NOVEL DUAL CULTURE METHOD FOR CULTURING IMMUNE CELLS AND NATURAL KILLER CELLS, AND USE THEREOF
20250154464 ยท 2025-05-15
Inventors
Cpc classification
C12N2500/33
CHEMISTRY; METALLURGY
International classification
Abstract
Provided is a method for the dual culture of immune cells used for immunotherapy, and more specifically, to a dual culture method in which immune cells, e.g., immune cells (PBMCs), natural killer cells, and gamma delta T cells, which are highly effective in treating malignant tumors and the like, are selectively and efficiently amplified and activated by culturing lymphocytes derived from human peripheral blood, wherein the culture efficiency and activity can be increased when immune cells are cultured in combination after applying different stimuli for a predetermined period of time.
Claims
1. A dual culture method for immune cells including lymphocytes or natural killer cells comprising: dividing immune cells into at least two groups; stimulating the cells of the respective groups with different combinations of antibodies or cytokines for a predetermined period of time during an immune cell culture period; and culturing the cells of the groups in combination during a next culture period.
2. The dual culture method according to claim 1, wherein the medium used for the dual culture comprises: a BM solution containing L-glutamine, IL-2, and a medium for cell culture; a medium containing cytokines; and a medium containing antibodies, the medium containing cytokines comprises: a C2 solution containing IL-2 and having a higher IL-2 content than the BM solution; a C12 solution containing IL-12; a C15 solution containing IL-15; a C17 solution containing IL-17; a C18 solution containing IL-18; and a C21 solution containing IL-21, and the medium containing antibodies comprises: an A3 solution containing anti-CD3 antibodies; an A16 solution containing anti-CD16 antibodies; and an A56 solution containing anti-CD56 antibodies.
3. The method according to claim 1, comprising: equally dividing lymphocytes or natural killer cells (NK cells) into at least two groups, adding different combinations of a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, and a C21 solution to a medium containing a BM solution and at least one of an A3 solution, an A16 solution, or an A56 solution to two groups to stimulate the cells with different cytokines; and culturing the cells in combination.
4. The method according to claim 1, comprising: equally dividing lymphocytes or natural killer cells (NK cells) into at least two groups; adding at least one of an A3 solution, an A16 solution, or an A56 solution to a culture medium of one group containing a BM solution and two or more of a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, and a C21 solution, and adding at least one of an A3 solution, an A16 solution, and an A56 solution to the culture medium of another group, and stimulating the solutions added to the two groups with different combinations of solutions containing different antibodies; and culturing the cells in combination.
5. The method according to claim 1, wherein the BM solution contains IL-2 at a concentration of 100 to 2,000 IU/mL, the C2 solution contains IL-2 at a concentration of 250 to 4,000 IU/mL, the C12 solution contains IL-12 at a concentration of 0.01 to 10 g/mL, the C15 solution contains IL-15 at a concentration of 0.01 to 10 g/mL, the C17 solution contains IL-17 at a concentration of 0.01 to 10 g/mL, the C18 solution contains IL-18 at a concentration of 0.01 to 10 g/mL, the C21 solution contains IL-21 at a concentration of 0.01 to 10 g/mL, the A3 solution contains anti-CD3 antibodies at a concentration of 0.1 to 20 g/mL, the A16 solution contains anti-CD16 antibodies at a concentration of 0.1 to 20 g/mL, and the A56 solution contains anti-CD56 antibodies at a concentration of 0.1 to 20 g/mL.
6. The method according to claim 1, comprising: (1) stimulating natural killer cells (NK cells) separated from blood or peripheral blood mononuclear cells (PBMCs) with a medium additive kit for culturing immune cells collected from blood or natural killer cells collected from blood, containing a BM solution and an A3 solution, an A16 solution, and an A56 solution, further containing two or more of a C12 solution, a C15 solution, a C17 solution, a C18 solution, and a C21 solution, and further containing autologous plasma; (2) separating the medium used in step 1 to stabilize the cells; (3) accelerating proliferation of lymphocytes or natural killer cells containing two or more of the C2 solution, the C12 solution, the C15 solution, the C17 solution, the C18 solution, and the C21 solution, and supplementing with the autologous plasma; and (4) mass-proliferatively culturing lymphocytes or natural killer cells in the medium including the BM solution and autologous plasma.
Description
DESCRIPTION OF DRAWINGS
[0033]
[0034]
BEST MODE
[0035] In the following description of the present invention, detailed descriptions of known functions and configurations incorporated herein will be omitted when the same may obscure the subject matter of the present invention. The terms which will be described below are defined in consideration of functions in the present invention and may be changed depending on intentions of users or judicial precedents, and thus the definitions should be understood based on the contents throughout the specification for describing the present invention.
[Example 1] Preparation of Medium (Culture Kit) for Culture
[0036] 100 mL of a 200 mM L-glutamine solution was dissolved in a basic medium for culturing suspension cells, IL-2 was added to reach 250 IU/mL, and a final volume of 10 L was obtained which was used as a BM solution.
[0037] IL-2 was dissolved at a concentration of 1,000 IU/mL in a BM solution to prepare a C2 solution.
[0038] IL-12 was dissolved at a concentration of 1 g/mL in a BM solution to prepare a C12 solution.
[0039] IL-15 was dissolved at a concentration of 1 g/mL in a BM solution to prepare a C15 solution.
[0040] IL-17 was dissolved at a concentration of 1 g/mL in a BM solution to prepare a C17 solution.
[0041] IL-18 was dissolved at a concentration of 1 g/mL in a BM solution to prepare a C18 solution.
[0042] IL-21 was dissolved at a concentration of 1 g/mL in a BM solution to prepare a C21 solution.
[0043] An anti-CD3 antibody was dissolved at a concentration of 1 g/mL in a BM solution to prepare an A3 solution.
[0044] An anti-CD16 antibody was dissolved at a concentration of 1 g/mL in a BM solution to prepare an A16 solution.
[0045] An anti-CD56 antibody was dissolved at a concentration of 1 g/mL in a BM solution to prepare an A56 solution.
[Example 2] Single Culture Method
[0046] Lymphocytes and autologous plasma were prepared from blood of a patient and then lymphocytes containing natural killer cells were cultured using the cell culture medium addition kit prepared in Example 1 as follows.
1. Lymphocyte Extraction and Autologous Plasma Preparation
[0047] 30 mL of peripheral blood of patient A was injected into a 50 mL conical tube and centrifuged, the autologous plasma of the upper layer was placed in a heparin tube and treated, injected into a novel 50 mL conical tube and centrifuged again, and the plasma component of the upper layer was prepared as autologous plasma.
[0048] Then, the blood tube from which the plasma was removed was filled with PBS to obtain a volume of 30 ml, thoroughly mixed, transferred to a tube containing the prepared Ficoll-Paque Plus, centrifuged at 800g for 15 minutes, the second layer, the buffy coat layer containing lymphocytes, was separated and collected in a 50 ml conical tube, and the volume was adjusted to 50 ml with PBS and then mixed. Then, the supernatant was discarded through 2 to 3 centrifugations to separate the lymphocytes.
2. Step 1: Initial Culture
[0049] A BM solution, a C12 solution, a C18 solution, an A3 solution, an A16 solution, an A56 solution, and an autologous plasma were added to 0.3010.sup.7 to 2.010.sup.7 cells taken from the separated lymphocytes and initial culture was performed for 4 days, from day 1, the day when culture began, to day 5, in a T25 or T75 flask in a CO.sub.2 incubator (37 C. 5% CO.sub.2).
3. Step 2: Stabilization
[0050] After the initial culture, on the 5th day of culture, the culture flask was scraped, transferred to a conical tube, centrifuged (400 g, 5 min) in PBS, washed once more with PBS, and then transferred to a T75 flask using the culture solution containing the BM solution and C2 solution and stabilized.
4. Step 3: Proliferation Culture
[0051] The cells stabilized in the T75 flask were added with a solution containing a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, a C21 solution, and a culture medium containing an A16 solution and an A56 solution, and continuously cultured in a CO2 incubator (37 C. 5% CO.sub.2) from the 5th to the 6th day of culture. As the number of cells increased, on the 7th day of culture, the cells were transferred to a T150 or T175 flask using a solution containing a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, and a C21 solution, and a culture medium containing an A16 solution and an A56 solution, and the culture medium was continuously added until the 8th day, and continuously cultured in a CO.sub.2 incubator (37 C. 5% CO.sub.2). (a total of 4 days)
5. Step 4: Mass Culture
[0052] After step 3 of proliferation culture, the entire culture product was injected into a 1 L CO.sub.2-permeable culture bag containing the cell culture medium, 5 to 10 mL of autologous plasma was added thereto, and the mixture was massaged to mix well with the culture medium and mass culture was performed in a CO.sub.2 incubator (37 C. 5% CO.sub.2) for 5 days. The culture medium was massaged every 24 hours to mix well.
6. Harvesting of Natural Killer Immune Cells
[0053] The final culture medium after culture was placed in a centrifuge tube and the cells were harvested through several centrifugations, and the harvested cells were packaged in a saline bag and stored in the refrigerator or frozen.
[Example 3] Dual Culture Method (A) Initial Step Dual Culture
[0054] In the initial culture step 1 in the single culture method of Example 2, the prepared cells were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C12 solution, and the C18 solution in common, the A3 solution was added to one flask, and the A16 solution and the A56 solution were added to the other flask, followed by separate culture, stabilization of step 2 and combination culture in the proliferation culture of step 3.
[Example 4] Dual Culture Method (B) Initial Step Dual Culture
[0055] In the initial culture step 1 in the single culture method of Example 2, the prepared cells were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C12 solution, and the C18 solution in common, the A3 solution and the A16 solution were added to one flask, and the A3 solution and the A56 solution were added to the other flask, followed by separate culture, stabilization of step 2 and combination culture in the proliferation culture of step 3.
[Example 5] Dual Culture Method (C) Intermediate Step Dual Culture
[0056] In the proliferation culture step 3 in the single culture method of Example 2, the cultured cells stabilized in step 2 were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C2 solution, the C12 solution, the C15 solution, the C18 solution and the C21 solution in common, the A16 solution was added to one flask, and the A56 solution were added to the other flask, followed by separate culture and then combination culture in the mass culture of step 4.
[Example 6] Comparison of Single Culture Method with General Culture Method
[0057] The culture method using Example 2 was compared with the culture method using cell mass culture medium from KOHJIN BIO. The culture was compared using KOHJIN BIO's NK Kit (NKCC-1, NKCC-2, NKCC-b, NKCC-c), known as an NK cell culture kit for 14 days. The result showed that Example 2 exhibited higher immune cell count and natural killer cell ratio. The results are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Immune Immune NK cell count cell count cell Culture method Subject before culture before culture rate KOHJIN BIO Healthy 0.41 10.sup.7 126 10.sup.7 32% KIT Patient 0.42 10.sup.7 88 10.sup.7 24% Example 2 Healthy 0.40 10.sup.7 448 10.sup.7 98% method Patient 0.38 10.sup.7 413 10.sup.7 92%
[Example 7] Comparison of the Number of Cultured Cells and Anticancer Activity of the Single Culture Method and the Dual Culture Method
[0058] The single culture method of Example 2 was compared with the dual culture methods of Examples 3, 4, and 5. The result showed that the number of cells in the dual culture method was higher than that in the single culture. Meanwhile, the K562 cancer cell line was stained with Cacein AM, immune cells or natural killer cells were added at a ratio of 1:30 and reacted in a CO.sub.2 incubator (37 C., 5% CO.sub.2) for 4 hours, and anticancer activity was analyzed by fluorescence colorimetry. At this time, all the methods exhibited high anticancer activity of more than 90% and the results are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Initial Immune immune cell count Cytotoxicity Item cell count before culture (K562) Single culture method 0.44 10.sup.7 460 10.sup.7 94% Dual culture method A 0.46 10.sup.7 476 10.sup.7 98% Dual culture method B 0.45 10.sup.7 478 10.sup.7 95% Dual culture method C 0.45 10.sup.7 522 10.sup.7 96%
[Example 8] Analysis of Immune Cell Rates from PBMCs Using Single Culture Method and Dual Culture Method
[0059] When the cells are cultured using PBMCs isolated from blood, the ratio of proliferated immune cell subtypes may vary. Blood was collected from several donors who agreed to participate in the study, and PBMCs were isolated and cultured using different culture methods, namely, single culture method, dual culture method A, dual culture method B, and dual culture method C. The cultured cells were analyzed by FACS. In this case, the dual culture method exhibited a higher natural killer cell ratio. In particular, the initial culture of Step 1 in dual culture methods A and B exhibited a higher NK content than the single culture method, and the results are shown in Table 3 below.
TABLE-US-00003 TABLE 3 NK cell NK cell Culture rate before rate after Item method culture culture Subject 1 Single culture 11.0% 88% method Dual culture 98% method A Dual culture 95% method B Dual culture 90% method C Subject 2 Single culture 7.8% 76% method Dual culture 94% method A Dual culture 91% method B
[Example 9] Changes in the Number of Cultured Cells of NK Cells Isolated from PBMC Using Single Culture Method and Dual Culture Method
[0060] Natural killer cells were cultured as follows using the cell culture medium addition kit prepared in Example 1.
1. Lymphocyte Extraction and Autologous Plasma Preparation
[0061] 30 mL of peripheral blood of patient A was injected into a 50 mL conical tube and centrifuged, the autologous plasma of the upper layer was placed in a heparin tube and treated, injected into a novel 50 mL conical tube and centrifuged again, and the plasma component of the upper layer was prepared as autologous plasma.
[0062] Only natural killer cells (NK cells) with a purity of 99.9% were isolated from peripheral blood mononuclear cells (PBMCs) separated from blood using an NK isolation kit (STEM Cell).
2. Step 1: Initial Culture
[0063] A BM solution, a C12 solution, a C18 solution, an A3 solution, an A16 solution, an A56 solution, and an autologous plasma were added to 0.3010.sup.7 to 2.010.sup.7 cells taken from the separated natural killer cells and initial culture was performed for 4 days, from day 1, the day when culture began, to day 5, in a T25 or T75 flask in a CO2 incubator (37 C. 5% CO.sub.2).
3. Step 2: Stabilization
[0064] After the initial culture, on the 5th day of culture, the culture flask was scraped, transferred to a conical tube, centrifuged (400 g, 5 min) in PBS, washed once more with PBS, and then transferred to a T75 flask using the culture solution containing the BM solution and C2 solution and stabilized.
4. Step 3: Proliferation Culture
[0065] The cells stabilized in the T75 flask were added with a solution containing a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, a C21 solution, and a culture medium containing an A16 solution and an A56 solution, and continuously cultured in a CO2 incubator (37 C. 5% CO.sub.2) from the 5th to the 6th day of culture. As the number of cells increased, on the 7th day of culture, the cells were transferred to a T150 or T175 flask using a solution containing a C2 solution, a C12 solution, a C15 solution, a C17 solution, a C18 solution, and a C21 solution, and a culture medium containing an A16 solution and an A56 solution, and the culture medium was continuously added until the 8th day, and continuously cultured in a CO.sub.2 incubator (37 C. 5% CO.sub.2). (a total of 4 days)
5. Step 4: Mass Culture
[0066] After step 3 of proliferation culture, the entire culture product was injected into a 1 L CO.sub.2-permeable culture bag containing the cell culture medium, 5 to 10 mL of autologous plasma was added thereto, and the mixture was massaged to mix well with the culture medium and mass culture was performed in a CO.sub.2 incubator (37 C. 5% CO.sub.2) for 5 days. The culture medium was massaged every 24 hours to mix well.
6. Harvesting of Natural Killer Cells
[0067] The final culture medium after culture was placed in a centrifuge tube and the cells were harvested through several centrifugations, and the harvested cells were packaged in a saline bag and stored in the refrigerator or frozen.
[Example 10] Dual Culture Method (A) Initial Step Dual Culture
[0068] In the initial culture step 1 in the single culture method of Example 9, the prepared cells were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C12 solution, and the C18 solution in common, the A3 solution was added to one flask, and the A16 solution and the A56 solution were added to the other flask, followed by separate culture, stabilization of step 2 and combination culture in the proliferation culture of step 3.
[Example 11] Dual Culture Method (B) Initial Step Dual Culture
[0069] In the initial culture step 1 in the single culture method of Example 9, the prepared cells were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C12 solution, and the C18 solution in common, the A3 solution and the A16 solution were added to one flask, and the A3 solution and the A56 solution were added to the other flask, followed by separate culture, stabilization of step 2 and combination culture in the proliferation culture of step 3.
[Example 12] Dual Culture Method (C) Intermediate Step Dual Culture
[0070] In the proliferation culture step 3 in the single culture method of Example 9, the cultured cells stabilized in step 2 were divided equally into two halves with the same cell number and cultured in two flasks. At this time, the medium contained the BM solution, the C2 solution, the C12 solution, the C15 solution, the C18 solution and the C21 solution in common, the A16 solution was added to one flask, and the A56 solution were added to the other flask, followed by separate culture and then combination culture in the mass culture of step 4.
[0071] When natural killer cells were cultured using the single culture method of Example 9 and the dual culture methods of Examples 10 to 12, the dual culture method A exhibited a higher culture efficiency and the results are shown in Table 4 below.
TABLE-US-00004 TABLE 4 Initial NK NK cell count Item cell count after culture Single culture method 0.041 10.sup.7 5.14 10.sup.7 Dual culture method A 0.040 10.sup.7 8.88 10.sup.7 Dual culture method B 0.040 10.sup.7 8.68 10.sup.7 Dual culture method C 0.040 10.sup.7 9.24 10.sup.7
[0072] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
INDUSTRIAL APPLICABILITY
[0073] The novel dual culture method for culturing immune cells and natural killer cells of the present invention and the use thereof have industrial applicability.