JP2022151225A - Separation method of cells with different degrees of undifferentiation using laminated adsorbents - Google Patents

Abstract

To provide technologies for easily and efficiently separating cells with different expression levels of undifferentiated markers from cell populations with different expression levels of undifferentiated markers present on the surface of cells.SOLUTION: The above problem is solved by preparing a plurality of adsorbents with different immobilization of fucose-binding proteins on water-insoluble carriers, bringing cell populations with different expression levels of undifferentiated sugar chain markers first into contact with an adsorbent with a low immobilization amount of fucose-binding proteins, then, gradually into contact with an adsorbent with a high immobilization amount of fucose-binding proteins, and separating and collecting cells with different expression levels of undifferentiated sugar chain markers present on the surface of cells.SELECTED DRAWING: Figure 2

Description

The present invention is a cell separation method using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier. The present invention relates to a method for separating cell populations with different degrees of undifferentiation by bringing the cell populations into contact stepwise and collecting fractions of cells flowing out from the column.

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 cell separation method using an adsorbent in which BC2LCN is immobilized on a water-insoluble carrier, a cell separation method that removes undifferentiated cells such as human iPS cells is known (Patent Document 3). A method of peeling and collecting bound undifferentiated cells is known (Patent Document 4). 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 2018-134073 A JP 2019-000063 A

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 for separating and purifying cell populations with different degrees of undifferentiation from a large amount of cells in a short period of time in the preparation of cells for regenerative medicine applications, specifically, an undifferentiation marker present on the cell surface. is to provide a technique for separating cell populations with different expression levels of
More specifically, a cell population is brought into stepwise contact with a column in which a plurality of adsorbents having different amounts of immobilized fucose-binding proteins on the carrier are layered, and then the cells that do not bind to the adsorbent are separated. An object of the present invention is to provide a technique for individually, simply and efficiently separating cell populations with different degrees of undifferentiation by collecting fractions.

As a result of intensive studies to solve the above problems, the present inventors found that expression of a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, which are undifferentiated markers present on the cell surface. Cell populations with different amounts are first brought into contact with an adsorbent having a low amount of immobilized fucose-binding protein, and then brought into contact with an adsorbent having a higher amount of immobilized fucose-binding protein step by step for adsorption. By collecting a fraction of cells that were not adsorbed to the agent, it was found that cell populations with different expression levels of undifferentiated markers present on the cell surface could be separated and collected from the original cell population, and the present invention was completed. came to.

That is, the present invention includes the inventions described in [1] to [8] below.
[1]
A cell separation method using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, wherein cell populations with different degrees of undifferentiation are adsorbed by a plurality of adsorptions with different immobilized amounts of the fucose-binding protein. A method for separating cells with different degrees of undifferentiation, comprising a step of binding to an agent, and a step of obtaining cells not bound to the adsorbent.
[2]
Cell populations with different degrees of undifferentiation are adsorbed on multiple adsorbents with different amounts of immobilized fucose-binding proteins, from adsorbents with low amounts of immobilized fucose-binding proteins to adsorbents with high immobilized amounts of fucose-binding proteins. The method for separating cells with different degrees of undifferentiation according to [1] above, comprising the step of stepwise binding to the agent, and the step of obtaining cells that have not bound to the adsorbent.
[3]
Use of a plurality of adsorbents having a fucose-binding protein immobilized amount ranging from 50 mg/L-adsorbent to 1000 mg/L-adsorbent and having a different fucose-binding protein immobilized amount of 100 mg/L-adsorbent or more. The method for separating cells with different degrees of undifferentiation according to [1] or [2] above, characterized in that:

[4]
from [1] above, characterized in that the cell populations with different degrees of undifferentiation express sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the surface of the cells. The method for separating cells with different degrees of undifferentiation according to any one of [3].
[5]
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 [4] above.
[6]
The method according to any one of [1] to [5] above, wherein the fucose-binding protein is any one of (a) to (d) below.
(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.

[7]
The method according to any one of [1] to [5], 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

[8]
The method according to any one of [1] to [7] above, wherein a column filled with a plurality of adsorbents having different amounts of immobilized fucose-binding proteins is used.

The present invention will be described in detail below.

In the method of separating cells with different degrees of undifferentiation using the laminated adsorbent of the present invention (hereinafter sometimes abbreviated as the cell separation method of the present invention), the fucose-binding protein is immobilized on a water-insoluble carrier. A method for separating cells using an adsorbent comprising a cell population in which a cell population is brought into stepwise contact with a column stacked with a plurality of adsorbents having different amounts of fucose-binding protein immobilized on the carrier. A method for separating cells with different degrees of undifferentiation, characterized by including a step of obtaining fractions of cells that did not bind to the adsorbent.

In the cell separation method of the present invention, the undifferentiated marker is a molecule that is present on the cell surface and serves as an indicator of the degree of undifferentiation. from Fucα1-2Galβ1-3GlcNAc with carbohydrates comprising H-type 1-type glycans, H-type 3-type glycans, Lewis Y-type glycans, and/or Lewis b-type glycans known as chains and/or a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc. 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, It may be SSEA-4, SSEA-5, or the like.

In the cell separation method of 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, the above-described cell populations with different degrees of undifferentiation are first brought into contact with an adsorbent with a low amount of immobilized fucose-binding protein, and then adsorbed with a higher amount of immobilized fucose-binding protein. A method of cell separation comprising stepwise contacting with an agent and recovering the fraction of cells not bound to the adsorbent.

In this case, at least two kinds of adsorbents prepared with different immobilized amounts of fucose-binding protein may be used, and the order of contacting the cell population with the adsorbent is such that the immobilized amount of fucose-binding protein is low. The number of kinds of adsorbents to be used is not limited as long as it is a form in which the adsorbent is changed from a high agent to a high adsorbent. In this case, the fucose protein to be immobilized on the adsorbent may be of the same type of amino acid sequence or may be functionally improved by amino acid substitution as described below. Alternatively, a plurality of types of adsorbents may be prepared by immobilizing fucose-binding proteins having different amino acid sequences in different immobilized amounts, and two or more adsorbents may be arbitrarily selected from among these and used in combination. It is possible.

In the cell separation method of the present invention, cell populations with different degrees of undifferentiation are first brought into contact with an adsorbent having a low immobilized amount of fucose-binding protein, thereby selecting a cell population with a high degree of undifferentiation contained in the cell population. can be effectively trapped on the adsorbent.

Next, by bringing the remaining cell population into contact with an adsorbent with a higher amount of immobilized fucose-binding protein in a stepwise manner, the less undifferentiated cell population contained in the cell population is transferred to the adsorbent. can be captured. Therefore, for example, when the cell separation method of the present invention is performed using a column filled with an adsorbent, the adsorbent with a low amount of immobilized fucose-binding protein is placed in the upper part of the column, and the fucose-bound protein is placed in the lower part of the column. By stacking adsorbents with a high amount of immobilized proteins, an adsorbent with a low amount of immobilized fucose-binding proteins packed in the upper part of the column when cell populations with different degrees of undifferentiation are brought into contact. A cell population with a high degree of undifferentiation is selectively adsorbed to the column, and a cell population with a low degree of undifferentiation is selectively adsorbed to the adsorbent packed at the bottom of the column and having a large amount of immobilized fucose-binding protein.

In this case, based on the difference in the expression level of undifferentiated sugar chain markers on the surface of individual cells, cells with high expression levels of undifferentiated marker sugar chains, i. It is considered that the cell distribution is formed by the difference in the expression level of the undifferentiated sugar chain marker, such as cells with high expression level of the undifferentiated sugar chain marker, ie, cells with low degree of undifferentiation. However, the cells that first flow out from the bottom end of the column are less undifferentiated cell populations, and the cells that flow out later are considered to be more undifferentiated cell populations. By finely collecting fractions of cell populations with different degrees of differentiation, it is possible to separate cell populations with different degrees of undifferentiation.

In this case, the binding between the undifferentiated sugar chain marker on the cell surface and the fucose-binding protein of the adsorbent is reversible, and is affected by the gravity of the cells themselves and the shear force of the solution flow introduced from the top of the column. Therefore, it is considered that cells repeatedly bind to and desorb from the adsorbent. It is thought that the adsorbent with the amount of binding protein immobilized forms a cell distribution such that the closer to the top of the column the more undifferentiated the cell population and the farther from the top the less undifferentiated the cell population. .

This phenomenon is demonstrated, for example, in an anti-CD34 antibody-immobilized cell rolling column, where cells with lower CD34 antigen expression levels migrate faster and flow out faster, while cells with higher CD34 antigen expression levels migrate slower. It is considered to be based on the same principle as the phenomenon that outflow is slow.

In the cell separation method of the present invention, it is possible to use an adsorbent with an immobilized amount of fucose-binding protein of 50 mg/L-adsorbent to 1000 mg/L-adsorbent. It is preferred to use multiple adsorbents that differ by more than 100 mg/L-adsorbent. As a form of the cell separation method of the present invention, for example, first, the cell population is brought into contact with an adsorbent having an immobilized amount of fucose-binding protein of 100 mg/L-adsorbent, and the cell population after contact is then treated with the fucose-binding protein. Contact with an adsorbent with an immobilized amount of 200 mg / L-adsorbent, and contact with an adsorbent with an immobilized amount of fucose-binding protein of 300 mg / L-adsorbent. can. The type of fucose-binding protein in the multiple adsorbents used, the amount of immobilized fucose-binding protein, the combination of adsorbents, the amount of each adsorbent used (ratio of adsorbents) It may be appropriately adjusted according to the degree of undifferentiation of the differentiated cells, the range (variation), and the like.

As the best embodiment of the cell separation method of the present invention, an adsorbent with a low amount of immobilized fucose-binding protein is placed in a vertical column, and an adsorbent with a high amount of immobilized fucose-binding protein is placed in the upper part of the column. A plurality of adsorbents may be laminated and packed such that the adsorbents are positioned at the bottom of the column. In this case, the method of packing the adsorbent is not particularly limited as long as the amount of fucose-binding protein immobilized on the surface of the packed adsorbent has a concentration gradient. Multiple layers of the agent may be packed and used, or the adsorbent may be packed so that the concentration gradient of the fucose-binding protein immobilized on the adsorbent is linear.

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.

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 3 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 the fucose-binding protein of the present invention on a water-insoluble carrier is not particularly limited as long as it is a general functional group for protein immobilization, and includes an epoxy group, a formyl group, a carboxyl group, an active Examples include an ester group, 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.

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, and is described in Patent Document 3, for example. You can do it according to the method.

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 amount of unreacted fucose-binding protein of the present invention from the amount of protein.

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 of the present invention 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.

In the cell separation method of the present invention, a plurality of adsorbents with different immobilized amounts of fucose-binding proteins are used, and cell populations with different degrees of undifferentiation are brought into contact stepwise to obtain Fucα1-2Galβ1-3GlcNA and/or Fucα1-2Galβ1-3GlcNA. Alternatively, it is an effective cell separation method for separating and purifying cells having a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GalNAc based on the difference in the expression level of the sugar chain. In the cell separation method of the present invention, it is possible to separate and recover cells based on the difference in the expression level of the undifferentiated marker sugar chain. Since it is useful for separation according to the amount of undifferentiated marker expression, cells with a particularly low degree of undifferentiation, i.e., undifferentiated deviant cells, can be efficiently removed and sorted from undifferentiated cells such as human iPS cells. It is an extremely effective means for the preparation of high-quality highly undifferentiated cells for regenerative medicine and the functional analysis of undifferentiated deviant cells.

In addition, the cell separation method of the present invention can easily process a large amount of cells compared to the conventional cell separation method using magnetic beads, cell sorting using a cell sorter, and cell separation method using a cell rolling column. It is also an extremely effective means for large-scale purification of different cell populations.

2 is a graph showing the 2102Ep cell recovery rate of each cell fraction in Example 1, Comparative Example 1, and Reference Example 1. FIG. 1 is a graph showing the BC2LCN positive rate of 2102Ep cells of each cell fraction in Example 1, Comparative Example 1, and Reference Example 1. FIG.

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 adsorbents 156GGC-A, B, C, and D According to the method described in Examples 1 to 3 of JP-A-2019-000063, by changing the reaction amount of immobilized BC2LCN during preparation of the adsorbent, fucose bond Four types of adsorbents (adsorbents 156GGC-A, B, C, and D) with different immobilized amounts of sex protein 156GGC (hereinafter referred to as BC2LCN) were prepared. The amount of BC2LCN immobilized on each adsorbent was 146 mg/L-adsorbent for adsorbent 156GGC-A, 307 mg/L-adsorbent for adsorbent 156GGC-B, and 412 mg/L-adsorbent for adsorbent 156GGC-C. Agent 156GGC-D was 682 mg/L-adsorbent (Table 1).

Example 1 Separation of cell populations with different degrees of undifferentiation from 2012Ep cells using columns laminated with adsorbents 156GGC-A, B, C, and D 2102Ep cells (Embryonal Carcinoma Cells Cl.4/ This invention relates to a method for separating cell populations with different degrees of undifferentiation from D3 cells (obtained from Cosmo Bio).
(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). One was produced. Next, after replacing the adsorbent 156GGC-D prepared in Preparation Example 1 with MACS buffer (manufactured by Miltenyi Biotech), the 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 of the suspension was added to the prepared column to fill the column with adsorbent 156GGC-D (adsorbent capacity: 0.5 mL). Next, in the same procedure, the adsorbent 156GGC-C prepared in Preparation Example 1 was filled above the adsorbent 156GGC-D with an adsorbent capacity of 0.5 mL. Next, in the same procedure, the adsorbent 156GGC-B prepared in Preparation Example 1 was filled above the adsorbent 156GGC-C with an adsorbent capacity of 0.5 mL. Next, in the same procedure, the adsorbent 156GGC-A prepared in Preparation Example 1 was filled above the adsorbent 156GGC-B with an adsorbent capacity of 0.5 mL. In this way, from the top of the column, adsorbent 156GGC-A, adsorbent 156GGC-B, adsorbent 156GGC-C, and adsorbent 156GGC-D are stacked in this order so that each has a packing capacity of 0.5 mL. A packed column was prepared. This column prepared is referred to as column X below.
(2) Culture of 2102Ep Cells and Preparation of Cell Suspension 2102Ep cells are prepared in D-MEM medium supplemented with 10% FBS (manufactured by Biological Industries) and an antibiotic solution (penicillin-streptomycin solution, manufactured by Fujifilm Wako Pure Chemical Industries). (High Glucose, manufactured by Fujifilm Wako Pure Chemical Industries, 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 placed under a 5% CO2 atmosphere for 37 days. ℃. Next, 2102Ep cells were fluorescently stained using Cell Tracker Orange (manufactured by Thermo Fisher Scientific) by the following method. After discarding the medium in the petri dish during which 2102Ep cells were being cultured, serum-free RPMI 1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries) 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, 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 supplemented with 10% FBS and an antibiotic solution, a new D-MEM medium supplemented with 10% FBS and an antibiotic solution was added and placed at 37° C. in a 5% CO atmosphere. was cultured overnight.

Next, cell collection and cell suspension preparation were performed by the following methods. After discarding the D-MEM medium supplemented with 10% FBS and antibiotic solution in the petri dish during cell culture and adding D-PBS (-) (manufactured by Cell Science Research Institute), the cells were washed and D- PBS (-) was discarded. Next, Accutase (manufactured by Innovative Cell Technology) was added and allowed to stand for several minutes to detach the 2102Ep 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 procedure twice, the cells were suspended in MACS buffer and filtered using a cell strainer (manufactured by Corning, opening 40 μm) to prepare a cell suspension of 2102Ep cells stained with Cell Tracker Orange. did. A portion of the obtained 2102Ep cell suspension was taken, diluted 10-fold, and the cell density was calculated using a hemocytometer. Based on this cell density, the number of cells added to the column was calculated from the value of the amount of cell solution applied to the column x the cell density.
(3) Separation of populations with different BC2LCN positive rates from 2012Ep cells using a column packed with adsorbent layered 2012Ep prepared in (2) while the column X prepared in (1) is vertically set. The cell suspension was added to the top of the column in a mixed cell volume of 0.1 mL so that the number of cells added per column was 7.8×10 ⁶ . Next, 1.0 mL of MACS buffer was gently added from the top of the column, and a total of 1.1 mL of effluent from the needle was collected in another container (hereinafter referred to as cell fluid Fr.1). . Next, 1.0 mL of MACS buffer was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell fluid Fr.2). . Next, 1.0 mL of MACS buffer was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell fluid Fr.3). . Next, 1.0 mL of MACS buffer was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell fluid Fr.4). . Next, 1.0 mL of MACS buffer containing 0.2 M fucose and 10 mM EDTA (hereinafter referred to as cell detachment solution) was gently added from the top of the column, and the total effluent from the needle was 1.0 mL. was collected in a separate container (hereinafter referred to as cell fluid Fr.5). Next, 1.0 mL of cell detachment solution was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell solution Fr.6). . Next, 1.0 mL of cell detachment solution was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell solution Fr.7). . Next, 1.0 mL of cell detachment solution was gently added from the top of the column, and a total of 1.0 mL of effluent from the needle was collected in another container (hereinafter referred to as cell solution Fr.8). .
(4) Each cell fluid Fr. Measurement of cell recovery rate and BC2LCN positive rate of 2012Ep cells in the cell solution Fr. For 1 to 8, 0.5 mL of the cell solution was diluted with 2 mL of MACS buffer, then dispensed into a 5 mL polystyrene round tube (manufactured by Corning) with a cell strainer cap, and CountBright Absolute was used as an internal standard bead for counting cells. After adding 50 μL of 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). Each cell Fr. The number of cells contained in was calculated by proportional calculation based on the number of particles of the internal standard beads obtained by dot plotting. Each cell Fr. The cell recovery rate of each cell Fr. was calculated by dividing the number of cells contained in each by the number of added cells (7.8×10 ⁶ cells) described in (3) above.

In addition, cell fluid Fr. 1 to 8, each cell fluid Fr. The BC2LCN positive rate of cells contained in was measured as follows. Each Fr. 0.5 mL each of the cell solution was collected in a 1.5 mL Eppendorf tube, centrifuged, the supernatant was discarded, and the cells were washed by suspending in 1.0 mL of MACS buffer. After centrifugation, the cell pellet obtained by discarding the supernatant was suspended in 1 mL of MACS buffer containing 0.5% (vol./vol.) BC2LCN-FITC (manufactured by Fujifilm Wako Pure Chemical Industries) and placed in the dark. and allowed to stand at room temperature for 30 minutes. After reacting for 30 minutes, the cells were washed with the above MACS buffer in an Eppendorf tube, and the resulting cell pellet was suspended in 2 mL of MACS buffer. After transferring 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 50 μL of 7-AAD as a cell viability determination reagent were added. Then, the fluorescence intensity of the cell population was analyzed using a cell sorter BD FACSAria (manufactured by Becton Dickinson). The BC2LCN positive rate of cells is determined by defining the 2102Ep cells unstained with BC2LCN-FITC as a negative cell population, and the cell population with increased FITC fluorescence intensity due to the reaction with a fluorescent reagent as a positive cell population, BC2LCN positive rate = ( It was calculated as the number of positive viable cells contained in each cell fluid Fr.)/(total viable cell count contained in each cell Fr.). 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 FACS analysis, each cell fluid Fr. The recovery of 2102p cells in Fr. 1=1.2%, Fr. 2=2.4%, Fr. 3=4.3%, Fr. 4=5.8%, Fr. 5=33.3%, Fr. 6=20.5%, Fr. 7=5.6%, Fr. 8=7.1%. Also, each cell Fr. The BC2LCN positive rate of 2102Ep cells in Fr. 1=31.9%, Fr. 2=38.9%, Fr. 3=58.6%, Fr. 4=60.0%, Fr. 5=82.3%, Fr. 6=81.0%, Fr. 7=80.1%, Fr. 8=76.7%. Each cell fluid Fr. Table 3 and FIG. 2 show the recovery rate of 2102p cells in each cell fluid Fr. BC2LCN positive rate of cells contained in.

From this result, four types of adsorbents with different immobilized amounts of fucose-binding protein were selected from the top of the column: adsorbent 156GGC-A (146 mg BC2LCN immobilized/mL-adsorbent), adsorbent 156GGC-B (307 mg BC2LCN immobilized/mL- adsorbent), adsorbent 156GGC-C (412 mg BC2LCN fixed/mL-adsorbent), and adsorbent 156GGC-D (682 mg BC2LCN fixed/mL-adsorbent) stacked in this order (column X), The effluent cell Fr. The undifferentiated degree (BC2LCN positive rate) of Fr. 1=31.9%, Fr. 2=38.9%, Fr. 3=58.6%, Fr. 4 = 60.0%, and the 2102Ep cell populations with different degrees of undifferentiation were subdivided from cell populations with low degrees of undifferentiation to cell populations with high degrees of undifferentiation, respectively. It became clear that it can be fractionated as

As a result, the amount of fucose-binding protein immobilized on the adsorbent packed in the direction from the top of the column to the bottom of the column has a concentration gradient from small to large, resulting in a highly undifferentiated cell population. By selectively binding the packing material in the upper part of the column and the cell population with low undifferentiated degree to the packing material in the lower part of the column, the distribution of cells due to the difference in the degree of undifferentiation occurs, and the outflow of cells from the column by MACS buffer Fr . First, cells with a low degree of undifferentiation flow out to the cell Fr. It can be interpreted that this is the result of the gradual outflow of highly undifferentiated cells with each fractionation. Therefore, adsorbents with different amounts of immobilized fucose-binding protein are packed in layers in a column, and cell populations with different degrees of undifferentiation are brought into contact stepwise from adsorbents with low levels of immobilized fucose-binding proteins to adsorbents with high levels of fucose-binding proteins. As a result, cells with different degrees of undifferentiation become cells Fr. It was confirmed that fractionation is possible as

Comparative Example 1 Separation of cell populations with different degrees of undifferentiation from 2012Ep cells using a column packed with adsorbent 156GGC-B ), prepare a 50% suspension of the adsorbent so that the sedimentation volume of the adsorbent after standing for 12 hours or more is 50%, add 4.0 mL to the prepared column, and adsorb The column was packed with agent 156GGC-B (adsorbent capacity 2.0 mL). This column prepared is referred to as column Y below. 2102Ep cells were prepared, the solution was passed through the column, and the obtained cells Fr. was performed by FACS analysis.

As a result of FACS analysis, each cell Fr. The recovery of 2102p cells in Fr. 1=13.4%, Fr. 2=14.3%, Fr. 3=3.2%, Fr. 4=3.3%, Fr. 5=10.3%, Fr. 6=8.1%, Fr. 7=2.3%, Fr. 8=1.2%. Also, each cell Fr. The BC2LCN positive rate of 2102Ep cells in Fr. 1=71.2%, Fr. 2=74.5%, Fr. 3=80.8%, Fr. 4=85.6%, Fr. 5=88.5%, Fr. 6=83.5%, Fr. 7=80.2%, Fr. 8=87.3%. Each cell fluid Fr. Table 3 and FIG. 2 show the recovery rate of 2102p cells in each cell fluid Fr. BC2LCN positive rate of cells contained in.

From this result, in the column (column Y) filled with only the adsorbent 156GGC-B (307 mg/L-adsorbent) with a single fucose-binding protein immobilization amount, the effluent cells obtained by passing the MACS buffer solution Fr. The undifferentiated degree (BC2LCN positive rate) of Fr. 1=71.2%, Fr. 2=74.5%, Fr. 3=80.8%, Fr. 4 = 85.6%, and although it is gradually increasing, the BC2LCN positive rate Fr. The difference between them was smaller than in Example 1, and unlike in Example 1, it was not possible to individually separate a cell population with a low degree of undifferentiation with a BC2LCN positive rate of about 30% to 60%. Therefore, when a column is filled with an adsorbent having a single amount of immobilized fucose-binding protein and brought into contact with cell populations with different degrees of undifferentiation, cells with greatly different degrees of undifferentiation are classified into cell Fr. It was confirmed that it could not be fractionated as

Reference Example 1 Separation of cell populations with different degrees of undifferentiation from 2012Ep cells using a column packed with Toyopearl HW-40EC ), prepare a 50% suspension of the adsorbent so that the sedimentation volume of the adsorbent after standing for 12 hours or more is 50%, add 4.0 mL to the prepared column, and adsorb The column was packed with the agent Toyopearl HW-40EC (adsorbent capacity: 2.0 mL). This column thus prepared will be referred to as column Z below. 2102Ep cells were prepared, the solution was passed through the column, and the obtained cells Fr. was performed by FACS analysis.

As a result of FACS analysis, each cell Fr. The recovery of 2102p cells in Fr. 1=22.5%, Fr. 2=33.0%, Fr. 3=3.6%, Fr. 4=0.8%, Fr. 5=1.7%, Fr. 6=2.9%, Fr. 7=0.9%, Fr. 8=1.9%. Also, each cell Fr. The BC2LCN positive rate of 2102Ep cells in Fr. 1=79.5%, Fr. 2=74.0%, Fr. 3=84.0%, Fr. 4 = 82.7% (Fr. 5-8 not measured). Table 2 and FIG. 1 show each cell fluid Fr. Table 3 and FIG. 2 show the recovery rate of 2102p cells in each cell fluid Fr. BC2LCN positive rate of cells contained in.

From this result, in the column (column Z) filled with the adsorbent Toyopearl HW-40EC on which no fucose-binding protein was immobilized, the effluent cells Fr. The undifferentiated degree (BC2LCN positive rate) of Fr. 1=79.5%, Fr. 2=74.0%, Fr. 3=84.0%, Fr. 4 = 82.7%, and as a result of the 2102Ep cells passing through the adsorbent without being adsorbed, it was not possible to individually separate cell populations with different degrees of undifferentiation. Therefore, when a column is filled with an adsorbent on which no fucose-binding protein is immobilized and cell populations with different degrees of undifferentiation are brought into contact, cells with different degrees of undifferentiation are classified into cell Fr. It was confirmed that it could not be fractionated as

Claims (8)

A cell separation method using an adsorbent in which a fucose-binding protein is immobilized on a water-insoluble carrier, wherein cell populations with different degrees of undifferentiation are adsorbed by a plurality of adsorptions with different immobilized amounts of the fucose-binding protein. A method for separating cells with different degrees of undifferentiation, comprising a step of binding to an agent, and a step of obtaining cells not bound to the adsorbent. Cell populations with different degrees of undifferentiation are adsorbed on multiple adsorbents with different amounts of immobilized fucose-binding proteins, from adsorbents with low amounts of immobilized fucose-binding proteins to adsorbents with high immobilized amounts of fucose-binding proteins. 2. The method for separating cells with different degrees of undifferentiation according to claim 1, comprising a step of stepwise binding to an agent, and a step of obtaining cells that have not bound to the adsorbent. Use of a plurality of adsorbents having a fucose-binding protein immobilized amount ranging from 50 mg/L-adsorbent to 1000 mg/L-adsorbent and having a different fucose-binding protein immobilized amount of 100 mg/L-adsorbent or more. The method for separating cells with different degrees of undifferentiation according to claim 1 or 2, characterized by: Claims 1 to 3, characterized in that the cell populations with different degrees of undifferentiation express sugar chains containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc on the surface of the cells. 3. The method for separating cells with different degrees of undifferentiation according to any one of . 5. 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 4. 6. The method according to any one of claims 1 to 5, 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. 6. The method according to any one of claims 1 to 5, 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 8. The method according to any one of claims 1 to 7, wherein a column filled with a plurality of adsorbents having different immobilized amounts of fucose-binding protein is used.

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