Culture optimization process and application of high-purity CD4 + T cells

Abstract

The invention discloses a culture optimization process and application of high-purity CD4 + T cells, and belongs to the field of cell culture.The culture optimization process comprises the following steps that firstly, peripheral blood mononuclear cells are separated; step 2, carrying out magnetic bead sorting to obtain CD4 + T cells; 3, carrying out CD4 + T cell amplification by adopting a gradient amplification culture system; 4, through multiple rounds of centrifugal collection and detection, obtaining CD4 + T cells with the purity larger than or equal to 95%; wherein in the step of magnetic bead sorting, CytoSintCD magnetic beads are adopted, a buffer solution is PBS (Phosphate Buffer Solution) containing 0.5% of human albumin, and a step-by-step replacement strategy of a GIBCO culture solution and a Concosale T cell culture solution is adopted in the culture process. The method has the advantages of high purity and high amplification rate.

Description

Technical Field

[0001] The present invention relates to the field of cell culture technology, and specifically to an optimized culture process and application of high-purity CD4+ T cells, which is particularly suitable for medical fields such as the preparation of tumor immunotherapy drugs and autologous cell transfusion. Background Art

[0002] CD4+ T cells play a key role in the human immune system. They not only assist B cells in producing antibodies but also enhance the killing function of cytotoxic T cells (CD8+ T cells). They have great potential for application in tumor immunotherapy and the treatment of autoimmune diseases. However, current CD4+ T cell culture technologies are plagued by numerous challenges.

[0003] Traditional CD4+T cell culture methods are difficult to meet the requirements of high purity and high amplification efficiency at the same time. On the one hand, during the cell separation stage, the commonly used separation methods cannot effectively remove impurity cells, resulting in a low purity of the final CD4+T cells, which affects the subsequent treatment effect; on the other hand, during the culture process, a single culture medium system cannot meet the needs of cells at different growth stages, resulting in limited cell amplification multiples and difficulty in obtaining a sufficient number of cells for clinical treatment. In addition, the existing culture process is not effective in maintaining cell activity, and cells are prone to apoptosis or functional abnormalities during the culture process. Therefore, there is an urgent need for an optimized culture process that can improve the purity, amplification efficiency and activity of CD4+T cells. Summary of the Invention

[0004] The purpose of the present invention is to provide an optimized culture process and application of high-purity CD4+ T cells. By optimizing a series of steps such as cell separation, sorting, culture and collection, the problems of low purity, low amplification efficiency and poor activity of CD4+ T cell culture in the existing technology are solved, thereby obtaining high-purity, high-activity and sufficient CD4+ T cells to meet the needs of medical applications such as tumor immunotherapy.

[0005] A high-purity CD4+ T cell culture optimization process comprises the following steps: Step 1: Isolation of peripheral blood mononuclear cells; Step 2: Obtain CD4+ T cells by magnetic bead sorting; Step 3: CD4+ T cell expansion using a gradient amplification culture system; Step 4: Collect and test cells through multiple rounds of centrifugation to obtain CD4+ T cells with a purity of ≥95%; The magnetic bead sorting uses CytoSinctCD magnetic beads, the buffer is PBS containing 0.5% human serum albumin, and the culture process adopts a step-by-step replacement strategy of GIBCO culture medium and Ecosai T cell culture medium.

[0006] As an improvement, the step of isolating peripheral blood mononuclear cells includes: (1) Whole blood was centrifuged at 1800 rpm for 15 minutes, the saturation was increased by 9 and decreased by 7, and the plasma was separated and inactivated at 56°C for 30 minutes, allowed to stand at 4°C for 30 minutes, and centrifuged at 3000 rpm for 5 minutes; (2) After the blood cell pellet is resuspended with physiological saline, it is added to the lymphocyte separation solution and centrifuged at 2000 rpm for 20 minutes, with the saturation increased to 9 and the saturation decreased to 0, to collect the peripheral blood mononuclear cells.

[0007] As an improvement, it is characterized in that the step of magnetic bead sorting CD4+ T cells includes: (1) Every 10 7 Resuspend the mononuclear cells in 100 μL buffer, add 10 μL CytoSinctCD magnetic beads, and incubate at 2-8°C for 15 minutes; (2) When processing the magnetic separation column, first empty it with 500 μL of buffer, then add the cell suspension, wash it three times with 500 μL of buffer, and then quickly punch it with 1 mL of buffer to collect CD4+ T cells.

[0008] As an improvement, the gradient amplification culture system includes: (1) Initially, add GIBCO complete culture medium containing anti-CD3 / anti-CD28 antibodies, inactivated plasma, and interleukin-2 to the T75 culture flask; (2) Transfer to T175 culture flasks and 2L culture bags in sequence, add culture medium and plasma step by step, and gradually expand the culture volume from 30mL to 1800mL; (3) During the culture process, the cells were dispersed by tapping the culture flask. On the 10th day, 6 mL of cell suspension was collected for sterility testing.

[0009] As an improvement, the cell collection step includes: (1) Centrifuge at 2000 rpm for 7 minutes three times, and resuspend the cells by tapping after each centrifugation; (2) After merging the cells, wash them twice with physiological saline, finally add NKT4 solution and transfer them to a blood bag.

[0010] As an improvement, the culture process further comprises: (1) If the blood cannot be processed immediately, store it at 10°C and return it to room temperature half an hour before the experiment; (2) Resuspend at most 10 μL of buffer 8 monocytes; (3) Detection of CD3 by flow cytometry + CD4 + The cell purity was ≥95.81%, and the CD4 / CD8 ratio was ≥24.19.

[0011] As an improvement, after the plasma is inactivated, platelets are removed by centrifugation at 3000 rpm to reduce the impact on cell activation. This treatment increases the subsequent CD4+ T cell expansion efficiency by more than 30%.

[0012] As an improvement, magnetic bead sorting was performed by incubating at 2-8°C and then washing with centrifugation at 300g.

[0013] As an improvement, the culture medium was replaced with: GIBCO culture medium containing 10% inactivated plasma was used for the first 10 days, and replaced with Icosai T cell culture medium after the 11th day. This strategy enabled the cell expansion to reach more than 2,000 times, which was 15 times higher than that of a single culture medium.

[0014] An application of the culture optimization process is to use high-purity CD4+ T cells to prepare tumor immunotherapy drugs or autologous cell transfusion preparations.

[0015] Beneficial effects High purity: Through unique plasma inactivation treatment and low-temperature magnetic bead sorting technology, impurity cells and interfering factors are effectively removed. After plasma inactivation treatment, centrifugation is used to remove platelets, reducing the impact on cell activation and increasing the subsequent CD4+T cell expansion efficiency by more than 30%; during magnetic bead sorting, low-temperature incubation at 2-8°C combined with 300g centrifugation washing is used to achieve a specific binding rate of magnetic beads to CD4+T cells of 98% and a non-specific binding rate of <1%. Finally, CD3 + CD4 + The cell purity is ≥95.81%, significantly higher than that of traditional methods.

[0016] High expansion efficiency: The gradient amplification culture system is combined with a step-by-step culture medium replacement strategy. GIBCO culture medium (containing 10% inactivated plasma) is used for the first 10 days to promote cell adhesion and initial growth. After the 11th day, it is replaced with Icosai T cell culture medium to meet the needs of cell suspension expansion. This strategy enables the cell expansion multiple to reach more than 2000 times, which is 15 times higher than that of a single culture medium, and can obtain a large number of CD4+ T cells that meet clinical applications.

[0017] High activity: Multiple rounds of low-speed centrifugation (2000 rpm for 7 minutes) combined with palm-patting resuspension reduce 40% of cell mechanical damage compared to traditional 3000 rpm centrifugation, and increase cell activity maintenance from 80% to over 95%, ensuring that cultured CD4+ T cells have good function and activity.

[0018] High application value: The high-purity, high-activity and sufficient number of CD4+T cells obtained can be directly used to prepare tumor immunotherapy drugs or autologous cell transfusion preparations. The CD4 / CD8 ratio is ≥24.19, which significantly improves the immune synergistic effect and reduces the risk of immune rejection. It has broad application prospects in medical fields such as tumor treatment and autoimmune disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flowchart of the present invention; Figure 2 This is the CD3 / CD4 purity diagram of the present invention. DETAILED DESCRIPTION

[0020] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0021] Example 1: Culture of high-purity CD4+ T cells Isolation of peripheral blood mononuclear cells (PBMCs) Peripheral blood samples were collected from healthy volunteers. Whole blood was processed within 6 hours of collection. The whole blood was transferred to a centrifuge tube and centrifuged at 1800 rpm for 15 minutes (speed setting 9, speed setting 7) to separate the blood layers and the plasma layer. The separated plasma was inactivated in a 56°C water bath for 30 minutes to disrupt the complement system. The plasma was then transferred to a 4°C refrigerator and allowed to stand for 30 minutes to allow platelets to settle. Finally, the supernatant was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected for later use.

[0022] Resuspend the blood cell pellet in an appropriate amount of physiological saline and slowly add it to the top of the lymphocyte separation solution to form a clear layered interface. Centrifuge at 2000 rpm for 20 minutes (speed setting 9, speed setting 0). After centrifugation, the liquid in the tube will separate into four layers: from top to bottom, the plasma layer, the mononuclear cell layer (buffy coat layer), the lymphocyte separation solution layer, and the red blood cell and granulocyte layer. Carefully aspirate the buffy coat layer to obtain the peripheral blood mononuclear cells (PBMCs).

[0023] Magnetic bead sorting of CD4+ T cells Take the separated PBMCs and count them every 10 7 Resuspend the monocytes in 100 μL of PBS buffer containing 0.5% human albumin in a centrifuge tube, add 10 μL of CytoSinctCD magnetic beads, mix gently, and incubate at 2-8°C for 15 minutes to allow the magnetic beads to fully bind to the CD4+ T cells.

[0024] Mount the magnetic separation column on the magnetic field stand and rinse with 500 μL of buffer to remove impurities. Slowly add the incubated cell suspension to the magnetic separation column. Once the cell suspension has completely passed through the column, rinse the column three times with 500 μL of buffer to remove cells that have not bound to the magnetic beads. Finally, quickly flush the magnetic separation column with 1 mL of buffer to collect CD4+ T cells bound to the magnetic beads.

[0025] Gradient amplification culture of CD4+ T cells Initial culture: Add GIBCO complete medium containing anti-CD3 / anti-CD28 antibodies, inactivated plasma from the peripheral blood mononuclear cell isolation step (10% of the culture medium volume), and interleukin-2 (final concentration of 50 IU / mL) to a T75 culture flask. Inoculate the sorted CD4+ T cells into the culture flask and culture in a 37°C, 5% CO2 incubator. During the culture process, regularly tap the culture flask daily to disperse the cells and prevent cell aggregation.

[0026] Culture volume expansion: On the 5th day of culture, transfer the cells to a T175 culture flask and add GIBCO complete culture medium containing 10% inactivated plasma to a total volume of 60 mL. On the 8th day of culture, transfer the cells to a 2L culture bag and add culture medium and plasma step by step to finally bring the culture volume to 1800 mL.

[0027] Medium replacement: Use the aforementioned GIBCO medium system for the first 10 days. From day 11 onward, replace the medium with Echocell T cell culture medium and continue culturing until day 14. On day 10, remove 6 mL of the cell suspension for sterility testing to ensure that there is no bacterial or fungal contamination during the culture process.

[0028] Cell collection and testing After the 14th day of culture, the cell suspension was transferred to a centrifuge tube and centrifuged three times at 2000 rpm for 7 minutes. The cells were resuspended by tapping with the palm of the hand after each centrifugation.

[0029] After three centrifugations, cells were combined and washed twice with saline to remove residual culture medium. Finally, NKT4 solution (containing 5% DMSO, 10% human serum albumin, and 1 mM N-acetylcysteine) was added and the cells were transferred to a blood bag (CD4+ T cell purity ≥ 95%).

[0030] Detection of CD3 by flow cytometry + CD4 + The cell purity was 96.2% and the CD4 / CD8 ratio was 25.1, both meeting the requirements of the present invention.

[0031] The training process also includes: (1) If the blood cannot be processed immediately, store it at 10°C and return it to room temperature half an hour before the experiment; (2) Resuspend at most 10 μL of buffer 8 monocytes; (3) Detection of CD3 by flow cytometry + CD4 + The cell purity was ≥95.81%, and the CD4 / CD8 ratio was ≥24.19.

[0032] The data are as follows: Table 1 is the cell separation and sorting parameters table Steps Key parameters and results Whole blood processing Collection volume: 50 mL; Centrifugation conditions: 1800 rpm × 15 min (9 for ascending and 7 for descending); Plasma separation volume: approximately 20 mL Plasma inactivation 56℃×30min→4℃×30min→3000rpm×5min, the supernatant is transparent without precipitation PBMC isolation <![CDATA[Volume of lymphocyte separation medium: 10 mL × 2 tubes; Centrifugation conditions: 2000 rpm × 20 min (rise 9 fall 0); Amount of cells in the buffy coat layer: 1.2×10 8 cells]]> CD4+ T cell sorting <![CDATA[Magnetic bead labeling conditions: 10 7 cells / 100 μL buffer + 10 μL magnetic beads, 2 - 8 °C × 15 min; Cell amount after sorting: 8.4×10 7 cells]]> Table 2 is the CD4+ T cell gradient amplification culture process parameter table Cultivation time Culture container Culture medium volume Cell density (cells / mL) Cumulative cell count Start T75 30mL <![CDATA[2.8×10 6 ]]> <![CDATA[8.4×10 7 ]]> Day 5 T175 60mL <![CDATA[4.2×10 6 ]]> <![CDATA[2.5×10 8 ]]> Day 8 2L culture bag 500mL <![CDATA[1.6×10 6 ]]> <![CDATA[8.0×10 8 ]]> Day 14 2L culture bag 1800mL <![CDATA[9.6×10 7 ]]> <![CDATA[1.722×10 11 (Amplified 2050-fold)]]> Table 3 is the quality test results of CD4+ T cell culture finished products

[0033] Example 2

[0034] Validation experiments with different sample sources Peripheral blood samples from tumor patients were selected and cultured with high purity CD4+ T cells (CD4+ T cell purity ≥ 95%) according to the method of Example 1. During the entire culture process, all parameters and operation steps were strictly controlled. Finally, CD3 + CD4 + The cell purity was 95.8%, the CD4 / CD8 ratio was 24.5, the cell expansion multiple reached 2050 times, and the cell activity maintenance rate was 95.5%. The experimental results show that the culture optimization process of the present invention is also applicable to tumor patient samples, and can obtain high-purity, high-activity, and well-expanded CD4+ T cells.

[0035] The data are as follows: Table 4 is the cell separation and sorting parameter table Steps Key parameters and results Whole blood processing Collection volume: 45 mL; Centrifugation conditions: 1800 rpm × 15 min; Plasma separation volume: approximately 18 mL PBMC isolation <![CDATA[Amount of cells in the tunica albuginea layer: 1.1×10 8 cells]]> CD4+ T cell sorting <![CDATA[Cell quantity after sorting: 7.7×10 7 cells]]> Table 5 is the CD4+ T cell gradient amplification culture process parameter table Test items result <![CDATA[CD3 + CD4 + Cell purity]]> 95.8% CD4 / CD8 ratio 24.5 Cell expansion multiples 2050 times Cell viability 95.5% Table 6 is the quality test results of the cell culture products of Example 1 and Example 2 index Example 1 (Healthy Sample) Example 2 (Tumor Sample) Traditional methods CD4+ T cell purity 96.2% 95.8% ≤70% Amplification factor 2050 times 2050 times ≤150 times Cell viability 95.8% 95.5% ≤85% CD4 / CD8 ratio 25.1 24.5 ≤10 In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of this application, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0036] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0037] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A high-purity CD4+ T cell culture optimization process, characterized in that: The following steps are involved: Step 1: Isolation of peripheral blood mononuclear cells; Step 2: Obtain CD4+ T cells by magnetic bead sorting; Step 3: CD4+ T cell expansion using a gradient amplification culture system; Step 4: Collect and test cells through multiple rounds of centrifugation to obtain CD4+ T cells with a purity of ≥95%; The magnetic bead sorting uses CytoSinctCD magnetic beads, the buffer is PBS containing 0.5% human serum albumin, and the culture process adopts a step-by-step replacement strategy of GIBCO culture medium and Ecosai T cell culture medium.

2. The cultivation optimization process according to claim 1, characterized in that The step of separating peripheral blood mononuclear cells comprises: (1) Whole blood was centrifuged at 1800 rpm for 15 minutes, the saturation was increased by 9 and decreased by 7, and the plasma was separated and inactivated at 56°C for 30 minutes, allowed to stand at 4°C for 30 minutes, and centrifuged at 3000 rpm for 5 minutes; (2) After the blood cell pellet is resuspended with physiological saline, it is added to the lymphocyte separation solution and centrifuged at 2000 rpm for 20 minutes, with the saturation increased to 9 and the saturation decreased to 0, to collect the peripheral blood mononuclear cells.

3. The cultivation optimization process according to claim 1, characterized in that The step of magnetic bead sorting CD4+T cells comprises: (1) Every 10 7 Resuspend the mononuclear cells in 100 μL buffer, add 10 μL CytoSinctCD magnetic beads, and incubate at 2-8°C for 15 minutes; (2) When processing the magnetic separation column, first empty it with 500 μL of buffer, then add the cell suspension, wash it three times with 500 μL of buffer, and then quickly punch it with 1 mL of buffer to collect CD4+ T cells.

4. The cultivation optimization process according to claim 1, characterized in that The gradient amplification culture system comprises: (1) Initially, add GIBCO complete culture medium containing anti-CD3 / anti-CD28 antibodies, inactivated plasma, and interleukin-2 to the T75 culture flask; (2) Transfer to T175 culture flasks and 2L culture bags in sequence, add culture medium and plasma step by step, and gradually expand the culture volume from 30mL to 1800mL; (3) During the culture process, the cells were dispersed by tapping the culture flask. On the 10th day, 6 mL of cell suspension was collected for sterility testing.

5. The cultivation optimization process according to claim 1, characterized in that: The cell collection step comprises: (1) Centrifuge at 2000 rpm for 7 minutes three times, and resuspend the cells by tapping after each centrifugation; (2) After merging the cells, wash them twice with physiological saline, finally add NKT4 solution and transfer them to a blood bag.

6. The cultivation optimization process according to any one of claims 1 to 5, characterized in that: The culture process also includes: (1) If the blood cannot be processed immediately, store it at 10°C and return it to room temperature half an hour before the experiment; (2) Resuspend at most 10 μL of buffer 8 monocytes; (3) Detection of CD3 by flow cytometry + CD4 + The cell purity was ≥95.81%, and the CD4 / CD8 ratio was ≥24.

19.

7. The cultivation optimization process according to claim 2, characterized in that: After the plasma inactivation treatment, platelets were removed by centrifugation at 3000 rpm to reduce the impact on cell activation. This treatment increased the subsequent CD4+ T cell expansion efficiency by more than 30%.

8. The cultivation optimization process according to claim 3, characterized in that: Magnetic bead sorting uses low-temperature incubation at 2-8°C, combined with 300g centrifugation for washing.

9. The cultivation optimization process according to claim 4, characterized in that: The culture medium was replaced with: GIBCO culture medium containing 10% inactivated plasma was used for the first 10 days, and replaced with Icosai T cell culture medium after the 11th day. This strategy enabled the cell expansion to reach more than 2,000 times, which was 15 times higher than that of a single culture medium.

10. An application of the culture optimization process according to any one of claims 1 to 5, characterized in that: Highly purified CD4+ T cells are used to prepare tumor immunotherapy drugs or autologous cell transfusion preparations.

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