WO1998052039A1 - Method for separating cells - Google Patents

Abstract

A method for efficiently separating desired cells from other cells with the use of the cell specificity of a relatively less expensive lectin and the specific interaction between the lectin and an antibody. A lectin (3) capable of binding specifically to a specific cell (1) is bonded to the specific cell to form a lectin/cell complex. An antibody (4) having a specific interaction with the above lectin is immobilized on a separating agent (5) to give a separating agent carrying the antibody immobilized thereon. Next, the resultant separating agent is brought into contact with a solution of the above lectin-cell complex so that the lectin is bonded to the antibody due to the specific interaction thereof, thus separating the lectin/cell complex.

Description

 Techniques for separation of written cells

 The present invention relates to a cell separation method utilizing specific binding between a lectin and a sugar chain on a cell surface. Background technology

 Various specific reactions, such as enzyme reactions and antigen-antibody reactions, exist in living organisms, forming sophisticated biological systems.Today, research on biomimetic systems that model these biological reactions Are being actively conducted in various fields.

 In particular, remarkable progress has been made in the field of research on the relationship between sugar chains and recognition methods in the recognition between cells and between cells, between cells and proteins, and between cells and foreign substances. Typically, proteodalican on the cell wall of plant cells, which is the first recognition site of the cell with the outside world and contributes to cell stabilization, affects cell differentiation, proliferation, adhesion, migration, etc. Glycolipids and glycoproteins involved in cell-cell interactions and cell recognition have been studied.

More recently, methods for efficiently separating cells having functions such as stem cells, T cells, B cells, and macrophages, and using the cells themselves as medicines to treat diseases such as cancer and leukemia have become active. At this time, methods to separate specific cells using sugar chain recognition of cells have been studied [for example, Urashima et al., “Today's transplant”, 7, 447 (1994)] ₀

 The present inventors have focused on the recognition of sugar chains by lectins on the cell surface, and have been conducting intensive studies. For example, lectins on hepatocyte surfaces and Asiatic glycoprotein receptor Yuichi (ASGPR) A synthetic design of a galactose-containing polymer (PVLA) recognized specifically was performed [M. Goto, et. Al., J. Controlled Release, 28, 223 (1994)]. .

 In other words, by using the above petri dish coated with PVLA, the hepatic parenchymal cells selectively bind to PVLA via the specific affinity between PVLA and the lipoprotein glycoprotein receptor on the surface of hepatic parenchymal cells. And found that it could be separated from other non-parenchymal liver cells [Akira Kobayashi et al., “Artificial Organs”, 21, 1060 (1992)].

 In addition, lectins having cell specificity interact with PVLA to bind to PVLA, and specific binding of specific cells can be achieved by utilizing the specific binding between the lectin and sugar chains on the cell surface. [See Akira Kobayashi et al., “Artificial Organs” above].

Lectins are proteins that exist in plants and animals and are deeply involved in the recognition mechanism of living organisms, such as cell-cell adhesion and recognition, and their functions are diverse, such as specifically activating specific cells. However, recently, it has been used for cell separation. However, in the conventional cell separation method, since lectin is immobilized on a column or petri dish, lectin activity is reduced and side reactions occur, and specific binding between lectin and specific cells is performed efficiently. As a result, it was difficult to effectively separate cells. On the other hand, a method of separating these cells using an antibody against cells having functions such as stem cells, T cells, B cells, and macrophages has been developed, and the function of the cells separated by this method has also been developed. It is well maintained and shows high yields. However, the antibody itself is very small and expensive, and the available system is small. Therefore, in order to separate and recover the required number of cells, it is necessary to use the small system several times. Disclosure of the invention

 Therefore, the present invention provides a novel and highly efficient method for separating desired cells from other cells by utilizing the cell specificity of lectins and the specific interaction between lectins and antibodies. It is intended to provide an improved method for separating cells.

 The method for separating cells according to the present invention comprises preparing a lectin-one-cell complex by binding a lectin that specifically binds to a specific cell to the specific cell, and using an antibody that specifically interacts with the lectin as a separating agent. An antibody-immobilized separating agent is prepared by immobilization, and the antibody-immobilized separating agent is brought into contact with the lectin-one-cell-combined liquid to bind the lectin and the antibody by their specific interaction. Further, a lectin-cell complex is separated.

The contact between the antibody-immobilized separating agent and the lectin-cell conjugate is preferably performed by passing the lectin-cell conjugate solution through a column filled with the antibody-immobilized separating agent. Can In the present invention, specific cells that specifically bind to lectin can be separated as cells to be finally recovered, or specific cells that specifically bind to lectin can be separated from other cells that do not bind to lectin. Separation and removal can be performed, and finally other cells that do not bind to the lectin can be recovered.

 According to the present invention, the lectin is specifically bound to specific cells in a free state without being fixed to a column or a Petri dish as in the prior art. The lectin-cell conjugate can be efficiently produced. By contacting the lectin-cell conjugate thus obtained with an antibody-immobilized separating agent, a specific interaction between the lectin and the antibody can be expressed, and as a result, an effective lectin-cell Selective separation of cell conjugates becomes possible. Brief explanation of drawings

 FIG. 1 is a schematic explanatory view showing the concept of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

The concept of the present invention will be described with reference to the schematic diagram of FIG. 1.Lectin 3, which recognizes and specifically binds to sugar chain 2 on a specific cell 1, binds to cell 1, and binds lectin-one cell. It is a conjugate. On the other hand, an antibody 4 that specifically interacts with lectin 3 is chemically bonded to a separating agent 5 to prepare an antibody-immobilized separating agent, which is packed in a column. By passing the solution containing the lectin-cell conjugate through this column, the specificity of lectin 3 and antibody 4 can be increased. By a simple interaction, the two can be bound together and a specific cell 1 can be separated from other cells.

 As the lectin used in the present invention, peanut lectin, soybean lectin, wheat germ lectin, kidney bean lectin, castor bean lectin, concanapalin A, and the like can be appropriately selected and used. These lectins specifically recognize and bind to monosaccharides and oligosaccharides such as glucose, galactose, mannose, N-acetyldarcosamine, and N-acetylgalactosamine on the cell surface. .

 Antibodies used in the present invention are those which specifically recognize and interact with the above-mentioned peanut lectin, soybean lectin, wheat germ lectin, kidney bean lectin, castor bean lectin, concanavalin A and the like. Any antibody can be used. In particular, an antibody derived from a chicken egg that is inexpensive, can be prepared in large quantities, and has high activity is preferably used.

 Examples of the separating agent used in the present invention include dextran, polyacrylamide, agarose, polystyrene, porous glass, and their cross-linked products and derivatives. Materials can be used. In the present specification, these materials are collectively referred to as a separating agent. Among these separating agents, agarose-based Sepharose 6 MB with low cell specificity can be used particularly preferably.

The cell specifically bound to lectin to form a lectin-one-cell complex is not particularly limited as long as it can specifically bind to lectin, and is required from blood cells, skin cells, and the like. To A specific cell may be selected accordingly. In addition, the cells finally separated and recovered may be specific cells that specifically bind to lectin to form a lectin-cell complex, or may not bind to lectin, and thus may bind to lectin-cell complex. It is also possible to finally separate and collect other cells that do not form. Among the cells that can be separated by the method of the present invention, stem cells are particularly useful because they can be used for cancer treatment and the like.

 In practicing the present invention, to prepare a lectin-cell conjugate, the cell solution and the lectin solution are mixed and mixed at 4 to 37 ° (preferably at 35 to 37 ° C for several minutes). The cell solution can be easily prepared by treating it for several tens of minutes, and various methods can be used to prepare the cell solution depending on the source of the cells. In the case of cells, a method of pre-concentrating a desired cell group by centrifuging blood can be adopted, and it is desirable to remove a small amount of erythrocytes by washing.

 On the other hand, when preparing the antibody-immobilized separating agent, the antibody is added to a dispersion in which the activated separating agent is dispersed in a neutral or weakly alkaline buffer, and the antibody is added at a low temperature of 4 to 10 ° C. By reacting for up to 10 hours, an antibody-immobilized separating agent gel can be obtained.

The thus-obtained antibody-immobilized separating agent gel is packed in a column, and the lectin-cell conjugate solution is passed through this column. The conjugate is adsorbed to the column of the antibody-immobilized separating agent gel, and other cells that do not bind to lectin flow out of the column, so that specific cells that specifically bind to lectin and do not bind to lectin other Can be separated from cells.

 The cells can also be separated in a batch system by mixing the antibody-immobilized separating agent with the lectin-cell conjugate solution without packing the column and then washing by filtration. As a washing solution, a phosphate buffer solution or the like can be used.

 In the method of the present invention, for example, the density of antibody lectin can be easily adjusted by appropriately changing the concentration and number composition of antibodies, lectins, and cells according to their types and characteristics, thereby improving the cell separation efficiency. Can be done.

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Immobilization of antibody on adsorbent

4 g of CNB r activation ceph § loin 6 MB (manufactured off Arumashia Co.), 0.0 containing 5 MN a C l. L MN a HC_〇 ₃ buffer (PH 8) separated in addition to 2 0 ml A solution of soybean lectin (SBA) (1 O mg) was added to this separating agent solution, and the mixture was reacted at 4 ° C. for 2 hours. After the reaction was completed, the obtained gel was washed with the above buffer solution, added with a 0.2 M glycine solution, and left at 4 ° C for 1 hour. After the gel was washed again with the above buffer, 6 ml of the gel was filled in a 10 ml syringe to obtain a 6 MB column of Sepharose immobilized with anti-SBA antibody.

 Preparation of cell solution

The necessary bone marrow cells are separated from the heparin-supplemented bone marrow blood by Ficoll-Hypaque centrifugation, and the PBS (—) solution (Ca) ² + -free phosphate buffered saline), then dissolve small amounts of contaminating red blood cells with 0.83% ammonium chloride-Tris solution, remove by washing, and remove bone marrow cells. A liquid was obtained.

Preparation of lectin solution

 SBA (Vector, Lot No. # L1010) was dissolved in Hanks buffer (HBBS, pH 7.4) supplemented with 0.1% albumin, and SBA lectin solution (concentration: lmg / ml).

Preparation of lectin-cell conjugate

Cell solution 0 containing 2 XI 0 ⁶ or bone marrow cells. 2 5 to m 1, 1 by adding the same amount of 0.2 5 1 1 3 8 eight lectin solution (concentration 1 11 8 ml) 0 By treating at 37 ° C. for 37 minutes, an SBA lectin-one cell conjugate solution was prepared.

Adsorption operation of lectin-cell conjugate to antibody-immobilized column

PBS (+) solution to the anti-SBA antibody immobilized ceph § b Ichisu 6 MB column equilibrated with (C a ^{2 +-phosphate} buffer containing), a SBA lectin single cell bound body fluid obtained above The liquid was passed. Next, this column was incubated at 37 ° C for 1 hour, and 4 ml of HBSS buffer (pH 7.4) was passed through to remove unadsorbed cells from the column. .

For comparison, the SBA lectin-cell conjugate was passed through a Sepharose 6 MB column immobilized with an antibody other than the anti-SBA antibody (anti-ConA antibody), and the HBSS buffer ( Unadsorbed cells were allowed to flow out of the column by passing through pH 7.4). Table 1 shows the results of calculating the number of unadsorbed cells in the effluent. [table 1 ]

 Example Comparative example

Number unadsorbed cells passing through the column prior to cell number 2 X 10 ⁶ 2 X 10 ⁶ effluent cell liquor ^{0. 4 X 10 6 1. 5 X} 10 6 column fraction 80% 25% Table 1 adsorption cells As can be seen from the figure, by the method of the present invention in which the SBA lectin-cell conjugate is passed through an anti-SBA antibody-bound column, cells that specifically bind to SBA lectin can be replaced by other cells that do not bind to SBA lectin. Can be efficiently separated from

Measurement of stem cell count

The bone marrow cell fluid collected from bone marrow blood contains a group of cells such as T cells and B cells that interact with SBA, and stem cells that do not interact with SBA. Most cell groups, such as cells and B cells, are specifically adsorbed to the anti-SBA antibody-immobilized Sepharose 6 MB column, and stem cells, etc., flow out without adsorbing to the column. The results of measuring the number of T cells and the number of stem cells in the cell solution and column effluent before passing through the column are shown in Table 2.The number of T cells and the number of stem cells were measured at 0.01% Dispersed in HBSS buffer (pH 7.4) supplemented with serum albumin (BSA) to transform CD34 that specifically interacts with stem cells and CD2 that specifically interacts with stem cells. Double staining was performed using each of those labeled with fluorescence, and the fluorescence intensity of CD34-positive cells (stem cells) and CD2-positive cells (T cells) was measured using flow cytometry. [Table 2]

 T cells-Number Stem cells

Cell solution 0.9X 10 ⁶ 0.09 X 10 ⁶ Column effluent before passing through the column 0.25 X 10 ⁶ 0.07 X 10 ⁶ As can be seen from Table 2, the bone marrow cell solution was treated with anti-SBA antibody By passing the solution through a 6 MB column, cells containing T cells in the bone marrow cells are selectively adsorbed to the column, while stem cells in the bone marrow cells are hardly adsorbed and flow out of the column. The stem cells can be relatively enriched therein. That is, according to the method of the present invention, it is found that stem cells useful for treating cancer and leukemia can be efficiently separated from other cell groups, concentrated and recovered. Industrial applicability

 As described in detail above, according to the present invention, the lectin is specifically bound to specific cells in a free state without being fixed to a column or a Petri dish as in the prior art. Without the occurrence of lectin-cell conjugates can be efficiently produced.

The solution containing the lectin-cell conjugate obtained in this manner is passed through a packed column of an antibody-immobilized separating agent in which an antibody that specifically interacts with lectin is immobilized on the separating agent. The specific interaction with the antibody allows the two to bind to each other, allowing the selective separation of lectin-cell conjugates, and as a result This makes it possible to efficiently separate cells that specifically bind to lectin from other cells that do not bind to lectin.

Claims

The scope of the claims A lectin that specifically binds to a specific cell is bound to the specific cell to prepare a lectin-cell conjugate, and an antibody that specifically interacts with the lectin is immobilized on a separating agent to immobilize and separate the antibody. A lectin-cell conjugate by contacting the antibody-immobilized separating agent with the lectin-cell conjugate to bind lectin and the antibody by their specific interaction. A method for separating cells, comprising separating cells. The contact between the antibody-immobilized separating agent and the lectin-cell conjugate is performed by passing a lectin-cell conjugate solution through a column filled with the antibody-immobilized separating agent. The method for separating cells according to claim 1, wherein The method according to claim 1, wherein the lectin is selected from peanut lectin, soybean lectin, wheat germ lectin, kidney bean lectin, castor bean lectin, or concanano 'lin A. The method for separating cells according to the above. The antibody is selected from an anti-pine nut lectin antibody, an anti-soybean lectin antibody, an anti-wheat germ lectin antibody, an anti-green bean lectin antibody, an anti-castor bean lectin antibody or an anti-concanapalin A antibody 2. The method for separating cells according to claim 1, wherein the method comprises the steps of: The separating agent is characterized in that the separating agent is selected from dextran, polyacrylamide, agarose, polystyrene, porous glass, and a cross-linked or derivative thereof. 2. The method for separating cells according to claim 1. 2. The method according to claim 1, wherein a soybean lectin is used as the lectin, and an anti-soybean lectin antibody is used as the antibody.