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WO1998052039A1 - Procede de separation de cellules - Google Patents

Procede de separation de cellules Download PDF

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Publication number
WO1998052039A1
WO1998052039A1 PCT/JP1998/000460 JP9800460W WO9852039A1 WO 1998052039 A1 WO1998052039 A1 WO 1998052039A1 JP 9800460 W JP9800460 W JP 9800460W WO 9852039 A1 WO9852039 A1 WO 9852039A1
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WO
WIPO (PCT)
Prior art keywords
lectin
antibody
cell
cells
separating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1998/000460
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English (en)
Japanese (ja)
Inventor
Mitsuaki Goto
Izumi Yamada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIO QUEST RESEARCH Ltd
Original Assignee
BIO QUEST RESEARCH Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BIO QUEST RESEARCH Ltd filed Critical BIO QUEST RESEARCH Ltd
Publication of WO1998052039A1 publication Critical patent/WO1998052039A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography

Definitions

  • the present invention relates to a cell separation method utilizing specific binding between a lectin and a sugar chain on a cell surface.
  • 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.
  • 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)]. .
  • 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)].
  • 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.
  • 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.
  • it has been used for cell separation.
  • 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.
  • 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.
  • 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
  • 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 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.
  • 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.
  • 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.
  • an antibody-immobilized separating agent 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.
  • FIG. 1 is a schematic explanatory view showing the concept of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
  • 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.
  • 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.
  • 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.
  • 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.
  • stem cells are particularly useful because they can be used for cancer treatment and the like.
  • 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.
  • 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.
  • the antibody 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.
  • a washing solution a phosphate buffer solution or the like can be used.
  • 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.
  • 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.
  • 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.
  • 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 ]
  • 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.
  • BSA serum albumin
  • 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.
  • the bone marrow 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
  • 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
  • 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.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé permettant de séparer de façon efficace des cellules désirées d'autres cellules, selon lequel on exploite la spécificité cellulaire d'une lectine relativement moins chère et l'interaction spécifique entre la lectine et un anticorps. Une lectine (3) pouvant se lier spécifiquement à une cellule spécifique (1) est liée à cette cellule pour former un complexe lectine/cellule. Un anticorps (4) présentant une interaction spécifique avec ladite lectine est immobilisé sur un agent de séparation (5), de sorte que l'on obtient un agent de séparation portant l'anticorps qui est immobilisé sur lui. Ensuite, l'agent de séparation obtenu est mis en contact avec une solution du complexe lectine-cellule susmentionné, de sorte que la lectine se lie à l'anticorps étant donné l'interaction spécifique de celui-ci avec la lectine, ce qui entraîne la séparation du complexe lectine/cellule.
PCT/JP1998/000460 1997-05-15 1998-02-04 Procede de separation de cellules Ceased WO1998052039A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP12537597A JPH10319014A (ja) 1997-05-15 1997-05-15 細胞の分離方法
JP9/125375 1997-05-15

Publications (1)

Publication Number Publication Date
WO1998052039A1 true WO1998052039A1 (fr) 1998-11-19

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PCT/JP1998/000460 Ceased WO1998052039A1 (fr) 1997-05-15 1998-02-04 Procede de separation de cellules

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JP (1) JPH10319014A (fr)
WO (1) WO1998052039A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104961827A (zh) 2010-04-06 2015-10-07 阿格罗塞文公司 农用化学品的特异性输送

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0373852A (ja) * 1989-05-24 1991-03-28 Eiji Ishikawa 特異的糖鎖を有する物質の測定法
JPH03287067A (ja) * 1990-04-03 1991-12-17 Terumo Corp リンパ球の分離剤、分離装置および分離方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0373852A (ja) * 1989-05-24 1991-03-28 Eiji Ishikawa 特異的糖鎖を有する物質の測定法
JPH03287067A (ja) * 1990-04-03 1991-12-17 Terumo Corp リンパ球の分離剤、分離装置および分離方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GRIFFAUT B, BETAIL G, COULET M: "PHYSIOLOGIE CELLULAIRE VEGETALE.-EFFECT DU COMPLEMENT DANS UN SYSTEME LECTINE-ANTILECTINE SUR LES CELLULES MERISTEMATIQUES DE SOJA PLANT CELL PHYSIOLOGY.-EFFECT OF COMPLEMENT ON THE SOYBEAN (GLYCINE MAX) MERISTEMATIC CELLS", COMPTES RENDUS DES SEANCES DE L'ACADEMIE DES SCIENCES.SERIE I: MATHEMATIQUES., EDITIONS SCIENTIFIQUES & MEDICALES ELSEVIER., FR, vol. 294, no. 15, 1 January 1982 (1982-01-01), FR, pages 779 - 781, XP002912308, ISSN: 0764-4442 *

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Publication number Publication date
JPH10319014A (ja) 1998-12-04

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