

[0054] The binding affinity of the fucose-binding protein of the present invention to sugar chains can be evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, or the like. Surface plasmon resonance will be described as an example. Binding affinity evaluation by surface plasmon resonance can be performed, for example, using a Biacore T200 instrument (GE Healthcare) with a fucose-binding protein as the analyte and a glycan (a glycan containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc) as the solid phase. Sensor chips with immobilized glycans can be prepared using biotin-labeled glycans, either on a streptavidin-coated sensor chip (Sensor Chip SA, GE Healthcare) or on a dextran-coated sensor chip (Sensor Chip CM5, GE Healthcare) on which streptavidin has been immobilized. Binding affinity evaluation can also be performed using the kinetic analysis program provided with the instrument. [Effects of the Invention]

[0055] The present invention produces a water-insoluble carrier with particle size and particle size distribution controlled within a specific range, immobilizes a hydrophilic substance by covalent bonding to the functional groups of the carrier to make the carrier hydrophilic, and further immobilizes a ligand that specifically recognizes target cells, thereby obtaining a cell adsorbent that can specifically adsorb target cells, suppress non-specific adsorption of non-target cells, and further allows non-target cells to be smoothly discharged from the system through the gaps in the carrier.

[0056] Furthermore, by passing a cell suspension containing a mixture of target cells and non-target cells through a cylindrical container containing the cell adsorbent of the present invention, the target cells are adsorbed onto the cell adsorbent in the container, while the non-target cells are discharged from the outlet, thereby facilitating so-called cell separation. For example, a cell adsorbent having a fucose-binding protein immobilized on a water-insoluble carrier can adsorb and remove small amounts of hiPS cells present in a cell suspension during the process of culturing cells induced to differentiate from hiPS cells. Because it allows for easy processing of large amounts of cells, it is an extremely effective means, particularly for the mass purification of hiPS cells for regenerative medicine applications. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a diagram showing the state of particles in a closest packed state. [Figure 2] FIG. 10 is a diagram illustrating particle diameter measurement results. [Example]

[0058] The present invention will be described in more detail below with reference to Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these.

[0059] Example 1 Preparation of Water-Insoluble Carrier-1 3.8 g of PVA (polyvinyl alcohol, suspension stabilizer) with a saponification rate of 88% and a degree of polymerization of 3,500 and 2,520 g of pure water were charged into a reactor equipped with a stirrer, and the PVA was dissolved in the pure water by stirring well to prepare a uniform aqueous solution. The aqueous solution was adjusted to 60°C. Next, a mixed solution consisting of 123.5 g of glycidyl methacrylate, 25.2 g of ethylene glycol dimethacrylate, 138.6 g of chlorobenzene, and 0.8 g of azobisisobutyronitrile was prepared. While stirring the aqueous solution at 60°C, the mixed solution was added dropwise little by little until the entire amount was charged. The charging ratio was glycidyl methacrylate / ethylene glycol dimethacrylate = 87.2 mol% / 12.8 mol%. While continuing to stir, the suspension was polymerized at 60°C for 6 hours. The reactor was cooled to room temperature, and the granular gel obtained by filtration was washed several times with 60°C warm water and then further washed with 1,4-dioxane. After thorough washing with pure water, the mixture was subjected to ultrasonic classification using a 355 μm mesh sieve in a water bath, and the particles that fell through the sieve were collected. This was then subjected to ultrasonic classification in a water bath using a sieve with 180 μm openings, and 60 g of particles remaining on the sieve (weight converted into dry gel) were collected.

[0060] Particle size distribution measurement The particles were spherical, and the particle size distribution in a wet state was measured using a Microtrac MT3200II. The results were: 10% cumulative particle size = 160 μm, 50% cumulative particle size = 190 μm, 90% cumulative particle size = 210 μm, and ε = 0.26. Furthermore, the proportion of particles smaller than 105 μm was 0.5%.

[0061] Epoxy group measurement The resulting slurry was filtered through a glass filter, and 2.0 g of the wet gel was weighed into a conical beaker. 17 mL of 0.2 mol / L HCl (Nacalai) was added and reacted at room temperature for 3 hours. The remaining HCl was then titrated with 0.05 mol / L NaOH. The amount of epoxy groups immobilized on the support was 800 mmol / L wet support.

[0062] Measurement of exclusion limit molecular weight and pore volume The resulting slurry was packed into a stainless steel column with an inner diameter of 10.7 mm and a length of 150 mm to achieve close packing. The column was then installed in a Tosoh HLC-803D equipped with a Tosoh RI-8000 detector. Subsequently, dextran with a molecular weight of 40 million and pullulan of various molecular weights were used as standards, and standards of various molecular weights were injected at a flow rate of 0.5 mL / min. The exclusion limit molecular weight was determined from the elution volume. The pore volume was also determined from the elution volumes of dextran and ethylene glycol and the column volume. The exclusion limit molecular weight of the particles was 870,000, the pore volume was 40% by volume, and the pore diameter was 90 nm.

[0063] Example 2 Preparation of Water-Insoluble Carrier-2 3.4 g of PVA (suspension stabilizer) with a saponification rate of 88% and a degree of polymerization of 3,500 and 2,287 g of pure water were charged into a reactor equipped with a stirrer, and the PVA was dissolved in the pure water by thorough stirring to prepare a uniform aqueous solution. The temperature of the aqueous solution was adjusted to 60°C, and then a mixed solution consisting of 123.5 g of glycidyl methacrylate, 7.3 g of ethylene glycol dimethacrylate, 30.1 g of chlorobenzene, and 0.7 g of azobisisobutyronitrile was prepared, and while stirring the aqueous solution at 60°C, the mixed solution was added dropwise little by little until the entire amount was charged. The charging ratio was glycidyl methacrylate / ethylene glycol dimethacrylate = 95.9 mol% / 4.1 mol%. The suspension was polymerized at 60°C for 6 hours with continued stirring. The reactor was cooled to room temperature, and the resulting granular gel was collected by filtration and washed several times with 60°C warm water, followed by further washing with 1,4-dioxane. After thorough washing with pure water, 120 g of the collected wet gel was thoroughly mixed with 500 mL of 0.5N aqueous sulfuric acid solution. This was heated to 90°C in a water bath and reacted for 5 hours to hydrolyze the epoxy groups. After thorough washing with pure water, the mixture was ultrasonically classified in a water bath using a 355 μm mesh sieve, and the particles that remained on the sieve were collected. This was further ultrasonically classified in a water bath using a 180 μm mesh sieve, and 40 g of the particles remaining on the sieve (weight equivalent to dry gel) were collected. The particles obtained were spherical in shape, and the particle size distribution in a water-wet state was measured using a Microtrac MT3200II. The results were a cumulative 10% particle size of 150 μm, a cumulative 50% particle size of 180 μm, a cumulative 90% particle size of 220 μm, and ε of 0.39. Furthermore, the proportion of particles smaller than 105 μm was 1.5%. Epoxy group analysis using the same method as in Example 1 did not detect any epoxy groups. The particles also had an exclusion limit molecular weight of 100,000, a pore volume of 20% by volume, and a pore diameter of 10 nm.

[0064] Comparative Example 1 Preparation of Water-Insoluble Carrier-3 Particles were prepared in the same manner as in Example 2 up to the step immediately before the sieve classification step, and then ultrasonically classified in a water bath using a sieve with 500 μm openings, and 100 g of particles (weight in terms of dry gel) that fell through the sieve were collected. The particles were spherical, and the particle size distribution in a wet state was measured using a Microtrac MT3200II. The results were: 10% cumulative particle size = 100 μm, 50% cumulative particle size = 150 μm, 90% cumulative particle size = 210 μm, and ε = 0.73. Furthermore, the proportion of particles smaller than 105 μm was 13%.

[0065] No epoxy groups were detected when analyzed using the same method as in Example 1. The particles had an exclusion limit molecular weight of 100,000, a pore volume of 20% by volume, and a pore diameter of 10 nm.

[0066] Comparative Example 2 Preparation of Water-Insoluble Carrier-4 3.8 g of PVA (suspension stabilizer) with a saponification rate of 88% and a degree of polymerization of 3,500 and 2,520 g of pure water were charged into a reactor equipped with a stirrer and stirred thoroughly to dissolve the PVA in the pure water, preparing a uniform aqueous solution. The aqueous solution was adjusted to 60°C, and then a mixed solution consisting of 123.5 g of glycidyl methacrylate, 25.2 g of ethylene glycol dimethacrylate, 400 g of chlorobenzene, and 0.8 g of azobisisobutyronitrile was prepared. While stirring the 60°C aqueous solution, the mixed solution was added dropwise until the entire amount was charged. The charging ratio was glycidyl methacrylate / ethylene glycol dimethacrylate = 87.2 mol% / 12.8 mol%. The reaction and post-treatment were then carried out in the same manner as in Example 2, resulting in the recovery of 40 g of particles (weight equivalent to dry gel). The particles obtained were spherical, but irregularly shaped particles were also present. The particle size distribution in a wetted state was measured using a Microtrac MT3200II, resulting in a cumulative 10% particle size of 160 μm, a cumulative 50% particle size of 185 μm, a cumulative 90% particle size of 230 μm, and ε of 0.38. Furthermore, the proportion of particles smaller than 105 μm was 2.3%. Epoxy groups were analyzed using the same method as in Example 1, but no epoxy groups were detected. The particles had an exclusion limit molecular weight of 2,000,000, a pore volume of 85% by volume, and a pore diameter of 150 nm.

[0067] [Table 1]

[0068] Example 3 Preparation of cell adsorbent-1 Hydrophilization 100 g of the water-insoluble carrier-1 prepared in Example 1 was placed in a 500 mL glass flask equipped with a stirrer, and 150 g of a 30% aqueous solution of dextran (SIGMA Aldrich, Dextran 500) with a number average separation amount of 50,000 was added and stirring was initiated. 10.5 mL of 48% NaOH was slowly added dropwise, and stirring was continued at 30 ° C for 18 hours. The reaction slurry was diluted with 200 mL of pure water and filtered through a G3 glass filter. 250 mL of pure water was poured onto the wet gel on the filter, and the mixture was stirred and washed with a medicine spoon, then filtered again. This washing procedure was repeated five times, and the pH of the filtrate was confirmed to be less than 7.5. It was determined that no NaOH remained, and 105 g of wet gel (48 g of dry gel equivalent) was recovered. A portion of the wet gel was heated and dried to a liquid content of less than 1%, and 2.0 g was weighed into an Erlenmeyer flask. 20 mL of 2.0 mol / L HCl (Nacalai) was added and the mixture was allowed to react at 91°C for 3 hours to hydrolyze the dextran. The entire supernatant after the reaction was filtered and collected, neutralized to pH 7-8 with aqueous NaOH, and then diluted to 100 mL. Measurement of this sample using the phenol-sulfuric acid method revealed that the amount of dextran immobilized on the carrier was 800 mg / L wet carrier.

[0069] Linker introduction 100 g of the wet gel, hydrophilized by immobilizing dextran in the previous step, was collected in a 500 mL glass flask equipped with a stirrer, and 150 mL of a 10% (w / v) aqueous solution of polyethylene glycol diglycidyl ether (Epolite 200E, Kyoeisha Chemical Co., Ltd.) was added and stirring was initiated. 2.1 mL of 48% NaOH was slowly added dropwise, and stirring was continued at 50 °C for 8 hours. The resulting slurry was filtered through a G3 glass filter, and the wet gel on the filter was washed with 200 mL of purified water and stirred with a medicine spoon, then filtered again. This washing procedure was repeated five times, and the pH of the filtrate was confirmed to be below 7.5. It was determined that no NaOH remained, and 100 g of wet gel (46 g of dry gel equivalent) was collected. Analysis using the same method as described above for quantifying epoxy groups revealed that the amount of epoxy groups immobilized on the support was 50 mmol / L wet support.

[0070] Amino group introduction 100 g of the wet gel, which had been previously linked, was placed in a 500 mL glass flask equipped with a stirrer. 200 g of purified water and then 6.0 mL of anhydrous ethylenediamine were added, and the mixture was stirred at 40 °C for 15 hours. The resulting slurry was filtered through a G3 glass filter, and 300 mL of purified water was poured onto the wet gel on the filter. The wet gel was washed by stirring with a medicine spoon, and then filtered again. This washing procedure was repeated 10 times, and the pH of the filtrate was confirmed to be less than 7.5. 100 g of wet gel (45 g of dry gel equivalent) was recovered. Analysis by titration with 0.05 mol / L HCl indicated that the amount of amino groups immobilized on the support was 45 mmol / L wet support.

[0071] Maleimide group introduction 100 g of the wet gel, which had been amino-functionalized in the previous step, was collected in a 500 mL glass flask equipped with a stirrer. 200 mL of a DMSO solution of BMPS (Kishida Chemical, N-β-maleimidopropionyloxysuccinimide ester) prepared at 10 mg / mL was added, and the mixture was stirred at 35 °C for 4 hours. The resulting slurry was filtered through a G3 glass filter. 200 mL of DMSO was poured onto the wet gel on the filter, washed by stirring with a spoon, and then filtered again to remove excess BMPS. 300 mL of purified water was poured onto the wet gel, washed by stirring with a spoon, and then filtered again. This washing procedure was repeated five times, and 100 g of wet gel (45 g of dry gel equivalent) was collected. Analysis revealed that the amount of maleimide introduced was 25 mmol / L of the wet carrier.

[0072] Ligand (BC2LCN lectin) immobilization Using the method described in JP 2020-25535 A (Patent Document 5), Escherichia coli W3110 strain genetically modified to express the modified lectin described in SEQ ID NO: 4 was cultured, and the extract was purified using a column packed with a cation exchange chromatography resin (Toyopearl GigaCap CM-650M, manufactured by Tosoh). The purified lectin solution was then concentrated using an ultrafiltration membrane (Amicon Ultra-15, manufactured by Merck, molecular weight cutoff = 10,000) and buffer exchanged with D-PBS(-) (manufactured by Cell Science Institute). The resulting purified lectin solution was sterilized through a 0.2 μm filter, sealed, and stored in a refrigerator at 4 °C. The concentration of this purified lectin solution was 10.2 mg / mL (absorbance at 280 nm). Next, a buffer solution for the immobilization reaction was prepared. The buffer solution consisted of 0.20 M phosphate, 0.50 M NaCl, and 0.02 M EDTA, pH 7.4. The immobilization reaction solution was prepared by uniformly mixing 20.0 mL of purified lectin solution, 122.4 mL of immobilization reaction buffer, 9.6 mL of D-PBS(-), and 1.3 mL of 0.1 M TCEP. The mixture was then allowed to stand at 23°C for 4 hours. The lectin concentration in this immobilization reaction solution was 1.33 mg / mL. TCEP (Nacalai Tesque, tris(2-carboxyethyl)phosphine hydrochloride) is a reducing agent used to cleave the double bond (disulfide bond) of the cysteine ​​tag at the end of the lectin molecule. Reduction of the disulfide bond exposed the -SH group of the cysteine ​​tag, significantly improving reactivity with the maleimide group immobilized on the water-insoluble support.

[0073] 100 g of wet gel, which had been previously maleimide-modified, was placed in a 500 mL glass flask equipped with a stirrer. 131 mL of a buffer solution of BC2LCN lectin, previously prepared at a concentration of 1.33 mg / mL, was added and the mixture was stirred slowly at 35 °C for 15 hours. The resulting slurry was filtered through a G3 glass filter, and 200 mL of D-PBS(-) was poured onto the wet gel on the filter and gently stirred and washed with a medicine spoon. This washing procedure was repeated five times to recover 100 g of wet gel, which was dispersed in D-PBS(-) to form cell adsorbent-1 (the volume of the cell adsorbent in the slurry was 130 mL). The amount of BC2LCN lectin in the supernatant before and after the reaction was analyzed using Pierce Protein Assay (Thermo). Assuming that all of the BC2LCN lectin lost was consumed in the immobilization reaction, the amount of BC2LCN lectin immobilized on cell adsorbent-1 was estimated to be 300 mg / L of wet carrier.

[0074] Example 4 Preparation of cell adsorbent-2 Epoxidation 100 g (50 g dry gel equivalent) of the water-insoluble carrier-2 prepared in Example 2 was placed in a 500 mL glass flask equipped with a stirrer, and 48.5 g of pure water, 52.0 g of DMSO (dimethyl sulfoxide, Kishida Pure Chemical Industries, Ltd.), and 47.0 g of epichlorohydrin (Kishida Pure Chemical Industries, Ltd.) were added and slowly stirred. 102 mL of a 5.0 mol / L aqueous NaOH solution (Nacalai Tesque, Ltd.) was added dropwise, and stirring was continued at 30°C for 3 hours. The NaOH concentration in the reaction solution was approximately 2.2 mol / L. Analysis revealed that the amount of epoxy groups immobilized on the water-insoluble carrier was 500 mmol / L-wet carrier.

[0075] Hydrophilization 100 g of the wet gel into which epoxy groups had been introduced in the previous step was reacted with an aqueous solution of dextran (Dextran 500) with a number average separation amount of 50,000 in the same manner as in Example 3. The amount of dextran immobilized on the carrier was 900 mg / L-wet carrier.

[0076] Linker introduction 100 g of the wet gel reacted with dextran in the previous step was reacted with polyethylene glycol diglycidyl ether (Epolite 200E) in the same manner as in Example 3. The amount of epoxy groups immobilized on the carrier was 60 mmol / L-wet carrier.

[0077] Amino group introduction 100 g of the wet gel that had been reacted with the linker in the previous step was reacted with anhydrous ethylenediamine in the same manner as in Example 3. The amount of amino groups on the carrier was 58 mmol / L-wet carrier.

[0078] Maleimide group introduction 100 g of the wet gel into which amino groups had been introduced in the previous step was reacted with BMPS in the same manner as in Example 3. The amount of maleimide introduced into the carrier was 30 mmol / L-wet carrier.

[0079] Ligand (BC2LCN lectin) immobilization 45.3 mL of the purified lectin solution used in Example 3, 125.8 mL of the buffer solution for immobilization reaction, and 2.6 mL of 0.1 M TCEP were uniformly mixed to prepare an immobilization reaction solution, which was then allowed to stand for 4 hours at 23° C. The lectin concentration in this immobilization reaction solution was 2.66 mg / mL.

[0080] 100 g of the wet gel into which maleimide groups had been introduced in the previous step was added with 150 mL of a buffer solution of BC2LCN lectin, which had been prepared in advance at a concentration of 2.66 mg / mL, and the mixture was stirred slowly at 35°C for 15 hours. After filtering and washing in the same manner as in Example 3, 100 g of wet gel was recovered and dispersed in D-PBS(-) to obtain cell adsorbent 2. Analysis indicated that the amount of BC2LCN lectin immobilized on the carrier in cell adsorbent 2 was estimated to be 700 mg / L-wet carrier.

[0081] Comparative Example 3: Preparation of Cell Adsorbent-3 Epoxidation 100 g (50 g in terms of dry gel) of the water-insoluble carrier-3 prepared in Comparative Example 1 was reacted with epichlorohydrin in the same manner as in Example 4. The amount of epoxy groups immobilized on the carrier was 380 mmol / L-wet carrier.

[0082] Hydrophilization 100 g of the wet gel into which epoxy groups had been introduced in the previous step was reacted with an aqueous solution of dextran (Dextran 500) with a number average separation amount of 50,000 in the same manner as in Example 3. The amount of dextran immobilized on the carrier was 650 mg / L-wet carrier.

[0083] Linker introduction 100 g of the wet gel reacted with dextran in the previous step was reacted with polyethylene glycol diglycidyl ether (Epolite 200E) in the same manner as in Example 3. The amount of epoxy groups immobilized on the carrier was 55 mmol / L-wet carrier.

[0084] Amino group introduction 100 g of the wet gel that had been reacted with the linker in the previous step was reacted with anhydrous ethylenediamine in the same manner as in Example 3. The amount of amino groups on the carrier was 48 mmol / L-wet carrier.

[0085] Maleimide group introduction 100 g of the wet gel into which amino groups had been introduced in the previous step was reacted with BMPS in the same manner as in Example 3. The amount of maleimide introduced into the carrier was 25 mmol / L-wet carrier.

[0086] Ligand (BC2LCN lectin) immobilization 100 g of the wet gel into which maleimide groups had been introduced in the previous step was reacted with a buffer solution of BC2LCN lectin in the same manner as in Example 3, and the resulting wet gel was dispersed in D-PBS(-) to obtain cell adsorbent-3. Analysis indicated that the amount of BC2LCN lectin immobilized on the carrier in cell adsorbent-3 was estimated to be 240 mg / L-wet carrier.

[0087] Comparative Example 4: Preparation of Cell Adsorbent-4 Epoxidation 100 g (50 g in terms of dry gel) of the water-insoluble carrier-4 prepared in Comparative Example 2 was reacted with epichlorohydrin in the same manner as in Example 3. The amount of epoxy groups immobilized on the carrier was 600 mmol / L-wet carrier.

[0088] Hydrophilization 100 g of the wet gel into which epoxy groups had been introduced in the previous step was reacted with an aqueous solution of dextran (Dextran 500) with a number average separation amount of 50,000 in the same manner as in Example 3. The amount of dextran immobilized on the carrier was 830 mg / L-wet carrier.

[0089] Linker introduction 100 g of the wet gel reacted with dextran in the previous step was reacted with polyethylene glycol diglycidyl ether (Epolite 200E) in the same manner as in Example 3. The amount of epoxy groups immobilized on the carrier was 70 mmol / L-wet carrier.

[0090] Amino group introduction 100 g of the wet gel that had been reacted with the linker in the previous step was reacted with anhydrous ethylenediamine in the same manner as in Example 3. The amount of amino groups on the carrier was 65 mmol / L-wet carrier.

[0091] Maleimide group introduction 100 g of the wet gel into which amino groups had been introduced in the previous step was reacted with BMPS in the same manner as in Example 3. The amount of maleimide introduced into the carrier was 40 mmol / L-wet carrier.

[0092] Ligand (BC2LCN lectin) immobilization 100 g of the wet gel into which maleimide groups had been introduced in the previous step was reacted with a buffer solution of BC2LCN lectin in the same manner as in Example 3, and the resulting wet gel was dispersed in D-PBS(-) to obtain cell adsorbent-4. Analysis indicated that the amount of BC2LCN lectin immobilized on the carrier in cell adsorbent-4 was estimated to be 420 mg / L-wet carrier.

[0093] [Table 2]

[0094] Reference Example 1 Cell adsorption evaluation Reference Example 1 relates to the evaluation of cell adsorption and separation with Cell Adsorbent-1 using a mixed cell suspension of the 201B7 strain, an hiPS cell line that has H-type 1 glycans and / or H-type 3 glycans on the cell surface (purchased under license from iPS Academia Japan, Inc.; hereafter abbreviated as 201B7 cells), and K562 cells (JCRB0019; human chronic myeloid leukemia cells; obtained from the JCRB Cell Bank), which do not have the above glycans. (1) Preparation of a column packed with adsorbent A filter for preventing adsorbent leakage (a polypropylene resin washer (Hirosugi Seisakusho, PPW0610-10, 10 mm diameter, 6.4 mm inner diameter, 1.0 mm thickness) with a 30 μm mesh nylon filter (Merck, NY30, hydrophilic) welded to one side) was attached to the bottom of a syringe-shaped polypropylene container with an inner diameter of 10 mm and a height of 30 mm. The D-PBS(-) suspension of cell adsorbent-1 prepared in Example 3 was packed into the container so that the sedimentation volume was 0.5 mL. An additional filter for preventing adsorbent leakage, similar to that described above, was inserted on top of the container, forming a cell adsorbent-1-packed column. Furthermore, 10 mL of MACS buffer (Miltenyi Biotec) was passed through the cell adsorbent-1-packed column, and the column was equilibrated. (2) Cultivation of 201B7 cells and preparation of cell suspension 201B7 cells were cultured in adherent culture dishes (Corning) as follows. A solution of pre-prepared iMatrix-511 (Nippon) diluted to 3 μg / mL in D-PBS was added to the dish and left overnight at 4°C to coat the culture surface of the dish with iMatrix-511. After discarding the iMatrix-511 solution from the coated dish, the dish was washed with StemFit AK02N medium (Ajinomoto), an iPS cell culture medium. 201B7 cells thawed from a cryovial were suspended in the same medium supplemented with 10 μM lock inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) and seeded. After overnight culture, the StemFit AK02N medium containing Y-27632 was discarded and replaced with StemFit AK02N medium without Y-27632. When the appropriate cell density was reached, the cells were collected and passaged.

[0095] Cells were harvested from the dish as follows: D-PBS(-) was added to the dish to wash the cells, and then the D-PBS(-) was discarded. This process was repeated twice to wash the cells. After that, a detachment solution consisting of a 1:1 mixture of CTS TrypLE Select Enzyme (Thermo Fisher Scientific) and Versene Solution (Thermo Fisher Scientific) was added and the dish was left to stand at 37°C for 1 minute in a 5% CO2 atmosphere. After confirming that the cells were detaching in a round shape, the detachment solution was discarded, and StemFit AK02N medium containing 10 μM Y-27632 was added. The cells were detached with a cell scraper and collected in a 50 mL tube. The number of cells in the collected cells was counted using a hemocytometer, and the cells were seeded at a concentration of 104–105 / mL in StemFit AK02N medium containing Y-27632. Culture was continued in StemFit AK02N medium without Y-27632 until an appropriate cell density was reached.

[0096] Next, fluorescent staining of 201B7 cells using Cell Tracker Orange (Thermo Fisher Scientific) was performed as follows. First, the medium in the Petri dish was discarded, and D-PBS(-) was added to rinse the cells. The D-PBS(-) was then aspirated and discarded. Next, a solution of Cell Tracker Orange dissolved in serum-free RPMI 1640 medium at a final concentration of 20 μM was added, and the cells were cultured for 1 hour at 37°C under a 5% CO2 atmosphere. After discarding the fluorescent reagent solution, StemFit AK02N medium was added, and the cells were cultured for 1 hour at 37°C under a 5% CO2 atmosphere. After discarding the medium, StemFit AK02N medium was added, and the cells were cultured overnight at 37°C under a 5% CO2 atmosphere. Next, the cells were harvested and a cell suspension was prepared as follows. The cells were rinsed with D-PBS(-) and discarded twice. A detachment solution consisting of a 1:1 mixture of CTS TrypLE Select Enzyme (Thermo Fisher Scientific) and Versene Solution (Thermo Fisher Scientific) was added and incubated at 37°C for 1 minute under a 5% CO2 atmosphere. After confirming that the cells were detaching in a rounded shape, the detachment solution was discarded, StemFit AK02N medium was added, and the cells were detached using a cell scraper and collected in a 50 mL tube. The collected cells were centrifuged to settle, then suspended in MACS buffer, centrifuged again, and the supernatant discarded. After washing twice, the cells were suspended in MACS buffer and filtered through a cell strainer to prepare a cell suspension of 201B7 cells stained with Cell Tracker Orange. This 201B7 cell suspension was dispersed uniformly, and a portion of the liquid was taken and diluted 10-fold, and the cell density was calculated using a hemocytometer. (3) Cultivation of K562 cells and preparation of cell suspension K562 cells were cultured as follows: K562 cells, which are suspension cells, were seeded in RPMI 1640 medium supplemented with 10% FBS (Biological Industries) and antibiotic solution (penicillin-streptomycin solution, Fujifilm Wako Pure Chemical Industries) onto 6-cm or 10-cm diameter suspension culture dishes (Sumitomo Bakelite) and cultured at 37°C in a 5% CO2 atmosphere. K562 cells were stained with Cell Tracker Green (Thermo Fisher Scientific).

[0097] Next, cells were harvested and a cell suspension was prepared as follows. K562 cells were harvested from a culture dish and placed in a 50 mL tube. The harvested cells were centrifuged to precipitate and the supernatant discarded. MACS buffer was added, gently resuspended, and then centrifuged again, discarding the supernatant. This cell washing procedure was repeated twice, after which the cells were suspended in MACS buffer and filtered through a cell strainer to prepare a K562 cell suspension. The K562 cell suspension was uniformly dispersed, and a portion of the liquid was taken and diluted 10-fold. The cell density was calculated using a hemocytometer. (4) Preparation of cell suspension for evaluation The 201B7 cell suspension and the K562 cell suspension were uniformly dispersed and then separated to prepare cell suspensions for evaluation at a cell density of 1.0 x 10^7 cells / mL. (5) Evaluation of the cell suspension passing through a column packed with Cell Adsorbent-1 A column packed with Cell Adsorbent-1 was placed vertically, and 1.0 mL of the 201B7 and K562 cell suspensions prepared as described above was gently added. Next, 4 mL of MACS buffer was added from the top of the column, and the effluent from the tip of the syringe was collected in a separate container (hereafter, this cell solution will be referred to as effluent cell solution-1). Similarly, 4 mL of MACS buffer was added from the top of the column, and the effluent from the tip of the syringe was collected in a separate container (hereafter, this cell solution will be referred to as effluent cell solution-2). This procedure was repeated, and effluent cell solutions-3 and -4 were collected. The cell suspension for evaluation added to the column and the collected effluent cell solutions-1 to -4 were resuspended, and 2 mL of each was dispensed into a 5 mL polystyrene round tube (Corning, 40 μm mesh) with a cell strainer and cap. 50 μL of CountBright Absolute Counting Beads (Invitrogen) were added as internal standard beads for cell counting, and 50 μL of 7-AAD (Funakoshi) was added as a cell viability assay reagent. Cell counts were then measured using a BD FACSAria cell sorter (Becton Dickinson). The dot plots were then analyzed based on the type of orange or green fluorescence intensity and the ratio to the internal standard beads, and the dot populations were calculated to calculate the number of 201B7 and K562 cells. From these values, the efflux rate of each cell type in efflux cell solutions 1 to 4 was calculated as follows: efflux rate of each cell type = number of effluxed cells of each cell type / number of added cells of each cell type × 100 (%).

[0098] When cell suspensions were passed through a column packed with Cell Adsorbent-1, the efflux rate of 201B7 cells was less than 1% for all of Cell Effluents-1 to -4, confirming that they were well adsorbed to the Cell Adsorbent-1 column. Meanwhile, the efflux rates for K562 cells were Cell Effluent-1: 80%, Cell Effluent-2: 8%, Cell Effluent-3: 1%, and Cell Effluent-4: 0%, confirming that the majority of cells were expelled from the column in the early stages of efflux. Therefore, Cell Adsorbent-1 produced by the production method of the present invention not only possesses high selectivity and adsorption capacity for hiPS cells, but also demonstrates the ability to efficiently expel control cells.

[0099] Reference Example 2 Cell adsorption evaluation Reference Example 2 relates to the evaluation of cell adsorption and separation with Cell Adsorbent-2, using the same cell suspension as in Reference Example 1. (1) Preparation of a column packed with adsorbent A cell adsorbent column was prepared by filling the D-PBS(-) suspension of cell adsorbent-2 prepared in Example 4 into a polypropylene syringe-shaped container and a leak-proof filter having the same specifications as those in Reference Example 1. This was used as a cell adsorbent-2-packed column. Furthermore, 10 mL of MACS buffer (Miltenyi Biotec) was passed through the cell adsorbent-2-packed column to equilibrate the column. (2) Cultivation of 201B7 cells and preparation of cell suspension A suspension of 201B7 cells was prepared in the same manner as in Reference Example 1. (3) Cultivation of K562 cells and preparation of cell suspension A suspension of K562 cells was prepared in the same manner as in Reference Example 1. (4) Preparation of cell suspension for evaluation The 201B7 cell suspension and the K562 cell suspension were uniformly dispersed and then separated to prepare cell suspensions for evaluation at a cell density of 1.0 x 10^7 cells / mL. (5) Evaluation of the cell suspension flow through a column packed with Cell Adsorbent-2 As in Reference Example 1, a suspension of 201B7 cells and K562 cells prepared by the above method was uniformly dispersed in a vertically placed cell adsorbent-2 packed column, and 1.0 mL of the suspension was gently added. Next, 4 mL of MACS buffer was added to the top of the column four times, and the effluent from the tip of the syringe after each addition was collected in a separate container. These are referred to as effluent cell solutions-1 to -4. The cell suspension for evaluation added to the column and the recovered effluent cell solutions-1 to -4 were resuspended, and the cell counts were measured using a cell sorter BD FACSAria (Becton Dickinson) in the same manner as in Reference Example 1. From the numbers of 201B7 cells and K562 cells in each of the obtained effluent cell solutions, the efflux rate of each cell in effluent cell solutions-1 to -4 was calculated as follows: "efflux rate of each cell = number of effluent cells of each cell type / number of added cells of each cell type × 100 (%)."

[0100] When cell suspensions were passed through a column packed with Cell Adsorbent-2, the efflux rate of 201B7 cells was less than 1% for all of Cell Effluent-1 to Cell Effluent-4, confirming that they were well adsorbed to the Cell Adsorbent-2 column. On the other hand, the efflux rates of K562 cells were Cell Effluent-1: 75%, Cell Effluent-2: 11%, Cell Effluent-3: 3%, and Cell Effluent-4: 0%, confirming that the majority of cells were expelled from the column in the early stages of efflux. Therefore, Cell Adsorbent-2 produced by the production method of the present invention not only possesses high selectivity and adsorption capacity for hiPS cells, but also demonstrates the ability to efficiently expel control cells.

[0101] Reference Example 3 Cell adsorption evaluation Reference Example 3 relates to the evaluation of cell adsorption and separation with Cell Adsorbent-3, using the same cell suspension as in Reference Example 1. (1) Preparation of a column packed with adsorbent A cell adsorbent column was prepared by filling the D-PBS(-) suspension of cell adsorbent-3 prepared in Comparative Example 3 into a polypropylene syringe-shaped container and a leak-proof filter having the same specifications as those in Reference Example 1. This was used as a cell adsorbent-3-packed column. Furthermore, 10 mL of MACS buffer (Miltenyi Biotec) was passed through the cell adsorbent-3-packed column to equilibrate the column. (2) Cultivation of 201B7 cells and preparation of cell suspension A suspension of 201B7 cells was prepared in the same manner as in Reference Example 1. (3) Cultivation of K562 cells and preparation of cell suspension A suspension of K562 cells was prepared in the same manner as in Reference Example 1. (4) Preparation of cell suspension for evaluation The 201B7 cell suspension and the K562 cell suspension were uniformly dispersed and then separated to prepare cell suspensions for evaluation at a cell density of 1.0 x 10^7 cells / mL. (5) Evaluation of the cell suspension passing through a column packed with Cell Adsorbent-3 As in Reference Example 1, a 1.0 mL suspension of 201B7 and K562 cells prepared as described above was uniformly dispersed in a vertically placed cell adsorbent-3 column, and then gently added. Next, 4 mL of MACS buffer was added to the top of the column four times. After each addition, the effluent from the tip of the syringe was collected in a separate container. These are referred to as effluent cell solutions-1 to -4. The cell suspension added to the column for evaluation and the collected effluent cell solutions-1 to -4 were resuspended, and the cell counts were measured using a BD FACSAria cell sorter (Becton Dickinson) as in Reference Example 1. Based on the number of 201B7 and K562 cells in each effluent cell solution, the efflux rate of each cell type in effluent cell solutions-1 to -4 was calculated as follows: "efflux rate of each cell type = effluent cell count of each cell type / added cell count of each cell type × 100 (%)."

[0102] When cell suspensions were passed through a column packed with Cell Adsorbent-3, the efflux rate of 201B7 cells was less than 1% for all of Cell Effluent-1 to Cell Effluent-4, confirming good adsorption to the Cell Adsorbent-2 column. On the other hand, the efflux rates of K562 cells were Cell Effluent-1: 23%, Cell Effluent-2: 6%, Cell Effluent-3: 2%, and Cell Effluent-4: 2%, resulting in significantly lower cell recovery rates compared to Reference Example 1 (Cell Adsorbent-1) and Reference Example 2 (Cell Adsorbent-2). Therefore, with Cell Adsorbent-3 produced by the manufacturing method of the present invention, K562 cells, which are non-adsorbed cells, clogged and were difficult to remove. Although hiPS cell adsorption ability was demonstrated, these results suggest the possibility of column clogging. These results reveal that Cell Adsorbent-3 is insufficient for the separation of 201B7 and K562 cells.

[0103] Reference Example 4 Cell adsorption evaluation Reference Example 4 relates to the evaluation of cell adsorption and separation by Cell Adsorbent-4, using the same cell suspension as in Reference Example 1. (1) Preparation of a column packed with adsorbent A cell adsorbent column was prepared by filling the D-PBS(-) suspension of cell adsorbent-4 prepared in Comparative Example 4 into a polypropylene syringe-shaped container and a leak-proof filter having the same specifications as those in Reference Example 1. This was used as a cell adsorbent-4-packed column. Furthermore, 10 mL of MACS buffer (Miltenyi Biotec) was passed through the cell adsorbent-4-packed column to equilibrate the column. (2) Cultivation of 201B7 cells and preparation of cell suspension A suspension of 201B7 cells was prepared in the same manner as in Reference Example 1. (3) Cultivation of K562 cells and preparation of cell suspension A suspension of K562 cells was prepared in the same manner as in Reference Example 1. (4) Preparation of cell suspension for evaluation The 201B7 cell suspension and the K562 cell suspension were uniformly dispersed and then separated to prepare cell suspensions for evaluation at a cell density of 1.0 x 10^7 cells / mL. (5) Evaluation of the cell suspension passing through a column packed with Cell Adsorbent-4 As in Reference Example 1, a 1.0 mL suspension of 201B7 and K562 cells prepared as described above was uniformly dispersed and gently added to a vertically placed cell adsorbent-4 column. Next, 4 mL of MACS buffer was added to the top of the column four times. After each addition, the effluent from the tip of the syringe was collected in a separate container. These are referred to as effluent cell solutions-1 to -4. The cell suspension for evaluation added to the column and the collected effluent cell solutions-1 to -4 were resuspended, and the cell counts were measured using a BD FACSAria cell sorter (Becton Dickinson) as in Reference Example 1. Based on the number of 201B7 and K562 cells in each effluent cell solution, the efflux rate of each cell type in effluent cell solutions-1 to -4 was calculated as follows: "efflux rate of each cell type = effluxed cell count of each cell type / added cell count of each cell type × 100 (%)."

[0104] When a cell suspension was passed through a column packed with cell adsorbent-4, the efflux rate of 201B7 cells was higher (5% and 2%) in cell effluents-1 and -2 compared to the other columns, confirming that the cell adsorbent-4 column and the adsorption performance of 201B7 cells were slightly inferior. This is thought to be because the pore size of the water-insoluble carrier-4, the carrier of cell adsorbent-4, was large (150 nm), causing BCNLCN lectin, the ligand that recognizes 201B7 cells, to be immobilized within the pores, reducing the probability of contact with 201B7 cells and resulting in a decrease in performance as a cell adsorbent. On the other hand, the efflux rate of K562 cells showed an excellent value, almost equivalent to that of Reference Example 1 (cell adsorbent-1).

[0105] Therefore, the cell adsorbent-4 produced by the manufacturing method of the present invention had a reduced ability to capture the target cells, 201B7 cells, suggesting that the pore size of the carrier is an important characteristic.

[0106] [Table 3]

Claims

1. A water-insoluble carrier for a cell adsorbent, characterized in that the carrier is a polymer particle obtained by polymerizing a vinyl monomer, and when wetted with water, the proportion of particles having a diameter of less than 105 μm is 3.0% by weight or less of the total, the pore size of the polymer particles is 100 nm or less, and the polymer has hydroxyl groups and / or amino groups and / or epoxy groups as functional groups.

2. 2. The water-insoluble carrier for a cell adsorbent according to claim 1, wherein the water-insoluble carrier has a volume average particle size of 130 μm or more in a water-wet state.

3. 3. The water-insoluble carrier for a cell adsorbent according to claim 1, wherein the water-insoluble carrier has a particle size distribution width ε of less than 0.7 in a water-wet state. ε = (cumulative 90% particle size - cumulative 10% particle size) ÷ cumulative 50% particle size

4. 4. The water-insoluble carrier for a cell adsorbent according to claim 1, wherein the polymer particles are a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer.

5. A water-insoluble carrier for a cell adsorbent according to claims 1 to 5, characterized in that a hydrophilic polymer is covalently bonded via a functional group of the polymer, and the polymer particles are hydrophilically modified.

6. 6. The water-insoluble carrier for a cell adsorbent according to claim 5, wherein the hydrophilic polymer is a neutral polysaccharide having a number-average molecular weight of 10,000 or more but less than 1,000,000.

7. 6. The water-insoluble carrier for a cell adsorbent according to claim 5, wherein the hydrophilic polymer is polyethylene glycol polyglycidyl ether having a number average molecular weight of 100 or more.

8. A water-insoluble carrier for cell adsorption material according to claims 1 to 7, characterized in that a maleimide group and / or a haloacetyl group is introduced into the polymer in order to immobilize a ligand that specifically recognizes target cells.

9. The water-insoluble carrier for a cell adsorbent according to any one of claims 1 to 8, further comprising a ligand that specifically recognizes target cells and has binding affinity for a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc, A cell adsorbent on which a fucose-binding protein is immobilized, the fucose-binding protein having the amino acid sequence shown in SEQ ID NO: 1 and comprising any of the amino acid sequences (a) to (d) below: (a) an amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, where X is an integer of 110 or more and 155 or less; (b) an amino acid sequence comprising one or more deletions, substitutions, insertions, and / or additions of amino acid residues in the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 110 or more and 155 or less; (c) an amino acid sequence having 90% or more homology to the amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 110 or more and 155 or less; (d) an amino acid sequence comprising specific amino acid substitutions in the amino acid sequence of any one of (a) to (c), wherein the specific amino acid substitutions are one or more amino acid substitutions selected from the amino acid substitutions of (1) to (5) below; (1) substitution of an amino acid residue corresponding to the 39th glutamine residue in the amino acid sequence represented by SEQ ID NO: 1 with an amino acid residue other than glutamine; (2) Substitution of an amino acid residue corresponding to the 72nd cysteine ​​residue in the amino acid sequence represented by SEQ ID NO: 1 with an amino acid residue other than a cysteine ​​residue; (3) Substitution of an amino acid residue corresponding to the 65th glutamine residue in the amino acid sequence represented by SEQ ID NO: 1 with an amino acid residue other than glutamine; (4) Substitution of the amino acid residue corresponding to the glutamic acid residue at position 81 of the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glutamic acid; (5) Substitution of the amino acid residue corresponding to the 36th glycine residue in the amino acid sequence shown in SEQ ID NO: 1 with an amino acid residue other than glycine.

10. A cell adsorbent in which a fucose-binding protein in which the amino acid substitutions described in (1) to (5) are the amino acid substitutions described in (6) to (10) below, respectively, is immobilized on a water-insoluble carrier for cell adsorbents described in claim 9. (6) Substitution of the amino acid residue corresponding to the glutamine residue at position 39 of the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue or a methionine residue; (7) Substitution of the amino acid residue corresponding to the 72nd cysteine ​​residue in the amino acid sequence shown in SEQ ID NO: 1 with a glycine residue or an alanine residue; (8) Substitution of the amino acid residue corresponding to the glutamine residue at position 65 of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue; (9) Substitution of the amino acid residue corresponding to the glutamic acid residue at position 81 of the amino acid sequence represented by SEQ ID NO: 1 with a cysteine ​​residue, a glutamine residue, a histidine residue, a methionine residue, a valine residue, a lysine residue, a serine residue, an isoleucine residue, a tyrosine residue, a glycine residue, a proline residue, a leucine residue, or an asparagine residue; (10) Substitution of the amino acid residue corresponding to the 36th glycine residue in the amino acid sequence shown in SEQ ID NO: 1 with a cysteine ​​residue.

Images