A method for culturing and preparing anti-tumor CTL cells

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a method for culturing and preparing anti-tumor CTL cells, the culturing method comprising: separating mononuclear cells from blood by density gradient centrifugation; using culture bottles for coating, using lymphocyte serum-free medium for mononuclear cells, adding GMP-grade recombinant proteins or humanized antibodies and blood sample serum for 18 days; after the cell culture is completed, centrifugation is performed to collect cells, CTL cells are washed 3 times with saline, and then the cells are resuspended with saline and fixed to 200 ml after adding 5% human serum albumin. The present invention establishes a high-purity cell culture technology that meets GMP standards, which is not only suitable for peripheral blood but also for the culture of cord blood CTL. Only 50 ml of peripheral blood or cord blood samples need to be collected, and all operations are completed under GMP conditions. The number of cells cultured in a short period of 18 days can also reach the clinical treatment application standard.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for culturing and preparing anti-tumor CTL cells. Background Art

[0002] In recent years, with the increasing maturity of in vitro large-scale expansion of immune cells, the clinical application of adoptive transfer of allogeneic or autologous immune cells has also increased. Cytotoxic T lymphocytes (CTL) are a subpopulation of white blood cells and a specific CD8+T cell. They can secrete various cytokines to participate in immune regulation and have certain antiviral effects. Adoptive transfer of CTL cells expanded in vitro can restore or enhance the body's antiviral ability. When CTL cells are expanded in vitro, specific CD8+T cells can be activated and expanded. After being transferred back into the body, they can be distributed to the body to play a certain antiviral and anti-tumor effect.

[0003] CTL is one of the most promising adoptive immunotherapy strategies and plays a major role in the anti-tumor immune process. CTL is a tumor-specific killer T cell that is amplified by DC cells activated by tumor antigens to stimulate initial T cells. It kills tumor cells mainly through direct mechanisms such as granzymes and perforins, as well as indirect mechanisms such as secretion of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). However, the in vitro preparation of CTL cells is cumbersome, with a long culture cycle, low yield, and easy contamination. A large amount of peripheral blood or umbilical cord blood is required to cultivate sufficient cell volume, and the killing rate of tumor cells is low. Some methods require the use of animal-derived fetal bovine serum, which has certain safety risks.

[0004] The present invention improves the existing technology and cultivates tens of billions of CTL cells that meet GMP standards and are applicable to clinical treatment, achieving the advantages of obvious expansion effect, high cell viability, high tumor cell killing rate and high safety. Summary of the invention

[0005] The purpose of the present invention is to provide a method for culturing and preparing anti-tumor CTL cells. The method can stably cultivate high-purity CTL cells of the order of tens of billions that meet GMP standards and are suitable for clinical treatment applications. The method is not only suitable for the cultivation of peripheral blood but also cord blood CTL cells. It has the advantages of high purity, obvious amplification effect, high cell viability, high tumor cell killing rate, and high safety. It can be used for the adjuvant treatment of hepatitis B, tumors, etc.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for culturing CTL cells, comprising the following steps:

[0008] (1) Mononuclear cells were isolated from blood using density gradient centrifugation;

[0009] (2) Using culture bottles for coating, monocytes are cultured in lymphocyte serum-free medium, GMP-grade recombinant proteins or humanized antibodies, and blood sample serum for 18 days; during the culture process, cell counts are performed every 2-3 days, and corresponding GMP-grade recombinant proteins or humanized antibodies, autologous serum, and lymphocyte serum-free medium are added, and microbial, mycoplasma, and endotoxin safety tests are performed after the last rehydration;

[0010] (3) After the cell culture was completed, the cells were collected by centrifugation, and the CTL cells were washed three times with physiological saline. The cells were then resuspended in physiological saline and fixed to 200 ml after adding 5% human serum albumin.

[0011] Furthermore, in step (1), the blood is umbilical cord blood or peripheral blood.

[0012] Furthermore, in step (1), the specific steps of separation are:

[0013] (1.1) Collect umbilical cord blood or peripheral blood and store it in a collection bag for later use;

[0014] (1.2) After the collection bag in step (1.1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use;

[0015] (1.3) Add lymph separation fluid to a high-efficiency centrifuge tube. Add PBS to the centrifuge tube containing cells in step (1.2) and resuspend the tube. Then add the tube to the high-efficiency centrifuge tube and centrifuge. After centrifugation, there are 4 layers of liquid in the high-efficiency centrifuge tube. Discard the first layer on the top. Discard the second white CBMC layer and transfer it to a new centrifuge tube. Add D-PBS to wash and centrifuge. Discard the supernatant. Add D-PBS to wash again and centrifuge to obtain mononuclear cells.

[0016] Furthermore, in step (1.2), the centrifugation parameters for centrifugation and collection of supernatant plasma are 700g, 20min; the water bath inactivation and standing followed by centrifugation are 56°C water bath inactivation for 30min, followed by standing at 4°C for 15min, and then centrifugation at 1200g, 15min.

[0017] Furthermore, in step (1.3), the centrifugation parameters when adding to the high-efficiency centrifuge tube are 800g, 20min; and the centrifugation parameters when adding PBS for washing and centrifugation are 500g, 10min.

[0018] Furthermore, in step (2), the specific steps of culturing are:

[0019] (2.1) Adding D-PBS coating solution containing CD137 and Retronectin to the culture flask containing the monocytes, incubating in a 37°C, 5% CO2 incubator for 2 hours, discarding the coating solution, washing the cells with D-PBS, and then inoculating the cells in a T175 culture flask, adding lymphocyte serum-free medium containing OK-432, IL-15, IL-2, IL-21 and 10% inactivated plasma to make the cell concentration 1-3*10^6 / ml, and continuing to culture in a 37°C, 5% CO2 incubator for 4 days;

[0020] (2.2) On the 4th day after the culture in step (2.1), a serum-free medium containing IL-2, IL-15, IL-21 and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were placed in an incubator and cultured for 2 more days;

[0021] (2.3) On the 6th day after the culture in step (2.2), a serum-free medium containing IL-2 and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 more days;

[0022] (2.4) On the 8th day after the culture in step (2.3), the CTL cells are transferred from the culture flask to the cell culture bag for large-scale expansion, and the bag is replenished or expanded every 2-3 days according to the cell growth. Each time the fluid is replenished, a lymphocyte serum-free culture medium containing IL-2 is added to keep the cell concentration in the range of 1-3*10^6 / ml.

[0023] Furthermore, in step (3), the centrifugation parameters are 1500 rpm, 5 min.

[0024] Furthermore, the lymphocyte serum-free culture medium also includes 1% NEAA and 1% L-glutamine.

[0025] Furthermore, the final concentrations of CD137 and Retronectin in the culture system are 0.25-1 μg / ml and 10-20 μg / ml, respectively;

[0026] The final concentrations of OK432, IL-2, IL-15, IL-21 and PHA-L in the culture system are 100-1000 ng / ml, 500-1000 U / ml, 20-50 ng / ml, 50-100 ng / ml and 1-10 μg / ml, respectively.

[0027] Furthermore, the final concentrations of CD137 and Retronectin in the culture system were 0.5 μg / ml and 20 μg / ml, respectively;

[0028] The final concentrations of OK432, IL-2, IL-15, IL-21 and PHA-L in the culture system are 500 ng / ml, 1000 U / ml, 25 ng / ml, 100 ng / ml and 5 μg / ml, respectively.

[0029] Beneficial effects:

[0030] In the present invention, serum-free culture medium is used in the whole process of CTL cell culture, antibiotics are not added, and animal-derived components such as fetal bovine serum are not added. Inactivated serum of the blood sample itself is used as a culture medium additive, which is safer.

[0031] The present invention establishes a CTL cell culture technology that complies with the GMP standard, which is not only applicable to the culture of peripheral blood but also to the culture of cord blood CTL. Only 50 ml of peripheral blood or cord blood samples need to be collected, and all operations are completed under GMP conditions. The number of cells cultured in a short period of 18 days can also meet the clinical treatment application standard.

[0032] The CTL cells cultured by the present invention have high purity, high viability and good tumor killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The figure is a graph of the CTL cell growth curve in Examples 1-2 of the present invention, wherein the cord blood CTL is the CTL cell growth curve in Example 1, and the peripheral blood CTL is the CTL cell growth curve in Example 2;

[0035] Figure 2 The figure is a CTL cell viability graph in Example 1-2 of the present invention, wherein the cord blood CTL is the CTL cell viability curve in Example 1, and the peripheral blood CTL is the CTL cell viability curve in Example 2;

[0036] Figure 3 The figure is a graph showing the killing effect of CTL cells on tumor cells prepared by the present invention, wherein A is the killing effect of CTL cells with different effector targets on human chronic myeloid leukemia cells K562, B ​​is the killing effect of CTL cells with different effector targets on human breast cancer cells MCF-7, and C is the killing effect of CTL cells with different effector targets on human colorectal adenocarcinoma cells SW620;

[0037] Figure 4Figures 1 and 2 are flow cytometric phenotype detection diagrams of CTL cells cultured for 18 days in Example 1-2 of the present invention, A is flow cytometric detection of phenotype of CTL cells in cord blood, and B is flow cytometric detection of phenotype of CTL cells in peripheral blood. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Some commonly used terms in the present invention are described as follows:

[0044] CD137: is a new co-stimulatory signal molecule that participates in the activation, proliferation, differentiation, apoptosis, signal transduction and extracellular adhesion of T cells. It also acts as a receptor to regulate the function of dendritic cells (DC). Activated CD137 can induce monocyte survival, proliferation and adhesion;

[0045] RetroNectin: A recombinant human fibronectin fragment that has physiological activities involved in cell attachment, extension, differentiation and proliferation, and can promote the adhesion and proliferation of lymphocytes;

[0046] IL-2: Interleukin 2, a cytokine that promotes the growth and proliferation of T cells, activates T cells to produce proliferation, and promotes the growth, proliferation, and differentiation of lymphocytes;

[0047] IL-21: Interleukin 21, which regulates B cell proliferation, promotes the proliferation of bone marrow precursor cells, enhances the proliferation, differentiation and cytotoxic activity of CTL cells, can induce T cell-stimulated B cells to differentiate into plasma cells and memory B cells, promote the production of IgG, and promote the anti-tumor activity of CD8+T cells and CTL cells;

[0048] IL-15: Interleukin 15; it has the function of activating T cells and mediating the proliferation and survival of T cells. It can activate, maintain and amplify CD8+ memory T cells;

[0049] PHA-L: Phytohemagglutinin-L, has mitotic and potential cell agglutination activity, can bind to T cell membrane, stimulate cell division and metabolic activity, can activate close contact between cell membranes, and efficiently induce human immune cells. The experimental instruments and experimental consumables used in the present invention are shown in Table 1 and Table 2.

[0050] Table 1 Experimental instruments used in the present invention

[0051] Carbon dioxide incubator Panasonic Biological Safety Cabinets Sujing Antai Centrifuge (Thermo Fisher)(KUBOTA) Electric pipette Eppendorf Manual pipette Eppendorf

[0052] Table 2 Experimental consumables used in the present invention

[0053]

[0054]

[0055] The method for culturing CTL cells in the present invention specifically comprises the following steps:

[0056] (1) Collecting umbilical cord blood or peripheral blood and placing it in a collection bag for storage and standby use;

[0057] (2) After the collection bag in step (1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use;

[0058] (3) Add lymph separation fluid to a high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells in step (2), resuspend the tube, and then add it to the high-efficiency centrifuge tube for centrifugation. After centrifugation, there are 4 layers of liquid in the high-efficiency centrifuge tube. The first layer on the top is discarded by suction, and the second white CBMC layer is removed and transferred to a new centrifuge tube. Add D-PBS to wash and centrifuge, discard the supernatant, add D-PBS to wash again, and centrifuge to obtain mononuclear cells.

[0059] (4) adding D-PBS coating solution containing CD137 and Retronectin to the culture flask containing the monocytes, incubating in a 37°C, 5% CO2 incubator for 2 hours, discarding the coating solution, washing the cells with D-PBS, and inoculating the cells in a T175 culture flask, adding lymphocyte serum-free medium containing OK-432, IL-15, IL-2, IL-21 and 10% inactivated plasma to make the cell concentration 1-3*10^6 / ml, and continuing to culture in a 37°C, 5% CO2 incubator for 4 days;

[0060] (5) On the 4th day after the culture in step (4), a serum-free medium containing IL-2, IL-15, IL-21 and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were placed in an incubator and cultured for 2 more days;

[0061] (6) On the sixth day after the culture in step (5), a serum-free medium containing IL-2 and 10% plasma was added to the cells to continue the culture, so that the cell concentration was within the range of 1-3*10^6 / ml, and the cells were cultured in a 37°C, 5% CO2 incubator for another 2 days;

[0062] (7) On the 8th day after the culture in step (6), the CTL cells are transferred from the culture flask to a cell culture bag for large-scale expansion, and the fluid is replenished or the bag is expanded every 2-3 days according to the cell growth condition, and each time the fluid is replenished, a lymphocyte serum-free culture medium containing IL-2 is added to make the cell concentration within the range of 1-3*10^6 / ml;

[0063] (8) After the cell culture was completed, the cells were collected by centrifugation, and the CTL cells were washed three times with physiological saline. The cells were then resuspended in physiological saline and fixed to 200 ml with 5% human serum albumin.

[0064] In the present invention, the final concentrations of CD137 and Retronectin in the culture coating system are 0.25-1 μg / ml and 10-20 μg / ml, respectively, and more preferably 0.5 μg / ml and 20 μg / ml;

[0065] The final concentrations of OK4432, IL-2, IL-15, IL-21 and PHA-L in the culture system are 100-1000 ng / ml, 500-1000 U / ml, 20-50 ng / ml, 50-100 ng / ml, 1-10 μg / ml, more preferably 500 ng / ml, 1000 U / ml, 25 ng / ml, 100 ng / ml, 5 μg / ml, respectively;

[0066] When IL-2 is added, the final concentration in the culture system is 200-1000 U / ml, more preferably 1000 U / ml.

[0067] The technical solution of the present invention will be described in detail below by way of embodiments.

[0068] Example 1

[0069] In this embodiment, umbilical cord blood is used to culture CTL cells, which specifically includes the following steps:

[0070] 1. Cord Blood Collection

[0071] Conduct a survey on the health status of the mothers and fill out the "Health Status Questionnaire" to exclude the risks of infectious diseases such as hepatitis A, hepatitis B, hepatitis C, and syphilis. After the mother and her family sign the "Informed Consent for Cord Blood Disposal", the doctor will collect the cord blood of the full-term newborn, place it in a cord blood collection bag, store it in a refrigerator at 2-8℃, and transport it to the laboratory for processing within 24 hours. The maternal blood and cord blood will be handed over to a third party for infectious disease testing.

[0072] 2. Isolation of Cord Blood Mononuclear Cells (CBMNCs)

[0073] 2.1 Plasma preparation

[0074] Disinfect the outer surface of the cord blood collection bag with 75% alcohol and place it in a biosafety cabinet; wipe the entire cord blood collection bag with an alcohol cotton ball and place it on a test tube rack. Use a 50ml syringe to transfer the cord blood to a 50ml centrifuge tube; centrifuge at 700g for 20min. After centrifugation, collect the supernatant plasma on the upper layer, keep 5ml of the sample for infectious source microorganism testing, and inactivate the remaining plasma in a 56℃ water bath for 30min; then let it stand at 4℃ for 15min; after standing, centrifuge at 1200g for 15min. Take the supernatant plasma and place it at 4℃ for later use.

[0075] 2.2 Extraction and separation of cord blood mononuclear cells (CBMNCs)

[0076] Add 13ml of lymph separation fluid to the high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells to 35ml, fully resuspend, and slowly add to the high-efficiency centrifuge tube; centrifuge at 800g for 25min. After centrifugation, there are 4 layers of liquid in the high-efficiency centrifuge tube. Discard the first layer on top, and transfer the second white CBMC layer to a new 50ml centrifuge tube, add 30ml D-PBS to wash, and centrifuge at 500g for 10min. After centrifugation, discard the supernatant, add 30ml D-PBS to wash, and centrifuge at 500g for 10min.

[0077] 3. Cultivation of Cord Blood CTL Cells

[0078] 3.1 Counting of cord blood mononuclear cells (CBMNCs)

[0079] After centrifugation, add 40 ml of culture medium to resuspend, take 100 ml of cell suspension and put it into a 1.8 ml cryotube for counting; mix thoroughly, take 50 ml of cells and place them in 50 ml of AOPI fluorescent dye, blow and beat until uniform, add 25 ml of cell suspension to each well, and count twice for each group.

[0080] 3.2 Induction, differentiation and expansion of cord blood CTL cells

[0081] Day 1: Place the T175 cell culture flask in a biosafety cabinet, wipe the entire flask with an alcohol cotton ball, then add D-PBS coating solution containing CD 137 and Retronectin, seal the flask with a sealing film and gently shake it to cover the entire surface, and incubate it in a 37°C, 5% CO2 incubator for 2 hours. Then discard the coating solution, wash it with 15ml D-PBS again, inoculate the cells in a T175 culture flask, add lymphocyte serum-free medium containing OK-432, IL-15, IL-2, 1L-21, and 10% inactivated plasma, so that the cell concentration is 1-3*10^6 / ml, and continue to culture in a 37°C, 5% CO2 incubator. The basal culture medium is pre-added with 1% NEAA and 1% L-glutamine).

[0082] Day 4: Add lymphocyte serum-free culture medium containing IL-2, IL-15, IL-21 and 10% plasma and continue culturing until the cell concentration is in the range of 1-3*10^6 / ml, and place in a 37°C, 5% CO2 incubator for continued cultivation.

[0083] Day 6: Add lymphocyte serum-free medium containing IL-2 and 10% plasma to continue culturing, so that the cell concentration is within the range of 1-3*10^6 / ml, and continue culturing in a 37°C, 5% CO2 incubator. The subsequent culture system is lymphocyte serum-free medium supplemented with IL-2, 1% NEAA and 1% L-glutamine.

[0084] Day 8: Transfer CTL cells from the culture flask to a cell culture bag for large-scale expansion, and replenish or expand the bag every 2-3 days according to the cell growth. Add 500-1000ml of lymphocyte serum-free culture medium each time to make the cell concentration within the range of 1-3*10^6 / ml.

[0085] Day 18: Transfer the cell culture bag to the biosafety cabinet, collect the cells into a 225ml centrifuge tube and centrifuge at 1500rpm for 5min. After centrifugation, discard the supernatant, resuspend the cells with an appropriate amount of 0.9% sodium chloride injection, and add 5% human albumin to make the volume 200ml. Keep a sample of the cell suspension for AOPI staining count, bacteria and fungi, mycoplasma and endotoxin, and flow cytometry phenotyping.

[0086] Example 2

[0087] In this embodiment, CTL cells are cultured using peripheral blood, specifically comprising the following steps:

[0088] 1. Peripheral Blood Collection

[0089] Conduct a health status survey and fill out the "Health Status Questionnaire" to exclude the risks of infectious diseases such as hepatitis A, hepatitis B, hepatitis C, and syphilis. After the volunteer signs the "Informed Consent for Peripheral Blood Disposal", the doctor will collect 50 ml of peripheral blood, place it in an anticoagulant tube, store it in a 2-8℃ refrigerator, and transport it to the laboratory for processing within 24 hours.

[0090] 2. Isolation of Peripheral Blood Mononuclear Cells (PBMNC)

[0091] 2.1 Plasma preparation

[0092] Disinfect the outer surface of the peripheral blood collection tube with 75% alcohol and place it in a biosafety cabinet; wipe the entire peripheral blood collection tube with an alcohol cotton ball and place it on a test tube rack. Use a 50ml syringe to transfer peripheral blood to a 50ml centrifuge tube; centrifuge at 700g for 20min. After centrifugation, collect the supernatant plasma on the upper layer, keep 5ml of the sample for infectious source microorganism testing, and inactivate the remaining plasma in a 56℃ water bath for 30min; then let it stand at 4℃ for 15min; after standing, centrifuge at 1200g for 15min. Take the supernatant plasma and put it at 4℃ for later use.

[0093] 2.2 Extraction and separation of peripheral blood mononuclear cells (PBMNC)

[0094] Add 13ml of lymph separation fluid to the high-efficiency centrifuge tube, add PBS to the centrifuge tube containing cells to 35ml, fully resuspend, and slowly add to the high-efficiency centrifuge tube; centrifuge at 800g for 20min. After centrifugation, there are 4 layers of liquid in the high-efficiency centrifuge tube. Discard the first layer on top, and transfer the second white PBMC layer to a new 50ml centrifuge tube, add 30ml D-PBS to wash, and centrifuge at 500g for 10min. After centrifugation, discard the supernatant, add 30ml D-PBS to wash, and centrifuge at 500g for 10min.

[0095] 3. Cultivation of Peripheral Blood CTL Cells

[0096] 3.1 Counting of peripheral blood mononuclear cells (PBMNCs)

[0097] After centrifugation, add 40 ml of culture medium to resuspend, take 100 ml of cell suspension and put it into a 1.8 ml cryotube for counting; mix thoroughly, take 50 ml of cells and place them in 50 ml of AOPI fluorescent dye, blow and beat until uniform, add 25 ml of cell suspension to each well, and count twice for each group.

[0098] 3.2 Induction, differentiation and expansion of peripheral blood CTL cells

[0099] Day 1: Place the T175 cell culture flask in a biosafety cabinet, wipe the entire flask with an alcohol cotton ball, then add D-PBS coating solution containing CD137 and Retronectin, seal the flask with a sealing film and gently shake it to cover the entire surface, and incubate it in a 37°C, 5% CO2 incubator for 2 hours. Then discard the coating solution, wash it again with 15ml D-PBS, inoculate the cells in a T175 culture flask, add lymphocyte serum-free culture medium containing OK-432, IL-15, IL-2, IL-21, and 10% inactivated plasma, so that the cell concentration is 1-3*10^6 / ml, and continue to culture in a 37°C, 5% CO2 incubator. The basal culture medium is pre-added with 1% NEAA and 1% L-glutamine).

[0100] Day 4: Add lymphocyte serum-free culture medium containing IL-2, IL-15, IL-21 and 10% plasma and continue culturing until the cell concentration is in the range of 1-3*10^6 / ml, and place in a 37°C, 5% CO2 incubator for continued cultivation.

[0101] Day 6: Add lymphocyte serum-free medium containing IL-2 and 10% plasma to continue culturing, so that the cell concentration is within the range of 1-3*10^6 / ml, and continue culturing in a 37°C, 5% CO2 incubator. The subsequent culture system is lymphocyte serum-free medium supplemented with IL-2, 1% NEAA and 1% L-glutamine.

[0102] Day 8: Transfer CTL cells from the culture flask to a cell culture bag for large-scale expansion, and replenish or expand the bag every 2-3 days according to the cell growth. Add 500-1000ml of lymphocyte serum-free culture medium each time to make the cell concentration within the range of 1-3*10^6 / ml.

[0103] Day 18: Transfer the cell culture bag to the biosafety cabinet, collect the cells into a 225ml centrifuge tube and centrifuge at 1500rpm for 5min. After centrifugation, discard the supernatant, resuspend the cells with an appropriate amount of 0.9% sodium chloride injection, and add 5% human albumin to make the volume 200ml. Keep a sample of the cell suspension for AOPI staining count, bacteria and fungi, mycoplasma and endotoxin, and flow cytometry phenotyping.

[0104] Test example

[0105] 1. Determination of CTL cell growth and proliferation

[0106] Using the AO / PI dual fluorescence cell counting method, 1 ml of samples were collected for counting on days 0, 3, 6, 9, 12, 15, and 18 of CTL cell culture, and the counts were repeated twice. The CTL cell concentration and volume were obtained based on the counting results, thereby obtaining the CTL cell number. The measurement results are shown in Figure 1 As shown by Figure 1 It can be seen that under the same culture technology system, after 18 days of culture, the number of CTL cells in Examples 1-2 reached more than 2*10^10, which meets the clinical application level.

[0107] 2. CTL cell viability determination

[0108] Using the AO / PI dual fluorescence cell method, 1 ml of samples were taken on the 0th, 3rd, 6th, 9th, 12th, 15th, and 18th days of CTL cell culture to calculate the viability, and the counts were repeated twice to obtain the cell viability of CTL cells at different time points. The results are shown in Figure 2 As shown by Figure 2 It can be seen that the cell viability of the CTL cells prepared in Examples 1-2 of the present invention after the completion of the culture was above 90%, maintaining a high viability state, and the cells may have apoptosis of some non-target cells during the early stage of the induction culture, so that the cell viability was slightly reduced, and then after a large number of expansion, the cells did not lose cell viability during the culture and proliferation process and maintained a high viability.

[0109] 3. Determination of the killing effect of CTL cells on tumor cells K562, MCF-7 and SW620

[0110] (1) Subcultured human chronic myeloid leukemia cells K562, human breast cancer cells MCF-7, and human colorectal adenocarcinoma cells SW620 were prepared into cell suspensions, and the cell concentrations of K562, MCF-7, and SW620 were counted and adjusted to 5×10^4 cells / ml.

[0111] (2) The CTL cells prepared in Example 1 were set as effector cells E, and the tumor cell line was set as target cell T. The CTL cells with a culture period of 18 days were taken to prepare cell suspensions of different concentrations. The final concentrations of CTL cells were adjusted to 5×10^4 cells / ml, 2.5×10^5 cells / ml, 5×10^5 cells / ml, and 1×10^6 cells / ml according to the effector-target ratio (1:1, 5:1, 10:1, and 20:1) required by the experiment.

[0112] (3) The cells were inoculated into 96-well cell culture plates at effector-target ratios of 1:1, 5:1, 10:1, and 20:1 (i.e., experimental groups), and target cell control group and effector cell control group were set up, with 3 replicate wells in each group and a total volume of 200 μl in each well.

[0113] (4) After plating, the cells were cultured in a 37°C, 5% CO2 incubator. CCK-8 reagent was added after 4 h, 24 h, and 48 h of co-culture, respectively. The cells were cultured in a cell culture incubator for another 2 h, and the OD450 value (i.e., A value, the absorbance of the test substance at a wavelength of 450 nm) was measured using an ELISA reader.

[0114] (5) Calculate the killing rate: Killing rate = [1-(A value of the experimental group-A value of the effector cell control group) ÷ A value of the target cell control group] × 100%.

[0115] The results of the test are as follows Figure 3 As shown by Figure 3 It can be seen that the killing effect of CTL on tumor cells increases with the action time and the increase of effector cells, and the killing effect of CTL gradually increases with the increase of effector-target ratio. In addition, the killing effect of CTL on solid tumor cells SW620 and MCF-7 is stronger than that on blood tumor K562, and the killing effect can reach about 90% after 24 hours.

[0116] 4. Determination of CTL cell phenotype by flow cytometry

[0117] The two types of CTL cells cultured for 20 days were washed with sodium chloride and prepared into a 5×10^6 cell suspension. Antibodies to CD3, CD4, and CD8 were added. After incubation at room temperature in the dark for 15 minutes, the suspension was tested by flow cytometry to analyze the proportion of CD3+CD8+ double positive cells. The final test results were as follows: Figure 4As shown, according to flow cytometry phenotyping analysis, the CD3+ T cells of the CTL cells of Example 1-2 were all above 98%, and the CD3+CD8+ double positive cells were all above 80%.

[0118] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for culturing CTL cells, characterized in that: The following steps are involved: (1) Isolate mononuclear cells from blood using density gradient centrifugation; (2) Use culture flasks for coating, culture the monocytes in lymphocyte serum-free medium, add GMP-grade recombinant proteins or humanized antibodies and blood sample serum for 18 days; count the cells every 2-3 days during the culture process, add corresponding GMP-grade recombinant proteins or humanized antibodies, autologous serum and lymphocyte serum-free medium, and perform microbial, mycoplasma and endotoxin safety tests after the last rehydration; (3) After the cell culture was completed, the cells were collected by centrifugation, and the CTL cells were washed three times with saline. The cells were then resuspended in saline and the volume was fixed to 200 ml after adding 5% human serum albumin.

2. The culture method according to claim 1, characterized in that In step (1), the blood is cord blood or peripheral blood.

3. The culture method according to claim 1, characterized in that In step (1), the specific steps of separation are: (1.1) Collect umbilical cord blood or peripheral blood and store it in a collection bag for later use; (1.2) After the collection bag in step (1.1) is disinfected, the umbilical cord blood or peripheral blood is placed in a centrifuge tube, centrifuged and the supernatant plasma is collected, inactivated in a water bath, allowed to stand, and then centrifuged for later use; (1.3) Add lymph separation fluid to the high-efficiency centrifuge tube. Add PBS to the centrifuge tube containing cells in step (1.2) and resuspend it. Then add it to the high-efficiency centrifuge tube and centrifuge it. After centrifugation, there are 4 layers of liquid in the high-efficiency centrifuge tube. Discard the first layer on the top, and transfer the second white CBMC layer to a new centrifuge tube. Add D-PBS to wash and centrifuge. Discard the supernatant, add D-PBS to wash again, and centrifuge to obtain mononuclear cells.

4. The culture method according to claim 3, characterized in that In step (1.2), the centrifugation parameters for centrifugation and collection of supernatant plasma are 700g, 20min; the water bath inactivation and standing followed by centrifugation are 56°C water bath inactivation for 30min, followed by standing at 4°C for 15min, and then centrifugation at 1200g, 15min.

5. The culture method according to claim 3, characterized in that: In step (1.3), the centrifugation parameters for adding the sample into the high-efficiency centrifuge tube are 800 g for 20 min; and the centrifugation parameters for adding PBS for washing and centrifugation are 500 g for 10 min.

6. The culture method according to claim 1, characterized in that In step (2), the specific steps of culturing are: (2.1) Adding D-PBS coating solution containing CD137 and Retronectin to the culture flask containing the monocytes, incubating in a 37°C, 5% CO2 incubator for 2 hours, discarding the coating solution, washing the cells with D-PBS, and then inoculating the cells in a T175 culture flask, adding lymphocyte serum-free medium containing OK-432, IL-15, IL-2, IL-21 and 10% inactivated plasma to make the cell concentration 1-3*10^6 / ml, and continuing to culture in a 37°C, 5% CO2 incubator for 4 days; (2.2) On the 4th day after the culture in step (2.1), add lymphocyte serum-free medium containing IL-2, IL-15, IL-21 and 10% plasma to continue the culture, so that the cell concentration is in the range of 1-3*10^6 / ml, and continue the culture in an incubator for 2 days; (2.3) On the 6th day after the culture in step (2.2), add lymphocyte serum-free medium containing IL-2 and 10% plasma to continue the culture, so that the cell concentration is in the range of 1-3*10^6 / ml, and continue the culture in a 37°C, 5% CO2 incubator for 2 days; (2.4) Based on the culture in step (2.3), on the 8th day, the CTL cells are transferred from the culture flask to the cell culture bag for large-scale expansion, and the bag is replenished or expanded every 2-3 days according to the cell growth situation. Each time the fluid is replenished, lymphocyte serum-free culture medium containing IL-2 is added to keep the cell concentration in the range of 1-3*10^6 / ml.

7. The culture method according to claim 1, characterized in that In step (3), the centrifugation parameters are 1500 rpm, 5 min.

8. The culture method according to claim 1, characterized in that The lymphocyte serum-free culture medium also includes 1% NEAA and 1% L-glutamine.

9. The culture method according to claim 6, characterized in that: The final concentrations of CD137 and Retronectin in the culture system are 0.25-1 μg / ml and 10-20 μg / ml respectively; The final concentrations of OK432, IL-2, IL-15, IL-21 and PHA-L in the culture system are 100-1000 ng / ml, 500-1000 U / ml, 20-50 ng / ml, 50-100 ng / ml and 1-10 μg / ml, respectively.

10. The culture method according to claim 9, characterized in that: The final concentrations of CD137 and Retronectin in the culture system are 0.5 μg / ml and 20 μg / ml respectively; The final concentrations of OK432, IL-2, IL-15, IL-21 and PHA-L in the culture system are 500 ng / ml, 1000U / ml, 25 ng / ml, 100 ng / ml and 5 μg / ml, respectively.

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