JP2022151517A - Cell purification method - Google Patents

Abstract

To provide a method for purifying a cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc.SOLUTION: The above problems are solved by a method comprising the following steps (1) to (3): (1) a step of contacting a cell mixture containing at least one type of cell with an adsorbent comprising a fucose-binding protein immobilized on a water-insoluble carrier to obtain a complex in which the adsorbent and cells are bound; (2) a step of separating and removing cells that did not bind to the adsorbent from the complex obtained in (1) above; and (3) a step of recovering the cells desorbed from the adsorbent by bringing a desorption liquid into contact with the complex obtained in (1) after the step (2) is completed.SELECTED DRAWING: Figure 5

Description

The present invention relates to a method for purifying cells having sugar chains containing fucose using an adsorbent comprising a fucose-binding protein immobilized on a water-insoluble carrier.

In recent years, research and development on regenerative medicine has been active, and technology using stem cells is at the core. Among stem cells, pluripotent stem cells such as human iPS cells and ES cells have the ability to differentiate into various cells such as muscle cells such as cardiomyocytes and neural cells such as neural stem cells. Practical application of regenerative medicine using therapeutic cells and tissues derived from sexual stem cells is expected (for example, Patent Document 1). However, the current situation is that systems such as safety evaluation, quality control, and large-scale supply have not been sufficiently established for therapeutic cells and tissues that are induced to differentiate from pluripotent stem cells. Toward the practical application of medical treatment, we will develop techniques for removing tumorigenic undifferentiated cells remaining in therapeutic cells, and maintain the functions of pluripotent stem cells, such as their ability to differentiate into therapeutic cells and their proliferative properties. Therefore, it is essential to develop a production technology for pluripotent stem cells that can be mass-cultured.

Techniques for removing tumorigenic undifferentiated cells include, for example, H type 1 sugar chains (sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc), which are undifferentiated sugar chain markers present on the surface of human iPS cells, and H A fusion protein that utilizes the BC2LCN lectin that binds to a type 3 sugar chain (a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc) and binds the catalytic domain of the Pseudomonas aeruginosa-derived exotoxin to the C-terminus of the lectin. has been reported to be able to remove tumorigenic undifferentiated cells by adding iPS cells to differentiated cells during differentiation induction (Patent Document 2). In addition, as a mass culture technique for pluripotent stem cells, for example, it has been reported that mass culture of human iPS cells is possible by a floating agitation culture method using an aerated agitation bioreactor (Patent Document 3). .

On the other hand, human iPS cells are produced by introducing genes such as Oct3/4 into cells collected from skin or blood (for example, Non-Patent Document 1), but iPS cells with a low degree of undifferentiation into human iPS cells and undifferentiated When cells that deviate from the state (for example, Non-Patent Document 2) are mixed, the function of human iPS cells as pluripotent stem cells is reduced. A method for removing out-of-state cells has been reported (eg, Patent Document 4). Therefore, in the production of therapeutic cells and tissues differentiated from pluripotent stem cells, human iPS cells having functions as pluripotent stem cells are selected from among the produced human iPS cells at a stage prior to mass culture of human iPS cells. There is a demand for a technique that allows simple and large-scale purification of cells.

WO2016/043168 WO2014/126146 WO2015/122528 WO2014/104207

Takahashi, K et al., Cell. 2007, 131(5):861-872. Watanabe, T. et al., Biochemical and Biophysical Research Communications, 2020, 529(3):575-581.

An object of the present invention is to develop human pluripotent stem cells and cancer cells that have fucose-containing sugar chains and H-type 1 sugar chains and/or H-type 3 sugar chains that are undifferentiated marker sugar chains on the cell surface. , to provide a technique for refining to a high degree of purity.

As a result of intensive studies by the present inventors to solve the above problems, (1) cells are brought into contact with an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, and the adsorbent and cells bind to each other. (2) separating and removing cells that did not bind to the adsorbent from the complex; and (3) contacting the complex with a desorption liquid to recover the cells desorbed from the adsorbent. The inventors have found that cells having H-type 1 sugar chains and/or H-type 3 sugar chains on the cell surface can be highly purified by a method including three steps, and have completed the present invention.

That is, the present invention includes the inventions described in [1] to [12] below.
[1]
A method for purifying cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, comprising the following steps (Z1) to (Z3):
(Z1) contacting a sample solution containing at least one type of cell with an adsorbent comprising any one of the following (a) to (d) fucose-binding proteins immobilized on a water-insoluble carrier, A step of obtaining a complex in which the adsorbent and cells are bound.
(a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more and 140 or less. Fucose-binding protein.
(b) an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising a fucose-binding protein, and having a binding property to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more and 140 or less, Fucose-binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1), wherein X is an integer of 120 or more and 140 or less, containing an amino acid sequence containing the above, substitution of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue ( 2) replacement of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) of the amino acid sequence shown by SEQ ID NO: 1 Substitution of the 65th glutamine residue with a leucine residue (d) in the amino acid sequence of the fucose-binding protein of (c) above, in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 Containing an amino acid sequence in which a plurality of amino acid residues are deleted, substituted, inserted or added, and having binding properties to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc , fucose-binding protein.
(Z2) A step of separating and removing cells that did not bind to the adsorbent from the complex obtained in (Z1) above.
(Z3) A step of recovering cells desorbed from the adsorbent by bringing a desorption liquid into contact with the complex obtained in (Z1) after the completion of (Z2).
[2]
The method for purifying cells according to [1] above, wherein the cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are human pluripotent stem cells.
[3]
The method for purifying cells according to [1] above, wherein the purified human pluripotent stem cells have a BC2LCN positive rate of 90% or more.
[4]
The method for purifying cells according to [1] above, wherein the cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are cancer cells.
[5]
The method for purifying cells according to [4] above, wherein the purified cancer cells have a BC2LCN positive rate of 70% or more.
[6]
[5] above, wherein the sample solution containing at least one type of cell contains cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. ] Purification method of the cell according to any one of.
[7]
A sample solution containing at least one type of cell is a cell having a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, and a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and The method for purifying cells according to any one of [1] to [6] above, which comprises both cells having no sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc.
[8]
The fucose-binding protein is a fucose-binding protein having an additional amino acid sequence at its N-terminus and/or C-terminus, wherein the additional amino acid sequence comprises an oligopeptide containing cysteine residues and/or a polyhistidine sequence. The method for purifying cells according to any one of [1] to [7] above, wherein the oligopeptide comprises
[9]
The method for purifying cells according to any one of [1] to [8] above, wherein the fucose-binding protein is a fucose-binding protein comprising the amino acid sequence shown in any one of SEQ ID NO:2 to SEQ ID NO:9.
[10]
The method for purifying cells according to any one of [1] to [9] above, wherein the desorption solution is an aqueous solution containing fucose and/or fucose-containing sugar chains.
[11]
The concentration of fucose and/or fucose-containing sugar chains in the desorption solution is 0.01 mol/L or more and 1.0 mol/L or less, according to any one of [1] to [10] above. Cell purification method.
[12]
The method for purifying cells according to any one of [1] to [11] above, wherein an adsorbent packed in a column is used.

The present invention will be described in detail below.

The method for purifying cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc of the present invention (hereinafter sometimes abbreviated as cell purification method) comprises the following (Z1): to (Z3) are included.
(Z1) contacting a sample solution containing at least one type of cell with an adsorbent comprising any one of the following (a) to (d) fucose-binding proteins immobilized on a water-insoluble carrier, A step of obtaining a complex in which the adsorbent and cells are bound.
(Z2) A step of separating and removing cells that did not bind to the adsorbent from the complex obtained in (Z1) above.
(Z3) A step of recovering cells desorbed from the adsorbent by bringing a desorption liquid into contact with the complex obtained in (Z1) after the completion of (Z2).
Details of each step from (Z1) to (Z3) are described below.

1. Process (Z1)
The step (Z1) in the cell purification method of the present invention comprises a sample solution containing at least one type of cell and any of the following fucose-binding proteins (a) to (d) immobilized on a water-insoluble carrier. It is a step of contacting an adsorbent formed by sintering to obtain a complex in which the adsorbent and cells are bound. A sample solution, cells contained in the sample solution, a fucose-binding protein, an adsorbent in which the protein is immobilized on a water-insoluble carrier, and a method of contacting the sample solution with the adsorbent will be described below.

2. Sample solution The sample solution used in step (Z1) contains cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, and the cells are not destroyed, and the cells and Substances other than the cells may be contained, for example, from Fucα1-2Galβ1-3GlcNAc, as long as an adsorbent-bound complex comprising a fucose-binding protein immobilized on a water-insoluble carrier can be obtained. Cells having neither a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc nor a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc, media and buffers for suspending cells, osmotic pressure regulation to prevent destruction of cells Agents, substances to prevent cell clumping may also be included. Specifically, examples of the medium include Dulbecco's Modified Eagle Medium (hereinafter sometimes abbreviated as D-MEM medium), RPMI medium, and StemFit medium (manufactured by Ajinomoto Co.).

Phosphate-buffered saline (hereinafter sometimes abbreviated as PBS) and Tris-buffered saline (hereinafter abbreviated as TBS) having a buffering capacity in the range of pH 7.0 to 8.0 are used as buffers. ) and Hepes-buffered saline (hereinafter sometimes abbreviated as HBS) can be exemplified.

The concentration of the buffer component in the buffer solution is not particularly limited as long as it can suppress cell destruction, and may be appropriately selected within the range of 1 mmol/L or more and 100 mmol/L or less. Examples of osmotic pressure regulators include inorganic salts such as sodium chloride and potassium chloride, and sugar alcohols such as erythritol, sorbitol, xylitol and mannitol. The concentration of the osmotic pressure regulator is not particularly limited as long as it can suppress cell destruction, and may be appropriately selected within the range of 10 mmol/L or more and 1000 mmol/L or less. Substances for preventing cell aggregation include proteins such as human serum albumin (hereinafter sometimes abbreviated as HSA) and bovine serum albumin (hereinafter sometimes abbreviated as BSA), ethylenediaminetetraacetic acid ( Hereinafter, it may be abbreviated as EDTA.) and ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (hereinafter also abbreviated as EGTA.) Chelating agents, non-ionics such as Triton-X100 and Tween 20 can be exemplified by surfactants. The concentration of the substance for preventing cell aggregation is not particularly limited as long as it can suppress cell aggregation. It may be appropriately selected in the range of 0.001 mmol/L or more and 100 mmol/L or less, and the nonionic surfactant in the range of 0.00001 to 1%. The substance used for preparing the sample solution used in step (Z1) preferably contains BSA in terms of reducing damage to cells and suppressing non-specific adsorption of cells to the adsorbent, and preventing cell aggregation. In that respect, it is preferable to contain a chelating agent. For example, a commercially available MACS buffer containing 0.5% (w/v) BSA and 2 mM EDTA in PBS may be used. The buffer solution, osmotic pressure regulator, and substance for preventing aggregation of cells preferably do not exhibit cytotoxicity when culturing cells purified by the cell purification method of the present invention.

In step (Z1), cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc and an adsorbed fucose-binding protein immobilized on a water-insoluble carrier Since this is a step of obtaining a complex bound with an agent, it is essential that the sample solution used in step (Z1) does not contain a substance that inhibits the formation of the complex. Substances that inhibit the formation of the complex include, for example, fucose, which is a monosaccharide, fucosyllactosamine (Fucα1-2Galβ1-4Glc), H type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc), H type 3 sugar chain (Fucα1-2Galβ1-3GalNAc), Lewis X sugar chain (Galβ1-4(Fucα1-3)GlcNAc), Lewis Y sugar chain (Galβ1-4(Fucα1-3)GlcNAc), Lewis b sugar chain ( Examples include fucose-containing sugar chains such as Fucα1-2Galβ1-3(Fucα1-4)GlcNAc) and glycoproteins and glycolipids to which these fucose-containing sugar chains are bound.

3. Cells Contained in Sample Solution As described above, the sample solution used in step (Z1) contains cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, and further, Fucα1- Cells having neither a sugar chain containing a structure consisting of 2Galβ1-3GlcNAc nor a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc may be included. Specific examples of cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc include pluripotent stem cells such as human iPS cells and human ES cells, and non-patent literatures. 2 and the like can be exemplified by cells that have deviated from the undifferentiated state. In addition, described in patent documents (eg, JP-A-2020-025535, WO2017/061449) and non-patent documents (eg, J. Biomark.2013:960862.doi:10.1155/2013/960862.) human embryonic cancer cells such as 2102Ep and NT2/D1, human lung adenocarcinoma cells such as PC-9, human pancreatic cancer cells such as Capan-1, and human colon cancer cells such as HT29. can be done. The number of types of cells contained in the sample solution is not particularly limited, and may be one type, two types, or more. In addition, the cells may be living cells or dead cells. For example, when culturing pluripotent stem cells or cancer cells purified by the cell purification method of the present invention, living cells are required. When used only for analysis such as proteome analysis and gene analysis, they may be live cells or dead cells.

Cells having neither a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc nor a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc include, specifically, body cells such as human fibroblasts. Cells, differentiated cells generated by inducing differentiation from pluripotent stem cells such as human iPS cells and human ES cells can be exemplified. Furthermore, Fucα1-2Galβ1-3GlcNAc such as human Burkitt's lymphoma cells (Ramos cells) and human chronic myelogenous leukemia cells (K562 cells) described in JP-A-2018-134073 and JP-A-2020-025535, and Cancer cells that do not have any sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc can be exemplified. There is no particular limit to the number of types of cells that have neither the sugar chain containing the structure consisting of Fucα1-2Galβ1-3GlcNAc nor the sugar chain containing the structure consisting of Fucα1-2Galβ1-3GalNAc contained in the sample solution. or two or more types, and may be live cells or dead cells.

4. Fucose-binding protein The fucose-binding protein in the cell purification method of the present invention includes H type 1 sugar chain, H type 3 sugar chain, Lewis X sugar chain, Lewis Y sugar chain, Lewis b sugar chain, and the like. is a protein that has binding properties to fucose-containing sugar chains of Specifically, (a): the amino acid sequence of the recombinant BC2LCN lectin shown in SEQ ID NO: 1 (matches the amino acid sequence from 2nd to 156th of the amino acid sequence registered with GenPept as accession number WP_006490828). A protein containing an amino acid sequence from the first proline to the X-th amino acid, wherein X is an integer of 120 or more and 140 or less, or (b): the first amino acid sequence shown in SEQ ID NO: 1 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence from proline to the Xth amino acid, and from Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A protein having a binding property to a sugar chain containing a structure and X being an integer of 120 or more and 140 or less is expressed as a recombinant protein in a transformant of Escherichia coli.

As long as the fucose-binding protein in the cell purification method of the present invention has binding properties to the fucose-containing sugar chain, particularly to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. , in the amino acid sequence from the first proline to the Xth amino acid in the amino acid sequence shown by SEQ ID NO: 1, one or more amino acids may be deleted, substituted or added, for example 10 or less, preferably may have up to 5 amino acids deleted, substituted or added. Moreover, X may be 120 or more and 140 or less, and may be 125 or more and 135 or less. As disclosed in Japanese Unexamined Patent Application Publication No. 2020-25535, the fucose-binding protein in the cell purification method of the present invention has multiple amino acid residues on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1 deleted. As a result, the productivity (expression level) can be improved when produced in a transformant of E. coli compared to when the amino acid residue is not deleted.

In addition, the fucose-binding protein in the cell purification method of the present invention is characterized by (i) replacement of the 39th glutamine residue in the amino acid sequence shown by SEQ ID NO: 1 with a leucine residue in terms of improving the stability against heat. , (ii) substitution of the 72nd cysteine residue of the amino acid sequence shown in SEQ ID NO: 1 with one type of amino acid residue selected from glycine and / or alanine residues, (iii) shown in SEQ ID NO: 1 Substitution of the 65th glutamine residue in the amino acid sequence described with a leucine residue.

As disclosed in JP-A-2020-25535, the amino acid substitutions described in (i) to (iii) above improve the heat stability of the fucose-binding protein in the cell purification method of the present invention. can be improved. The amino acid substitutions described in (i) to (iii) above are effective in improving the stability against heat whether they are used alone or in combination, but they can further improve the stability against heat. , It is more preferable to combine multiple amino acid substitutions described in (i) to (iii) above. Furthermore, the fucose-binding protein in the cell purification method of the present invention is SEQ ID NO. In the amino acid sequence from the 1st proline to the Xth amino acid in the amino acid sequence shown, one or more amino acid residues are deleted in the region other than the positions substituted by the substitutions (i) to (iii). Deletions, substitutions or insertions may be made, for example no more than 10, preferably no more than 5 amino acid residues may be deleted, substituted or inserted.

As long as the fucose-binding protein in the cell purification method of the present invention has a binding property to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, its N-terminal side and/or Alternatively, it may have an additional amino acid sequence on the C-terminal side that is useful for detecting fucose-binding proteins. The additional amino acid sequences include oligopeptides containing a polyhistidine sequence, glutathione S-transferase (hereinafter referred to as GST), maltose binding protein, cellulose binding domain, myc tag, FLAG tag and the like. Among these additional amino acid sequences, the productivity is high when produced using E. coli, and the fucose-binding protein can be easily detected by using a fluorescently labeled anti-polyhistidine antibody or anti-GST antibody. , preferably an oligopeptide or GST containing a polyhistidine sequence, more preferably an oligopeptide containing a polyhistidine sequence.

The number of histidine repeat sequences in an oligopeptide containing a polyhistidine sequence is not particularly limited. connectivity may be compromised. Therefore, the length of the repeating sequence of histidines in an oligopeptide containing a polyhistidine sequence is preferably a repeating sequence consisting of 5 to 15 histidines, more preferably a repeating sequence consisting of 5 to 10 histidines. preferable.

The position at which the oligopeptide containing the polyhistidine sequence is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. However, it is preferable that the oligopeptide containing the polyhistidine sequence is added to the N-terminal side of the fucose-binding protein in terms of efficient detection with an anti-polyhistidine antibody.

Furthermore, the fucose-binding protein in the cell purification method of the present invention has a cysteine residue or lysine residue on its N-terminal side and/or C-terminal side, which are useful for immobilizing the fucose-binding protein on a water-insoluble carrier. It may have an additional amino acid sequence consisting of an oligopeptide containing residues (hereinafter referred to as carrier immobilization tag). By immobilizing a fucose-binding protein on a carrier, for example, an undifferentiated cell adsorbent for removing undifferentiated cells such as human iPS cells described in JP-A-2020-25535 can be produced. can. The length of the carrier-immobilizing tag is not particularly limited as long as the fucose-binding protein has the ability to bind to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. do not have.

As the tag for carrier immobilization, an oligopeptide consisting of 2 to 10 amino acid residues containing one or more cysteine residues is preferable, since immobilization to a water-insoluble carrier can be performed with high selectivity and high efficiency. includes an oligopeptide consisting of 3 amino acid residues of "Gly-Gly-Cys", an oligopeptide consisting of 5 amino acid residues of "Ala-Ser-Gly-Gly-Cys" and an oligopeptide consisting of 5 amino acid residues of "Gly-Gly-Gly-Ser- An oligopeptide consisting of 7 amino acid residues "Gly-Gly-Cys" can be exemplified. The position at which the oligopeptide containing one or more cysteines is attached to the fucose-binding protein is not particularly limited, and may be both the N-terminal side and the C-terminal side, or either the N-terminal side or the C-terminal side. , the fucose-binding protein can be efficiently immobilized on a carrier, and the binding activity is less likely to be inhibited because the fucose-binding protein is away from the active center of the fucose-binding protein. The peptide is preferably added to the C-terminal side of the fucose-binding protein.

Specific examples of the fucose-binding protein in the cell purification method of the present invention include SEQ ID NO: 2 (amino acid sequence from 1st to 127th of the amino acid sequence shown by SEQ ID NO: 1), SEQ ID NO: 3 (72 of SEQ ID NO: 2) amino acid sequence in which the cysteine residue of SEQ ID NO: 2 is replaced with a glycine residue), SEQ ID NO: 4 (the 39th glutamine residue of SEQ ID NO: 2 is replaced with a leucine residue, and the 72nd cysteine residue of SEQ ID NO: 2 is replaced with a glycine residue) sequence), SEQ ID NO: 5 (an amino acid sequence obtained by substituting a leucine residue for the 39th glutamine residue, a leucine residue for the 65th glutamine residue, and a glycine residue for the 72nd cysteine residue in SEQ ID NO: 2) ), SEQ ID NO: 6 (amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by SEQ ID NO: 2 and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 7 (SEQ ID NO: An amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence shown by 3 and an oligopeptide containing a cysteine residue to the C-terminus), SEQ ID NO: 8 (N of the amino acid sequence shown by SEQ ID NO: 4) an oligopeptide containing a polyhistidine sequence at the terminal and an oligopeptide containing a cysteine residue at the C-terminus) and SEQ ID NO: 9 (an oligo containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 5) Peptides include fucose-binding proteins represented by any of the amino acid sequences obtained by adding an oligopeptide containing a cysteine residue to the C-terminus.

Among them, SEQ. 7, SEQ ID NO: 8 and SEQ ID NO: 9 are preferred, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8 are more preferred, and SEQ ID NO: 4 and SEQ ID NO: 8 are particularly preferred.

A signal peptide may be added to the N-terminal side of the fucose-binding protein in the cell purification method of the present invention to promote efficient expression in the host. Examples of the signal peptide when the host is Escherichia coli include signal peptides such as PelB, DsbA, MalE, and TorT that secrete proteins into the periplasm. A DNA encoding a fucose-binding protein in the cell purification method of the present invention can be prepared by a known method. As the method for preparing the DNA, a method of converting the amino acid sequence of the fucose-binding protein into a base sequence in the cell purification method of the present invention and artificially synthesizing DNA containing the base sequence, or a method of artificially synthesizing DNA containing the base sequence A method of directly and artificially preparing a DNA encoding a fucose-binding protein or a method of preparing it from Burkholderia cenocepacia genomic DNA or the like using a DNA amplification method such as PCR can be exemplified. In the preparation method, when designing the base sequence, it is preferable to consider the frequency of codon usage in E. coli to be transformed. Ile), ATA, leucine (Leu), CTA, glycine (Gly), GGA, and proline (Pro), CCC, are rare codons (rare codons). Selective conversion is preferred. Analysis of codon usage frequency is performed using a public database (for example, Codon Usage Database on the homepage of Kazusa DNA Research Institute, http://www.kazusa.or.jp/codon/, access date: May 7, 2020). It is also possible by using In order to transform Escherichia coli with the DNA encoding the fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. It is preferable to insert the DNA into an appropriate position in a vector based on a commonly used bacteriophage, cosmid, plasmid, or the like to form an expression vector, and to transform using the expression vector, since stable transformation can be performed. .

Here, the appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the region involved in transmissibility. When inserting the DNA into a vector, it is preferable to insert into the vector in a state of being linked to a functional DNA such as a promoter required for expression. The vector used as the expression vector is not particularly limited as long as it can stably exist and replicate in the host, and pET vector, pUC vector, pTrc vector, pCDF vector, pBBR vector and the like can be exemplified.

Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and λ phage λ PL promoter and λ PR promoter. Transformation of the host Escherichia coli with the expression vector can be carried out by a method commonly used by those skilled in the art. When Escherichia coli W3110 strain or the like is selected, methods described in known literature (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

Next, a method for producing a fucose-binding protein in the cell purification method of the present invention will be explained. The fucose-binding protein in the cell purification method of the present invention comprises a step of producing a fucose-binding protein by culturing the transformant (hereinafter referred to as the first step), and It can be produced by a process including a process of recovering the protein (hereinafter referred to as the second process). In the present specification, the culture includes the cultured transformant cells themselves and cell secretions, as well as the medium used for culture. In the first step, the transformant may be cultured in a suitable medium. For example, when E. coli is used as a host, Terrific Broth (TB) medium or Luria-Bertani (LB) medium supplemented with necessary nutrients. etc. is preferably used. When the expression vector contains a drug resistance gene, selective growth of the transformant becomes possible by adding a drug corresponding to the gene to the medium and carrying out the first step. For example, the expression vector contains a kanamycin resistance gene kanamycin is preferably added to the medium.

The culture temperature may be any temperature that is generally known for the host to be used. It can be determined appropriately while taking into account the above. In addition, the pH of the medium may be within the generally known pH range for the host to be used. For example, when the host is Escherichia coli, the pH is in the range of pH 6.8 to pH 7.4, preferably around pH 7.0. , may be appropriately determined in consideration of the production amount of the fucose-binding protein and the like. When an inducible promoter is introduced into the expression vector, the expression can be induced by adding an inducer to the medium under conditions that allow the fucose-binding protein to be produced well.

Preferred inducers include, for example, isopropyl-β-D-thiogalactopyranoside (IPTG) when using the tac promoter or lac promoter, and the concentration to be added is in the range of 0.005 mM to 1.0 mM, preferably 0 0.01 mM to 0.5 mM. Induction of expression by adding IPTG may be performed under conditions generally known for the host to be used. In the second step, the fucose-binding protein is recovered from the culture obtained in the first step by a generally known recovery method. For example, when the fucose-binding protein is secreted and produced in the culture medium, the cells may be separated by centrifugation, and the fucose-binding protein may be recovered from the resulting culture supernatant. ), the cells are collected by centrifugation, the cells are disrupted by adding an enzyme treatment agent or surfactant, etc., and the fucose-binding protein can be recovered from the cell lysate. . Moreover, when it is desired to improve the purity of the fucose-binding protein, a method known in the art may be used, and an example thereof is a separation and purification method using liquid chromatography. As liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like are preferably used, and a combination of these chromatographies is more preferred. In addition, the purity and molecular weight of the fucose-binding protein purified by the chromatography may be examined using methods known in the art. Examples include SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Graphical methods may be mentioned.

Next, the evaluation of the binding properties of fucose-binding proteins to sugar chains in the cell purification method of the present invention will be described. The binding property of a fucose-binding protein to sugar chains can be evaluated by a surface plasmon resonance method. Binding affinity evaluation by the surface plasmon resonance method is performed, for example, using a Biacore T200 instrument (manufactured by Cytiva), using a fucose-binding protein as an analyte and a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc as a solid phase. can be measured as a sugar chain containing The sugar chain-immobilized sensor chip is produced by using a biotin-labeled sugar chain to prepare a streptavidin-coated sensor chip (Sensor Chip SA, manufactured by Cytiva) or a dextran-coated sensor chip (Sensor Chip CM5, Cytiva). (manufactured by Cytiva) to which streptavidin has been immobilized in advance. In addition, binding evaluation can be performed using a kinetics analysis program attached to the device.

5. Adsorbent The adsorbent in the cell purification method of the present invention is an adsorbent in which the fucose-binding protein is immobilized on a water-insoluble carrier. There are no particular restrictions on the raw material of the water-insoluble carrier. Inorganic carriers such as silica gel and glass deposited with a thin gold film, water-insoluble polysaccharide carriers made from polysaccharides such as agarose, cellulose, chitin and chitosan, and Crosslinked polysaccharide carriers obtained by crosslinking them with a crosslinking agent, crosslinked polysaccharide carriers obtained by crosslinking water-soluble polysaccharides such as dextran, pullulan, starch, alginate, and carrageenan with a crosslinking agent, poly(meth)acrylate, polyvinyl alcohol, polyurethane, Synthetic polymer carriers such as polystyrene and crosslinked synthetic polymer carriers obtained by crosslinking these with a crosslinking agent can be exemplified.

Among these carriers, uncharged polysaccharide carriers such as agarose, cellulose, dextran, and pullulan, which have hydroxyl groups and can be easily modified with a hydrophilic polymer to be described later, and their cross-linking agents. Crosslinked crosslinked polysaccharide carriers, hydrophilic synthetic polymer carriers such as poly(meth)acrylate and polyurethane, and crosslinked hydrophilic synthetic polymer carriers obtained by crosslinking these with a crosslinking agent are preferred. In addition, the water-insoluble carrier used as the adsorbent is preferably modified with a hydrophilic polymer on the surface of the carrier from the viewpoint of suppressing non-specific adsorption of cells, and the hydrophilic polymer is covalently bonded to the carrier. Fixed is more preferred. Hydrophilic polymers that modify the surface of water-insoluble carriers include neutral polysaccharides such as agarose, cellulose, dextran, pullulan and starch, and hydroxyl groups such as poly(2-hydroxyethyl (meth)acrylate) and polyvinyl alcohol. can be exemplified by synthetic polymers having Among these hydrophilic polymers, neutral polysaccharides such as dextran, pullulan and starch are preferred, and dextran and pullulan are more preferred, because they are highly hydrophilic and can be easily fixed to the surface of an insoluble carrier by covalent bonding. . Although the molecular weights of dextran and pullulan are not particularly limited, those having a number average molecular weight of 10,000 to 1,000,000 are preferred in terms of sufficient hydrophilic modification of the surface of the insoluble carrier.

The shape of the water-insoluble carrier used for the adsorbent is not particularly limited, and specific examples include particulate, monolithic, spongy, flat sheet, plate, hollow, and fibrous carriers. Furthermore, the presence or absence of pores in the water-insoluble carrier used for the adsorbent is not particularly limited, and the carrier may be either a porous carrier or a non-porous carrier. A porous particulate carrier, a sponge-like carrier having through holes, or a monolithic carrier are preferred in terms of efficient adsorption of cells to the adsorbent.

When the water-insoluble carrier used as the adsorbent in the cell purification method of the present invention is a particulate carrier, the average particle diameter (median diameter) in the state of being swollen in water is It is preferably 100 μm or more and 500 μm or less, more preferably 100 μm or more and 500 μm or less, in that the cells to be separated are in sufficient contact with the adsorbent surface when filled in the adsorbent, and the cells that do not bind to the adsorbent can pass through the gaps between the adsorbents without stagnation. is 150 μm or more and 400 μm or less. If the particle size is less than 100 μm, it becomes difficult for cells that do not bind to the adsorbent to pass through the gaps between the adsorbents, resulting in a low cell recovery rate.

On the other hand, if the particle size exceeds 500 μm, the contact between the adsorbent-bound cells and the surface of the adsorbent is insufficient, and the separation efficiency between the adsorbent-bound cells and the non-bound cells is reduced. The particle size of the water-insoluble carrier can be measured, for example, using a precision particle size distribution analyzer (product name: "Multisizer 3") manufactured by Beckman Coulter, Inc., or the like. In addition, after taking an image of a graduated slide glass using an optical microscope, images of a plurality of particles to be measured are taken at the same magnification, and the particle sizes of the multiple carriers taken are measured using a ruler. , can also be obtained by calculating the average value. Furthermore, the particulate carrier used in the adsorbent of the present invention may be a commercially available product, for example, Toyopearl (manufactured by Tosoh) using poly(meth)acrylate as a raw material, Sepharose (manufactured by Cytiva) using agarose as a raw material, Celphia (manufactured by Asahi Kasei Co., Ltd.) using cellulose as a raw material can be used.

The adsorbent in the cell purification method of the present invention comprises a step of producing a reactive carrier from a water-insoluble carrier (hereinafter referred to as step Y1), and immobilization by reacting a fucose-binding protein on the reactive water-insoluble carrier. It can be manufactured by a process including a process of converting (hereinafter referred to as process Y2). The details of process Y1 and process Y2 will be described below. Step Y1 is a step of producing a reactive carrier by introducing a reactive functional group for immobilizing a fucose-binding protein into a water-insoluble carrier.

The reactive functional group for immobilizing a fucose-binding protein on a water-insoluble carrier is not particularly limited as long as it is a general functional group for protein immobilization, and includes epoxy group, formyl group, carboxyl group, active ester group, Examples include an amino group, a maleimide group, a haloacetyl group, and the like. The method for introducing the functional group into the carrier is not particularly limited as long as it is a general method for introducing the functional group, and the method for introducing the epoxy group includes the hydroxyl group of the carrier and epichlorohydrin, epibromohydrin, or the like. halohydrins, 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, resorcinol diglycidyl Epoxy groups such as diglycidyl ethers such as ethers, triglycidyl ethers such as glycerol triglycidyl ether, erythritol triglycidyl ether, diglycerol triglycidyl ether, tetraglycidyl ethers such as erythritol tetraglycidyl ether and pentaerythritol tetraglycidyl ether A method of reacting the contained compounds can be exemplified.

Moreover, when the hydroxyl group of the carrier and the epoxy group-containing compound are reacted, the reaction is preferably carried out under basic conditions in order to increase the reaction efficiency. Examples of methods for introducing formyl groups into the carrier include a method of reacting a hydroxyl group of the carrier with a bifunctional aldehyde such as glutaraldehyde, and a method of reacting the carrier with an oxidizing agent such as sodium periodate. . In addition, a water-insoluble carrier into which an epoxy group has been introduced by the above-described method is reacted with a compound such as D-glucamine, N-methyl-D-glucamine, α-thioglycerol, etc., to obtain a carrier into which adjacent hydroxyl groups have been introduced. A method of reacting with an oxidizing agent such as sodium iodate can be exemplified. As a method for introducing a carboxyl group into a water-insoluble carrier, in addition to a method of reacting a hydroxyl group of the carrier with a haloacetic acid such as monochloroacetic acid or monobromoacetic acid under basic conditions, an epoxy group was introduced by the above-mentioned method. 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 are used as carriers. and a method of reacting under basic conditions can be exemplified.

Further, the carboxyl group introduced into the water-insoluble carrier is reacted with N-hydroxysuccinimide in the presence of a condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDC). Thus, a method of deriving an N-hydroxysuccinimide ester, which is an active ester group, can be exemplified. As a method for introducing an amino group into a water-insoluble carrier, a compound having at least two amino groups such as ethylenediamine, diethylenetriamine, tris(2-aminoethyl)amine, etc. A method of reacting with can be exemplified. A method for introducing a maleimide group into a water-insoluble carrier includes a water-insoluble carrier having a hydroxyl group and/or an amino group, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, 4-(N-maleimide A method of reacting carboxylic acids having a maleimide group such as methyl)cyclohexanecarboxylic acid in the presence of a condensing agent such as EDC can be exemplified. Furthermore, a method of reacting an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester of a carboxylic acid having a maleimide group as described above can be exemplified. As a method for introducing a haloacetyl group into a carrier, for example, a carrier having a hydroxyl group or a carrier having an amino group introduced by the above method is reacted with an acid halide such as chloroacetic chloride, bromoacetic acid chloride, or bromoacetic acid bromide. and a method of reacting halogenated acetic acid such as chloroacetic acid, bromoacetic acid and iodoacetic acid in the presence of a condensing agent such as EDC. Furthermore, a method of reacting the aforementioned N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of halogenated acetic acid can be mentioned.

Step Y2 is a step of immobilizing the fucose-binding protein of the present invention on the reactive carrier produced in step Y1. The method for immobilizing the fucose-binding protein on the reactive carrier obtained in step Y1 is not particularly limited as long as it is a general immobilization method for proteins, and for example, coordination bonding or affinity bonding is used. a method of immobilizing a protein on a carrier without forming a covalent bond; A method of reacting the introduced active functional group for immobilization with the protein to form a covalent bond to immobilize it on a carrier can be mentioned. As a method for immobilizing a protein on a carrier without forming a covalent bond, an avidin-biotin affinity bond is used, and a biotin-introduced protein is immobilized with avidin such as Streptavidin Sepharose High Performance (manufactured by Cytiva). A method of immobilization on a carrier can be exemplified. Methods for introducing biotin into proteins include a method of reacting a biotinylation reagent having an active ester group such as 9-(biotinamide)-4,7-dioxanonanoic acid-N-succinimidyl with an amino group of the protein; A method of reacting a biotinylation reagent having a maleimido group such as biotinyl-N'-[2-(N-maleimido)ethyl]piperazine hydrochloride with a mercapto group of a protein can be exemplified. In addition, as a method of immobilizing by reacting an active functional group for immobilization introduced into a protein with a carrier to form a covalent bond, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N - The active ester group of a compound having both a maleimide group and an active ester group, such as hydroxysuccinimide ester sodium salt, is reacted with the amino group of the protein to introduce the maleimide group into the protein, followed by reaction with the carrier into which the mercapto group has been introduced. can be exemplified. Furthermore, as a method of immobilizing the protein by reacting the active functional group for immobilization introduced into the carrier, an epoxy group, a formyl group, a carboxyl group, an active ester group such as an N-hydroxysuccinimide ester introduced into the carrier and the protein can be immobilized. , a method of reacting an amino group introduced into a carrier with a carboxyl group of a protein, and a method of reacting an epoxy group, maleimide group, haloacetyl group or haloalkyl group introduced into a carrier with a mercapto group of a protein. can do. Among these immobilization methods, a method of reacting a formyl group or an active ester group introduced into a carrier with an amino group of a protein, and a method of reacting a protein with an amino group in the support, in that the protein can be immobilized onto the carrier in a short period of time and at a high yield. A method of reacting a maleimide group or a haloacetyl group introduced into the carrier with a mercapto group of the protein is preferred, and the immobilization reaction can be carried out at around neutral pH and protein denaturation can be suppressed. A method of reacting a maleimide group or a haloacetyl group introduced into a carrier with a mercapto group of a protein is more preferred, and a method of reacting a maleimide group introduced into a carrier with a mercapto group of a protein is more preferred in terms of high stability of the functional group. .

The adsorbent in the cell purification method of the present invention can be produced by reacting the carrier into which the immobilization functional group has been introduced and the fucose-binding protein dissolved in a buffer. The buffer for dissolving the fucose-binding protein is not particularly limited, and may be acetate buffer, phosphate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfone. Commercially available buffers such as acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and D-PBS(−) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) can be exemplified. For the purpose of increasing the efficiency of the immobilization reaction, an inorganic salt such as sodium chloride or a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added to the buffer.

The reaction temperature and pH at which the fucose-binding protein is immobilized on the carrier are determined in consideration of the reactivity of the active functional group and the stability of the fucose-binding protein. may be appropriately set within the range of pH 4 or higher and pH 10 or lower, and from the viewpoint of suppressing the deactivation of the fucose-binding protein, the reaction temperature should be in the range of 15°C or higher and 40°C or lower, and the pH should be in the range of pH 5 or higher and pH 9 or lower. preferable. The amount of fucose-binding protein immobilized on the water-insoluble carrier may be appropriately set in consideration of the binding property between the cells to be purified and the fucose-binding protein in the cell purification method of the present invention. It is preferably 0.01 mg or more and 10 mg or less, more preferably 0.05 mg or more and 5 mg or less per carrier.

In addition, the amount of fucose-binding protein immobilized on the carrier can be appropriately adjusted by adjusting the amount of fucose-binding protein used during the immobilization reaction and the amount of active functional group introduced into the carrier. The amount of fucose-binding protein immobilized on the carrier was determined by recovering the immobilization reaction solution and the post-reaction washing solution to determine the amount of unreacted fucose-binding protein, and then determining the amount of fucose-binding protein used in the immobilization reaction. can be calculated by subtracting the amount of unreacted fucose-binding protein from In addition, as described above, the carrier used as the adsorbent in the cell purification method of the present invention preferably has a hydrophilic polymer fixed by a covalent bond in order to suppress non-specific adsorption of cells. When producing an adsorbent, it is also possible to immobilize a hydrophilic polymer on the carrier with a covalent bond before introducing the functional group for immobilizing the fucose-binding protein in step Y1. The method for covalently immobilizing the hydrophilic polymer on the carrier is not particularly limited as long as it is a general covalent bond formation reaction. A method of introducing an epoxy group into a carrier by reacting an epoxy group-containing compound such as 4-butanediol diglycidyl ether under basic conditions, and then reacting the epoxy groups with the hydroxyl groups of the hydrophilic polymer under basic conditions. can be mentioned.

6. Method of Contacting Sample Liquid with Adsorbent In step (Z1), a sample liquid containing cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc and a fucose-binding protein are added to water. The method of contacting the adsorbent immobilized on an insoluble carrier is not particularly limited as long as a complex in which the cells and the adsorbent are bound can be obtained. For example, the adsorbent is added to a container containing the sample solution. Alternatively, the sample liquid may be added to a container containing the adsorbent and contacted. Moreover, in order to improve the contact efficiency between the sample liquid and the adsorbent, the container containing the sample liquid and the adsorbent may be stirred or shaken. The shape of the container is not particularly limited, and examples thereof include a container with an open top, such as a glass tube, a plastic tube, a Petri dish, and a plate having 6 to 96 wells. Further, the sample liquid may be added to the column filled with the adsorbent and brought into contact therewith.

The shape of the column is not particularly limited, and examples thereof include a column whose upper end is open and a column whose both ends are closed. Among the above-mentioned contact methods, it is possible to avoid re-release of cells bound to the adsorbent and damage to the cells due to excessive contact with the adsorbent. The same applies to steps (Z2) and (Z3) to be described later. In addition, there are no particular restrictions on the shape of the column, such as the inner diameter and length, and it may be set appropriately according to the number of cells described above. A cylindrical column is preferable in that it can be suppressed. Furthermore, the material of the column is not particularly limited as long as non-specific adsorption of cells to the column does not occur, and synthetic polymer columns such as polypropylene, polystyrene, polycarbonate, etc., and glass columns can be used. In addition, a commercially available column may be used, for example, a Terumo syringe (manufactured by Terumo) and an injection needle may be used in combination.

The adsorbent in the cell purification method of the present invention has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, provided that the immobilized amount of fucose-binding protein is 0.05 mg or more and 5 mg or less per 1 mL of the adsorbent. At least 1,000,000 cells having fucose-containing sugar chains, such as sugar chains containing Therefore, the amount of adsorbent used in the cell purification method of the present invention, that is, the amount packed into the column, depends on the number of cells bound to the adsorbent and Fucα1-2Galβ1- Considering the number of cells having a sugar chain containing a structure consisting of 3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, it may be set as appropriate. In addition, the amount of the sample solution used in step (Z1) is not particularly limited, and may be appropriately selected depending on the shape and capacity of the column described above. From the viewpoint of increasing the contact efficiency of cells having a sugar chain containing a structure, the amount is preferably 0.01 mL or more and 100 mL or less, more preferably 0.05 mL or more and 50 mL or less per 1 mL of the adsorbent swollen with water. Further, the temperature at which the sample liquid and the adsorbent are brought into contact is preferably 2° C. or higher and 40° C. or lower, more preferably 2° C. or higher and 30° C. or lower, in order to suppress destruction of cells. In addition, the contact time between the sample liquid and the adsorbent is preferably 1 minute or more and 60 minutes or less, more preferably 1 minute or more and 30 minutes or less, in that the working time can be shortened by obtaining the complex in a short time.

7. Process (Z2)
Step (Z2) in the cell purification method of the present invention is a step of separating and removing cells that did not bind to the adsorbent from the complex obtained in step (Z1), more specifically, step (Z1 ), a separation step of separating the complex obtained in step 1) from the sample solution, and a washing step of washing the complex with a washing liquid.

8. Separation step in step (Z2) The separation method in the separation step of step (Z2) is not particularly limited as long as it is a separation method in the separation and purification operation of biological substances such as cells and proteins. You can choose. Specifically, a method of separating and removing cells that did not bind to the adsorbent contained in the liquid using a pipette or the like from a mixture of the sample liquid and the adsorbent in a container, and a column filled with the adsorbent By removing the sample solution added to the bottom of the column, the cells not bound to the adsorbent can be separated and removed.

9. Washing Step in Step (Z2) The washing step in step (Z2) is a step of washing the complex obtained in the separation step using a washing liquid. By performing the washing step, it is possible to remove cells that have not bound to the adsorbent and contaminants in the sample solution that have bound to the adsorbent. The washing solution used in the washing step in the step (Z2) is not particularly limited as long as the cells are not released from the complex obtained in the step (Z1). The medium and buffer contain osmotic pressure regulators such as inorganic salts and sugar alcohols to prevent cell destruction, and substances to prevent cell aggregation such as proteins, chelating agents, and nonionic surfactants. An aqueous solution can be exemplified. Suitable concentrations of these substances are as described in "1. Sample solution" above.

The washing method in the washing step of step (Z2) is also not particularly limited as long as it is a washing method in the separation and purification of biological substances such as cells and proteins, and may be appropriately selected from methods commonly used by those skilled in the art. Specifically, the above-described washing solution is added to the container containing the complex obtained in step (Z1), and the washing solution is removed using a pipette or the like, or the complex obtained in step (Z1) is A method of adding the washing liquid described above to a packed column and removing it from the bottom of the column can be exemplified. In addition, the amount of the washing solution used in step (Z2) is not particularly limited. It is preferably 2 mL or more and 20 mL or less, more preferably 3 mL or more and 10 mL or less per 1 mL of the adsorbent swollen with water. In addition, the time and temperature for performing the operation of step (Z2) are not particularly limited, and the time may be appropriately adjusted within 1 minute or more and 60 minutes or less, preferably 1 minute or more and 30 minutes or less, and the temperature is 2°C. Above 40° C., preferably above 2° C. and below 30° C. may be appropriately adjusted.

10. Process (Z3)
The step (Z3) in the cell purification method of the present invention is a step of recovering the cells desorbed from the adsorbent by bringing the complex obtained in the step (Z1) into contact with a desorption liquid after the step (Z2). More specifically, by contacting the complex obtained in step (Z1) with a desorption solution containing fucose and/or fucose-containing sugar chains, Fucα1-2Galβ1-3GlcNAc and/or This is a step of detaching cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc and collecting the detached cells. The desorption liquid, the method for contacting the complex with the desorption liquid, the method for recovering the desorbed cells, and the method for analyzing the recovered cells are described below.

11. Desorption liquid The desorption liquid used in step (Z3) is composed of an adsorbent in which the fucose-binding protein is immobilized on a water-insoluble carrier in the complex obtained in step (Z1), Fucα1-2Galβ1-3GlcNAc and / Or binding to cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc, that is, a sugar chain containing a structure consisting of the aforementioned fucose-binding protein and Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc Used to dissociate the bond with Therefore, the desorption solution must contain fucose and/or fucose-containing sugar chains to which the above-described fucose-binding proteins have binding properties. Fucose-containing sugar chains such as H type 1 sugar chains, H type 3 sugar chains, Lewis X sugar chains, Lewis Y sugar chains, and Lewis b sugar chains, and glycoproteins and sugars to which these fucose-containing sugar chains are bound Must contain lipids.

Among these fucose and/or fucose-containing sugar chains, fucose and fucosyllactosamine, which are monosaccharides, are preferable, and fucose, which is a monosaccharide, is more preferable, because they are easily available in large amounts. The concentration of fucose in the desorption solution is not particularly limited as long as the cells are not destroyed and the cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc can be desorbed from the adsorbent. is preferably 0.01 mol/L or more and 1.0 mol/L or less, more preferably 0.05 mol/L or more and 0.5 mol/L or less.

The desorption solution used in step (Z3) is the above-mentioned fucose and/or fucose-containing sugar chain of cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc from the adsorbent. Substances other than fucose and/or fucose-containing sugar chains may be contained as long as they do not inhibit desorption. Examples include aqueous solutions containing osmotic pressure regulators such as inorganic salts and sugar alcohols to prevent destruction of cells, substances to prevent cell aggregation such as proteins, chelating agents, and nonionic surfactants. can. Suitable concentrations of these substances are as described in "1. Sample solution" above. The desorption solution used in step (Z3) preferably contains a protein such as HSA or BSA in terms of reducing damage to cells and suppressing non-specific adsorption of cells to the adsorbent. It is preferable that a chelating agent is contained in terms of aggregation prevention. Further, the desorption solution may be a commercially available buffer solution to which fucose and/or fucose-containing sugar chains are added. Those with added chains can be mentioned. The buffer solution, osmotic pressure regulator, and substance for preventing aggregation of cells preferably do not exhibit cytotoxicity when culturing cells purified by the cell purification method of the present invention.

12. Method for Contacting Desorbed Liquid and Complex and Method for Collecting Detached Cells The method for contacting the desorbed liquid and the complex and the method for collecting desorbed cells in the step (Z3) is to remove Fucα1-2Galβ1-3GlcNAc and/or Fucα1- from the adsorbent. There is no particular limitation as long as cells having a sugar chain containing a structure consisting of 2Galβ1-3GalNAc are detached and the cells can be recovered. A method of recovering a suspension containing cells desorbed from the adsorbent using a pipette or the like by adding the desorption solution obtained by adding the desorption solution described above to the column filled with the complex washed in step (Z2). is added to collect a suspension containing detached cells from the bottom of the column.

The amount of desorption solution used in step (Z3) is not particularly limited. 2 mL or more and 20 mL or less, preferably 3 mL or more and 10 mL or less per 1 mL of the water-swollen adsorbent. Furthermore, the time and temperature for performing the operation in step (Z3) are not particularly limited, and the time may be appropriately adjusted within 1 minute or more and 60 minutes or less, preferably 1 minute or more and 30 minutes or less, and the temperature is 2°C. Above 40° C., preferably above 2° C. and below 30° C. may be appropriately adjusted.

The number of times the desorption solution is added in step (Z3) is as long as cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc can be desorbed from the adsorbent and collected. There is no particular limitation, and the desorption solution containing fucose and/or fucose-containing sugar chains at a specific concentration may be added once or multiple times, and the desorption solution containing different concentrations of fucose and/or fucose-containing sugar chains may be added multiple times. may be added.

13. Method for analyzing collected cells In step (Z3), cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are collected by the method described above. Confirmation of whether or not the cell has a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc may be appropriately selected from methods commonly used by those skilled in the art, such as rBC2LCN-FITC, After staining the cells with a fluorescence-labeled BC2LCN lectin such as rBC2LCN-547 and rBC2LCN-635 (both manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), the BC2LCN positive rate calculated by a conventional immunoassay method can be confirmed. . In addition, both the cells in the sample solution in step (Z1) and the cells collected in step (Z3) were stained with a fluorescently labeled BC2LCN lectin, and a conventional immunoassay and/or flow cytometer was used. The degree of purification of the collected cells can be evaluated by analyzing the cells and comparing the BC2LCN positive rate. As shown in the examples below, human pluripotent stem cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are purified from 90% or more BC2LCN-positive human pluripotent stem cells by the cell purification method of the present invention. can be refined at a rate In addition, cancer cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc can be purified with a BC2LCN positive rate of 70% or more by the cell purification method of the present invention. In addition, when the cells to be purified are pluripotent stem cells such as human iPS cells, the cells in the sample solution in step (Z1) and the cells collected in step (Z3) are treated with undifferentiated markers other than fluorescently labeled BC2LCN lectin. After staining the cells with a fluorescently-labeled antibody against such as a fluorescently-labeled anti-TRA-1-60 antibody, a fluorescently-labeled anti-SSEA-3 antibody, a fluorescently-labeled anti-SSEA-4 antibody, etc., it is analyzed by a conventional immunoassay method. By doing so, the difference in undifferentiated degree between the cells in the sample solution and the collected cells can be evaluated. After analyzing the expression level of sugar chains containing structures composed of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc in the cells collected in step (Z3) by these methods and the undifferentiated marker positive rate, differentiation induction and Only necessary fractions of the cells collected in step (Z3) may be aggregated and used for induction of differentiation, reculturing, etc., depending on the intended use of the cells after purification such as reculture.

In the cell purification method of the present invention, human pluripotent stem cells such as human iPS cells that can be induced to differentiate into therapeutic cells used in regenerative medicine can be purified by a simple method. Specifically, highly undifferentiated pluripotent stem cells can be obtained by removing low-undifferentiated pluripotent stem cells contained in human pluripotent stem cells. In particular, according to the cell purification method of the present invention, a fucose-binding protein having binding properties to H-type 1 sugar chains and/or H-type 3 sugar chains, which are undifferentiated sugar chain markers, is added to a water-insoluble carrier. By using an immobilized adsorbent, it is possible to purify a large amount of human pluripotent stem cells in a short period of time, compared to the conventional method for purifying pluripotent stem cells using a flow cytometer/cell sorter.

By culturing the purified human pluripotent stem cells and inducing differentiation into target cells, therapeutic cells and tissues used in regenerative medicine can be produced. As described above, the adsorbent in the cell purification method of the present invention is Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1- if the immobilized amount of fucose-binding protein is 0.05 mg or more and 5 mg or less per 1 mL of the adsorbent. It is possible to bind approximately one million cells having fucose-containing sugar chains such as sugar chains containing a structure consisting of 3GalNAc. Assuming that 100 million human iPS cells are required for the production of therapeutic cells and tissues used in regenerative medicine, 100 mL of the adsorbent can be used to purify 100 million human iPS cells.

In addition, by the cell purification method of the present invention, iPS cells produced from patients with intractable rare diseases, which are useful in drug discovery screening, can be highly purified. Furthermore, according to the cell purification method of the present invention, it is also possible to purify cancer cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the cell surface. For example, cancer cells having the sugar chains on their cell surfaces can be selectively recovered from a sample containing cells collected from a subject, and the presence or absence and amount of cancer cells can be identified.

1 is a diagram showing a method for purifying human iPS cells using an adsorbent in Examples 1 and 2. FIG. 10 is a graph showing the undifferentiated marker-positive rate of iPS cells in the iPS cell suspension added to the adsorbent, the outflow cell fluid, and each desorbed cell fluid in Example 3. FIG. 10 is a graph showing the undifferentiated marker-positive rate of cells contained in the cell suspension added to the adsorbent, the effluent cell sap, and each desorbed cell sap in Example 3. FIG. Graph showing undifferentiated marker positive rate of iPS cells in cell suspension containing undifferentiated deviant cells added to adsorbent and iPS cells contained in outflow cell fluid and desorbed cell fluid in Example 7 is. Graph showing undifferentiated marker positive rate of iPS cells in cell suspension containing undifferentiated deviant cells added to adsorbent and iPS cells contained in outflow cell fluid and desorbed cell fluid in Example 8 is.

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

Preparation Example 1 Preparation of Adsorbent 127C72G and Adsorbent 127Q39L/C72G After producing a fucose-binding protein 127C72G (amino acid sequence represented by SEQ ID NO: 7) according to the method described in Example 12 of JP-A-2020-025535, Adsorbent 127C72G comprising fucose-binding protein 127C72G immobilized on a water-insoluble carrier was produced according to the method described in Example 26 of the publication. Further, after producing the fucose-binding protein 127Q39L/C72G (amino acid sequence represented by SEQ ID NO: 8) according to the method described in Example 33 of JP-A-2020-025535, the method described in Example 34 of the same publication. An adsorbent 127Q39L/C72G was prepared according to the method, in which the fucose-binding protein 127Q39L/C72G was immobilized on a water-insoluble carrier.

Example 1 Purification of Human iPS Cells Using Adsorbent 127C72G It concerns refining. In Examples 1 to 4, Example 7, Example 8 and Comparative Example 1, human iPS cells 201B7 strain (hereinafter abbreviated as 201B7 cells) purchased under license from iPS Academia Japan, Inc. were used as human iPS cells. in some cases) was used.

(1) Preparation of column filled with adsorbent 127C72G A column equipped with a 40 μM hydrophilic nylon mesh filter (manufactured by Corning) between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G). was made. Next, after substituting the adsorbent 127C72G produced in Preparation Example 1 with MACS buffer (manufactured by Miltenyi Biotech), the adsorbent was adjusted so that the sedimentation volume of the adsorbent after standing for 12 hours or more was 50%. A 50% suspension was prepared, 1.0 mL of which was added to the prepared column, and the column was packed with adsorbent 127C72G (adsorbent capacity: 500 μL).

(2) Culture of 201B7 Cells and Preparation of Cell Suspension 201B7 cells were cultured by the following method. A solution obtained by diluting iMatrix-511 (manufactured by Nippi) at a rate of 3 μg/mL in D-PBS(-) (manufactured by Cell Science Research Institute) is added to a petri dish for adhesion culture (manufactured by Corning) and incubated at 4° C. overnight or more. By allowing to stand, the culture surface of the petri dish was coated with iMatrix-511. Next, StemFit AK02N medium (manufactured by Ajinomoto) was added to wash the petri dish, and then 201B7 cells thawed from the frozen vial were suspended in the same medium supplemented with 10 μM of a lock inhibitor (Y-27632: manufactured by Fujifilm Wako Pure Chemical Industries). Seeded in turbidity. After culturing overnight at 37°C in a 5% CO2 atmosphere in a CO2 incubator, the medium was changed to StemFit AK02N medium containing no lock inhibitor, and when the cell density reached an appropriate level, cells were harvested and subcultured. .

Next, 201B7 cells were fluorescently stained using Cell Tracker Orange (manufactured by Thermo Fisher Scientific) by the following method. After culturing the 201B7 cells, the medium in the petri dish was discarded, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to wash the cells, and then the medium was aspirated and discarded. Next, a solution obtained by dissolving Cell Tracker Orange in serum-free RPMI 1640 medium at a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, StemFit AK02N medium was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the medium, StemFit AK02N medium was added and cultured overnight at 37°C in a 5% CO2 atmosphere. Next, cell collection and cell suspension preparation were performed by the following methods. After repeating the operation of adding D-PBS (-) to the petri dish to wash the cells and discarding D-PBS (-) twice, CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) and Versene Solution ( (Thermo Fisher Scientific) was added and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere. After confirming that the cells were detaching from the culture surface, the cells were detached from the culture surface by repeating pipetting and collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating this washing operation twice, the cells were resuspended in MACS buffer and filtered using a cell strainer (manufactured by Corning, opening 40 μm) to obtain a cell suspension of 201B7 cells stained with Cell Tracker Orange. A liquid was prepared. A portion of the resulting 201B7 cell suspension was collected, diluted 10-fold, and then the cell density was calculated using a hemocytometer. The number of cells added to the column was calculated by dividing by .

The undifferentiated marker-positive rate of 201B7 cells obtained by the above method was measured by the following method. 1 mL of the 201B7 cell suspension was placed in a 1.5 mL Eppendorf tube (manufactured by Eppendorf), the supernatant was discarded by centrifugation, and the cells were washed by resuspending in 1 mL of MACS buffer. . After centrifugation again, the resulting cell precipitate was suspended in 1 mL of MACS buffer containing 0.5% (vol./vol.) BC2LCN-635 (manufactured by Fujifilm Wako Pure Chemical Industries) and placed in the dark. Cells were stained by incubating for 30 minutes at room temperature. After the reaction, the cells were washed with MACS buffer in an Eppendorf tube in the same manner as above, and the resulting cell precipitate was suspended in a total of 2 mL of MACS buffer (suspended twice with 1 mL of MACS buffer). ). After transferring 2 mL of the cell suspension to a 5 mL polystyrene round tube (manufactured by Corning), 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as internal standard beads for counting cells and 7- as a cell viability determination reagent 50 μL of AAD was added, and the fluorescence intensity of the cell suspension was analyzed using a cell sorter (FACSAria: manufactured by Becton Dickinson). Cells unstained with BC2LCN-635 are defined as negative cell populations, cell populations with increased fluorescence intensity due to reaction with BC2LCN-635 are defined as positive viable cell populations, and the number of cells in the positive viable cell population is divided by the total viable cell count. The undifferentiated marker positive rate (BC2LCN positive rate) of 201B7 cells calculated by the above was 97.5%. Table 1 shows the undifferentiated marker positive rate of cultured 201B7 cells.

(3) Purification of 201B7 cells Suspension of 201B7 cells prepared in (2) above, with the column prepared in (1) upright, so that the number of added cells is 4.0×10 ⁶ . A liquid (0.5 mL) was added from the top of the column to obtain a complex in which the adsorbent and 201B7 cells were bound (corresponding to step (Z1) of the cell purification method of the present invention). Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container (corresponding to the step (Z2) of the cell purification method of the present invention). Next, a MACS buffer solution (8.0 mL) containing 0.2 mol/L fucose is added as a desorption solution from the top of the column, and the complexes of the desorption solution, the adsorbent, and 201B7 cells are brought into contact with each other. , Desorbed cell solution (8.0 mL) containing cells desorbed from the adsorbent from the column bottom (desorbed cells) was collected in a container different from the above operation (corresponding to step (Z3) of the cell purification method of the present invention. ).

(4) Measurement of cell outflow rate and undifferentiated marker-positive rate of outflowing cells The outflow rate of outflowing cells (cell outflow rate) in (3) above was measured by the following method. The effluent cell fluid (2 mL) obtained by the operation of (3) was collected in a 5 mL polystyrene round tube (manufactured by Corning, opening 40 μm) with a cell strainer cap, and CountBright was used as an internal standard bead for counting cells. After adding 50 μL of Absolute Counting Beads and 50 μL of 7-AAD, the number of cells in the effluent cell solution was measured using a cell sorter (FACSAria). The number of viable cells contained in the effluent cell fluid is calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting, and this number of viable cells is divided by the number of added cells described in (3) above. The cell outflow rate calculated by was 0.6%. In addition, the undifferentiated marker positive rate of outflow cells calculated by the method described in (2) above was 77.8%. Therefore, the 201B7 cells cultured by the method described in (2) above contain a cell population with a low undifferentiated marker positive rate, and the 201B7 cells cultured by the cell purification method of the present invention using the adsorbent 127C72G It was found that cells with a low undifferentiated marker-positive rate can be removed. Table 1 shows the cell outflow rate and the undifferentiated marker-positive rate of the outflow cells.

(5) Measurement of Cell Detachment Rate and Undifferentiated Marker Positive Rate of Detached Cells The detachment rate of detached cells (cell detachment rate) in (3) above was measured by the following method. Using the desorbed cell solution obtained in the operation (3) above, the cell detachment rate was calculated by the same method as the measurement of the cell outflow rate in (4) above, resulting in a cell detachment rate of 91.9%. . In addition, the undifferentiated marker positive rate of detached cells calculated by the method described in (2) above was 99.8%. As a result, the undifferentiated marker positive rate of 201B7 cells purified by the cell purification method of the present invention is higher than that of 201B7 cells before purification. It was found that human iPS cells can be highly purified. Table 1 shows the cell detachment rate and the undifferentiated marker-positive rate of the detached cells.

(6) Culture of effluent cells and desorbed cells and measurement of undifferentiated marker positive rate Using the effluent cell sap and desorbed cell sap (each 1 mL) collected in (3), effluent cells by the method described in (2) above After culturing the detached cells for 9 days, the undifferentiated marker-positive rate of the outflow cells and detached cells after culture was calculated by the method described in (2) above. In addition, the undifferentiated marker positive rate after culturing, in addition to the BC2LCN positive rate using the BC2LCN-635, TRA-1-60 using anti-human TRA-1-60 Alexa Fluor 488 antibody (manufactured by R&D Systems) The 1-60 positive rate was also calculated. Cell staining with the anti-human TRA-1-60 Alexa Fluor 488 antibody was performed in the same manner as with BC2LCN-635. The BC2LCN positive rate and the TRA-1-60 positive rate of the outflow cells calculated by the above method were 87.5% and 87.5%, respectively. On the other hand, the BC2LCN-positive rate and the TRA-1-60-positive rate of detached cells calculated by the above method were 99.8% and 98.1%, respectively. Therefore, it was revealed that detached cells, that is, purified human iPS cells can maintain a high degree of undifferentiation even when cultured. In addition, it was clarified that culturing outflow cells with a low degree of undifferentiation increases the degree of undifferentiation, but the undifferentiation marker-positive rate is not as high as that of purified human iPS cells. Table 1 shows the undifferentiated marker-positive rate of outflow cells and detached cells after culture.

Example 2 Purification of human iPS cells using adsorbent 127Q39L/C72G-1
Example 2 relates to purification of human iPS cells using adsorbent 127Q39L/C72G.

(1) Culture of 201B7 Cells and Preparation of Cell Suspension Culture of 201B7 cells and preparation of cell suspension were performed by the method described in Example 1 (2). The undifferentiated marker positive rate (BC2LCN positive rate) of the cultured 201B7 cells was 97.5%. Table 2 shows the undifferentiated marker positive rate of cultured 201B7 cells.

(2) Purification of 201B7 cells A column (adsorbent capacity: 500 μL) filled with the adsorbent 127Q39L/C72G prepared in (1) of Example 1 was placed vertically, and the number of added cells was 4.0 × 10. The 201B7 cell suspension (0.5 mL) prepared in (1) above was added from the top of the column so that there were ⁶ cells, to obtain a complex in which the adsorbent and 201B7 cells were bound. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (8.0 mL) containing 0.2 mol/L fucose is added as a desorption solution from the top of the column, and the complexes of the desorption solution, the adsorbent, and 201B7 cells are brought into contact with each other. A desorbed cell solution (8.0 mL) containing cells (desorbed cells) desorbed from the adsorbent from the bottom of the column was collected in a container different from the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the method described in (4) of Example 1, the efflux rate of effluent cells (cell efflux rate) in (2) above was calculated. The cell efflux rate was 1.0%. In addition, the undifferentiated marker-positive rate of outflow cells calculated by the method described in Example 1 (2) was 46.6%. Therefore, in the cell purification method of the present invention, it was clarified that even when the adsorbent 127Q39L/C72G was used, cells with a low undifferentiated marker-positive rate could be removed from the cultured 201B7 cells. Table 2 shows the cell outflow rate and the undifferentiated marker-positive rate of the outflow cells together with the results of Example 1.

(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells By the method described in (5) of Example 1, the detachment rate of detached cells (cell detachment rate) in (2) above was calculated. The cell detachment rate was 97.5%. In addition, the undifferentiated marker positive rate of the detached cells calculated by the method described in Example 1 (2) was 99.8%. As a result, it was revealed that human iPS cells, which are human pluripotent stem cells, can be highly purified even when the adsorbent 127Q39L/C72G is used in the cell purification method of the present invention. Table 2 shows the cell detachment rate and the undifferentiated marker-positive rate of the detached cells together with the results of Example 1.

(5) Cultivation of effluent cells and desorbed cells and measurement of undifferentiated marker positive rate By the same method as in (6) of Example 1, the effluent cell fluid and desorbed cell fluid (1 mL each) collected in (2) above were After culturing the outflow cells and desorbed cells for 9 days, the undifferentiated marker positive rate of the outflow cells and desorbed cells after culture was calculated by the method described in (2) of Example 1. As a result, the outflow cells were BC2LCN positive. The rate was 90.7% and the TRA-1-60 positive rate was 88.9%. On the other hand, the detached cells had a BC2LCN positive rate of 99.8% and a TRA-1-60 positive rate of 98.1%. Therefore, it was revealed that human iPS cells purified using the adsorbent 127Q39L/C72G can maintain a high degree of undifferentiation even when cultured. Table 2 shows the undifferentiated marker-positive rate of outflow cells and detached cells after culture together with the results of Example 1.

Comparative Example 1 Purification of Human iPS Cells Using Adsorbent 155 In Comparative Example 1, the amino acid sequence of recombinant BC2LCN consisting of 155 amino acid residues shown in SEQ ID NO: 1 has a polyhistidine sequence at the N-terminus and a cysteine at the C-terminus. Recombinant BC2LCN(155)cys added with an oligopeptide sequence containing BC2LCN(155)cys is related to the purification of human iPS cells using an adsorbent 155 in which it is immobilized on a water-insoluble carrier.

(1) Preparation of adsorbent 155 and column packed with adsorbent 155 Recombinant BC2LCN (155) cys (amino acid sequence shown by SEQ ID NO: 10) was prepared according to the method described in Comparative Example 1 of JP-A-2020-025535. After production, according to the method described in Reference Example 3 of the publication, an adsorbent 155 was produced in which recombinant BC2LCN(155)cys was immobilized on a water-insoluble carrier. Also, a column filled with the adsorbent 155 was prepared by the method described in (1) of Example 1 (capacity of adsorbent: 500 μL).

(2) Culture of 201B7 Cells and Preparation of Cell Suspension Culture of 201B7 cells and preparation of cell suspension were performed by the method described in Example 1 (2). The undifferentiated marker positive rate of the cultured 201B7 cells was 96.2% for BC2LCN positive rate and 98.5% for TRA-1-60 positive rate. Table 3 shows the undifferentiated marker positive rate of cultured 201B7 cells.

(3) Purification of 201B7 cells using adsorbent 155 Prepared in (1) so that the number of cells added is 5.0 × 10 ⁶ with the column prepared in (1) standing vertically A suspension of 201B7 cells (0.5 mL) was added from the top of the column to obtain a complex in which the adsorbent and 201B7 cells were bound. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container.

(4) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the method described in (4) of Example 1, the efflux rate of effluent cells (cell efflux rate) in (3) above was calculated. The cell efflux rate was 8.0%. In addition, the undifferentiated marker positive rate of outflow cells calculated by the method described in (2) of Example 1 was 93.7% for BC2LCN positive rate and 93.5% for TRA-1-60 positive rate. . Comparing the results of Examples 1 and 2 with the results of Comparative Example 1, the cell outflow rate was 1% or less in Examples 1 and 2, whereas it was 8% in Comparative Example 1. In addition, while the undifferentiated marker-positive rate of outflow cells is 80% or less in Examples 1 and 2, it is 90% or more in Comparative Example 1. Therefore, in the cell purification method of the present invention, when the adsorbent 155 is used, cells with a high undifferentiated marker-positive rate flow out in the step (Z2) of the cell purification method of the present invention. It became clear that it could not be purified to Table 3 shows the cell outflow rate, the undifferentiated marker positive rate of the cultured 201B7 cells and outflow cells in Examples 1, 2 and Comparative Example 1.

Example 3 Purification of human iPS cells using adsorbent 127Q39L/C72G-2
Example 3 relates to the purification of human iPS cells using adsorbent 127Q39L/C72G and desorption solutions with different concentrations of fucose.

(1) Culture of 201B7 Cells and Preparation of Cell Suspension Culture of 201B7 cells and preparation of cell suspension were performed by the method described in Example 1 (2). The undifferentiated marker positive rate of the cultured 201B7 cells was 98.9% for BC2LCN positive rate and 95.6% for TRA-1-60 positive rate. Table 4 and FIG. 2 show the undifferentiated marker positive rate of cultured 201B7 cells.

(2) Purification of 201B7 cells using desorption solutions with different fucose concentrations A column (adsorbent capacity: 500 μL) filled with the adsorbent 127Q39L/C72G prepared in (1) of Example 1 was vertically placed, The 201B7 cell suspension (0.25 mL) prepared in (1) above was added from the top of the column so that the number of added cells was 1.2 × 10 ⁷ cells, and the adsorbent and 201B7 cells bound to each other. got a body Next, MACS buffer (3.75 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (6.5 mL) containing 0.08 mol/L fucose was added as the desorption liquid-1 from the top of the column, and the desorption liquid-1 was brought into contact with the complex, thereby removing the Desorbed cell sap-1 (6.5 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from the above operation. Next, a MACS buffer solution (10.0 mL) containing 0.18 mol/L fucose was added from the top of the column as the desorption liquid-2, and the complex was brought into contact with the desorption liquid-2, thereby removing the Desorbed cell solution-2 (10.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from that used in the above operation. Finally, MACS buffer (8.0 mL) containing 0.50 mol/L fucose is added as desorbing liquid-3 from the top of the column, and the complex is brought into contact with desorbing liquid-3 to remove the complex from the bottom of the column. Desorbed cell suspension-3 (8.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the method described in (4) of Example 1, the efflux rate of effluent cells (cell efflux rate) in (2) above was calculated. The cell efflux rate was 1.0%. In addition, the undifferentiated marker positive rate of outflow cells calculated by the method described in (2) of Example 1 was 69.1% for BC2LCN positive rate and 51.2% for TRA-1-60 positive rate. . Table 4 shows the cell outflow rate, and Table 4 and FIG. 2 show the undifferentiated marker-positive rate of the outflow cells.

(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells By the method described in (5) of Example 1, the cell detachment rate of detached cell sap-1 to detached cell sap-3 in (2) above As a result, the cell detachment rate of desorbed cell sap-1 was 1.2%, the cell detachment rate of desorbed cell sap-2 was 92.8%, and the cell detachment rate of desorbed cell sap-3 was 3.6%. there were. In addition, the undifferentiated marker positive rate of desorbed cells in each desorbed cell liquid calculated by the method described in (2) of Example 1 was 72.7% for BC2LCN positive rate for desorbed cell liquid-1 and TRA-1. -60 positive rate was 75.0%, desorbed cell sap-2 had a BC2LCN positive rate of 99.1%, TRA-1-60 positive rate was 97.1%, desorbed cell sap-3 had a BC2LCN positive rate of 91.0%. 7%, and the TRA-1-60 positive rate was 92.4%. From the above results, the complex obtained by contacting the adsorbent 127Q39L/C72G with human iPS cells is contacted with a desorbed solution having a fucose concentration higher than 0.08 mol / L and 0.18 mol / L or less. It was clarified that human iPS cells, which are human pluripotent stem cells, can be highly purified by collecting the desorbed cell suspension-2. Table 4 shows the cell detachment rate and undifferentiated marker-positive rate of detached cells in each desorbed cell sap, and FIG. 2 shows the undifferentiated marker-positive rate of detached cells in each desorbed cell sap.

Example 4 Purification of human iPS cells using adsorbent 127Q39L/C72G-3
Example 4 shows human iPS cells using adsorbent 127Q39L/C72G and human fetal striated muscle that does not have a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the cell surface. This relates to purification of human iPS cells from a mixture of tumor-derived cells (obtained from KAC; hereinafter sometimes abbreviated as RD cells).

(1) Culture of 201B7 Cells and RD Cells and Preparation of Cell Suspension Culture of 201B7 cells and preparation of cell suspension were performed by the method described in Example 1 (2). RD cells were cultured at 37° C. in a 5% CO 2 atmosphere using a cell growth medium (KAC), seeded in a petri dish for adherent culture (manufactured by Corning). Next, RD cells were fluorescently stained using Cell Tracker Orange by the following method. After discarding the medium in the petri dish in which the RD cells were being cultured, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to wash the cells, and then the medium was aspirated and discarded. Next, a solution obtained by dissolving Cell Tracker Orange in RPMI 1640 medium at a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, the cell growth medium was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the medium, the cell growth medium was added again, and the cells were cultured overnight at 37°C in a 5% CO2 atmosphere.

Next, cell collection and cell suspension preparation were performed by the following methods. After repeating the operation of adding D-PBS(-) to the Petri dish to wash the cells and discarding the D-PBS(-) twice, Accutase (manufactured by Innovative Cell Technology) was added and allowed to stand for several minutes. , the cells were detached from the culture surface by repeated pipetting and collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating this washing operation twice, the cells were resuspended in MACS buffer and filtered using a cell strainer (40 μm opening) to prepare a cell suspension of RD cells stained with Cell Tracker Orange. did.

A portion of the prepared 201B7 cell and RD cell suspensions was collected and diluted 10-fold, the cell density was calculated using a hemocytometer, and both cell suspensions were mixed and treated as described below. Used for purification of 201B7 cells using adsorbents. The undifferentiated marker positive rate of 201B7 cells calculated by the same method as in Example 1 (2) was 96.2% for BC2LCN positive rate and 94.6% for TRA-1-60 positive rate. Table 5 shows the undifferentiated marker positive rate of 201B7 cells contained in the 201B7 cell suspension.

(2) Purification of 201B7 cells using desorption solutions with different fucose concentrations A column (adsorbent capacity: 500 μL) filled with the adsorbent 127Q39L/C72G prepared in (1) of Example 1 was vertically placed, The mixed cell suspension of 201B7 cells and RD cells prepared in ( ¹ ) above ( ¹ . 0 mL) was added from the top of the column to obtain a complex in which the adsorbent and cells were bound. Next, MACS buffer (3.0 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (6.5 mL) containing 0.08 mol/L fucose was added as the desorption liquid-1 from the top of the column, and the desorption liquid-1 was brought into contact with the complex, thereby removing the Desorbed cell sap-1 (6.5 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from the above operation. Next, a MACS buffer solution (10.0 mL) containing 0.18 mol/L fucose was added from the top of the column as the desorption liquid-2, and the complex was brought into contact with the desorption liquid-2, thereby removing the Desorbed cell solution-2 (10.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from that used in the above operation. Finally, MACS buffer (8.0 mL) containing 0.50 mol/L fucose is added as desorbing liquid-3 from the top of the column, and the complex is brought into contact with desorbing liquid-3 to remove the complex from the bottom of the column. Desorbed cell suspension-3 (8.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells 2 mL of the effluent cell solution obtained by the operation in (2) is aliquoted into a 5 mL polystyrene round tube (40 μm opening) with a cell strainer cap. Then, 50 μL of CountBright Absolute Counting Beads and 50 μL of 7-AAD were added as internal standard beads for counting cells, and the number of viable cells of 201B7 cells and RD cells contained in the effluent cell solution was measured using a cell sorter (FACSAria). did. The number of viable cells of each cell contained in the effluent cell fluid was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting, and the number of viable cells was calculated as the number of added cells described in (2) above. As a result of calculating the cell outflow rate of each cell by dividing, the cell outflow rate of 201B7 cells was 2.0%, and the cell outflow rate of RD cells was 87.0%. In addition, as a result of calculating the undifferentiated marker positive rate of 201B7 cells contained in the outflow cell fluid by the method described in (2) of Example 1, the BC2LCN positive rate was 0.1%, and the TRA-1-60 positive rate. was 2.4%. Table 5 shows the cell outflow rate, and Table 5 and FIG. 3 show the undifferentiated marker-positive rate of 201B7 cells contained in the outflow cell fluid.

(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells 201B7 cells and RD contained in detached cell sap-1 to detached cell sap-3 in (2) above by the same method as in (3) above As a result of calculating the cell detachment rate of the cells, the cell detachment rate of the 201B7 cells in the desorbed cell sap-1 was 0.9%, and the cell detachment rate of the RD cells was 2.4%. In the desorbed cell sap-2, the cell detachment rate of 201B7 cells was 70.4%, and the cell detachment rate of RD cells was 1.1%.

In addition, the cell detachment rate of 201B7 cells and RD cells in detached cell suspension-3 was 10.4% and 0.3%, respectively. Furthermore, by the method described in (2) of Example 1, the undifferentiated marker positive rate of 201B7 cells contained in desorbed cell sap-1 to desorbed cell sap-3 in (2) above was calculated. -1 had a BC2LCN positive rate of 38.5% and TRA-1-60 positive rate of 49.6%, and desorbed cell sap-2 had a BC2LCN positive rate of 97.2% and a TRA-1-60 positive rate of 96.5%. 4%, desorbed cell sap-3 had a BC2LCN positive rate of 92.4%, and a TRA-1-60 positive rate of 93.5%.

From the above results, human iPS cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the cell surface and RD cells not having the sugar chain on the cell surface are included. Desorbed cell sap-2 obtained by contacting the complex obtained by contacting the sample liquid with the adsorbent 127Q39L/C72G with a desorbed liquid having a fucose concentration higher than 0.08 mol/L and 0.18 mol/L or less. It was clarified that human iPS cells, which are human pluripotent stem cells having the sugar chain, can be highly purified by collecting the sugar chains. Table 5 shows the cell detachment rate and undifferentiated marker positive rate of 201B7 cells for each desorbed cell sap, and FIG. 3 shows the undifferentiated marker positive rate of 201B7 cells contained in each desorbed cell sap.

Example 5 Purification of cancer cells using adsorbent 127Q39L/C72G-1
Example 5 shows human lung adenocarcinoma-derived cells (PC-9 cells, obtained from DS Pharma Biomedical, ECACC strain number: 90071810).

(1) Culture of PC-9 cells and preparation of cell suspension PC-9 cells were added with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries). Using RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), cells were seeded in petri dishes for adhesion culture (manufactured by Corning) and cultured at 37° C. in a 5% CO 2 atmosphere. Next, PC-9 cells were fluorescently stained using Cell Tracker Orange by the following method. After culturing the PC-9 cells, the medium in the petri dish was discarded, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to wash the cells, and then the medium was aspirated and discarded. Next, a solution obtained by dissolving Cell Tracker Orange in serum-free RPMI 1640 medium at a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, RPMI 1640 medium supplemented with 10% FBS and antibiotic solution was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the medium, RPMI 1640 medium supplemented with 10% FBS and the antibiotic solution was added, and cultured overnight at 37°C in a 5% CO2 atmosphere. PC-9 cells stained with Cell Tracker Orange were detached from the culture surface using Accutase in the same manner as for RD cells described in Example 4, washed with MACS buffer, and the cell suspension was removed. prepared. A portion of the obtained PC-9 cell suspension was collected, diluted 10-fold, and then the cell density was calculated using a hemocytometer. Based on this cell density, the number of cells added to the column was determined. The number of cells added to the column was calculated by dividing by the cell density. As a result of calculating the BC2LCN positive rate of PC-9 cells by the same method as in Example 1 (2), the BC2LCN positive rate was 54.6%. Table 6 shows the BC2LCN positive rate of PC-9 cells.

(2) Purification of PC-9 cells A column (adsorbent capacity: 500 μL) filled with the adsorbent 127Q39L/C72G prepared in (1) of Example 1 was vertically placed, and the number of added cells was 2.7. The PC-9 cell suspension (0.5 mL) prepared in (1) above was added from the top of the column to obtain a complex of the adsorbent and PC-9 cells bound to each other so that the number of cells was ×10 ⁶ . rice field. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (8.0 mL) containing 0.4 mol/L fucose is added as a desorbing solution from the top of the column, and the desorbing solution is brought into contact with the complex in which the adsorbent and PC-9 cells are bound. As a result, the desorbed cell solution (8.0 mL) containing the cells (desorbed cells) desorbed from the adsorbent from the bottom of the column was collected in a container different from the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the method described in (4) of Example 1, the efflux rate of effluent cells (cell efflux rate) in (2) above was calculated. The cell efflux rate was 28.7%. In addition, the BC2LCN positive rate of outflow cells calculated by the method described in Example 1 (2) was 45.6%. Table 6 shows the cell outflow rate and the BC2LCN positive rate of outflow cells.

(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells By the method described in (5) of Example 1, the detachment rate of detached cells (cell detachment rate) in (2) above was calculated. The cell detachment rate was 70.0%. The BC2LCN positive rate of the detached cells calculated by the method described in Example 1 (2) was 75.0%. Therefore, according to the cell purification method of the present invention, a cell population having many sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the cell surface can be obtained from the cultured PC-9 cells. It became clear that Table 6 shows the cell detachment rate and the BC2LCN positive rate of detached cells.

Example 6 Purification of cancer cells using adsorbent 127Q39L/C72G-2
Example 6 uses adsorbent 127Q39L/C72G to produce 2102Ep cells, which are human embryonic carcinoma cells ( Embryonal Carcinoma Cells Cl.4/D3 cells (obtained from Cosmo Bio) and human mesenchymal stem cells (JCRB1133: human bone marrow-derived mesenchymal stem cells obtained from JCRB Cell Bank, which do not have the above-mentioned sugar chain on the cell surface). , sometimes abbreviated as hMSC).

(1) Cultivation of 2102Ep cells and hMSCs and preparation of cell suspension 2102Ep cells were added with 10% FBS and an antibiotic solution (penicillin-streptomycin solution) in D-MEM medium (high glucose, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ), the cells were seeded in a petri dish for adhesion culture (manufactured by Corning) with a diameter of 10 cm, and cultured at 37° C. in a 5% CO 2 atmosphere. Next, 2102Ep cells were fluorescently stained using Cell Tracker Green (manufactured by Thermo Fisher Scientific) by the following method. After discarding the medium in the petri dish in which the 2102Ep cells were being cultured, serum-free RPMI 1640 medium was added to wash the cells, and then the medium was aspirated and discarded.

Next, a solution obtained by dissolving Cell Tracker Green in RPMI 1640 medium at a final concentration of 0.03 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, D-MEM medium supplemented with 10% FBS and the antibiotic solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the medium, D-MEM medium supplemented with 10% FBS and the antibiotic solution was added again, and the cells were cultured overnight at 37° C. in a 5% CO 2 atmosphere. The 2102Ep cells stained with Cell Tracker Green were detached from the culture surface using Accutase in the same manner as for the RD cells described in Example 4, and then washed with MACS buffer to prepare a cell suspension.

For hMSCs, 10% FBS, antibiotic solution (penicillin-streptomycin solution), L-glutamine aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries) D-MEM medium (low glucose, manufactured by Fujifilm Wako Pure Chemical Industries) was used. Cells were seeded in a petri dish for adhesion culture (manufactured by Corning) and cultured at 37°C in a 5% CO2 atmosphere. Next, hMSCs were fluorescently stained using Cell Tracker Red (manufactured by Thermo Fisher Scientific) by the following method. After discarding the medium in the petri dish in which hMSCs were being cultured, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to wash the cells, and then the medium was aspirated and discarded. Next, a solution obtained by dissolving Cell Tracker Red in RPMI 1640 medium at a final concentration of 10 μM was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. After discarding the fluorescent reagent solution, D-MEM (low glucose) medium supplemented with 10% FBS, the antibiotic solution and L-glutamine aqueous solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the medium, D-MEM medium (low glucose) supplemented with 10% FBS, the antibiotic solution and L-glutamine aqueous solution was added again, and cultured overnight at 37°C in a 5% CO2 atmosphere. did. The hMSCs stained with Cell Tracker Red were detached from the culture surface using Accutase in the same manner as for the RD cells described in Example 4, and then washed with MACS buffer to prepare a cell suspension.

For each of the prepared 2102Ep cell and hMSC cell suspensions, the cell density was calculated using a hemacytometer in the same manner as for the PC-9 cells described in Example 4. It was mixed and used to purify cancer cells using the adsorbent described below. The BC2LCN positive rate of 2102Ep cells and hMSCs was calculated by the same method as in Example 1 (2). rice field. Table 7 shows the BC2LCN positive rate of 2102Ep cells and hMSCs.

(2) Purification of 2102Ep cells A column (adsorbent capacity: 500 µL) filled with the adsorbent 127Q39L/C72G prepared in (1) of Example 1 was placed vertically. The mixed cell suspension of 2102Ep cells and hMSCs (0.5 mL) prepared in (1) above was added from the top of the column so as to obtain 4×10 ⁵ cells and 2.6×10 ⁶ hMSCs, and adsorbed. A complex was obtained in which the agent and the cell were bound. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer (10.0 mL) containing 0.2 mol/L fucose is added as a desorption liquid from the top of the column, and the desorption liquid is brought into contact with the complex in which the adsorbent and cells are bound. A desorbed cell solution (10.0 mL) containing cells desorbed from the adsorbent from the bottom of the column (desorbed cells) was collected in a container different from the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the same method as in Example 4 (3), the cell efflux rate of 2102Ep cells and hMSCs contained in the effluent cell fluid was calculated. The cell efflux rate of cells was 20.0%, and the cell efflux rate of hMSC was 94.0%. In addition, the BC2LCN positive rate of 2102Ep cells and hMSCs contained in the effluent cell fluid was calculated by the method described in Example 1 (2). The rate was 0.8%%. Table 7 shows the cell efflux rate and BC2LCN positive rate of 2102Ep cells and hMSCs contained in the effluent cell fluid.
(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells By the same method as in Example 4 (3), the cell detachment rate of 2102Ep cells and hMSCs contained in the detached cell solution was calculated. The cell detachment rate of cells was 64.0%, and the cell detachment rate of hMSCs was 5.6%. In addition, the BC2LCN positive rate of 2102Ep cells and hMSCs contained in the desorbed cell solution was calculated by the method described in (2) of Example 1. As a result, the BC2LCN positive rate of 2102Ep cells was 97.7%, and the BC2LCN positive rate of hMSCs was 97.7%. was 2.0%. Therefore, according to the cell purification method of the present invention, cancer cells (2102Ep cells) having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc and a structure consisting of Fucα1-2Galβ1-3GlcNAc were purified. A cancer cell (2102Ep cell ) can be purified. Table 7 shows the cell efflux and cell detachment rates of 2102Ep cells and hMSCs.

Example 7 Purification of human iPS cells using adsorbent 127Q39L/C72G-5
Example 7 uses adsorbent 127Q39L/C72G to remove cells that have deviated from the undifferentiated state from human iPS cells that have deviated from the undifferentiated state (hereinafter sometimes abbreviated as deviated undifferentiated cells). and purification of human iPS cells maintaining a high degree of undifferentiation.

(1) Culture of 201B7 Cells Containing Undifferentiated Deviated Cells and Preparation of Cell Suspension The culture of 201B7 cells containing undifferentiated deviant cells was performed with reference to the method described in Non-Patent Document 2. Specifically, after heat-treating B solution and C solution, which are components of StemFit AK02N medium, at 65°C for 30 minutes, they are added to and mixed with A solution, which is a component of StemFit AK02N medium. was prepared in advance (hereinafter referred to as undifferentiated deviated medium). Next, a solution obtained by diluting iMatrix-511 (manufactured by Nippi) at a rate of 3 μg/mL in D-PBS(-) was added to the petri dish for adherent culture and allowed to stand overnight at 4° C. to culture the petri dish. The surface was coated with iMatrix-511. Next, StemFit AK02N medium was added to wash the petri dish, and then 201B7 cells thawed from the frozen vial were suspended in the same medium supplemented with 10 μM lock inhibitor (Y-27632) and seeded. After culturing overnight at 37° C. in a 5% CO 2 atmosphere in a CO 2 incubator, the medium was changed to StemFit AK02N medium containing no lock inhibitor. The next day, the medium was replaced with an undifferentiated deviated medium, and thereafter, the medium was replaced with an undifferentiated deviated medium once every 3 to 4 days, and the cells were harvested when an appropriate cell density was reached.

Next, cell collection and cell suspension preparation were performed by the following methods. After repeating the operation of adding D-PBS(-) to the petri dish to wash the cells and discarding D-PBS(-) twice, a stripping solution obtained by mixing CTS TrypLE Select Enzyme and Versene Solution at 1:1. was added and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere. After confirming that the cells were detaching from the culture surface, the cells were detached from the culture surface by repeating pipetting and collected in a 50 mL tube. After centrifuging and sedimenting the collected cells, the cells were suspended in MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating this washing operation twice, the cells were resuspended in MACS buffer and filtered using a cell strainer (40 μm opening) to prepare a cell suspension of 201B7 cells containing undifferentiated deviant cells. did. A portion of the resulting suspension of 201B7 cells containing undifferentiated deviant cells was collected, diluted 10-fold, and then the cell density was calculated using a hemocytometer. The number of cells added to the column was calculated by dividing the number of added cells by the cell density. The undifferentiated marker positive rate of 201B7 cells containing undifferentiated deviant cells calculated by the same method as in Example 1 (2) was 86.8% for BC2LCN positive rate and 83.3 for TRA-1-60 positive rate. %, and the undifferentiated marker positive rate was lower than that of the 201B7 cells cultured under the conditions described in Examples 1 to 4. Table 8 and FIG. 4 show the undifferentiated marker-positive rate of cultured 201B7 cells containing undifferentiated deviant cells.

(2) Removal of deviated undifferentiated cells and purification of 201B7 cells maintaining a high degree of undifferentiation In an upright state, the 201B7 cell suspension (0.5 mL) containing undifferentiated deviant cells prepared in (1) above was added from the top of the column so that the number of added cells was 1.0×10 ⁶ . Thus, a complex in which the adsorbent and cells were bound was obtained. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (4.0 mL) containing 0.2 mol/L fucose is added as a desorption liquid from the top of the column, and the desorption liquid is brought into contact with the complex in which the adsorbent and cells are bound, A desorbed cell solution (4.0 mL) containing cells (desorbed cells) desorbed from the adsorbent from the bottom of the column was collected in a container different from the above operation.

(3) Measurement of cell outflow rate and undifferentiated marker-positive rate of outflowing cells The outflow rate of outflowing cells (cell outflow rate) in (3) was calculated by the method described in (4) of Example 1. As a result, The cell efflux rate was 4.8%. In addition, the undifferentiated marker positive rate of outflow cells calculated by the method described in (2) of Example 1 was 76.9% for BC2LCN positive rate and 69.2% for TRA-1-60 positive rate. . Table 8 shows the cell outflow rate and the undifferentiated marker-positive rate of the outflow cells, and FIG. 4 shows the undifferentiated marker-positive rate of the outflow cells.

(4) Measurement of cell detachment rate and undifferentiated marker positive rate of detached cells As a result of calculating the cell detachment rate of the detached cells in (2) above by the method described in (5) of Example 1, the cell detachment rate was It was 76.0%. In addition, the undifferentiated marker positive rate of the detached cells in the detached cell solution calculated by the method described in (2) of Example 1 was 99.6% for BC2LCN positive rate and 98.6% for TRA-1-60 positive rate. 0%, showing the same level of undifferentiation as the cells in the desorbed cell suspension described in Examples 1-4. Therefore, it was clarified that human iPS cells maintaining a high degree of undifferentiation can be purified from human iPS cells containing deviated undifferentiated cells by the cell purification method of the present invention. Table 8 and FIG. 4 show the cell detachment rate and the undifferentiated marker-positive rate of the detached cells.

Example 8 Purification of human iPS cells using adsorbent 127Q39L/C72G-6
Example 8 relates to removal of cells that have deviated from an undifferentiated state from deviated undifferentiated cells and purification of human iPS cells that maintain a high degree of undifferentiation using the adsorbent 127Q39L/C72G.

(1) Culture of 201B7 Cells Containing Undifferentiated Deviant Cells and Preparation of Cell Suspension By the method described in Example 7 (1), an undifferentiated deviant medium was prepared. Similarly, by the method described in Example 7 (1), after coating iMatrix-511 on a petri dish for adherent culture, 201B7 cells were started to be cultured in StemFit AK02N medium, and cultured overnight at 37°C. Medium was changed to StemFit AK02N medium without lock inhibitor. The next day, the medium was replaced with an undifferentiated deviant medium, and then the medium was replaced with an undifferentiated deviant medium once every 3 to 4 days. Then, 201B7 cells were collected by the method described in Example 7 (1), and a cell suspension was prepared. A portion of the resulting suspension of 201B7 cells containing undifferentiated deviant cells was collected, diluted 10-fold, and then the cell density was calculated using a hemocytometer. The number of cells added to the column was calculated by dividing the number of added cells by the cell density. The undifferentiated marker positive rate of 201B7 cells containing undifferentiated deviated cells calculated by the same method as in Example 1 (2) was BC2LCN positive rate of 69.6% and TRA-1-60 positive rate of 54.2. %, and the undifferentiated marker-positive rate was lower than that of 201B7 cells containing undifferentiated deviant cells cultured under the conditions described in Example 7. Table 9 and FIG. 5 show the undifferentiated marker-positive rate of cultured 201B7 cells containing undifferentiated deviant cells.

(2) Removal of undifferentiated deviated cells using desorption solutions with different fucose concentrations and purification of 201B7 cells maintaining a high degree of undifferentiation Column filled with adsorbent 127Q39L/C72G prepared in (1) of Example 1 ( Adsorbent capacity: 500 μL) was placed vertically, and the suspension of 201B7 cells containing undifferentiated deviant cells prepared in ( ¹ ) above (0 .5 mL) was added from the top of the column to obtain a complex in which the adsorbent and cells were bound. Next, MACS buffer (3.5 mL) was added from the top of the column to wash the adsorbent, and the effluent cell solution (4.0 mL) containing cells that did not bind to the adsorbent (effluent cells) was removed from the bottom of the column. ) was collected in a container. Next, a MACS buffer solution (4.0 mL) containing 0.05 mol/L fucose was added as the desorbing liquid-1 from the top of the column, and the desorbing liquid-1 was brought into contact with the complex, thereby removing the complex from the bottom of the column. Desorbed cell solution-1 (4.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from the above operation. Next, a MACS buffer solution (4.0 mL) containing 0.20 mol/L fucose was added from the top of the column as the desorbing liquid-2, and the complex was brought into contact with the desorbing liquid-2. Desorbed cell solution-2 (4.0 mL) containing cells desorbed from the adsorbent (desorbed cells) was collected in a container different from that used in the above operation.

(3) Measurement of cell efflux rate and undifferentiated marker positive rate of effluent cells By the method described in (4) of Example 1, the efflux rate of effluent cells (cell efflux rate) in (2) above was calculated. The cell efflux rate was 4.1%. In addition, the undifferentiated marker positive rate of outflow cells calculated by the method described in (2) of Example 1 was 23.1% for BC2LCN positive rate and 23.1% for TRA-1-60 positive rate. . Table 9 shows the cell outflow rate and the undifferentiated marker-positive rate of the outflow cells, and FIG. 5 shows the undifferentiated marker-positive rate of the outflow cells.

(4) Measurement of cell detachment rate and detached cell undifferentiated marker positive rate As a result, the cell detachment rate of the desorbed cell sap-1 was 77.5%, and the cell detachment rate of the desorbed cell sap-2 was 8.6%. In addition, the undifferentiated marker positive rate of desorbed cells in each desorbed cell liquid calculated by the method described in (2) of Example 1 was 75.2% for BC2LCN positive rate for desorbed cell liquid-1, and 75.2% for TRA-1. -60 positive rate was 57.7%, BC2LCN positive rate of desorbed cell suspension-2 was 91.8%, and TRA-1-60 positive rate was 82.0%. From the above results, by the cell purification method of the present invention, 201B7 cells that maintain a high degree of undifferentiation are purified even from 201B7 cells containing undifferentiated deviant cells in which the BC2LCN positive rate, which is an undifferentiation marker, has decreased to 70% or less. It turned out to be possible. Table 9 shows the cell detachment rate and the undifferentiated marker-positive rate of each detached cell, and FIG. 5 shows the undifferentiated marker-positive rate of the detached cells.

Claims (12)

A method for purifying cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, comprising the following steps (Z1) to (Z3):
(Z1) contacting a sample solution containing at least one type of cell with an adsorbent comprising any one of the following (a) to (d) fucose-binding proteins immobilized on a water-insoluble carrier, A step of obtaining a complex in which the adsorbent and cells are bound.
(a) A fucose-binding protein comprising an amino acid sequence from the first proline residue to the X-th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1, wherein X is an integer of 120 or more and 140 or less. Fucose-binding protein.
(b) an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence from the first proline residue to the Xth amino acid residue in the amino acid sequence shown by SEQ ID NO: 1 A fucose-binding protein comprising a fucose-binding protein, and having a binding property to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more and 140 or less, Fucose-binding protein.
(c) any one of the amino acid substitutions described in (1) to (3) below in the 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 Fucose-binding protein (1), wherein X is an integer of 120 or more and 140 or less, containing an amino acid sequence containing the above, substitution of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue ( 2) replacement of the 72nd cysteine residue in the amino acid sequence shown by SEQ ID NO: 1 with one type of amino acid residue selected from glycine residues and alanine residues (3) of the amino acid sequence shown by SEQ ID NO: 1 Substitution of the 65th glutamine residue with a leucine residue (d) in the amino acid sequence of the fucose-binding protein of (c) above, in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 Containing an amino acid sequence in which a plurality of amino acid residues are deleted, substituted, inserted or added, and having binding properties to a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc , fucose-binding protein.
(Z2) A step of separating and removing cells that did not bind to the adsorbent from the complex obtained in (Z1) above.
(Z3) A step of recovering cells desorbed from the adsorbent by bringing a desorption liquid into contact with the complex obtained in (Z1) after the completion of (Z2). 2. The method for purifying cells according to claim 1, wherein the cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are human pluripotent stem cells. 2. The method for purifying cells according to claim 1, wherein the purified human pluripotent stem cells have a BC2LCN positive rate of 90% or more. 2. The method for purifying cells according to claim 1, wherein the cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc is a cancer cell. 5. The method for purifying cells according to claim 4, wherein the purified cancer cells have a BC2LCN positive rate of 70% or more. 6. Any one of claims 1 to 5, wherein the sample solution containing at least one type of cell contains cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. 2. A method for purifying cells according to claim 1. A sample solution containing at least one type of cell is a cell having a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, and a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and 7. The method for purifying cells according to any one of claims 1 to 6, comprising both cells having no sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc. The fucose-binding protein is a fucose-binding protein having an additional amino acid sequence at its N-terminus and/or C-terminus, wherein the additional amino acid sequence comprises an oligopeptide containing cysteine residues and/or a polyhistidine sequence. The method for purifying cells according to any one of claims 1 to 7, wherein the oligopeptide comprises 9. The method for purifying cells according to any one of claims 1 to 8, wherein the fucose-binding protein is a fucose-binding protein comprising the amino acid sequence shown in any one of SEQ ID NOS:2 to 9. 10. The method for purifying cells according to any one of claims 1 to 9, wherein the desorption solution is an aqueous solution containing fucose and/or fucose-containing sugar chains. Purification of cells according to any one of claims 1 to 10, characterized in that the concentration of fucose and/or fucose-containing sugar chains in the desorption solution is 0.01 mol/L or more and 1.0 mol/L or less. Method. 12. The method for purifying cells according to any one of claims 1 to 11, wherein an adsorbent packed in a column is used.

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