KR20150120260A - Method for cultivating and longterm preserving activated lymphocyte - Google Patents

Abstract

A method of cultivating activated lymphocyte of the present invention includes: separating mononuclear cells from peripheral blood of a healthy adult; growing and culturing activated lymphocyte by combining mononuclear cells with the interleukin-2 (IL-2), interleukin-15 (IL-15), and anti-CD3 antibody in vitro; freezing and storing the activated lymphocyte for a long period of time; and unfreezing and restoring the activated lymphocyte obtained in the freezing and storing step. Therefore, lymphocyte cultured and stored according to the present invention can be unfrozen and restored when an owner of the lymphocyte has a disease and thus can be used as a treating agent for diseases such as anticancer, antitumor, and antivirus agents.

Description

METHOD FOR CULTIVATING AND LONGTERM PRESERVING ACTIVATED LYMPHOCYTE BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 > [0002] <

The present invention relates to a technique for separating mononuclear cells from peripheral blood and proliferating, activating and cryopreserving lymphocytes in vitro in vitro.

Cancer can occur when any cell that constitutes the body abnormally proliferates, and more than 100 cancers are known to date. Therefore, the nature of cancer and treatment methods are very diverse. Tumor refers to the abnormal proliferation of cells and is divided into benign and malignant. Benign tumors, like skin warts, are localized at the site of development, do not penetrate into the surrounding normal tissue, and do not spread to other parts of the body. However, malignant tumors can penetrate into surrounding normal tissues and can spread throughout the body through the circulatory or lymphatic system, which is called cancer metastasis. Usually malignant tumors are regarded as cancer, and the reason why cancer is actually dangerous is the penetration and transfer ability of cancer cells.

Irregular proliferation of cancer cells in humans can be reproduced through cell culture. Proliferation of normal cells is suppressed in a density-dependent manner. Normal cells grow only until they reach a constant cell density limited by growth factors added to the medium as serum. When the cells stop growing, they stop at the G0 phase of the cell cycle. On the other hand, cancer cells are largely unrestrained by density-dependent inhibition and can continue to grow at higher densities. This is similar to the unsteady growth of cancer cells in the human body.

Tumor viruses are animal viruses that can cause cancer directly in experimental animals or humans. Viruses that cause cancer in humans include hepatitis B virus and hepatitis C virus, papillomavirus (cervical and other anal gonads), Epstein-Barr virus, Kaposi sarcoma, and human T-cell lymphotropic virus (adult T-cell leukemia). HIV also causes immune deficiency in AIDS patients and indirectly causes cancer.

NK cells (natural killer cells) are recognized by NK cells, which are natural killer cells with cytotoxicity, such as virus-infected cells or cancer cells. NK cells play an important role in viral infection or in the early stages of tumorigenesis before large numbers of active cytotoxic T lymphocytes are produced. Histologically, NK cells are large granular lymphocytes. Intracellular granules contain molecules that already have strong biological functions and are secreted when NK cells contact the target cells. Some of these molecules form holes in the membranes of the target cells to interfere with the cells and other molecules enter the target cells and increase the segments of nuclear DNA to cause apotosis or programmed cell death .

Therefore, NK cells can dissolve certain virus-infected cells and cancer cells without pre-stimulation. Unlike cytotoxic T lymphocytes that recognize antigen-specifically through the expression of TCR, NK cells lack antigen-specific receptors. NK cells express a killer cell inhibitory receptor (KIR) that binds MHC type I in normal cells. Murine cytotoxic receptors, when combined with MHC type I, produce intracellular signals that induce the inhibition of specific transcription factors. As a result, activation of NK cells, degradation and destruction of target cells are inhibited. Viral infected cells or cancer cells have greatly reduced MHC type I molecules on their surface. Therefore, when these cells meet NK cells, they are exposed to NK cell-mediated cytotoxicity and are solubilized because they do not bind effectively with killer cell inhibitory receptors.

In the prior art, cell-mediated immunotherapy activates CD4 T cells to activate antigen-specific lymphocytes and acquire immune responses that produce a memory cell capable of protecting the host from reinfection by the same infectious agent, thereby fusing and killing the infected cells .

This technique has a problem in treating cancer cells and tumor viruses that are not memorized even when long-term activated lymphocytes are stored.

The object of the present invention is to isolate mononuclear cells in the peripheral blood when a healthy person is healthy and to proliferate and activate the lymphocytes in vitro to inactivate the activated lymphocytes, It is possible to improve the immune function by returning healthy autologous lymphocytes to the blood by self-administration at a time when the immune function is decreased and needed to be utilized for malignant tumor or viral diseases, And also to utilize activated lymphocytes in these diseases.

Cancer patients or viral patients can be treated with passive cellular immunotherapy. When NK cells or T cells are cultured in the presence of IL-2 by such a therapeutic method, activated lymphocytes having a strong anticancer activity can be cultured to alleviate the patient's pain.

The present invention relates to a method for mass proliferation and long-term storage of an activated lymphocyte so as to facilitate utilization of a stored lymphocyte, comprising the steps of extracting mononuclear cells from a peripheral blood of a normal person, extracting the extracted mononuclear cells in the presence of an anti-CD3 antibody And culturing the activated lymphocytes under an incubation condition; and cryopreserving the activated lymphocytes.

The step of extracting the mononuclear cells from the peripheral blood of the normal person is a step of collecting and culturing the activated lymphocytes by separating the mononuclear cells from the peripheral blood of a normal person. The peripheral blood sampling method includes a step of collecting lymphocytes Although it can be used as a material, it is most preferable to collect blood in the pulmonary vein and median main vein. The amount of blood to be collected is preferably from about 10 ml to 100 ml, more preferably about 50 ml.

EDTA or heparin may be added to the peripheral blood to prevent coagulation, and heparin is preferably used. Mononuclear cells were isolated from the collected peripheral blood and cultured in vitro to obtain activated lymphocytes of the present invention.

In the present invention, an activated lymphocyte means to obtain an activated lymphocyte by putting it in a culture medium containing a nutrient source necessary for the proliferation of the lymphocyte in vitro, and the term "proliferation" means a quantitative increase of the lymphocyte.

The incubation step refers to a culturing step in which activated lymphocytes are proliferated. Interleukin 2, interleukin 15, and anti-CD3 antibody are added to activate NK cells and T cells evenly. Interleukin 2 stimulates proliferation of T cells and NK cells , Monoclonal antibodies were added so that they could participate in the role of antigen to express CD3 in immune cells. From the viewpoint of the anticancer effect, the combination of interleukin 2, interleukin 15 and anti-CD3 antibody is most preferable, and it is excellent for activation and proliferation of NK cells and T cells.

Interleukin 2 is produced when T cells are recognized and activated by an antigen. The interleukin gene is located on the fourth chromosome of the human chromosome and consists of four expression sites and three non-expression sites. Interleukins are mainly produced in T cells (anti-CD4, anti-CD8) but also in NK cells. In addition, IL-2 acts on T cells, B cells, NK cells, LAK cells, macrophage neutrophils and the like, and acts as a factor for promoting the cell cycle. Therefore, interleukin 2 promotes the growth of NK cells and T cells and enhances the injury ability of these cells.

Interleukin 15 is a 15 kDa glycoprotein that is produced when T cells are recognized and activated by the same antigen as interleukin 2. It is produced in T cells and epithelial cells and enhances the ability of T cells to be injured similarly to interleukin 2 .

The interleukin 2 and interleukin 15 used in the culturing step can be commercially available, and the medium for culture is preferably used at a concentration of 100-2000 U / ml, more preferably 500-1000 U / ml. Interleukin 2 and interleukin 15 can be used by dissolving in widely used culture media for cell culture, physiological saline, RPMI 1640, DMEM, alpha MEM, and the like. The present inventors used alpha MEM. The medium for culture was excellent in the proliferation effect by using fetal bovine serum, and a commercial product was used. In addition to the fetal bovine serum, an autologous serum may be used. The cultivation was carried out in a CO2 incubator, which is a general cell culture method, and the concentration was in the range of 1 to 10%, preferably in the range of 3 to 7%, more preferably in the concentration of 5%. The incubation temperature in the incubator is in the range of 30 to 40 占 폚, particularly 37 占 폚.

In the above culture, the anti-CD3 antibody is preferably cultured for 3 to 10 days, more preferably for 3 to 8 days, in order to stimulate transmission to the cells. During the incubation period, the cell concentration was observed with a microscope or the culture container was visually confirmed. When the culture solution became yellow, the culture solution was added additionally to increase the cell proliferation efficiency. The addition period of the culture solution is preferably 1 to 2 times between 3 and 5 days.

The anti-CD3 antibody used for the stimulation of lymphocytes in the culturing step may be added to the culture broth or may be introduced into a solidified culture broth to initiate the culture. Preferably, the incubation is carried out in a solidified culture broth. The anti-CD3 antibody may be purified using an animal or an antibody produced in a cell, or may be an OKT 3 antibody in the market. A commercially available cell culture flask can be used as an apparatus for solidifying the antibody, or a culture vessel of various materials can be used.

The solidification of the anti-CD3 antibody can be carried out by adding the antibody to the cell culture flask diluted with the antibody and left at 37 ° C for 2 hours. The anti-CD3 antibody was diluted to a concentration of 0.1 to 15 ug / ml, for example, in a buffer solution of the velocic acid, and the cell culture flask containing the diluted antibody was stored in a refrigerator (4 캜) until use. In use, the diluted antibody solution was removed and used.

The cultured activated lymphocytes are frozen and stored for a certain period of time. That is, as the step of cryopreserving the cultured activated lymphocytes for a predetermined period, the cell concentration was maintained at a density of 1 × 10 ⁶ cells / ml to 1 × ^{10 8} cells / ml in the frozen storage solution. As the cell-freezing storage solution, a commercially available preservation solution can be used, or it can be used by self-preparation. Dimethyl sulfoxide may be used as a component of the cell stock solution, such as serum, proteins, polysaccharides, etc., in a basic medium such as RPMI 1640, DMEM, and alpha MEM.

The number of cells to be frozen in the cryopreservation step is preferably 1 to 5 x 10 ⁷ cells per cell, and a cell-freezing test tube is preferably a commercially available freezing bag or a test tube.

The cell-freezing test tube to which the activated lymphocyte is added is preferably stored in a freezer at -80 ° C for 1 to 10 days, preferably 3 to 5 days, and after 3 to 5 days, -196 ℃ nitrogen tank for a long time.

In addition, when thawed and frozen cells are thawed and restored and they are required to be treated for anticancer, antiviral, etc., they can be very usefully used as a cell therapy agent such as an anti-cancer and antiviral treatment agent including activated lymphocytes.

The present invention can be effectively used as a cell therapy agent when an active lymphocyte is isolated and cultured in a peripheral blood of a normal person, and stored for a long period of time and then treated for anticancer, antiviral and the like.

In addition, the self-activated lymphocytes in a healthy state can be returned to the blood to enhance the immune function, and the prevention and treatment effects of diseases can be obtained.

In addition, cancer patients or viral patients can be treated through passive cellular immunotherapy. Therefore, when NK cells or T cells are cultured in the presence of IL-2 by this treatment, activated lymphocytes with strong anticancer activity are cultured This can alleviate the patient's pain.

FIG. 1 is a graph showing a monocyte layer separated from the peripheral blood of the present invention and examined by a flow cytometer. FIG.
FIG. 2 is a graph showing the immunophenotype of an activated lymphocyte cultured by the culture composition of the present invention by using a flow cytometer. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Collection of peripheral blood and separation of mononuclear cells

Blood 30 cc to 60 cc of peripheral blood is drawn into a vacuum test tube to which heparin has been added and transferred to a 50 ml test tube in a sterile workbench (Bio Safety Cabinet). 15 ml of Ficoll-Paque Plus solution is dispensed into a leucosep (BD) test tube which separates the mononuclear layer. Centrifuge at 2000 rpm for 30 seconds, and then 15 to 30 ml of peripheral blood are superimposed on a leucosep test tube. The blood layer was separated in a break-off state at a rotation speed of 2000 rpm for 20 minutes to recover the mononuclear layer. The recovered mononuclear cell suspension was washed with Dulbecco's buffer solution to obtain a cell suspension, and the cell culture medium was added thereto, and the cell number was counted on a hemocyte counter. The number of cells was measured as 1 × ^{10 7} to 1 × ^{10 8} .

Example 2: Activated lymphocyte culture

The anti-CD3 antibody was diluted to 1.5 ul / ml in phosphate buffer, and 10 ml of the solution was placed in a 175 cm ² cell culture flask and left in a 37 ° C incubator for 2 hours. The solution was discarded in a flask for cell culture, and a flask coated with the antibody was prepared. The above lymphocytes were stimulated to an anti-CD3 antibody-coated flask in a medium supplemented with interleukin-2 (700 U / ml), interleukin-15 (10 ng / ml) and 10% fetal bovine serum in an alpha-MEM medium.

On the third day after the culture, the cell growth concentration in the flask was checked, and 30-50 ml of the culture medium was added. 90 ml-150 ml of the culture medium was added on the 5th day after the start of the culture, CO ₂ concentration.

Example 3: Cryopreservation of activated lymphocytes

The number of cells of the activated lymphocyte under culturing in Example 2 was counted and centrifuged to obtain a cell precipitate. The number of cells was measured as 2 × 10 ⁸ to 2 × 10 ⁹ . The cell freezing solution was prepared by mixing 10 ml of Dimethy Sulfoxide and 20% albumin in 70 ml of alpha MEM. 10 ml of cell-freezing preservative solution was added to the activated lymphocyte precipitate, and 1 ml of the solution was dispensed into a cell-freezing test tube and transferred to a freezing treatment vessel.

The frozen treatment vessel was stored in a -80 ° C cryogenic freezer for 1 to 5 days, transferred to a -196 ° C nitrogen tank, and stored.

Example 4: Thawing and cell analysis of cryopreserved activated lymphocytes

After 14 days, the cell-freezing test tube containing the activated lymphocytes kept frozen in Example 3 was taken out and the activated lymphocytes were thawed and restored by heating in a 37 ° C digital dry bath for 3 minutes. 1 ml of thawed activated lymphocyte was transferred to 10 ml of phosphate buffer solution and centrifuged at 1200 rpm for 3 minutes to precipitate the cells. The cell suspension was suspended in 20 ml of culture medium, and the cells were stained with a trypan blue solution, and cell count and cell viability were confirmed by a microscope. The recovered cell viability was over 70% and the cell recovery rate was over 80%. The activated lymphocyte suspension was dispensed into a 75 cm ² flask to stabilize the cells for 2 days. After 2 days, cell count and cell viability of activated lymphocytes were confirmed. The survival rate and recovery rate were over 95%.

The activated lymphocytes were analyzed for cell surface antigens using a flow cytometry analyzer. The anti-CD3, TK cell marker of activated lymphocyte, anti-CD21 and anti-CD56 were attached to the activated lymphocyte as the NK cell marker. FIG. 1 shows the cell immunophenotype analyzed 5 days after the initiation of culturing. Anti-CD21 and anti-CD56 were identified on the Y axis and anti-CD3 was activated on the X axis to confirm the activity of T cells. In early lymphocyte culture, mononuclear cells were observed, but the activity of T cells and NK cells increased as the activated lymphocytes were cultured. As a result of T cell analysis in FIG. 2 (a), 89.8% increase was observed, and in the case of anti-CD21 and anti-CD56, 95.2% of NK cells were increased.

Claims (6)

Extracting mononuclear cells from a peripheral blood of a normal person;
An activated lymphocyte culture step in which the extracted mononuclear cells are cultured in the presence of an anti-CD3 antibody in a culture solution; and
And cryopreserving the activated lymphocyte. The method according to claim 1,
Wherein the step of culturing the activated lymphocyte comprises culturing the cultured medium after confirming the cell proliferation concentration in the flask and adding the culture medium several times. The method according to claim 1,
Wherein said culture medium contains a large amount of activated lymphocytes, characterized by comprising interleukin 2 (IL-2), interleukin 15 (IL-15), anti-CD3 antibody, L-glutamine and Fetal bovine serum And long-term storage methods. The method according to claim 1,
And thawing and restoring the cryopreserved cells. The present invention also provides a method for mass proliferation and long-term storage of activated lymphocytes. 5. An activated lymphocyte proliferated and stored according to any one of claims 1 to 4. An anticancer, antitumor, antiviral agent comprising the activated lymphocyte of claim 5.

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