KR20120127023A - The specific binding molecules-nanofibers complex and method for activating the same - Google Patents

Abstract

PURPOSE: A method for activating immunocytes using a specific binding molecule-nanofiber composite is provided to specifically and efficiently isolate a target cell and to obtain 95% or more nave cells without cell loss. CONSTITUTION: A method for preparing a specific binding molecule-nanofiber composite for cell isolation comprises: a step of electrospining a polymer solution to fabricate a nanofiber; a step of treating the nanofiber with 20-100%(v/v) of alcohol and prepare a nanofiber; and a step of mixing specific binding molecules, anti-CD4, or anti-CD4 antibody to the nanofiber. A method for isolating cells using the nanofibers conjugated with the specific binding molecules comprises: a step of mixing the composite and cells and culturing; and a step of isolating the cells conjugated to the composite.

Description

The specific binding molecules-nanofibers complex and method for activating the same}

The present invention relates to a specific binding molecule-nanofiber complex and a method for activating immune cells using the same.

Isolation of cells is one of the most important techniques in various fields, such as biological or biomedical applications in the diagnosis or treatment of diseases, and its use is increasingly being used to isolate specific cells of interest from a mixture of different cells. [McCloskey KE , et al., Anal Chem, 2003, Dec 15; 75 (24): 6868-6874; McCloskey KE, et al., Biotechnol Prog, 2003, May-Jun; 19 (3): 899-907. Therefore, because of this importance, many research groups are currently conducting a lot of research on the technology of cell separation.

In the case of separating cells having different specific gravity, they can be separated by speed sedimentation. On the other hand, when there is little difference between cells, such as distinguishing sensitized cells from unsensitized cells, it is necessary to separate the cells by information stained with fluorescent antibodies or by direct microscopic observation. For example, a cell sorter (Kamarck, M.E., Methods Enzymol.) That separates and collects specific cell populations by fractionating cells according to the label strengths by labeling with fluorescent dyes. Vol. 151, p150-165, 1987], and recently, a cell fractionation apparatus for separating microscopically observed microscopic particles flowing in a laminar flow generated in a microchannel made using microfabrication techniques [Micro Total Analysis, 98 , pp. 77-80, Kluwer Academic Publishers, 1998; Analytical Chemistry, 70, pp. 1909-1915, 1998].

Fluorescence activated cell sorter (FACS), one of the most widely used cell separation methods, is a device that analyzes the surface antigens of glass cells such as lymphocytes or separates cells by the presence or absence of surface antigens using laser beam. As the latex particles or cells pass through a certain sensing area, each particle or cell is quickly measured, the intensity of fluorescence of each cell is measured, and the specific cells are detected using the quantized intensity of fluorescence. Can be selectively separated. Fluorescent dyes or monoclonal antibodies can be used to determine the immune state of the surface and inside of the cells to isolate the cells. However, there are drawbacks to the complex operation techniques of the device, highly skilled technicians, time consuming and expensive machines [Lancioni CL, et al., J Immunol Methods, 2009, May 15; 344 (1): 15-25].

In addition, one of the currently used magnetic activated cell sorter (magnetic activated cell sorter) is to separate the specific cells desired by the force of the magnetic field using an antibody or a corresponding material attached to the magnetic particles (magnetic particle) In this case, there is a disadvantage in that a change in the state of a cell occurs because beads necessary for cell separation bind to the cell and enter the cell.

On the other hand, antibodies are widely used for the detection, isolation and measurement of biological substances using antibodies specific for cell surface antigens because of their specificity to antigens. Isolation of specific cells for cell therapy or detection of specific cells requires the establishment of a method for separating simpler and more stable high purity cells with the same cellular characteristics. In addition, the technology to develop in vitro nanoplatforms that can be applied to cancer and autoimmune diseases patients by activating the isolated cells in vitro without a separate process is a technology that is currently being mined worldwide.

An object of the present invention is to provide a specific binding molecule-nanofiber complex and a method for activating immune cells using the same.

Electrospun nanofibers have a good feature of having a large surface area in a small space, being durable, very easy to handle, made in various forms, and easy to chemically bond various materials. It provides a very good approach to the application of cell separation.

Accordingly, the present inventors have designed a simple and effective one-step cell separation and activation platform by devising the cell separation and cell activation in one platform. The dispersed nanofibers prepared by treating alcohol with nanofibers increased the amount of antibody bound by increasing the space between the fibers, and the dispersed nanofibers in which the antibodies were bound are effective and specifically targeted to the antibody. As well as combining, it was found to be easy and quick to use.

The present inventors also found that the activation and sustained release of the immune cells bound to the nanofibers can be regulated according to the type of antibody. For example, when the cells are separated by binding CD4 antibodies to normal nanofibers, the binding of the nanofibers to the isolated CD4 + T cells is so strong that the activated cells stay mainly in the nanofiber matrix and very small amounts of cells Slow release over time could be observed. However, when CD3 + T cells were isolated by incubating CD3 antibodies to nanofibers and then cultured, the affinity between CD3 antibodies and immune cells was low, thereby confirming that a larger amount of activated immune cells were released from the nanofibers. Was completed.

In order to achieve the above object,

1) preparing a nanofiber by electrospinning the polymer solution;

2) treating the nanofibers of step 1) with 20-100% (v / v) alcohol to prepare dispersed nanofibers; And

3) It provides a method for producing a specific binding molecule-nanofiber composite for cell separation, comprising the step of combining and binding specific binding molecules to the dispersed nanofibers of step 2).

The present invention also provides a specific binding molecule-nanofiber composite prepared according to the above method.

In addition,

1) culturing by mixing the cells with the specific binding molecule-nanofiber complex; And

2) provides a method for cell separation using nanofibers with specific binding molecules, including the step of separating the cells bound to the complex from the mixture of step 1).

3) A method of activating a cell using a specific binding molecule-nanofiber complex to which cells of step 2) are bound is provided.

Nanofibers to which specific binding molecules of the present invention are bound are not only simple and easy to use because the binding and separation of antibodies and target cells are fast and simple to use, but also to separate target cells specifically and efficiently, In particular, since about 95% or more of the cells in a naive state can be obtained without losing the number of cells, the nanofibers to which the specific binding molecules of the present invention are bound are disadvantageous of the cell separation method using the conventional beads (about 50% It can be useful for the removal of a high amount of antibody or aptamer-bound nanofibers, which can compensate for the cell loss), and for simple, rapid and effective cell separation. In addition, by immediately activating the cells thus isolated can be used very simply for cell activity experiments.

Figure 1 schematically shows the manufacturing process of nanofibers.
2 shows the results of Example 3, which is an optical microscopic image of cells released from nanofiber complexes bound with anti-CD4 or anti CD3 monoclonal antibodies.
FIG. 3 shows the activation and increase over time of T cells isolated from CD3 binding molecule-nanofiber complexes as a result of Example 4. FIG.

The following examples are only presented to more easily understand the present invention, whereby the scope or content of the present invention is reduced or limited and cannot be interpreted.

Example 1 Preparation of Materials

4-nitrophenyl butyrate (4-NPB), N, N-dimethylformamide (DMF) was purchased from Sigma (ST Louis, MO, USA) and polystyrene , PS, MW = 950,400) and styrenemaleic anhydride copolymer (poly (styrene-co-maleic anhydride), PSMA, MW = 224,000}, Tetrahydrofuran (THF) are Aldrich (Milwaukee, WI, USA). ) And ethanol from Deajung (Siheung, Korea). Seven-week-old C57BL / 6 mice were purchased from Narabiotech (Seoul, Korea) and bred in Korea University animal lab. All mice used in the experiments were 7-10 weeks of age. All procedures were conducted with the approval of Korea University Experimental Animal Operation Committee (KUIACUC-2009-105). Anti-mouse CD3 (145-2C11), CD4 (GK1.5, RM4-4), CD8 (53-6.7), NK1.1 with or without fluorescein isothiocyanate (FITC) bound (PK136) and CD25 (PC61.5) monoclonal antibodies (mAbs), phycoerythrin (PE), or allophycocyanin (APC) were purchased from eBioscience (San Diego, Calif.). Anti-mouse CD19 (6D5) mAb coupled to Peridinin Chlorophyll Protein Complex (PerCP) was purchased from BioLegend (San Diego, Calif.). All other reagents were purchased from Sigma and Aldrich at the highest commercially available grades.

Example 2 Preparation of Nanofibers

As shown in Figure 1, the polymer solution was prepared by dissolving a PS: PSMA mixture in tetrahydrofuran (THF) at room temperature in a 2: 1 weight ratio and dissolving it using tetrahydrofuran as an organic solvent for dissolution. The mixed solution was completely dissolved in the PS and PSMA mixture for 3 hours using a magnetic stirrer. Then, acetone solution was added to the polymer solution to reduce the viscosity of the polymer solution. The polymer solution was filled into a 5 mL plastic syringe equipped with a 30 gauge stainless steel injection needle. The operating conditions of the voltage using the high voltage supply was 7 kV and the solution was supplied at 0.1 mL / hour using a syringe pump (PHD-2000 Infusion, Harvard Apparatus, Holliston, Mass., USA). Electrospun nanofibers were collected on a clean aluminum foil (connected to the floor) placed at an appropriate distance (7-10 cm) from the needle tip.

Example 3 Optical Microscopy Image of Cells Released from Nanofiber Complex Bound to Anti-CD4 or Anti CD3 Monoclonal Antibodies

As shown in FIG. 2, cells bound to specific binding molecule-nanofiber complexes were incubated with 1 mg / mL of anti-CD3 monoclonal antibody 1 mg / mL for 7 days for optical microscopy. Next, experiments were conducted on whether the cells attached to the CD4 and CD3 nanoparticles-nanofibers could be activated or released from the scaffold. Attached T cells can be activated by the addition of anti-CD3, anti-CD28 1 mg / mL leading to the activation of T cells via stimulation of the T cell receptor / CD3 complex and the auxiliary stimulant CD28 by antibody-mediated receptor cross-reaction. However, when cells and CD4 binding molecule-nanofiber complexes were cultured for 7 days in the presence of soluble anti-CD3 and anti-CD28 monoclonal antibodies, most of the cells adhered to the CD4 binding molecule-nanofibers to collect fewer cells in the culture medium. It became. In contrast, when the CD3 binding molecule-nanofiber complex was cultured for 7 days, it was observed that the cells were separated from the CD3 binding molecule-nanofiber complex and showed a more activated form under the microscope.

Example 4 Activation and increase over time of T cells isolated from CD3 binding molecule-nanofiber complex

In order to confirm the activation level of the T cells released from the CD3 binding molecule-nanofiber complex, the CD25 level, which is an activation marker of the cells, was checked after 7 days of culture. As a result, as shown in Figure 3, it was confirmed that the level of CD25, which is an activation marker of T cells isolated from the CD3 binding molecule-nanofiber complex, is very high.

Claims (2)

1) preparing a nanofiber by electrospinning the polymer solution;
2) treating the nanofibers of step 1) with alcohol at a concentration of 20 to 100% (v / v) to produce dispersed nanofibers; And
3) mixing the specific binding molecule, anti-CD4 or anti-CD4 antibody to the dispersed nanofibers of step 2), comprising the step of combining and binding, specific binding molecule-nanofiber composite for cell separation. A method of activating a cell in vitro by using a specific binding molecule-nanofiber complex to which a cell according to claim 1 is bound.

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