CTL cell culture method
By using 4-1BBL to activate the nuclear factor-κB signaling pathway of CTL cells and the addition of cytokines, the problems of low efficiency and unsatisfactory CTL cell expansion are solved, and large-scale rapid proliferation and high-quality preparation of CTL cells are achieved, meeting the needs of tumor immunotherapy.
Patent Information
- Application Number
- CN202510545946.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, CTL cells have limited amplification efficiency, insufficient expansion fold, and unsatisfactory cell purity, making it difficult to obtain a sufficient number of high-quality CTL cells in a short time to meet the needs of clinical application.
4-1BBL is used to contact CTL cells, and by activating the nuclear factor-κB signaling pathway, it promotes the rapid proliferation and activation of CTL cells, and combines the use of cytokines such as IL-2 and IFN-γ to enhance the amplification effect and cytotoxic function of CTL cells.
It significantly improves the amplification fold and purity of CTL cells, so that they proliferate in a large amount in a short period of time, meets the needs of large-scale industrial preparation, and improves the effectiveness and safety of tumor immunotherapy.
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Figure CN120349964A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medicine and relates to a method for culturing CTL cells. Background Art
[0002] Cytotoxic T Lymphocytes (CTL) are a class of extremely important immune cells in the immune system. They are like the "special forces" in the body's immune defense line, specifically responsible for identifying and killing virus-infected cells, tumor cells, and other abnormal cells, playing a crucial role in maintaining the health and stability of the body. With the continuous in-depth study of immunology, CTL cells have shown broad application prospects in many fields such as disease treatment and vaccine development, becoming one of the current hotspots in immunology research.
[0003] The development of CTL cells begins with hematopoietic stem cells in the bone marrow. Hematopoietic stem cells go through a series of differentiation processes, gradually forming lymphoid progenitor cells, and then differentiating into T cell precursors. These T cell precursors enter the thymus and, under the regulation of the thymic microenvironment, undergo two key stages: positive selection and negative selection. Positive selection ensures that T cells can recognize self-major histocompatibility complex (MHC) molecules, and negative selection eliminates those T cell clones with high affinity for self-antigens, thus avoiding the occurrence of autoimmune reactions. T cells that pass through the selection process further differentiate into mature CD8 + CTL cells, which express the CD8 molecule, that is, CD3 + CD8 + and can recognize and bind to antigen peptides presented by MHC class I molecules, and then leave the thymus and enter the peripheral immune system, ready to respond to the invasion of pathogens or tumor cells at any time.
[0004] When the body is infected with a virus or there are tumor cells, CTL cells will be activated and exert their powerful immune function. The key to activating CTL cells lies in the role of antigen-presenting cells (APC). APCs, such as dendritic cells, can uptake, process the antigens of pathogens or tumor cells, and present antigen peptides on MHC class I molecules. When the T cell receptor (TCR) of CTL cells specifically binds to the antigen peptide-MHC class I molecule complex on the surface of APCs and simultaneously receives co-stimulatory signals (such as the interaction between CD80 / CD86 and CD28), CTL cells are activated. After activation, CTL cells undergo a series of changes, including cell proliferation, differentiation into effector CTL cells, and secretion of various cytokines.
[0005] Effector CTL cells have multiple mechanisms for killing target cells. Among them, the perforin-granzyme pathway is one of the main killing methods. CTL cells release perforin onto the surface of target cells through degranulation. Perforin forms pores on the target cell membrane, leading to the outflow of target cell contents, osmotic imbalance, and ultimately causing the lysis and death of target cells. At the same time, granzymes secreted by CTL cells can enter the target cells through the pores formed by perforin, activate the apoptotic pathway within the cells, and promote the programmed death of target cells. In addition, CTL cells can also bind to Fas molecules on the surface of target cells by expressing Fas ligand (FasL), initiating the Fas / FasL-mediated apoptosis signaling pathway and inducing apoptosis of target cells. These killing mechanisms cooperate with each other to ensure that CTL cells can efficiently eliminate infected cells and tumor cells, protecting the body from pathogens and tumors.
[0006] However, there is a problem of limited cell amplification efficiency in the amplification and culture of CTL cells under artificial conditions:
[0007] The natural proliferation rate of CTL cells is relatively slow, and it is difficult to obtain a large number of cells in a short time. For example, in some traditional culture methods, it may take a long time to reach a certain scale of cell quantity, which is a great limitation for clinical applications that require rapid acquisition of sufficient cells for treatment.
[0008] Limited amplification multiple: Although existing culture techniques can promote the amplification of CTL cells to a certain extent, their amplification multiples are still limited. For example, the reported amplification multiples in research are usually between dozens and hundreds. However, for the large number of cells required for the treatment of diseases (such as cancer), the more CTL cells, the better.
[0009] The cell purity is not ideal enough. Under conventional in vitro activation and amplification conditions (such as stimulation with anti-CD3 monoclonal antibody and IL-2), the proportion of CD3 + CD8 + cells usually increases significantly, but the specific proportion varies depending on the culture conditions and cell sources. After activation and amplification, the proportion of CD3 + CD8 + cells can usually reach 30% - 70%, but the cell purity is still not ideal enough.
[0010] Therefore, developing a method for rapidly amplifying CTL cells has become an urgent problem to be solved in the medical field. Summary of the Invention
[0011] To solve the above problems, the object of the present invention is to provide a method for culturing CTL cells, which improves the amplification multiple of CTL cells by using 4-1BBL, enabling CTL cells to achieve a large amount of proliferation in a short time.
[0012] To achieve the above object, the present invention provides a method for culturing CTL cells, comprising the following steps:
[0013] 1) Pre-coating of cell culture flask
[0014] Coat the cell culture flask with OKT-3. The coating concentration of OKT-3 is 1-5 μg / m, and the total volume is 10 mL. Incubate overnight at 4°C or incubate for at least 2 hours at 37°C;
[0015] 2) Plasma separation and inactivation
[0016] Collect fresh blood samples and add them to blood collection tubes containing anticoagulants. Separate the upper plasma by centrifugation and perform inactivation treatment at 56°C for 30 min; then perform high-speed centrifugation on the inactivated plasma to remove the precipitate part, retain the supernatant, and store it at 4°C;
[0017] 3) Isolation and washing of mononuclear cells
[0018] Isolate and collect mononuclear cells from peripheral blood using gradient density centrifugation technology;
[0019] 4) Cell activation and amplification
[0020] Resuspend the isolated mononuclear cells with serum-free immunocyte medium and inoculate them into the pre-coated cell culture flask; add 4-1BBL with a final concentration of 50-200 ng / mL, IFN-γ with a final concentration of 200-1000 U / mL, IL-1α with a final concentration of 200-1000 U / mL, and anti-CD28 monoclonal antibody with a final concentration of 50-200 ng / mL to the cell culture flask, and then add 1%-5% autologous plasma or 0.5%-1% platelet lysate. Then place the culture flask in an incubator at 37°C and 5% CO2 for culture; after 24 hours of culture, add IL-2 with a final concentration of 250-2000 U / mL and continue to culture in an incubator at 37°C and 5% CO2.
[0021] Furthermore, add step 6) further amplification of cells: Observe and supplement the serum-free immunocyte medium containing 250-2000 U / mL of IL-2 as a replenishing fluid daily; before subsequent addition of the replenishing fluid, sample and count to ensure that the cell density is 1×10 6 ~2.5×10 6 / mL; as the amount of replenishing fluid increases, the culture container is changed to a T225 cell culture flask or a cell culture bag, and sub-bottling or sub-bagging operations are performed when the maximum capacity of the container is exceeded, where the culture volume of the cell culture bag does not exceed 1500 mL / bag.
[0022] Furthermore, the cell culture flask described in step 1) is a T75 or T175.
[0023] The initial number of cultured cells is small, so a cell culture flask with a smaller volume is used to avoid waste.
[0024] Furthermore, the anticoagulant described in step 2) is heparin or sodium citrate.
[0025] Furthermore, the blood sample described in step 2) is cord blood or peripheral blood.
[0026] Furthermore, the method for separating and collecting in step 3) is: extracting mononuclear cells through lymphocyte separation medium, collecting the obtained mononuclear cell layer, and then centrifuging and washing with physiological saline or PBS, repeating 2 - 3 times.
[0027] Furthermore, the seeding amount of the mononuclear cells described in step 5) is 3×10 7 ~10×10 7 / flask, and the cell density is 1×10 6 ~2×10 6 / mL.
[0028] Furthermore, the serum-free medium for immune cells described in step 5) is KBM581 or X-VIVO15.
[0029] The beneficial effects of the present invention are as follows:
[0030] The present invention provides a method for culturing CTL cells. This method can effectively increase the amplification multiple of CTL cells by introducing 4-1BBL, enabling CTL cells to achieve a large number of proliferations in a short time and also improving the proportion of CTL cells. The method provided by the present invention meets the strict requirements for the quality of CTL cells in tumor immunocyte therapy and can achieve industrial-scale preparation of CTL cells. Description of the Drawings
[0031] Figure 1 It is a statistical chart of the increase in the number of cells cultured for 14 days by the CTL cell culture method provided by the present invention.
[0032] Figure 2 It is a flow cytometry diagram of the cell control group cultured for 14 days by the CTL cell culture method provided by the present invention. Among them, the control group was not treated with 4-1BBL, the abscissa is CD3-FITC, and the ordinate is CD8-APC.
[0033] Figure 3 It is a flow cytometry diagram of the cell experimental group cultured for 14 days by the CTL cell culture method provided by the present invention. Among them, the experimental group was treated with 4-1BBL, the abscissa is CD3-FITC, and the ordinate is CD8-APC. Detailed implementation mode
[0034] The embodiments of the present invention will be described in detail and perfectly below, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.
[0035] Tumor necrosis factor ligand superfamily member 9 (4-1BBL), also known as 4-1BB ligand CD137L or TNFSF9, is a cytokine that binds to TNFRSF9.
[0036] 4-1BB (CD137) is an inducible costimulatory receptor expressed on activated CD4 + and CD8 + T cells, NKT, NK cells, DC, macrophages, eosinophils, neutrophils, mast cells, and Tregs. 4-1BBL is a high-affinity ligand of 4-1BB and can induce the proliferation of activated T cells. Specifically, after 4-1BB signaling is activated, it can preferentially promote the proliferation, activation, enhanced cytotoxicity, and metabolic adaptation of CD8 + T cells, promote CD8 + T cells to produce IL-2 and IFN-γ, and can generate CD8 + memory T cells.
[0037] Signal pathway activation and cell proliferation
[0038] After 4-1BBL binds to 4-1BB, it rapidly activates the nuclear factor-κB signaling pathway, which plays a key role in cell proliferation regulation, can significantly promote the rapid proliferation of T cells, and provides a basis for the large-scale expansion of CTL. The activated nuclear factor-κB signaling pathway can also enhance the survival ability of T cells. By inhibiting the expression of apoptosis-related genes, CTL has a stronger anti-apoptotic ability during culture and its survival time is extended. Therefore, adding 4-1BBL during culture can significantly enhance the proliferation and activity of CTL, enabling it to maintain a high immune activity during culture.
[0039] Cytokine secretion and immune regulation
[0040] After 4-1BBL interacts with 4-1BB, it can significantly promote T cells to secrete cytokines such as IL-2 and IFN-γ. IL-2 can further stimulate the proliferation of T cells, forming a positive feedback mechanism and enhancing the amplification effect of CTL. IFN-γ has multiple immune regulatory functions. It can enhance the cytotoxic function of CTL, promote its killing effect on tumor cells, and at the same time can regulate the immune microenvironment and enhance the overall anti-tumor ability of the immune system.
[0041] Enhancement of CTL Activity and Anti-Tumor Ability
[0042] Co-stimulatory signals enhance the cytotoxic function of CTLs through multiple pathways, enabling them to more effectively recognize and kill tumor cells, thereby improving the efficiency of tumor cell clearance and providing a more powerful cellular weapon for tumor immunotherapy. The co-stimulatory signal provided by 4-1BBL can also enhance the antigen specificity of CTLs, making them more precise in recognizing and attacking tumor cells, reducing the accidental injury to normal cells, and improving the safety and effectiveness of treatment.
[0043] Example 1
[0044] 1. Pre-coating of culture flasks
[0045] Use T175 culture flasks for coating, with an OKT-3 coating concentration of 2.5 μg / mL, a total volume of 10 mL, coat 2 flasks, and place them at 37 °C for 2 h.
[0046] OKT-3 is a monoclonal antibody for T cell activation, belonging to a type of CD3 monoclonal antibody, specifically used for the activation of T cells.
[0047] 2. Plasma separation and inactivation
[0048] Centrifuge 80 mL of freshly collected peripheral blood in a heparin anticoagulant tube at 2000 rpm for 10 min; take out the upper plasma and inactivate it in a 56 °C water bath for 30 min; centrifuge the inactivated plasma at 3000 rpm for 10 min, discard the precipitate and retain the supernatant, and place it at 4 °C for later use.
[0049] 3. Isolation and washing of peripheral blood mononuclear cells
[0050] Dilute the remaining blood with physiological saline to 1.5 times the original blood volume. After ensuring thorough mixing, slowly add the blood to a Ficoll centrifuge tube containing 15 mL, about 30 mL / tube, for a total of 4 tubes; then, centrifuge at 2000 rpm for 20 min, with the acceleration set to 7 and the deceleration set to 4. After centrifugation, carefully collect the mononuclear cell layer, and wash the mononuclear cells twice with physiological saline by centrifugation, with the rotation speed decreasing to 1600 rpm and 1200 rpm successively for each centrifugation. Finally, take a sample for counting, and the number of cells obtained is approximately 1.24×10 8 cells, and the cell viability is about 93.4%.
[0051] 4. Cell activation and expansion
[0052] On Day 0, resuspend the isolated PBMC with Lonza X-VIVO15 serum-free immunocyte medium to a cell density of 1.5×10 6 / mL, and evenly inoculated into 2 coated T175 culture flasks, which were respectively labeled as: control group and experimental group. Among them, to the experimental group culture flasks, 4-1BBL with a final concentration of 100 ng / mL, IFN-γ with a final concentration of 1000 U / mL, IL-1α with a final concentration of 1000 U / mL, and anti-CD28 monoclonal antibody with a final concentration of 50 ng / mL were respectively added, and then 10% autologous plasma was added; while in the control group culture flasks, 4-1BBL was not added, and the other added components were the same. The two groups of cells were placed in an incubator at 37°C and 5% CO2 for culture.
[0053] The autologous plasma therein can also be replaced with commercial platelet lysate.
[0054] On Day 1, IL-2 with a final concentration of 1000 U / mL was added to both groups of culture flasks, and they were continued to be placed in an incubator at 37°C and 5% CO2 for culture.
[0055] After Day 3, the T cells have been activated and rapidly expanded, and daily observation and fluid replenishment are required. As the volume of fluid replenishment increases, the culture container is changed to a T225 culture flask, and then to a cell culture bag, and sub-bagging operation is carried out when necessary.
[0056] As Figure 1 shown in the statistical chart of the increase in the number of cells cultured for 14 days, from Figure 1 it can be seen the growth of the number of cells over time. Specifically, the number of cells in the blank group reached 9.78 billion on the 14th day, which was about 150 times higher than the initially inoculated PBMC; while the number of cells in the experimental group was 16.67 billion, which was more than 260 times higher than the initially inoculated PBMC. In addition, at the same culture time points, the number of cells in the experimental group was always higher than that in the blank group. Therefore, the addition of 4-1BBL significantly promoted the rapid proliferation of CTL, and this proliferative activity could last for a long time.
[0057] From Figure 2 and Figure 3 the flow cytometry plots of the control group and the experimental group, it can be seen that the proportion of CD3 + cells in the control group was 88.19%, among which the proportion of CD3 + CD8 + was 55.98%; while the proportion of CD3 + in the experimental group was 95.84%, among which the proportion of CD3 + CD8 + was 72.69%. Since the culture of CTL cells is related to the blood collected, but from the above comparison, it can be seen that the addition of 4-1BBL in the blood of the same source significantly promotes the increase in the proportion of CD3 + CD8 + CTL.
[0058] As can be seen from the above embodiments, a method for culturing CTL cells provided by the present invention not only significantly increases the amplification multiple of CTL cells based on the introduction of 4-1BBL, enabling them to achieve a large amount of proliferation in a short time, but also can increase the proportion of CTL cells. This progress provides new strategies and methods for adoptive immunocyte therapy, meets the strict requirements of tumor immunocyte therapy for the quality of CTL cells, and enables the large-scale preparation of CTL cells.
[0059] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, 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 appended claims.
Claims
1. A method for culturing CTL cells, characterized in that, It includes the following steps: 1) Pre-coating of cell culture flasks Use OKT-3 to coat the cell culture flasks. The coating concentration of OKT-3 is 1-5 μg / m, the total volume is 10 mL, and incubate overnight at 4°C or for at least 2 hours at 37°C. 2) Plasma separation and inactivation Collect fresh blood samples and add them to blood collection tubes containing anticoagulants. Separate the upper plasma by centrifugation and perform inactivation treatment at 56°C for 30 minutes. Then, perform high-speed centrifugation on the inactivated plasma to remove the precipitate and retain the supernatant, which is stored at 4°C. 3) Isolation and washing of mononuclear cells Isolate and collect mononuclear cells from peripheral blood using gradient density centrifugation technology. 4) Cell activation and amplification Resuspend the isolated mononuclear cells with serum-free immunocyte medium and inoculate them into the pre-coated cell culture flasks. Add 4-1BBL with a final concentration of 50-200 ng / mL, IFN-γ with a final concentration of 200-1000 U / mL, IL-1α with a final concentration of 200-1000 U / mL, and anti-CD28 monoclonal antibody with a final concentration of 50-200 ng / mL to the cell culture flasks. Then add 1%-5% of the autologous plasma separated and inactivated in step 2) or 0.5%-1% platelet lysate. Then place the culture flasks in an incubator at 37°C and 5% CO2 for culture. After 24 hours of culture, add IL-2 with a final concentration of 250-2000 U / mL and continue to culture in an incubator at 37°C and 5% CO2.
2. The CTL cell culture method according to claim 1, wherein Furthermore, add step 6) further expansion of cells: Observe and supplement the serum-free medium for immune cells containing 250 - 2000 U / mL of IL-2 as the replenishing fluid daily; before subsequent addition of the replenishing fluid, sample and count to ensure that the cell density is at 1×10 6 ~2.5×10 6 / mL; as the amount of the replenishing fluid increases, the culture container is changed to a T225 cell culture flask or a cell culture bag, and sub-bottling or sub-bagging operations are carried out when the maximum capacity of the container is exceeded, where the culture volume of the cell culture bag does not exceed 1500 mL / bag.
3. The CTL cell culture method according to claim 1, wherein The cell culture flasks described in step 1) are T75 or T175.
4. The CTL cell culture method according to claim 1, characterized in that, The anticoagulant described in step 2) is heparin or sodium citrate.
5. The CTL cell culture method according to claim 1, characterized in that, The blood samples described in step 2) are cord blood or peripheral blood.
6. The CTL cell culture method according to claim 1, wherein The method for separating and collecting described in step 3) is: extract mononuclear cells through lymphocyte separation medium, collect the obtained mononuclear cell layer, and then perform centrifugal washing with normal saline or PBS, repeating 2-3 times.
7. The CTL cell culture method according to claim 1, characterized in that, The seeding amount of the mononuclear cells in step 5) is 3×10 7 ~10×10 7 / bottle, and the cell density is 1×10 6 ~2×10 6 / mL.
8. The CTL cell culture method according to claim 1, wherein The serum-free immunocyte medium described in step 5) is KBM581 or X-VIVO15.