JP2022122595A - Method of separating cells with different undifferentiation degree - Google Patents

Abstract

To provide a technique for easily and efficiently separating target cells from a heterogeneous cell population with different expression levels of undifferentiated markers present on cell surfaces and/or a cell population with different expression levels of undifferentiated markers even in monotypic cells, in accordance with the expression level of the undifferentiated markers.SOLUTION: A heterogeneous cell population and/or a monotypic cell population with different expression levels of undifferentiated sugar chain markers present on cell surfaces are brought into contact with an adsorbent agent in which a fucose binding protein is immobilized on a water-insoluble carrier, and cells that bind with the absorbent agent are separated from cells that do not. Cells with different expression levels of undifferentiated sugar chain markers on cell surfaces are separated by sequentially applying a peeling solution in which the fucose concentration is increased stepwise to the cells bound to the adsorbent agent and recovering the cells by the fucose concentration.SELECTED DRAWING: None

Description

The present invention is a cell separation method using an adsorbent in which a fucose-binding protein is immobilized on an insoluble carrier. After the cells are brought into contact with the adsorbent, the cells are bound by sequentially acting a solution with a controlled fucose concentration. It relates to a method for separating heterologous cells with different degrees of undifferentiation and/or cell populations with different degrees of undifferentiation contained in the same cell type by exfoliating.

BC2LCN, derived from the N-terminal domain of BC2L-C lectin produced by Gram-negative bacteria (Burkholderia cenocepacia), is a protein having binding affinity to sugar chains containing fucose residues. In addition to H-type 1 sugar chain (Fucα1-2Galβ1-3GlcNAc) and H-type 3 sugar chain (Fucα1-2Galβ1-3GalNAc) known as undifferentiated sugar chain markers described in Document 1 and Patent Document 2, Lewis High binding affinity to multiple types of sugar chains containing fucose residues such as Y-type sugar chains (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and Lewis X-type sugar chains (Galβ1-4(Fucα1-3)GlcNAc) (Non-Patent Document 2).

In addition, BC2LCN binds to undifferentiated human iPS cells and human ES cells in which H type 1 and H type 3 sugar chains are highly expressed, but does not bind to human somatic cells. known (Non-Patent Document 3). Furthermore, since BC2LCN has binding properties to the undifferentiated glycan markers, it can be used, for example, to detect complex carbohydrates containing undifferentiated glycan markers and to detect undifferentiated cells such as human iPS cells and human ES cells. (Patent Document 1 and Patent Document 2). In addition, it is known that the H-type 1 sugar chain is highly expressed in specific cancer cells as SSEA-5 (Non-Patent Document 4).

Although BC2LCN has the same ability to detect undifferentiated stem cells as anti-Nanog antibody, which is a known antibody for detecting undifferentiated cells (Patent Document 2), the binding of BC2LCN and sugar chains of undifferentiated cells is due to electrostatic interaction. and the bond strength is affected by external environment such as solvent and salt concentration. Therefore, depending on the experimental conditions, in the detection of the undifferentiated cells and/or the glycoconjugate containing the undifferentiated sugar chain marker, the binding affinity between the sugar chain and BC2LCN may be low. There is a need for BC2LCN with improved binding affinity to undifferentiated glycan markers.

As a method for improving the function of a protein, a method of introducing amino acid mutations into the protein by protein engineering techniques to improve the desired function is known. For example, a specific amino acid residue is substituted with another amino acid residue. Antibody-binding proteins with improved stability against heat, acid and/or alkali are known (Patent Document 3). In addition, BC2LCN is known that has improved thermal stability and binding affinity to sugar chains by amino acid substitution at specific positions using a similar technique (Patent Document 4). Furthermore, BC2LCN with improved productivity when produced using microorganisms such as Escherichia coli is known (Patent Document 5). In addition, as a cell separation method using an adsorbent in which BC2LCN with improved functions such as heat stability and binding affinity to sugar chains and productivity by microbial expression is immobilized on a water-insoluble carrier, human iPS A cell separation method for removing undifferentiated cells such as cells is known (Patent Document 6), and a method for peeling and collecting undifferentiated cells bound to an adsorbent is also known (Patent Document 7).

However, in the methods disclosed in these patent documents, cells expressing H type 1 sugar chains and H type 3 sugar chains could be separated from cells not expressing them, but H Cells with different expression levels of H-type 1 and H-type 3 glycans are separately isolated from heterogeneous cell populations with different expression levels of type 1-type and H-type 3 glycans, or cells of the same species. I couldn't.

In general, as a method of separating cells using the expression level of undifferentiated markers on the cell surface and the difference in the expression level of markers such as cell surface antigens according to the CD (cluster of differentiation) classification that characterizes each cell type. , a cell separation method using magnetic beads (Non-Patent Document 5), a cell sorting method using a cell sorter (Non-Patent Document 5), and a cell separation method using a cell rolling column (Non-Patent Document 6). However, although the cell separation method using magnetic beads is suitable for processing a large number of cells, it is not possible to separate cells with small differences in expression levels of undifferentiated markers and CD antigens. There are quality control problems such as cell damage and foreign matter contamination due to this, and there is also concern about the influence on the proliferation and differentiation tropism of the collected cells. In addition, cell sorting by a cell sorter and cell separation by a cell rolling column require expensive equipment, and it takes a long time to process a large amount of cells. rice field.

However, at present, for regenerative medicine applications, there is a strong demand for the development of a technology for separating and purifying a large amount of cells with high accuracy in a short time based on differences in cell surface undifferentiated marker expression levels and CD antigen expression levels. had been

WO2013/065302 WO2013/128914 JP 2011-206046 A JP 2020-025535 JP 2020-058343 JP 2018-134073 JP 2019-000063

Tateno, H. et al., Stem Cells Transl Med. 2013, 2(4):265-273. Sulak, O. et al., Structure. 2010, 18(1):59-72. Tateno, H. et al., J Biol Chem. 2011, 286(23): 20345-20353. Tang, C et al., Nat Biotechnol. 2011, 29(9):829-835. Fong CY et al., Stem Cell Rev Rep. 2009, 5(1):72-80. Mahara, A. et al., J Biomater Sci Polym Ed. 2014, 25(14-15): 1590-601.

An object of the present invention is to provide a technique capable of separating and purifying a large amount of cells in a short period of time in the preparation of cells for use in regenerative medicine. From a heterogeneous cell population and/or a single species cell population with different expression levels of differentiation markers, such as sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, expression levels of these undifferentiated markers are determined. It is to provide a technique for separating different cell groups. More specifically, after bringing a cell population into contact with an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, the cells bound to the adsorbent are sequentially detached using a concentration-controlled fucose solution. , to provide a technique for easily and efficiently separating a target cell group.

As a result of intensive studies to solve the above problems, the present inventors have found that an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier contains undifferentiated markers Fucα1-2Galβ1-3GlcNAc and Fucα1-2Galβ1-3GlcNAc present on the cell surface. Heterogeneous cell populations and/or single cell populations with different expression levels of sugar chains containing a structure consisting of Fucα1-2Galβ1-3GalNAc are brought into contact, and the cell population expressing this undifferentiated marker is first adsorbed. A cell group that does not express the undifferentiated marker or that expresses a low amount of the undifferentiated marker is collected by binding to the agent.

Next, an aqueous solution in which fucose is dissolved at a low concentration is allowed to act on the adsorbent to which the cell group is bound, and a part of the bound cells is exfoliated and recovered. By collecting a plurality of cell fractions by sequential action, the inventors have found that it is possible to separate and collect cell groups with different expression levels of undifferentiated markers present on the cell surface from the cell population, and have completed the present invention. .

That is, the present invention includes the inventions described in [1] to [13] below.
[1]
A step of binding cells that bind to a fucose-binding protein from cell populations with different degrees of undifferentiation to an adsorbent; obtaining detached cells, wherein the adsorbent has a structure in which a fucose-binding protein is immobilized on a water-insoluble carrier.
[2]
[1] above, characterized in that a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc is expressed on the surface of the cell that binds to the fucose-binding protein. Method.
[3]
The method according to [2] above, wherein the cell populations with different degrees of undifferentiation are a mixture of heterogeneous cells with different expression levels of undifferentiation markers.
[4]
The method according to [2] above, wherein the cell populations with different degrees of undifferentiation are cells of the same type but with different expression levels of undifferentiated markers.
[5]
The method according to any one of [1] to [4] above, wherein the concentration of fucose solutions with different fucose concentrations is changed stepwise from low concentration to high concentration to obtain exfoliated cells.
[6]
A method for isolating cells with different expression levels of sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, using the method according to any one of [1] to [5] above.
[7]
Expression of sugar chains from a cell population having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, using the method according to any one of [1] to [5] above. A method for separating cells according to their quantity.
[8]
Using the method described in any one of [1] to [5] above, heterologous cell groups with different expression levels of sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are isolated. and a method of isolating cell groups having different sugar chain expression levels among single cell types having one or more sugar chains contained in a cell population to be separated.
[9]
The method according to any one of [1] to [8] above, wherein the cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc is a human pluripotent stem cell.
[10]
The method according to any one of the above [1] to [9], wherein the fucose-binding protein is any one of the following (a) to (d).
(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. sex 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 fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more 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) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution 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) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, Fucose-binding protein.
[11]
The method according to any one of the above [1] to [9], wherein the fucose-binding protein is any one of (e) to (h) below.
(e) to the 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, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.
(g) to the 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, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of
[12]
The method according to any one of [1] to [9] above, wherein a hydrophilic polymer is covalently fixed to a water-insoluble carrier.
[13]
The method according to any one of [1] to [9], wherein an adsorbent packed in a column is used.

The present invention will be described in detail below.

The method of the present invention for separating cells with different degrees of undifferentiation (hereinafter sometimes abbreviated as the cell separation method of the present invention) comprises contacting a cell population with an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier. After that, a cell group that did not bind to the adsorbent and a cell group that sequentially detached the cells bound to the adsorbent using fucose solutions with different fucose concentrations are obtained. It is a method of separating cells.

In the present invention, an undifferentiated marker is a molecule present on the cell surface that serves as an indicator of the degree of undifferentiation, specifically known as a fucose-containing sugar chain to which the fucose-binding protein can bind. Examples include H-type 1 sugar chains and H-type 3 sugar chains. In addition, the undifferentiated marker in the present invention is not limited to the above-mentioned sugar chain structure, and TRA-1-60, TRA-1-81 and SSEA-3, which are generally known as cell surface undifferentiated markers, Sugar chains such as SSEA-4 and SSEA-5 may also be used.

In the present invention, cells with different degrees of undifferentiation refer to cells with different expression levels of the undifferentiated markers on the cell surface.

Cells with different degrees of undifferentiation suggest, first of all, cells with clearly different expression levels of the above undifferentiated markers. In this case, the expression level of the undifferentiated marker refers to the ratio of cells expressing the undifferentiated marker molecule on the cell surface in the cell population, or the number of undifferentiated marker molecules present on the surface of each cell. Determined by strength of density.

Taking sugar chains to which the fucose-binding protein can bind as an undifferentiated marker, for example, K562 cells (human chronic myelogenous leukemia) and NHDF cells (human dermal fiber It is known that cells having a sugar chain that binds to the fucose-binding protein do not exist in the cell population of blast cells). In 2102Ep cells (human embryonic tumor cells), about 50% to 80% of the cell population expresses a sugar chain that binds to the fucose-binding protein, and human ES, which is a human pluripotent stem cell. It is known that nearly 100% of cells and human iPS cells express sugar chains that bind to the fucose-binding protein.

Thus, different cell types (hereinafter abbreviated as heterologous cells) are considered to have different proportions of cells having undifferentiated marker molecules. In comparison of the undifferentiated degree of heterologous cells, even if each cell type expresses the sugar chain that binds to the fucose-binding protein in the same number of cells, if the heterologous cell is a cell surface It is thought that the numbers and densities of undifferentiated marker molecules present in the cells are different.

Secondly, even if it is a single cell type, a cell population with different degrees of undifferentiation is a case where cells with different numbers and densities of undifferentiated marker molecules present on the cell surface are mixed for each individual cell. Point.

For example, when the undifferentiated marker is a sugar chain that can bind to the fucose-binding protein, 2102Ep cells exist in a wide variety of cells, ranging from cells with a low expression level of this sugar chain to cells with a high level of expression of this sugar chain. are also considered to be mixtures of cell populations with different levels of expression of undifferentiated markers, and are also included in cells with different degrees of undifferentiation in the present invention. In the case of human iPS cells, as mentioned above, almost 100% of the cells are known to express sugar chains that bind to the fucose-binding protein. It is known that less than 1% of undifferentiated deviant cells exist at a low degree. It can be considered as cells with different degrees of differentiation.

Furthermore, as a third form, it is a mixture of heterologous cells with different expression levels of the undifferentiated markers shown in the first above, and has a sugar chain that binds to one or more types of the fucose-binding proteins contained therein. Cells refer to all cell populations in which the numbers and densities of undifferentiated marker molecules present on the cell surface are different for each individual cell shown in the second.

In the present invention, cell mixtures of all forms from the first to the third can be separated.

The cell mixture to be separated in the cell separation method of the present invention may be a mixture of heterologous cells with different expression levels of undifferentiated markers, or a single cell that is an aggregate of cell populations with different expression levels of undifferentiated markers. It may be a seed, or a cell mixture in which both are mixed. In the present invention, heterologous cells with different levels of undifferentiated marker expression, and cell populations with different levels of undifferentiated marker expression even within a single cell type, are different depending on the cell type and the difference in undifferentiated marker expression level. It is possible to separate cells with different degrees of undifferentiation.

In this case, the number of types of heterologous cells with different undifferentiated marker expression levels and the number of types of cell populations with different undifferentiated marker expression levels among the single cell types may be two or more. good too. Note that the index of the difference in the expression level of the undifferentiated marker is, for example, specifically, the cell sorter BD FACSAria ( Becton, Dickinson), etc.) can be considered to be the degree of shift in the fluorescence intensity of the cell population that can be detected.

In the cell separation method of the present invention, after contacting a cell population with an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, the cell group that does not bind to the adsorbent is first separated, and then the cells bound to the adsorbent are separated. is brought into contact with fucose solutions having different fucose concentrations, and exfoliated cell groups are sequentially obtained according to the fucose concentration. In this cell detachment step, the weaker binding between the cells bound to the adsorbent and the fucose-binding protein is dissociated with a low-concentration fucose solution, and the stronger binding is dissociated with a high-concentration fucose solution. In this case, the fucose molecules in the solution bind to the fucose-binding sugar chains on the cell surface, so that the electrostatic binding between the fucose-binding protein of the adsorbent and the cells is released, and the bound cells are detached. Conceivable.

The solvent for dissolving fucose is not particularly limited as long as it does not interfere with the viability of the cells. Water, MACS buffer (0.5% (w / v ) bovine serum albumin (hereinafter abbreviated as BSA) and 2 mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) added), or various solvents such as mannitol aqueous solution can be used. In addition, the composition of the cell detachment solution can be adjusted as necessary. For example, if the cells are difficult to detach, the concentration of mannitol should be 0.5 to 0.7M to prevent cell contraction due to osmotic pressure. Detachment of cells can be promoted by adding about 10 mM EDTA to suppress non-specific adsorption of cells to the adsorbent. In addition, it is also possible to add a reagent that enhances cell viability, or to use a buffer solution as the base solution in order to suppress fluctuations in pH.

The cell separation method of the present invention includes a step of sequentially reacting the cells bound to the adsorbent with a solution having an appropriately adjusted fucose concentration as a cell detachment solution, and recovering the detached cells. It is possible to obtain a plurality of cell fractions with different degrees of undifferentiation by separately collecting fractions of cells detached when a concentrated cell detachment solution is applied. It is desirable to adjust the fucose concentration of the cell detachment solution to be used as appropriate depending on the binding force to the fucose-binding protein for each cell type. Specifically, for example, the concentration of fucose was gradually increased in the order of 0.0125 M → 0.025 M → 0.05 M → 0.1 M → 0.2 M → 0.4 M. By obtaining the exfoliated cells as a fraction, it is possible to separate cells with different degrees of undifferentiation.

In this case, at the stage of first introducing a detachment solution in which fucose is dissolved at a low concentration, cells with weak binding strength to the fucose-binding protein immobilized on the water-insoluble carrier, that is, cells with low undifferentiated marker expression levels ( That is, cells with a low degree of undifferentiation) are thought to be peeled off from the adsorbent and recovered. Next, at the stage where a fucose solution with a higher concentration was introduced as a cell detachment solution, cells with a stronger binding force to the fucose-binding protein immobilized on the water-insoluble carrier, that is, cells with a high expression level of undifferentiated markers (Highly undifferentiated cells) are thought to be exfoliated and recovered.

Therefore, in this case, as the concentration of fucose contained in the cell detachment solution is increased, it is possible to obtain stepwise from the cell fraction with low undifferentiated marker expression level to the cell fraction with high expression level. Further, each cell fraction obtained by the separation method of the present invention is centrifuged to obtain a cell pellet, and after preparing an appropriate cell suspension with MACS buffer or the like, the fucose-binding protein is again immobilized on a water-insoluble carrier. It goes without saying that by contacting with an adsorbent and obtaining a fraction of detached cells by repeating the same separation method, it is possible to perform more precise cell separation according to slight differences in the undifferentiated degree of cells. .

As a method for adjusting the fucose concentration of the cell detachment solution, in addition to the stepwise method described above (step gradient), a high-concentration fucose solution and a low-concentration fucose solution are supplied and mixed by separate pumps, and A method of varying concentrations (linear gradient) can be used. In this case, if it is possible to minimize the amount of the fraction to a droplet size and limit the number of cells contained in this droplet to one, it is theoretically possible to separate the cells one by one according to the order of the degree of undifferentiation. technically possible.

As the fraction recovery liquid, the exfoliated and recovered cells can be effectively used for property evaluation, reculturing, etc. by recovering in a centrifugation vessel, test tube, small tube, multi-well dish/plate, or the like.

Next, the fucose-binding protein in the cell separation method of the present invention will be explained. The fucose-binding proteins in the cell separation method of the present invention include H type 1 sugar chains (Fucα1-2Galβ1-3GlcNAc), H type 3 sugar chains (Fucα1-2Galβ1-3GalNAc), Lewis Y sugar chains (Fucα1- 2Galβ1-4(Fucα1-3)GlcNAc), Lewis b-type sugar chain (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc) and other proteins having binding properties to fucose-containing sugar chains, and the above-described recombinant BC2LCN Lectins are also included in fucose-binding proteins in the cell separation method of the present invention.

Specifically, the fucose-binding protein in the cell separation method of the present invention is (a): the amino acid sequence of the recombinant BC2LCN lectin shown in SEQ ID NO: 1 (2 of the amino acid sequence registered with GenPept as accession number WP_006490828). matches the amino acid sequence from th to 156th), wherein X is an integer of 120 or more, or (b): Consists of 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 to the Xth amino acid in the amino acid sequence shown by SEQ ID NO: 1, and H type 1 A protein having a binding property to type 3 sugar chains and/or H type 3 sugar chains and X being an integer of 120 or more is expressed as a recombinant protein in a transformant of Escherichia coli. As long as the fucose-binding protein in the cell separation method of the present invention has the ability to bind 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 15 or less, preferably may have 10 or fewer amino acids deleted, substituted or added. Moreover, X may be 120 or more and 155 or less, and may be 125 or more and 155 or less.

As disclosed in Japanese Unexamined Patent Application Publication No. 2020-25535, the fucose-binding protein in the cell separation method of the present invention is obtained by deleting a plurality of amino acid residues on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1. 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 separation 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 separation method of the present invention. be able to. 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 separation method of the present invention is represented by 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 15, preferably no more than 10 amino acid residues may be deleted, substituted or inserted.

Specific examples of the fucose-binding protein in the cell separation method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (amino acid sequences from 1st to 127th in the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 ( Amino acid sequence in which the 72nd 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 a leucine residue, and the 72nd cysteine residue is a glycine residue ), SEQ ID NO: 5 (the 39th glutamine residue of SEQ ID NO: 2 is a leucine residue, the 65th glutamine residue is a leucine residue, and the 72nd cysteine residue is a glycine residue ), 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: 1 and an oligopeptide containing a cysteine residue to the C-terminus), sequence No. 7 (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: 8 (shown by SEQ ID NO: 3) an oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 9 (an amino acid sequence with a polyhistidine sequence at the N-terminus and an oligopeptide containing a cysteine residue at the C-terminus), SEQ ID NO: 9 (poly An oligopeptide containing a histidine sequence, an amino acid sequence obtained by adding an oligopeptide containing a cysteine residue to the C-terminus) and SEQ ID NO: 10 (an oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence shown in SEQ ID NO: 5, and a fucose-binding protein represented by any of the amino acid sequences obtained by adding an oligopeptide containing a cysteine residue to the C-terminus.

Among them, SEQ. 8, SEQ ID NO: 9 and SEQ ID NO: 10 are preferred, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 9 are more preferred, and SEQ ID NO: 4 and SEQ ID NO: 9 are even more preferred.

As long as the fucose-binding protein in the cell separation 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 separation 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 Patent Document 4 can be produced. 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.

A signal peptide may be added to the N-terminal side of the fucose-binding protein in the cell separation 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 separation 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 separation 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 present invention will be described. The fucose-binding protein in the present invention is produced by culturing the transformant to produce the fucose-binding protein (hereinafter referred to as the first step) and recovering the fucose-binding protein from the resulting culture. It can be manufactured by a process including two processes (hereinafter referred to as a 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 medium suitable for the culture.

For example, when Escherichia coli is used as a host, it is preferable to use Terrific Broth (TB) medium, Luria-Bertani (LB) medium, etc. supplemented with necessary nutrients. 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 It ranges from 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.

The binding affinity of the fucose-binding protein to sugar chains in the present invention can be evaluated by Enzyme-linked immunosorbent assay method, surface plasmon resonance method, or the like.

As an example, the surface plasmon resonance method will be described. Binding affinity evaluation by the surface plasmon resonance method is performed, for example, using a Biacore T200 instrument (manufactured by GE Healthcare), using a fucose-binding protein as an analyte and a sugar chain (Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1 -3GalNAc structure). Sugar chain-immobilized sensor chips are produced by using biotin-labeled sugar chains to prepare streptavidin-coated sensor chips (Sensor Chip SA, manufactured by GE Healthcare) and dextran-coated sensor chips (Sensor Chip CM5). , GE Healthcare) to which streptavidin has been immobilized in advance. In addition, binding affinity evaluation can be performed using a kinetics analysis program attached to the instrument.

Next, the adsorbent in the cell separation method of the present invention will be explained. There are no particular restrictions on the raw materials for the water-insoluble carrier used as the adsorbent in the cell separation method of the present invention. Inorganic carriers such as silica gel and glass on which a thin gold film is deposited, and polysaccharides such as agarose, cellulose, chitin and chitosan. Water-insoluble polysaccharide carriers used as raw materials and crosslinked polysaccharide carriers obtained by crosslinking these 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, Synthetic polymer carriers such as poly(meth)acrylate, polyvinyl alcohol, polyurethane, polystyrene, etc., 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 for the adsorbent is preferably modified with a hydrophilic polymer on the surface of the water-insoluble carrier from the viewpoint of suppressing non-specific adsorption of cells, and the hydrophilic polymer is water-insoluble. More preferably, it is fixed to the carrier by covalent bonding.

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 may be particulate, sponge-like, flat membrane-like, flat plate-like, hollow, or fibrous. A particulate carrier is preferable, and a spherical particulate carrier is more preferable, because it can be carried out efficiently.

The average particle diameter (median diameter) of the water-insoluble carrier used as the adsorbent in the cell separation method of the present invention in a state of being swollen in water is the separation target when the adsorbent produced from the carrier is packed in a column. is preferably 100 μm or more and 1000 μm or less, more preferably 100 μm or more and 500 μm or less, in that the cells are in sufficient contact with the adsorbent surface and the cells that do not bind to the adsorbent can pass through the gaps between the adsorbents without stagnation. , more preferably 150 μm or more and 300 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. If the particle size exceeds 1000 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 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. Alternatively, after photographing an image of a slide glass with a scale using an optical microscope, images of a plurality of particles to be measured are photographed at the same magnification, and the particle size of the photographed plurality of carriers is measured using a ruler. , can be obtained by calculating the average value thereof.

There is no particular limitation on the presence or absence of pores in the water-insoluble carrier used for the adsorbent, and it may be either porous or non-porous. In addition, the water-insoluble carrier used in the adsorbent of the present invention is a particle having a hydroxyl group in that active functional groups can be easily introduced for immobilizing the fucose-binding protein used in the adsorbent of the present invention on the carrier. A shape carrier is preferred. Furthermore, the water-insoluble carrier used in the adsorbent of the present invention may be a commercially available product. (manufactured by Asahi Kasei Corp.), Cellophia (manufactured by Asahi Kasei Corp.) using cellulose as a raw material, and the like can be used.

The adsorbent in the cell separation method of the present invention comprises a step of producing a reactive water-insoluble carrier from a water-insoluble carrier (hereinafter referred to as step X), and a step of reacting the reactive water-insoluble carrier with a fucose-binding protein. It can be produced by a process including two processes of immobilization (hereinafter referred to as process Y). The details of process X and process Y are described below.

Step X is a step of introducing a reactive functional group for immobilizing a fucose-binding protein into a water-insoluble carrier to produce a reactive 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. Examples include an amino group, a maleimide group, a haloacetyl group, and the like.

The method for introducing the functional group into the water-insoluble carrier is not particularly limited as long as it is a general method for introducing the functional group. Halohydrins such as bromohydrin, 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 ether, etc.; triglycidyl ethers, such as glycerol triglycidyl ether, erythritol triglycidyl ether, diglycerol triglycidyl ether; A method of reacting an epoxy group-containing compound such as can be exemplified. Moreover, when reacting the hydroxyl group of the water-insoluble carrier with the epoxy group-containing compound, it is preferable to carry out the reaction under basic conditions in order to increase the reaction efficiency.

Examples of methods for introducing formyl groups into the water-insoluble carrier include a method of reacting a hydroxyl group of the water-insoluble carrier with a bifunctional aldehyde such as glutaraldehyde, and a method of reacting the carrier with an oxidizing agent such as sodium periodate. can do. Alternatively, a water-insoluble carrier into which an epoxy group has been introduced by the above method is reacted with a compound such as D-glucamine, N-methyl-D-glucamine, α-thioglycerol, etc. to obtain a water-insoluble carrier into which adjacent hydroxyl groups have been introduced. , a method of reacting with an oxidizing agent such as sodium periodate.

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 water-insoluble carrier with a haloacetic acid such as monochloroacetic acid or monobromoacetic acid under basic conditions, an epoxy group was introduced by the method described above. A water-insoluble carrier, 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; A method of reacting with carboxylic acids under basic conditions can be exemplified. Furthermore, 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). can exemplify a method of derivatization to an N-hydroxysuccinimide ester, which is an active ester group.

As a method for introducing an amino group into a water-insoluble carrier, the water-insoluble carrier into which an epoxy group has been introduced by the above method has at least two amino groups such as ethylenediamine, diethylenetriamine, and tris(2-aminoethyl)amine. A method of reacting with a compound 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-maleimidomethyl ) A method of reacting carboxylic acids having a maleimide group such as 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.

Methods for introducing a haloacetyl group into a water-insoluble carrier include, for example, a water-insoluble carrier having a hydroxyl group, a water-insoluble carrier into which an amino group has been introduced by the method described above, and chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, and the like. A method of reacting an acid halide and a method of reacting a halogenated acetic acid such as chloroacetic acid, bromoacetic acid and iodoacetic acid in the presence of a condensing agent such as EDC can be mentioned. Furthermore, a method of reacting the aforementioned N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of halogenated acetic acid can be mentioned.

Step Y is a step of immobilizing the fucose-binding protein of the present invention on the reactive water-insoluble carrier produced in step X. The method for immobilizing the fucose-binding protein on the reactive water-insoluble carrier obtained in step X is not particularly limited as long as it is a general protein immobilization method. A method of immobilizing a protein on a water-insoluble carrier without forming a covalent bond, and a method of introducing an immobilizing active functional group into a protein and then reacting the immobilizing active functional group with the carrier to form a water-insoluble carrier. Examples include a method of immobilization, and a method of immobilizing on a water-insoluble carrier by reacting an active functional group for immobilization introduced into a water-insoluble carrier with a protein to form a covalent bond.

As a method for immobilizing a protein on a water-insoluble carrier without forming a covalent bond, an avidin-biotin affinity bond is used, and a biotin-introduced protein is treated with an avidin such as Streptavidin Sepharose High Performance (manufactured by GE Healthcare). A method of immobilization on an immobilized water-insoluble 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 water-insoluble carrier to form a covalent bond, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo -The active ester group of a compound having both a maleimide group and an active ester group, such as N-hydroxysuccinimide ester sodium salt, is reacted with the amino group of the protein to introduce the maleimide group into the protein, and then the water into which the mercapto group is introduced. A method of reacting with an insoluble carrier can be exemplified.

Furthermore, as a method of reacting an active functional group for immobilization introduced into a water-insoluble carrier with a protein to immobilize it onto a water-insoluble carrier, epoxy groups, formyl groups, carboxyl groups, and N-hydroxysuccinimide introduced into the water-insoluble carrier can be used. A method of reacting an active ester group such as an ester with an amino group of a protein, a method of reacting an amino group introduced into a water-insoluble carrier with a carboxyl group of a protein, an epoxy group, a maleimide group, a haloacetyl group or a haloalkyl group introduced into a water-insoluble carrier. A method of reacting a group with a mercapto group of a protein can be exemplified.

Among these immobilization methods, a method of reacting a formyl group or an active ester group introduced into a water-insoluble carrier with an amino group of a protein is preferred in that protein can be immobilized onto a water-insoluble carrier in a short period of time with high yield. , and a method of reacting a maleimide group or a haloacetyl group introduced into a water-insoluble carrier with a mercapto group of a protein is preferable, and the immobilization reaction can be carried out at around neutral pH, and protein denaturation can be suppressed. Therefore, the method of reacting the maleimide group or haloacetyl group introduced into the water-insoluble carrier with the mercapto group of the protein is more preferable. More preferred is the method of reacting the mercapto groups.

The adsorbent of the present invention can be produced by reacting the water-insoluble carrier into which the functional group for immobilization has been introduced and the fucose-binding protein dissolved in a buffer solution. 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 water-insoluble carrier are determined in consideration of the reactivity of the active functional group and the stability of the fucose-binding protein. The pH may be appropriately set within the range of pH 4 or more and pH 10 or less, and in order to suppress deactivation of the fucose-binding protein, the reaction temperature should be 15°C or more and 40°C or less, and the pH should be pH 5 or more and pH 9 or less. A range is preferred.

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 separated and the fucose-binding protein in the cell separation method of the present invention. The amount per insoluble carrier is preferably 0.01 mg or more and 50 mg or less, more preferably 0.05 mg or more and 30 mg or less. In addition, the amount of fucose-binding protein immobilized on the water-insoluble carrier can be adjusted by adjusting the amount of the protein used during the immobilization reaction and the amount of active functional group introduced into the water-insoluble carrier. The amount of fucose-binding protein immobilized on the water-insoluble carrier was determined by recovering the immobilization reaction solution and the post-reaction washing solution to determine the amount of unreacted fucose-binding protein. It can be calculated by subtracting the unreacted fucose-binding protein amount from the protein amount.

In addition, as described above, the water-insoluble carrier used for the adsorbent in 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 agent, before introducing the functional group for immobilizing the fucose-binding protein in step X, the hydrophilic polymer can be immobilized on the water-insoluble carrier by covalent bonding. The method for covalently immobilizing a hydrophilic polymer on a water-insoluble carrier is not particularly limited as long as it is a general covalent bond formation reaction. Epoxy groups are introduced into the water-insoluble carrier by reacting epoxy group-containing compounds such as ether and 1,4-butanediol diglycidyl ether under basic conditions. A method of reacting under conditions can be mentioned.

The present invention uses an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier to obtain a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or a sugar containing a structure consisting of Fucα1-2Galβ1-3GalNAc. It is a particularly effective cell separation method for separating and purifying cells having chains, especially human iPS cells. By the cell separation method of the present invention, it is possible to remove undifferentiated deviant cells generated during the culture of human iPS cells, and to separate the cells according to the difference in the degree of undifferentiation, thereby purifying human iPS cells with excellent differentiation performance and proliferation. is possible. In addition, the cell separation method of the present invention can easily process a large amount of cells compared to existing methods such as a cell separation method using magnetic beads, a cell sorting method using a cell sorter, and a cell separation method using a cell rolling column. This is an extremely effective means for large-scale purification of human iPS cells for regenerative medicine applications.

2 is a graph showing fucose concentration-dependent 201B7 cell detachment rates in Examples 1 and 2. FIG. 2 is a graph showing the BC2LCN positive rate of 201B7 cells exfoliated at each fucose concentration in Examples 1 and 2. FIG. 2 is a graph showing outflow rate and detachment rate of 201B7 cells and PC-9 cells in Examples 3 and 4. FIG. FIG. 10 is a graph showing the BC2LCN positive rate of 201B7 cells and PC-9 cells flowed out and detached from the adsorbent in Examples 3 and 4. FIG. 10 is a graph showing four types of cell recovery rates in each cell fraction of Example 5. FIG. 10 is a graph showing four types of cell recovery rates in each cell fraction of Example 6. FIG.

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

Preparation Example 1 Preparation of Adsorbent 127C72G and Adsorbent 127Q39L/C72G According to the method described in Examples 12 and 34 of JP-A-2020-025535 (Patent Document 4), fucose-binding protein 127C72G (shown in SEQ ID NO: 8 Amino acid sequence) was immobilized on a water-insoluble carrier, and an adsorbent 127Q39L/C72G was prepared by immobilizing a fucose-binding protein 127Q39L/C72G (amino acid sequence shown in SEQ ID NO: 9) on a water-insoluble carrier.

Example 1 Separation of cells with different degrees of undifferentiation by adsorption/desorption to adsorbent-1
Examples 1 to 5 relate to separation of cells with different degrees of undifferentiation by cell binding and detachment using the adsorbent 127Q39L/C72G produced in Production Example 1 above. The cell separation methods in Examples 1 to 5 use 127C72G or 127Q39L/C72G as the adsorbent. The kind can be used without being limited.

Example 1 describes "Fucα1-2Galβ1-3GlcNAc and/or Fucα
1-2Galβ1-3GalNAc” human iPS cell strain 201B7 (abbreviated as 201B7 cells in this specification) (distributed from Kyoto University CiRA after concluding a patent license agreement and an MTA agreement) was used. It relates to the separation of 201B7 cell populations with different degrees of undifferentiation by cell adsorption/desorption to a cell adsorbent.

(1) Preparation of column filled with adsorbent A column equipped with a hydrophilic nylon mesh filter (manufactured by Corning) with an opening of 40 μM between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G). 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 the suspension was added to the prepared column, and the column was filled with the adsorbent (adsorbent capacity: 500 μL). Column is NO. Five samples 1 to 5 were prepared.

(2) Cultivation of 201B7 Cells and Preparation of Cell Suspension 201B7 cells were cultured using a petri dish for adhesion culture (manufactured by Corning) 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) was added to the petri dish and allowed to stand overnight at 4° C. to culture the petri dish. A coating of iMatrix-511 was applied to the surface. After discarding the iMatrix-511 solution in the coated petri dish, StemFit AK02N medium (manufactured by Ajinomoto Co., Ltd.), which is a human iPS cell culture medium, was added and washed. -27632: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was suspended in the same medium to which 10 μM was added and seeded. After culturing overnight at 37°C in a 5% CO2 atmosphere in a CO2 incubator, the StemFit AK02N medium containing Y-27632 was discarded, and the medium was replaced with StemFit AK02N medium not containing Y-27632 to obtain an appropriate cell density. Cells were collected and passaged at the time of growth.

Next, 201B7 cells were fluorescently stained using Cell Tracker Orange (manufactured by Thermo Fisher Scientific) by the following method. First, after discarding the medium in the petri dish, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to rinse 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 washing the cells by adding D-PBS (-) to the petri dish and rinsing the cells, discarding the D-PBS (-) was repeated twice, and then the cells were washed with CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific). and Versene Solution (manufactured by Thermo Fisher Scientific) were added at a ratio of 1:1 and left at 37° C. for 10 minutes in a 5% CO 2 atmosphere.

After confirming that the cells were detaching in a round shape, the cells were detached by repeating pipetting in the detachment solution 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 cell washing operation twice, the cell suspension of 201B7 cells stained with Cell Tracker Orange was obtained by suspending with MACS buffer and filtering using a cell strainer (manufactured by Corning, opening 40 μm). prepared. A portion of the obtained 201B7 cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Based on this cell density, the amount of cells to be added to the column was calculated from the value of the number of cells applied to the column divided by the cell density.

(3) Binding and detachment of 201B7 cells using a column filled with adsorbent A column filled with 500 μL of adsorbent 127C72G was placed vertically, and the amount of cells added was 3.8 × 10^6 cells/mL-adsorbent. The cell suspension of 201B7 cells prepared by the above method was used as NO. 1 to NO. 5 was added to the top of the column. Next, 4 mL of MACS buffer was gently added from the top of the column, and the effluent from the needle was collected in separate containers (hereinafter referred to as effluent cell effluents-1 to 5, respectively).

Next, NO. For column 1, 1 mL of 0.4 M mannitol aqueous solution containing 10 mM EDTA was added from the top of the column, and then 3 mL of MACS buffer was added to collect 4 mL of effluent from the needle in another container. (Hereinafter, this cell sap will be referred to as exfoliated cell sap-1).

Next, NO. For column 2, 1 mL of 0.4 M mannitol aqueous solution containing 0.05 M fucose and 10 mM EDTA was added from the top of the column, and then 3 mL of MACS buffer was added, and 4 mL of the effluent from the needle was collected in another container. recovered to. (Hereinafter, this cell sap will be referred to as exfoliated cell sap-2).

Next, NO. For the column No. 3, 1 mL of 0.4 M mannitol aqueous solution containing 0.10 M fucose and 10 mM EDTA was added from the top of the column, and then 3 mL of MACS buffer was added, and 4 mL of the effluent from the needle was collected in another container. recovered to. (Hereinafter, this cell sap is referred to as exfoliated cell sap-3).

Next, NO. For column No. 4, 1 mL of 0.4 M mannitol aqueous solution containing 0.20 M fucose and 10 mM EDTA was added from the top of the column, and then 3 mL of MACS buffer was added to remove 4 mL of the effluent from the needle in another container. recovered to. (Hereinafter, this cell sap is referred to as exfoliated cell sap-4).

Next, NO. For column No. 5, 1 mL of 0.4 M mannitol aqueous solution containing 0.40 M fucose and 10 mM EDTA was added, followed by the addition of 3 mL of MACS buffer, and 4 mL of the effluent from the needle was collected in another container. . (Hereinafter, this cell solution is referred to as exfoliated cell solution-5).

(4) Measurement of cell efflux rate of 201B7 cells The effluent cell fluids -1 to 5 obtained during the above cell adsorption operation were resuspended, and 2 mL of each was placed in a 5 mL polystyrene round tube with a cell strainer cap (manufactured by Corning). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a reagent for judging cell viability, the beads were sorted into a cell sorter BD FACSAria (Becton · Dickinson) was used to measure the number of cells. The number of viable cells contained in each of the effluent cell sap-1, 2, 3, 4 and 5 was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting. These viable cell numbers are defined as the effluent cell numbers -1, 2, 3, 4, and 5, respectively. The cell efflux rate was calculated by dividing the number of effluent cells - 1, 2, 3, 4 and 5 by the number of added cells described in (3).

(5) Measurement of cell detachment rate and BC2LCN positive rate of 201B7 cells Detached cell sap-1, 2, 3, 4, and 5 obtained by the above-described cell detachment procedure were each diluted to an appropriate volume with MACS buffer. After that, 2 mL was aliquoted into a 5 mL polystyrene round tube (manufactured by Corning) with a cell strainer cap, and 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as internal standard beads for counting cells and 7-AAD as a cell viability determination reagent. After adding 50 μL of the cell sorter BD FACSAria (manufactured by Becton Dickinson), the number of viable cells was measured. The number of viable cells contained in each of exfoliated cell sap-1, 2, 3, 4 and 5 was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting. The viable cell counts are defined as detached cell counts -1, 2, 3, 4, and 5, respectively. The cell detachment rate was calculated by dividing the number of detached cells -1, 2, 3, 4 and 5 by the number of added cells described in (3).

Next, the undifferentiated degree of cells was measured using a solution obtained by diluting the exfoliated cell solution-1, 2, 3, 4 and 5 with a MACS buffer solution to an appropriate volume. 1 mL of each cell suspension was dispensed into a 1.5 mL Eppendorf tube, centrifuged, the supernatant was discarded, and the cells were washed by resuspending in 1 mL of MACS buffer. The cell pellet obtained after centrifugation was suspended in 1 mL of MACS buffer containing 0.5% (vol./vol.) fluorescent reagent BC2LCN-635 (manufactured by Fujifilm Wako Pure Chemical Industries) and incubated at room temperature for 30 minutes in the dark. Let stand for 1 minute.

After 30 minutes of reaction, the cell pellet was washed with MACS buffer in the same manner as described above in an Eppendorf tube, and the obtained cell pellet was suspended in a total of 2 mL of MACS buffer (suspended twice with 1 mL of MACS buffer). turbidity). 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 an internal standard bead for counting cells and 7-AAD as a cell viability determination reagent were added. 50 µL of the solution was added, and the fluorescence intensity of the cell population was analyzed using a cell sorter BD FACSAria (manufactured by Becton Dickinson) in the same manner as the cell count measurement.

The undifferentiated degree of cells is BC2LCN-635 and unstained 201B7 cells are regarded as negative cell populations, and the cell populations with increased APC fluorescence intensity due to reaction with fluorescent reagents are regarded as positive live cell populations, BC2LCN positive rate =Number of positive cells contained in each measurement sample÷Total number of viable cells contained in each measurement sample. That is, the BC2LCN-positive rate indicates the ratio of the cell population having a sugar chain structure with BC2LCN reactivity, which is one of the undifferentiated markers.

As a result of the measurement, the cell efflux rate was effluent-1=0.4%, effluent-2=0.4%, effluent-3=0.4%, and effluent-4=0.4%. 5%, effluent cell fluid -5 = 0.6%, and it was confirmed that all columns bound 201B7 cells well (measurement results of cell efflux rate are shown in Table 1).

In addition, the cell detachment rate was detached cell sap -1 = 0.7%, detached cell sap -2 = 40.9%, detached cell sap -3 = 47.5%, detached cell sap -4 = 65.9%, Exfoliated cell sap -5 = 69.0%, and when a solution containing no fucose was applied to the cells bound to the cell adsorbent, almost no 201B7 cells were exfoliated, but when a mannitol solution containing fucose was applied, , the cell detachment rate increased in a fucose concentration-dependent manner (the results of measuring the cell detachment rate are shown in Table 1 and FIG. 1).

In addition, as a result of measuring the BC2LCN positive rate, it was 33.3% for exfoliated cell solution-1, 98.5% for exfoliated cell solution-2, 99.6% for exfoliated cell solution-3, and 99% for exfoliated cell solution-4. .1%, and 99.6% for exfoliated cell suspension-5. ).

This result shows that when the fucose concentration is low, the 201B7 cell population, which has a weak binding action to the fucose-binding protein 127C72G present on the adsorbent surface, is selectively peeled off easily, whereas when the fucose concentration is high, the adsorbent surface It can be interpreted that this is because the cells that strongly bind to the fucose-binding protein 127C72G present in the cells were detached.

That is, it was confirmed that it is possible to separate 201B7 cells with low degree of undifferentiation and 201B7 cells with high degree of undifferentiation by controlling the concentration of fucose when peeling off 201B7 cells bound to the cell adsorbent. was done.

Example 2 Separation of cells with different degrees of undifferentiation by adsorption/desorption to adsorbent-2
Example 2 relates to separation of 201B7 cell populations with different degrees of undifferentiation by cell adsorption/desorption to a cell adsorbent using 201B7 cells.

For Example 2, a NO. 6 to No. 5 columns of 10 were prepared and 201B7 cells were passed through them. Also, as an operation for cell detachment, NO. 6 column, 1 mL of MACS buffer containing 10 mM EDTA was added, and then 3 mL of MACS buffer was added. 7 column, 1 mL of MACS buffer containing 0.05 M fucose and 10 mM EDTA was added, and then 3 mL of MACS buffer was added. 8 column, 1 mL of MACS buffer containing 0.10 M fucose and 10 mM EDTA was added, and then 3 mL of MACS buffer was added. 9 column, 1 mL of MACS buffer containing 0.20 M fucose and 10 mM EDTA was added, and then 3 mL of MACS buffer was added. For 10 columns, 1 mL of MACS buffer containing 0.40 M fucose and 10 mM EDTA was added, followed by the addition of 3 mL of MACS buffer, and the fractions were collected in separate containers. I went with

Also obtained by this method, No. 6 to NO. The effluent cell sap from the 10 columns was designated as effluent cell sap -6 to 10, and the detached cell sap from the collected fractions was designated as detached cell sap -6 to 10. BC2LCN-positive rate of exfoliated cells was measured.

As a result of the measurement, the cell effluent rate was 0.5%, 7 cell effusion, 8 cell effusion, 0.6% effluent cell effluent, 9 cell effusion, 0.5% effluent cell effusion, 8 cell effusion, 0.6% cell effluent, 9 cell effluent, 0.5% cell effusion, 8 cell effluent, 0.6% cell effusion, 0.5% cell effusion, 8 cell effluent, 0.6% cell effusion, 6%, effluent cell fluid -10 = 0.5%, and it was confirmed that all columns bound 201B7 cells well (measurement results of cell efflux rate are shown in Table 1). In addition, the cell detachment rate was detached cell sap -6 = 0.3%, detached cell sap -7 = 21.0%, detached cell sap -8 = 46.0%, detached cell sap -9 = 58.6%, Exfoliated cell solution -10 = 73.4%, and when a solution containing no fucose was applied to the cells bound to the cell adsorbent, the 201B7 cells were hardly detached, but the MACS buffer solution containing fucose was applied. , the cell detachment rate increased in a fucose concentration-dependent manner (the results of measuring the cell detachment rate are shown in Table 1 and FIG. 1).

In addition, as a result of measuring the BC2LCN positive rate, it was 0.0% for exfoliated cell solution-6, 98.0% for exfoliated cell solution-7, 99.4% for exfoliated cell solution-8, and 98% for exfoliated cell solution-9. .8%, and 99.1% for exfoliated cell suspension-10. ). This result shows that when the fucose concentration is low, the 201B7 cell population, which has a weak binding action to the fucose-binding protein 127C72G present on the adsorbent surface, is selectively peeled off easily, whereas when the fucose concentration is high, the adsorbent surface It can be interpreted that this is because the cells that strongly bind to the fucose-binding protein 127C72G present in the cells were detached. That is, it was confirmed that it is possible to separate 201B7 cells with low degree of undifferentiation and 201B7 cells with high degree of undifferentiation by controlling the concentration of fucose when peeling off 201B7 cells bound to the cell adsorbent. was done.

Further, from the results of Examples 1 and 2, it is sufficient that the solution used for detaching the cells bound to the adsorbent contains fucose. Both of them can be used, and it was proved that it is possible to separate cell populations with different degrees of undifferentiation by controlling the concentration of fucose and performing cell detachment.

Example 3 Separation of cells with different degrees of undifferentiation by adsorption/desorption to adsorbent-3
Example 3 relates to separation of 201B7 cell populations with different degrees of undifferentiation by cell adsorption/desorption to a cell adsorbent using 201B7 cells. More specifically, to 201B7 cells bound to one column, a detachment solution in which the concentration of fucose solution was changed stepwise from low concentration to high concentration was added, and the recovery rate of the obtained cell recovery solution was and undifferentiated degree.

(1) Preparation of column filled with adsorbent A column equipped with a hydrophilic nylon mesh filter (manufactured by Corning) with an opening of 40 μm between a 2.5 mL syringe (manufactured by Terumo) and an injection needle (manufactured by Terumo, 22G). made. Next, after replacing the adsorbent 127Q39L/C72G produced in Preparation Example 1 with MACS buffer (manufactured by Miltenyi Biotech), adsorption was adjusted so that the sedimentation volume of the adsorbent after standing for 12 hours or more was 50%. A 50% suspension of the agent was prepared, and 1.0 mL was added to the prepared column to prepare one column filled with the adsorbent (adsorbent capacity: 500 μL).

(2) Culture of 201B7 Cells and Preparation of Cell Suspension 201B7 cells were cultured in the same manner as in Example 1, and a cell suspension of 201B7 cells stained with Cell Tracker Orange was prepared. A portion of the obtained 201B7 cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Based on this cell density, the amount of cells added to the column was calculated from the number of cells added to the column/cell density.

(3) Binding and detachment of 201B7 cells using a column filled with adsorbent A cell suspension of 201B7 cells prepared by the above method was added to a column filled with adsorbent 127Q39L/C72G in a vertical position. It was added so that the amount was 1.2×10̂7/mL-adsorbent. Next, 4 mL of MACS buffer was gently added from the top of the column, and 4 mL of effluent from the needle was collected in a container (hereinafter referred to as effluent cell solution).

Next, 1 mL of MACS buffer containing 0.0125 M fucose and 10 mM EDTA was added from the top of the column, and then 1 mL of MACS buffer was added to collect 2 mL of the effluent from the needle in another container. (Hereinafter, this cell recovery solution is referred to as exfoliated cell solution-11).

Subsequently, 1 mL of MACS buffer containing 0.025 M fucose and 10 mM EDTA was added to the same column from the top of the column, and then 1 mL of MACS buffer was added to separate 2 mL of the effluent from the needle into another container. The cells were recovered (hereinafter, this cell recovery solution is referred to as exfoliated cell solution-12).

Subsequently, 1 mL of MACS buffer containing 0.05 M fucose and 10 mM EDTA was added to the same column from the top of the column, and then 1 mL of MACS buffer was added to separate 2 mL of the effluent from the needle into another container. The cells were recovered (hereinafter, this cell recovery solution is referred to as exfoliated cell solution-13).

Subsequently, 1 mL of MACS buffer containing 0.4 M fucose and 10 mM EDTA was added to the same column from the top of the column, and then 1 mL of MACS buffer was added to separate 2 mL of the effluent from the needle into another container. The cell suspension was collected (hereinafter, this cell suspension is referred to as exfoliated cell suspension-14).

(4) Measurement of cell efflux rate of 201B7 cells 2 mL of the effluent cell fluid obtained by the above operation is aliquoted into a 5 mL polystyrene round tube (manufactured by Corning) with a cell strainer cap and used as internal standard beads for cell counting. After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) and 50 μL of 7-AAD as a cell viability determination reagent, the number of cells was measured using a cell sorter BD FACSAria (manufactured by Becton Dickinson). The number of viable cells 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. The cell efflux rate was calculated by dividing the number of effluent viable cells by the number of added cells respectively described in (3).

(5) Measurement of cell detachment rate and BC2LCN positive rate of 201B7 cells After diluting each of the detached cell sap-11, 12, 13, and 14 obtained by the above cell detachment operation with MACS buffer to an appropriate volume, Dispense 2 mL into a 5 mL polystyrene round tube (manufactured by Corning, opening 40 μm) with a cell strainer cap, add 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as internal standard beads for cell counting, and a cell viability determination reagent. After adding 50 μL of 7-AAD as a solution, the number of cells was measured using a cell sorter BD FACSAria (manufactured by Becton Dickinson). The number of viable cells contained in each of exfoliated cell sap-11, 12, 13, and 14 was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting. These viable cell counts are defined as detached cell counts -11, 12, 13, and 14, respectively. The cell detachment rate was calculated by dividing the number of detached cells -11, 12, 13 and 14 by the number of added cells described in (3).

Next, the undifferentiated degree of cells was measured using a solution obtained by diluting the exfoliated cell solutions-11, 12, 13, and 14 with a MACS buffer to an appropriate volume. 1 mL of each cell solution was collected in a 1.5 mL Eppendorf tube, centrifuged, the supernatant was discarded, and the cells were washed by suspending them in 1 mL of MACS buffer. The cell pellet obtained after centrifugation was suspended in 1 mL of MACS buffer containing 0.5% (vol./vol.) fluorescent reagent BC2LCN-635 (manufactured by Fujifilm Wako Pure Chemical Industries) and incubated at room temperature for 30 minutes in the dark. Let stand for 1 minute. After 30 minutes of reaction, the cell pellet was suspended in a total of 2 mL of MACS buffer (suspended twice with 1 mL of MACS buffer) by performing a washing operation with the above MACS buffer in an Eppendorf tube. . 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 an internal standard bead for counting cells and 7-AAD as a cell viability determination reagent were added. 50 µL of the solution was added, and the fluorescence intensity of the cell population was analyzed using a cell sorter BD FACSAria (manufactured by Becton Dickinson) in the same manner as the cell count measurement.

The undifferentiated degree of cells is BC2LCN-635 and unstained 201B7 cells are defined as negative cell populations, and the cell populations with increased APC fluorescence intensity due to reaction with fluorescent reagents are defined as positive cell populations, BC2LCN positive rate = ( It was calculated as (the number of positive viable cells contained in each measurement sample)/(the total number of viable cells contained in each measurement sample). That is, the BC2LCN-positive rate indicates the ratio of the cell population having a sugar chain structure with BC2LCN reactivity, which is one of the undifferentiated markers.

As a result of measurement, the cell outflow rate was 0.1%, confirming that the cell adsorbent binds 201B7 cells well. In addition, the cell detachment rate was detached cell sap -11 = 0.1%, detached cell sap -12 = 0.1%, detached cell sap -13 = 4.1%, and detached cell sap -14 = 56.1%. It was found that the 201B7 cells bound to the cell adsorbent began to gradually detach after the introduction of 0.05 M fucose solution, and most detached by the introduction of 0.4 M fucose solution (cell outflow rate and cell detachment rate were shown in Table 2 and Figure 3).

In addition, as a result of measuring the BC2LCN positive rate, it was 73.7% for the effluent cell sap, 62.5% for the exfoliated cell sap-1, 93.3% for the exfoliated cell sap-2, and 99.2 for the exfoliated cell sap-3. %, and 100.0% in exfoliated cell solution-4, and it was found that as the fucose concentration was increased, the cell population with a high BC2LCN positive rate gradually exfoliated (the BC2LCN positive rate is shown in Table 2 and FIG. 4). ).

As a result, the 201B7 cell population, which has a weak binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, is selectively peeled off by first applying a solution with a low fucose concentration, and then the fucose concentration increases sequentially. It can be interpreted that this is the result of selective detachment of the 201B7 cell population, which has a strong binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, by the action of the liquid. Therefore, even when 201B7 cells are bound to a single column filled with a cell adsorbent, the degree of undifferentiation can be determined by sequentially applying solutions with stepwise controlled fucose concentrations to obtain the fraction of each detached cell. different cell populations can be separated.

Example 4 Separation of cells with different degrees of undifferentiation by adsorption/desorption to adsorbent-4
In Example 4, human lung adenocarcinoma cells (PC-9 cells, obtained from DS Pharma Biomedical, ECACC strain No.: 90071810) is used to separate PC-9 cell populations with different degrees of undifferentiation by cell adsorption/desorption to a cell adsorbent. More specifically, it relates to the results of cell separation when using PC-9 cells, which express less undifferentiated glycan markers than 201B7 cells.

(1) Production of column filled with adsorbent One column filled with adsorbent 127Q39L/C72G was produced in the same manner as in Example 3 (adsorbent capacity: 500 µL).

(2) Cultivation of PC-9 cells and preparation of cell suspension for evaluation PC-9 cells, which are adherent cells, were mixed with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, Fujifilm Co., Ltd.). Using RPMI 1640 medium (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) supplemented with Kojunyaku Co., Ltd., cells were seeded in petri dishes for adherent culture (manufactured by Corning Co., Ltd.) and cultured at 37° C. in a 5% CO 2 atmosphere.

Next, fluorescent staining of PC-9 cells using Cell Tracker Orange was performed by the following method. After discarding the medium in the petri dish in which the PC-9 cells were being cultured, serum-free RPMI 1640 medium was added to wash the cells, and then this medium was discarded. Next, Cell Tracker Orange dissolved in serum-free RPMI 1640 medium to 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 the cells were cultured at 37° C. in a 5% CO 2 atmosphere for 1 hour. Next, after discarding the RPMI 1640 medium, a new RPMI 1640 medium supplemented with 10% FBS and an antibiotic solution 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 the RPMI 1640 medium in the petri dish during cell culture was discarded and D-PBS(-) (manufactured by Cell Science Research Institute) was added, the cells were washed and the D-PBS(-) was discarded. Next, an appropriate amount of Accutase (manufactured by Innovative Cell Technology) was added and left for several minutes to detach the PC-9 cells and collect them in a 50 mL tube. After centrifuging and sedimenting the cells, the cells were suspended in the aforementioned MACS buffer, centrifuged again, and the supernatant was discarded to wash the cells. After repeating the cell washing operation twice, the cells were suspended in MACS buffer and filtered using a cell strainer (manufactured by Corning, opening 40 μm) to obtain a cell suspension of PC-9 cells stained with Cell Tracker Orange. was prepared. A portion of the obtained PC-9 cell solution was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Based on this cell density, the amount of cell solution to be added to the column was calculated from the value of the number of cells applied to the column/cell density.

(3) Binding and Detachment of PC-9 Cells Using Adsorbent-Filled Column A cell suspension of PC-9 cells prepared by the above method was placed in a vertical column filled with adsorbent 127Q39L/C72G. The suspension was added to the top of the column so that the amount added was 5.4×10̂6 cells/mL-adsorbent. Next, 4 mL of MACS buffer was gently added from the top of the column, and the effluent from the needle was collected in a container (hereinafter referred to as effluent cell effluent).

Next, 1 mL of MACS buffer containing 0.0125 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (hereinafter, this cell suspension is referred to as exfoliated cell suspension-15).

Subsequently, 1 mL of MACS buffer containing 0.025 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (hereinafter, this cell suspension is referred to as exfoliated cell suspension-16).

Subsequently, 1 mL of MACS buffer containing 0.05 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (hereinafter, this cell suspension is referred to as exfoliated cell suspension-17).

Subsequently, 1 mL of MACS buffer containing 0.4 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (hereinafter, this cell suspension will be referred to as exfoliated cell suspension-18).

(4) Measurement of cell efflux rate of PC-9 cells The effluent cell fluid obtained by the above operation was suspended, and 2 mL of it was collected in a 5 mL polystyrene round tube with a cell strainer cap (manufactured by Corning, opening 40 μm). After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as an internal standard bead for counting cells and 50 μL of 7-AAD as a cell viability determination reagent, the number of viable cells was measured using a cell sorter BD FACSAria (manufactured by Becton Dickinson). was measured. The number of viable cells 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. The cell efflux rate was calculated by dividing the number of effluent viable cells by the number of added cells respectively described in (3).

(5) Measurement of cell detachment rate and BC2LCN positive rate of PC-9 cells Detached cell sap-15, 16, 17, and 18 obtained by the above cell detachment operation were each diluted to an appropriate volume with MACS buffer. After that, 2 mL was dispensed into a 5 mL polystyrene round tube (manufactured by Corning, opening 40 μm) with a cell strainer cap, 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as internal standard beads for cell counting, and cell life and death. After adding 50 μL of 7-AAD as a determination reagent, the number of viable cells was measured using a cell sorter BD FACSAria (manufactured by Becton Dickinson). The number of viable cells contained in each of exfoliated cell sap-15, 16, 17 and 18 was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting. These viable cell numbers are defined as detached cell numbers -15, 16, 17, and 18, respectively. The cell detachment rate was calculated by dividing the number of detached cells -15, 16, 17 and 18 by the number of added cells described in (3).

Next, the undifferentiated degree of cells was measured using a solution obtained by diluting the exfoliated cell solution-15, 16, 17, and 18 with a MACS buffer to an appropriate volume. 1 mL of each cell solution was collected in a 1.5 mL Eppendorf tube, centrifuged, the supernatant was discarded, and the cells were washed by suspending them in 1 mL of MACS buffer. The cell pellet obtained after centrifugation was suspended in 1 mL of MACS buffer containing 0.5% (vol./vol.) fluorescent reagent BC2LCN-635 (manufactured by Fujifilm Wako Pure Chemical Industries) and incubated at room temperature for 30 minutes in the dark. Let stand for 1 minute.

After 30 minutes of reaction, the cell pellet was suspended in a total of 2 mL of MACS buffer (suspended twice with 1 mL of MACS buffer) by performing a washing operation with the above MACS buffer in an Eppendorf tube. . 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 an internal standard bead for counting cells and 7-AAD as a cell viability determination reagent were added. 50 µL of the solution was added, and the fluorescence intensity of the cell population was analyzed using a cell sorter BD FACSAria (manufactured by Becton Dickinson) in the same manner as the cell count measurement.

The undifferentiated degree of cells is BC2LCN-635 and non-stained PC-9 cells are regarded as negative cell populations, and the cell populations with increased APC fluorescence intensity due to reaction with fluorescent reagents are regarded as positive cell populations, BC2LCN positive rate = (the number of positive viable cells contained in each measurement sample)/(the total number of viable cells contained in each measurement sample). That is, the BC2LCN-positive rate indicates the ratio of the cell population having a sugar chain structure with BC2LCN reactivity, which is one of the undifferentiated markers.

As a result of the measurement, the cell outflow rate was 38.7%, and it was confirmed that about 60% of the PC-9 cells added to the column were bound. In addition, the cell detachment rate was detached cell sap -15 = 50.3%, detached cell sap -16 = 11.8%, detached cell sap -17 = 5.4%, and detached cell sap -18 = 2.4%. It was found that most of the PC-9 cells bound to the cell adsorbent were detached by the introduction of 0.0125 M fucose solution (the cell outflow rate and cell detachment rate are shown in Table 3 and FIG. 3).

In addition, as a result of measuring the BC2LCN positive rate, it was 69.2% for the effluent cell sap, 71.4% for the exfoliated cell sap-15, 82.4% for the exfoliated cell sap-16, and 88.2 for the exfoliated cell sap-17. %, and 83.6% for exfoliated cell solution-18, and it was found that as the fucose concentration was increased, the cell population with a high BC2LCN positive rate was exfoliated (BC2LCN positive rate is shown in Table 3 and FIG. 4). .

As a result, the PC-9 cell population, which has a weak binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, was selectively peeled off by first acting with a solution having a low fucose concentration, and then the fucose concentration gradually increased. It can be interpreted that the PC-9 cell population, which has a strong binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, was selectively detached by the action of a solution with a high . Therefore, even when PC-9 cells are bound to a single column packed with a cell adsorbent, a fraction of each exfoliated cell can be obtained by sequentially applying a solution with a stepwise controlled fucose concentration. It was confirmed that cell populations with different degrees of differentiation could be separated.

Furthermore, in Examples 3 and 4, the difference in the results of the cell detachment rate when a solution with a specific fucose concentration was applied was due to the expression level of the undifferentiated sugar chain marker of the cell type added to the column, that is, the degree of undifferentiation. Most of the highly undifferentiated 201B7 cells were peeled off in a solution with a fucose concentration of 0.4M, and most of the PC-9 cells with a relatively low degree of undifferentiation were removed in a fucose solution of 0.0125M. As a result, it was peeled off. From this result, even when a mixture of multiple types of cells with different degrees of undifferentiation is added to the cell separation agent, by performing cell detachment while controlling the fucose concentration, heterogeneous cells can be separated from each other. Regarding cells, it was suggested that cell populations with different degrees of undifferentiation can be sorted.

Example 5 Separation of four types of cell mixture by adsorption/desorption to adsorbent-1
In Example 5, NHDF cells (manufactured by PromoCell), which are normal human skin fibroblasts that do not have a sugar chain containing a structure consisting of "Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc", and "Fucα1- 2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc” and 201B7 cells, which are human iPS cell lines having sugar chains, and a structure consisting of “Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc” Structure consisting of 2102Ep cells (Embryonal Carcinoma Cells Cl.4/D3 cells (obtained from Cosmo Bio), which are human embryonal cancer cells having sugar chains, and "Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc" Cell adsorption to a cell adsorbent using a mixed cell of four types of human lung adenocarcinoma cells (PC-9 cells, obtained from DS Pharma Biomedical, ECACC strain number: 90071810) having sugar chains containing It relates to the separation of heterogeneous cells by desorption.

(1) Preparation of column filled with adsorbent A column filled with adsorbent 127Q39L/C72G was prepared in the same manner as in Example 3 (adsorbent capacity: 500 µL).

(2) Culture of NHDF cells, 201B7 cells, 2102Ep cells, and PC-9 cells and preparation of cell suspension for evaluation 1 and in the same manner as described in Example 4. 201B7 cells were prepared by Cell Tracker Orange staining, and PC-9 cells were not fluorescently stained.

In addition, NHDF cells, which are adherent cells, were prepared using fibroblast growth medium 2 (manufactured by PromoCell). The cells were inoculated and cultured at 37°C in a 5% CO2 atmosphere. Next, fluorescent staining of NHDF cells using Cell Tracker Red (manufactured by Thermo Fisher Scientific) was performed by the following method. After discarding the medium in the petri dish during which NHDF 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 this medium was discarded. Next, Cell Tracker Red dissolved in serum-free RPMI 1640 medium to 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 fibroblast growth medium 2 was added and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding fibroblast growth medium 2, fresh fibroblast growth medium 2 was added again and cultured overnight at 37° C. in a 5% CO 2 atmosphere. NHDF cells stained with Cell Tracker Red were detached with Accutase in the same manner as in Example 4, then washed with MACS buffer to prepare a cell suspension.

Furthermore, 2102Ep cells, which are adherent cells, were grown in D-MEM medium (High Glucose, Fujifilm Co., Ltd.) supplemented with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Kojunyaku Co., Ltd.), the cells were seeded in a 6 cm diameter adherent culture petri dish (Corning) or a 10 cm diameter adherent culture petri dish (Corning) and cultured at 37°C in a 5% CO2 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 this medium was discarded. Next, Cell Tracker Green dissolved in serum-free RPMI 1640 medium to a final concentration of 0.03 μM was added, and cultured at 37° C. in a 5% CO 2 atmosphere for 1 hour. After discarding the fluorescent reagent solution, the D-MEM medium supplemented with 10% FBS and antibiotic solution was added, and cultured at 37° C. for 1 hour in a 5% CO 2 atmosphere. Next, after discarding the D-MEM medium, new D-MEM medium was added again and the cells were cultured overnight at 37° C. in a 5% CO 2 atmosphere. 2102Ep cells stained with Cell Tracker Green were detached with Accutase in the same manner as in Example 4, and then washed with MACS buffer to prepare a cell suspension.

The NHDF cells, 201B7 cells, 2102Ep cells, and PC-9 cells prepared as described above were each measured for cell density with a hemocytometer, mixed, and bound in a column filled with the adsorbent described below. and peel test.

(3) Binding and Detachment of Mixed Cell Solution of Four Types of Cells Using Column Filled with Adsorbent With the column filled with the adsorbent 127Q39L/C72G standing vertically, the four types of cells prepared by the above method were separated. Addition of mixed cell solution Cell amount NHDF cells: 1.6 x 10 ^ 6 / mL - adsorbent, 201B7 cells: 4.3 x 10 ^ 5 / mL - adsorbent, 2102Ep cells: 9.5 x 10 ^ 4 / mL - Adsorbent, PC-9 cells: 5.7 x 10^5 cells/mL - A cell liquid volume of 1 mL was gently added to the top of the column so as to become an adsorbent. Next, 1 mL of MACS buffer was slowly added from the top of the column, and a total of 2 mL of effluent from the needle was collected in a container (hereinafter referred to as cell effluent fraction No. 1: 0 to 2 mL effluent cell sap). ).

Subsequently, 1 mL of MACS buffer containing 0.0125 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (Hereinafter referred to as Cell Fluid Fraction No. 2: 2-4 mL effluent cell fluid).

Subsequently, 1 mL of MACS buffer containing 0.025 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to separate the effluent from the needle into another container. Collected (hereinafter referred to as cell fluid fraction No. 3: 4-6 mL effluent cell fluid).

Subsequently, 1 mL of MACS buffer containing 0.05 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (Hereinafter referred to as Cell Fluid Fraction No. 4: 6-8 mL effluent cell fluid).

Subsequently, 1 mL of MACS buffer containing 0.1 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (Hereinafter referred to as Cell Fluid Fraction No. 5: 8-10 mL effluent cell fluid).

Subsequently, 1 mL of MACS buffer containing 0.2 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (Hereinafter referred to as Cell Fluid Fraction No. 6: 10-12 mL effluent cell fluid).

Subsequently, 1 mL of MACS buffer containing 0.4 M fucose and 10 mM EDTA was added to the same column from the top of the column, and 1 mL of MACS buffer was added to collect the effluent from the needle in another container. (Hereinafter referred to as Cell Fluid Fraction No. 7: 12-14 mL effluent cell fluid).

As described above, 7 2 mL effluent cell fractions were each collected.

(4) Measurement of cell recovery rate of four types of cells contained in each cell fraction Cell fluid fraction NO. After diluting each of 1 to 7 with MACS buffer to an appropriate volume, 2 mL is aliquoted into a 5 mL polystyrene round tube with a cell strainer cap (manufactured by Corning, opening 40 μm), and internal standard beads for cell counting. After adding 50 μL of CountBright Absolute Counting Beads (manufactured by Invitrogen) as a cell viability determination reagent and 50 μL of 7-AAD as a cell viability determination reagent, NHDF cells contained in each cell fraction were analyzed using a cell sorter BD FACSAria (manufactured by Becton Dickinson). Viable cell numbers were measured for 201B7 cells, 2102Ep cells, and PC-9 cells. Discrimination of NHDF cells, 201B7 cells, 2102Ep cells, and PC-9 cells was performed by analyzing the cell populations developed by dot plots of any two of FITC, PE, APC, and Texas Red. . The number of cells contained in each cell fraction was calculated by proportional calculation based on the number of particles of internal standard beads obtained by dot plotting. The cell recovery rate of each cell in each cell fraction was calculated by dividing the number of each viable cell contained in each cell fraction by the number of added cells of each cell to the column described in (3). .

As a result of measuring the cell efflux rate of 4 types of cells in each cell fraction, cell fraction NO. 1 (0-2 mL effluent cell fluid) contains 54.3% NHDF cells, 1.2% 201B7 cells, 25.9% 2102Ep cells, 33.8% PC-9 cells, and 0.0125 M fucose. The cell fraction NO. 2 (2nd to 4 mL effluent cell fluid), 14.9% NHDF cells, 3.7% 201B7 cells, 49.8% 2102Ep cells, 19.3% PC-9 cells, 0.025M fucose The cell fraction NO. 3 (4-6 mL effluent cell fluid) contains NHDF cells at 6.4%, 201B7 cells at 23.9%, 2102Ep cells at 15.0%, PC-9 cells at 7.9%, and fucose at 0.05M. The cell fraction NO. 4 (6-8 mL effluent cell fluid) contains 6.0% NHDF cells, 43.1% 201B7 cells, 7.4% 2102Ep cells, 10.0% PC-9 cells, and 0.1 M fucose. The cell fraction NO. 5 (8-10 mL effluent cell fluid) contains 5.3% NHDF cells, 16.9% 201B7 cells, 3.7% 2102Ep cells, 13.8% PC-9 cells, and 0.2 M fucose. The cell fraction NO. 6 (10-12 mL effluent cell fluid) contains 3.6% NHDF cells, 4.5% 201B7 cells, 1.9% 2102Ep cells, 11.3% PC-9 cells, and 0.4 M fucose. The cell fraction NO. 7 (12-14 mL effluent cells), NHDF cells were 2.9%, 201B7 cells were 1.5%, 2102Ep cells were 0.6%, and PC-9 cells were 16.6% (each cell Cell recovery in fractions is shown in Table 4 and Figure 5).

From this result, cell fraction NO. NHDF cells and PC-9 cells, cell fraction NO. 2, 2102Ep cells, fraction NO. After 3, it was found that the cell outflow rate of 201B7 cells was high, and it was found that the distribution ratio of cells in each cell fraction was different. That is, it was clarified that 2102Ep cells and 201B7 cells can be separated from NHDF cells and PC-9 cells, respectively, by stepwise action of solutions with successively increased concentrations of fucose.

This result shows that the NHDF cells and PC-9 cell populations, which have a weaker binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, are selected by passing the cells through a solution that does not contain fucose. 2102Ep cells, which have a relatively strong binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, are selectively exfoliated, and further It can be interpreted that this is the result of detachment of 201B7 cells, which have a strong binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, by the action of a high-concentration fucose solution. Therefore, even when a mixed cell solution of four types of cells is bound and detached to a single column filled with a cell adsorbent, a solution with a stepwise control of fucose concentration is sequentially applied to obtain a fraction of each detached cell. By doing so, it was confirmed that different types of cells could be separated from each other.

Example 6 Separation of four types of cell mixture by adsorption/desorption to adsorbent-2
Example 6 relates to separation of heterologous cells by cell adsorption/desorption to a cell adsorbent using four types of heterogeneous mixed cells, NHDF cells, 201B7 cells, 2102Ep cells, and PC-9 cells, as in Example 5. be. The difference from Example 5 is that MACS buffer was used in Example 5 and 0.4M mannitol solution was used in Example 6 when preparing the stripping solution.

(1) Preparation of column packed with adsorbent By the same method as in Example 3, the column was packed with adsorbent 127Q39L/C72G (capacity of adsorbent: 500 µL).

(2) Cultivation of NHDF cells, 201B7 cells, 2102Ep cells, and PC-9 cells and preparation of cell suspension for evaluation The same procedure as in Example 5 (2) was performed.

(3) Binding and Detachment of Mixed Cell Solution of Four Types of Cells Using Column Filled with Adsorbent With the column filled with the adsorbent 127Q39L/C72G standing vertically, the four types of cells prepared by the above method were separated. Addition of mixed cell solution Cell amount NHDF cells: 1.6 x 10 ^ 6 / mL - adsorbent, 201B7 cells: 4.3 x 10 ^ 5 / mL - adsorbent, 2102Ep cells: 9.5 x 10 ^ 4 / mL -Adsorbent, PC-9 cells: 5.7 x 10^5 cells/mL -Added in a volume of 1 mL of cell liquid so as to be an adsorbent.

Next, 1 mL of MACS buffer was gently added from the top of the column, and a total of 2 mL of the effluent from the needle was collected in a container (hereinafter referred to as cell fluid fraction NO.8: 0 to 2 mL effluent cell fluid ).

Next, 1 mL of 0.4 M mannitol aqueous solution containing 0.0125 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. (Hereinafter referred to as Cell Fluid Fraction No. 9: 2-4 mL effluent cell fluid).

Next, 1 mL of 0.4 M mannitol aqueous solution containing 0.025 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. (Hereinafter referred to as Cell Fluid Fraction No. 10: 4-6 mL effluent cell fluid).

Next, 1 mL of 0.4 M mannitol aqueous solution containing 0.05 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. (Hereinafter referred to as Cell Fluid Fraction No. 11: 6-8 mL effluent cell fluid).

Next, 1 mL of a 0.4 M mannitol aqueous solution containing 0.1 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. (Hereinafter referred to as Cell Fluid Fraction No. 12: 8-10 mL effluent cell fluid).

Next, 1 mL of 0.4 M mannitol aqueous solution containing 0.2 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. (Hereinafter referred to as Cell Fluid Fraction No. 13: 10-12 mL effluent cell fluid).

Next, 1 mL of a 0.4 M mannitol aqueous solution containing 0.4 M fucose and 10 mM EDTA was added from the top of the column, followed by the addition of 1 mL of MACS buffer, and 2 mL of the effluent from the needle was transferred to another container. recovered to. (Hereinafter referred to as Cell Fluid Fraction No. 14: 12-14 mL effluent cell fluid).

As described above, 7 fractions of 2 mL of the effluent cell fluid were each collected.

(4) Measurement of cell recovery rate of 4 types of cells contained in each cell fraction The cell recovery rate of 4 types of cells was measured in the same manner as in Example 5(4). As a result of measuring the cell efflux rate of 4 types of cells in each cell fraction, cell fraction NO. 8 (0-2 mL effluent cell fluid) contains 48.2% NHDF cells, 1.0% 201B7 cells, 21.4% 2102Ep cells, 33.0% PC-9 cells, and 0.0125 M fucose. The cell fraction NO. 9 (2-4 mL effluent cell fluid) contains 33.1% NHDF cells, 21.9% 201B7 cells, 38.2% 2102Ep cells, 41.7% PC-9 cells, and 0.025M fucose. The cell fraction NO. 10 (4-6 mL effluent cell fluid) contains 7.9% NHDF cells, 52.4% 201B7 cells, 9.5% 2102Ep cells, 7.1% PC-9 cells, and 0.05M fucose. The cell fraction NO. 11 (6-8 mL effluent cell fluid) contains 3.3% NHDF cells, 23.9% 201B7 cells, 2.9% 2102Ep cells, 2.7% PC-9 cells, and 0.1 M fucose. The cell fraction NO. 12 (8-10 mL effluent cell fluid) contains 1.2% NHDF cells, 6.1% 201B7 cells, 0.8% 2102Ep cells, 1.4% PC-9 cells, and 0.2 M fucose. The cell fraction NO. In 13 (10-12 mL effluent cell fluid), NHDF cells are 1.2%, 201B7 cells are 3.6%, 2102Ep cells are 0.3%, PC-9 cells are 1.9%, and fucose is 0.4M. The cell fraction NO. 14 (12th to 14th mL effluent cell fluid) contained NHDF cells at 0.6%, 201B7 cells at 0.7%, 2102Ep cells at 0.0%, and PC-9 cells at 1.3% (each Cell recovery in cell fractions is shown in Table 5 and Figure 6).

From this result, cell fraction NO. NHDF cells, cell fraction NO. 2 PC-9 cells and 2102Ep cells, fraction NO. After 3, it was found that the cell outflow rate of 201B7 cells was high, and it was found that the distribution ratio of cells in each cell fraction was different. That is, it was clarified that NHDF cells and 201B7 cells can be separated from PC-9 cells and 2102Ep cells by stepwise action of solutions with successively increased fucose concentrations.

As a result, by passing the cells through a solution that does not contain fucose, NHDF cells, which have a weak binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, first selectively flow out, and then sequentially. By applying a solution with an increased fucose concentration, 2102Ep cells and PC-9 cell populations, which have a relatively strong binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent, are selectively detached. It can be interpreted that this is the result of detachment of 201B7 cells, which have a strong binding action to 127Q39L/C72G present on the surface of the adsorbent, by the action of a high-concentration fucose solution. Therefore, even when a mixed cell solution of four types of cells is bound and detached to a single column filled with a cell adsorbent, a solution with a stepwise control of fucose concentration is sequentially applied to obtain a fraction of each detached cell. By doing so, it was confirmed that different types of cells could be separated from each other.

In addition, the difference in the cell distribution ratio between each cell fraction in Example 5 and Example 6 depends on whether fucose dissolved in MACS buffer or 0.4 M mannitol aqueous solution is used as the cell detachment solution. In particular, the cell detachment of 2102Ep cells and PC-9 cells, which are considered to have a moderate binding action to the fucose-binding protein 127Q39L/C72G present on the surface of the adsorbent. It was thought that there was a difference in

Claims (13)

A step of binding cells that bind to a fucose-binding protein from cell populations with different degrees of undifferentiation to an adsorbent; obtaining detached cells, wherein the adsorbent has a structure in which a fucose-binding protein is immobilized on a water-insoluble carrier. 2. The method according to claim 1, wherein a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc is expressed on the surface of the cell that binds to the fucose-binding protein. . 3. The method according to claim 2, wherein the cell population with different degrees of undifferentiation is a mixture of heterogeneous cells with different expression levels of undifferentiation markers. 3. The method according to claim 2, wherein the cell populations with different degrees of undifferentiation are cell populations of the same type of cells but with different expression levels of undifferentiated markers. 5. The method according to any one of claims 1 to 4, wherein fucose solutions with different fucose concentrations are changed stepwise from low concentration to high concentration to obtain exfoliated cells. 6. A method for isolating cells with different expression levels of sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, using the method according to any one of claims 1 to 5. Using the method according to any one of claims 1 to 5, from a cell population having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, depending on the expression level of these sugar chains A method for isolating cells from cells. Using the method according to any one of claims 1 to 5, heterogeneous cell groups with different expression levels of sugar chains containing structures consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc are separated and separated. A method of separating cell groups with different expression levels of sugar chains from single cell types having one or more sugar chains contained in a target cell population. 9. The method according to any one of claims 1 to 8, wherein the cell having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc is a human pluripotent stem cell. 10. The method according to any one of claims 1 to 9, wherein the fucose-binding protein is any one of the following (a) to (d).
(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. sex 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 fucose and having binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, wherein X is an integer of 120 or more 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) substitution of leucine residue for glutamine residue at position 39 in the amino acid sequence shown in SEQ ID NO: 1, wherein X is an integer of 120 or more, comprising an amino acid sequence containing the above (2) Substitution 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) 65th amino acid sequence shown in SEQ ID NO: 1 (d) in the amino acid sequence of the fucose-binding protein of (c), one or more in regions other than 39th, 65th and 72nd of SEQ ID NO: 1 and has binding affinity to a sugar chain comprising a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, Fucose-binding protein. 10. The method according to any one of claims 1 to 9, wherein the fucose-binding protein is any of the following (e) to (h).
(e) to the 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, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is A fucose-binding protein consisting of an amino acid sequence to which a cysteine-containing oligopeptide is added, wherein X is an integer of 120 or more.
(f) 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 On the other hand, a sugar comprising an amino acid sequence to which a polyhistidine sequence is added to the N-terminus and an oligopeptide containing cysteine is added to the C-terminus, and which has a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having binding affinity to a chain, wherein X is an integer of 120 or greater.
(g) to the 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, an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and the C-terminus is In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the amino acid substitutions described in (4) to (6) below, wherein X is an integer of 120 or more, fucose-binding protein (4) replacement of the 39th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (5) substitution of the 72nd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1, Substitution of one amino acid residue selected from glycine residues and alanine residues (6) Substitution of the 65th glutamine residue in the amino acid sequence shown in SEQ ID NO: 1 with a leucine residue (h) The above ( An amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added to regions other than the 39th, 65th and 72nd regions of SEQ ID NO: 1 in the amino acid sequence of the fucose-binding protein of g) to which an oligopeptide containing a polyhistidine sequence is added at the N-terminus and an oligopeptide containing a cysteine sequence is added at the C-terminus, comprising an amino acid sequence, Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity to sugar chains comprising a structure of 10. The method according to any one of claims 1 to 9, wherein a hydrophilic polymer is covalently immobilized on a water-insoluble carrier. 10. The method according to any one of claims 1 to 9, wherein an adsorbent packed in a column is used.

Images