Method for amplifying and purifying CD8 + T cells, culture medium and application of culture medium

Abstract

The invention discloses a method for amplifying and purifying CD8 < + > T cells, a culture medium and application of the culture medium, and relates to the technical field of cell purification, the culture medium for amplifying and purifying the CD8 < + > T cells comprises the following components: 5-25ng / mL of interleukin-2, 1mM of sodium pyruvate, 2mM of L-alanyl-L-glutamine dipeptide, 55mu M of 2-mercaptoethanol and the balance of water. The culture medium comprises 50-400ng / mL of anti-CD3 antibody, a basic culture medium, serum and antibiotics. The culture medium provided by the invention can effectively activate and proliferate CD8 < + > T cells, but cannot effectively promote proliferation and activation of CD4 < + > T cells and other cells, thereby playing a role in purifying the CD8 < + > T cells. The CD8 + T cells can be amplified and purified by incubating the mouse lymph node cells with the culture medium, the operation is simple and convenient, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the technical field of cell purification, in particular to amplifying and purifying CD8 + T cell methods, culture media and their applications. Background Art

[0002] CD8 + T cells play a vital role in the body's surveillance of cell mutations and pathogen infection. In-depth research on their activation and response mechanisms will help us better understand how the immune system recognizes and eliminates abnormal cells. + T cells are crucial for clearing viruses and certain bacteria, and understanding their response mechanisms in different pathogen infections can not only drive vaccine design but also optimize infection control strategies. + T cells may also participate in the attack on autologous cells. Exploring their role in these diseases can reveal the underlying pathological mechanisms and provide important clues for the development of new treatment options. + T cells play a key role in anti-tumor immunity. Their function directly affects the effect of cancer immunotherapy. Focusing on their role in the tumor microenvironment can lay the foundation for the innovation of immunotherapy strategies, including tumor vaccines, immune checkpoint inhibitors and even CAR-T cell therapy (i.e., chimeric antigen receptor T cell therapy). + The anti-tumor activity of T cells can significantly improve the patient's treatment response. + T cells are widely used and have important research value. Currently, many research teams around the world are focusing on CD8 + T cell research.

[0003] Currently, CD8 + The separation and purification of T cells mainly rely on two technologies: magnetic bead sorting and flow cytometry sorting. However, these methods have obvious limitations. Although the magnetic bead sorting technology is relatively simple to operate, the magnetic microbeads used in its positive selection method may change the expression of T cell surface markers or affect the functional state of cells, and the problem of residual microbeads may interfere with subsequent experimental results; although the negative selection method can better maintain cell activity, its separation efficiency is low and the cost of single separation is high. Although flow cytometry sorting technology can obtain high-purity and high-activity CD8 + T cells, but the equipment investment is large, the maintenance cost is high, and the operation process is complicated, requiring professional operation. More importantly, during the sorting process, the cells may be affected by mechanical shearing and liquid flow pressure and in vitro operation time, resulting in decreased cell activity. It is worth noting that both methods have the problem of high operating costs. For some CD8 +T cell purity requirements are high but simple experiments (such as CD8 + For genetic testing of T cell-specific gene knockout or knock-in, existing methods are too expensive and complicated.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention aims to provide a method for amplifying and purifying CD8 + T cell methods, culture media and their applications are intended to solve the existing CD8 + The purification method of T cells is costly and complex to operate.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect of the present invention, there is provided a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The T cell expansion and purification medium contains the following components:

[0008] 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, basic culture medium, serum and antibiotics.

[0009] In a second aspect of the present invention, there is provided a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The T cell expansion and purification medium contains the following components:

[0010] 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

[0011] The third aspect of the present invention provides a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The culture medium for expanding and purifying T cells includes a first culture medium and a second culture medium;

[0012] The first culture medium includes the following components:

[0013] 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, and basal culture medium, serum, and antibiotics;

[0014] The second culture medium includes the following components:

[0015] 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

[0016] Optionally, the basal culture medium is RPMI-1640 culture medium.

[0017] Optionally, the antibiotics include penicillin and streptomycin, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 0.1 mg / mL;

[0018] and / or,

[0019] The volume of serum is the same as that used for CD8 + 9% to 12% of the volume of the culture medium for T cell expansion and purification.

[0020] The fourth aspect of the present invention provides a method for CD8 + T cell expansion and purification medium in purified CD8 + Application in T cells.

[0021] The fifth aspect of the present invention provides a method for amplifying and purifying CD8 + The method of producing T cells comprises the following steps:

[0022] provided mouse lymph node cells;

[0023] The mouse lymph node cells were incubated with culture medium to obtain CD8 + T cells;

[0024] The culture medium is the one for CD8 + T cell expansion and purification medium.

[0025] Optionally, the mouse lymph node cells include mouse inguinal lymph node cells and / or mouse axillary lymph node cells.

[0026] Optionally, when the mouse lymph node cells are incubated with the culture medium, the initial concentration of the mouse lymph node cells is 1×10 5 ~1×10 6 cells / ml.

[0027] Optionally, the culture medium is the one described above for CD8 + The culture medium for T cell expansion and purification was used to incubate the mouse lymph node cells to obtain CD8 + The T cell steps specifically include:

[0028] The mouse lymph node cells were incubated in the first culture medium for several days, and the initial concentration of the mouse lymph node cells was 1×10 5 ~1×10 6 cells / ml;

[0029] Then, the first culture medium was replaced with the second culture medium, and the incubation was continued for several days. During the incubation process, the second culture medium was replaced every day, and the initial concentration of mouse lymph node cells after the replacement of the second culture medium was 1.0×10 5 ~5.0×10 5 cells / ml, CD8 + T cells.

[0030] Beneficial effect: The culture medium provided by the present invention can make CD8 + T cells are effectively activated and proliferated, but CD4 + Proliferation and activation of T cells and other cells, thereby purifying CD8 + The present invention utilizes the culture medium to incubate mouse lymph node cells to achieve CD8 + The expansion and purification of T cells are simple to operate, low-cost, and have application prospects in scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For T cells, CD4 + T cells and CD8 + Flow cytometry of the proportion of T cells.

[0032] Figure 2 CD8 in Example 1 + Statistical graph of the initial and final proportions of T cells to total cells.

[0033] Figure 3 For T cells, CD4 + T cells and CD8 + Flow cytometry of the proportion of T cells.

[0034] Figure 4 CD8 in Example 2 + Statistical graph of the initial and final proportions of T cells to total cells.

[0035] Figure 5For T cells, CD4 + T cells and CD8 + Flow cytometry of the proportion of T cells.

[0036] Figure 6 CD8 in Example 3 + Statistical graph of the initial and final proportions of T cells to total cells.

[0037] Figure 7 CD8 in Example 3 + Statistical graph of the proliferation fold of T cells relative to the initial cell number at different time points.

[0038] Figure 8 CD8 in Example 3 + Flow cytometry of T cell apoptosis and statistical diagrams of each cell state, where (a) is the flow cytometry diagram and (b) is the statistical diagram of each cell state. DETAILED DESCRIPTION

[0039] The present invention provides amplified and purified CD8 + In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0040] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0041] If there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features.

[0042] The embodiment of the present invention provides a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The T cell expansion and purification medium contains the following components:

[0043] 5-25 ng / mL interleukin-2 (IL-2), 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, and basic culture medium, serum, and antibiotics.

[0044] The present invention can adjust the factors of the culture medium for T cell in vitro culture to make CD8 + T cells are effectively activated and proliferated, but CD4 + Proliferation and activation of T cells and other cells, thereby purifying CD8 + The present invention is the first to use culture medium to culture (or incubate) mouse lymph node cells to achieve CD8 + The expansion and purification of T cells are simple to operate, low-cost, and have application prospects in scientific research.

[0045] In the present embodiment, the role of anti-CD3 antibody and IL-2 is to act as cytokines to promote T cell activation and proliferation. The role of sodium pyruvate is to provide energy to support cell growth and proliferation. The role of L-alanyl-L-glutamine dipeptide is to provide amino acids to promote cell growth and proliferation. 2-mercaptoethanol is a reducing agent that removes a large amount of ROS (reactive oxygen species) produced during the rapid proliferation of T cells and reduces the damage of oxidative stress to cells. The role of serum is mainly to provide growth factors. The role of antibiotics is antibacterial.

[0046] The inventors found through research that although anti-CD28 antibodies can also stimulate CD8 + T cell proliferation, but it can also stimulate CD4 + T cells, CD8 + For the purification of T cells, only anti-CD3 antibody is added in the present invention.

[0047] In this embodiment, the concentration of IL-2 can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL or any value in the numerical range consisting of any two of the above values.

[0048] The concentration of the anti-CD3 antibody can be 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 320 ng / mL, 350 ng / mL, 380 ng / mL, 400 ng / mL, or any value in a range consisting of any two of the foregoing values.

[0049] The embodiment of the present invention also provides a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The T cell expansion and purification medium contains the following components:

[0050] 5-25 ng / mL IL-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

[0051] The embodiment of the present invention also provides a method for CD8 + T cell expansion and purification medium, wherein the CD8 + The culture medium for expanding and purifying T cells includes a first culture medium and a second culture medium;

[0052] The first culture medium includes the following components:

[0053] 5-25 ng / mL IL-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, and basal culture medium, serum, and antibiotics;

[0054] The second culture medium includes the following components:

[0055] 5-25 ng / mL IL-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

[0056] In this embodiment, the first culture medium and the second culture medium are combined for CD8 + The expansion and purification of T cells has a better purification effect and can obtain CD8 T cells with a purity of more than 95%. + T cells.

[0057] In this embodiment, the concentration of IL-2 can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL or any value in the numerical range consisting of any two of the above values.

[0058] The concentration of the anti-CD3 antibody can be 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 320 ng / mL, 350 ng / mL, 380 ng / mL, 400 ng / mL, or any value in a range consisting of any two of the foregoing values.

[0059] In some embodiments, the basal culture medium is RPMI-1640 medium.

[0060] In some embodiments, the antibiotics include penicillin and streptomycin, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 0.1 mg / mL.

[0061] In some embodiments, the volume of serum is the volume of serum used for CD8 + 9% to 12% of the volume of the culture medium for T cell expansion and purification.

[0062] In some embodiments, the serum is animal serum, and in some embodiments, the animal serum is fetal bovine serum.

[0063] The embodiment of the present invention further provides a method for CD8 + T cell expansion and purification medium in purified CD8 + Application in T cells.

[0064] The present invention also provides a method for amplifying and purifying CD8 + T cell method (which is also a method of isolating, expanding, and purifying CD8 T cells from mouse lymph nodes + T cell method), comprising the steps of:

[0065] S1, provide mouse lymph node cells;

[0066] S2, incubate the mouse lymph node cells with culture medium to obtain CD8 + T cells;

[0067] The culture medium is the one used for CD8 + T cell expansion and purification medium.

[0068] In the embodiment of the present invention, the mouse lymph node cells are incubated with culture medium, which can make CD8 + T cells are effectively activated and proliferated, but CD4 + Proliferation and survival of T cells and other cells, thereby purifying CD8+ The present invention utilizes the culture medium to incubate mouse lymph node cells to achieve CD8 + The expansion and purification of T cells is simple to operate and low in cost. It has application prospects in scientific research and is comparable to the currently commonly used magnetic bead sorting and flow cytometry purification of CD8 + Compared with the method of T cells, the present invention utilizes the + CD8 T cell expansion and purification culture medium was used to incubate mouse lymph node cells. + The purification of T cells is simple to operate, low-cost, and has application prospects in scientific research.

[0069] In step S1, in some embodiments, the mouse lymph node cells include mouse inguinal lymph node cells and / or mouse axillary lymph node cells.

[0070] In step S2, when the mouse lymph node cells are incubated with the culture medium, the initial concentration of the mouse lymph node cells is 1×10 5 ~1×10 6 cells / ml. This allows for better purification results. Because T cells proliferate rapidly, excessive concentrations can lead to excessive T cell density and apoptosis.

[0071] In some embodiments, the mouse lymph node cells are incubated with culture medium to obtain CD8 + The T cell steps specifically include:

[0072] S21, incubate the mouse lymph node cells in the first culture medium for several days, with the initial concentration of the mouse lymph node cells being 1×10 5 ~1×10 6 cells / ml;

[0073] S22, then replace the first culture medium with the second culture medium, continue incubation for several days, and replace the second culture medium every day during the incubation process, so that the initial concentration of mouse lymph node cells after replacing the second culture medium every day is 1.0×10 5 ~5.0×10 5 cells / ml, CD8 + T cells.

[0074] The present inventors have found through research that when only the first culture medium is used to incubate mouse lymph node cells, although the number of cells is greater, the CD8 + The proportion of T cells could not meet the requirement of being higher than 95% in all cultures on day 6. Therefore, in this embodiment, the mouse lymph node cells were first incubated with the first culture medium for several days, and then replaced with the second culture medium for further incubation, so as to increase the CD8 +The purity of T cells can achieve better purification effect.

[0075] In a specific embodiment, the mouse lymph node cells are incubated with culture medium to obtain CD8 + The T cell steps specifically include:

[0076] S21, incubate the mouse lymph node cells with the first culture medium for 2 days, the initial concentration of the mouse lymph node cells is 1×10 5 ~1×10 6 cells / ml (because T cells proliferate very quickly, if the concentration is too high, it will lead to excessive density of T cells and cause apoptosis, so ensure that the initial concentration of mouse lymph node cells is 1.0×10 5 ~1×10 6 cells / ml);

[0077] S22. From the end of the second day of incubation, the culture medium was replaced with the second culture medium every day and the incubation was continued for 4 days, and the initial concentration of mouse lymph node cells after replacing the new second culture medium every day was 1.0×10 5 ~5.0×10 5 cells / ml (because T cells proliferate very quickly, if the concentration is too high, it will lead to excessive density of T cells and cause apoptosis, so make sure that the initial concentration of mouse lymph node cells is 1.0×10 5 ~5.0×10 5 cells / ml), CD8 + T cells.

[0078] The present invention will be further described below with reference to specific examples.

[0079] In the following examples and comparative examples: RPMI-1640 culture medium (item number MA0215) was purchased from Dalian Meilun Biotechnology Co., Ltd.; IL-2 (item number 200-02-50UG) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; anti-CD3 antibody (item number 100340) was purchased from Biolegend; 2-mercaptoethanol (item number 21985023), L-alanyl-L-glutamine dipeptide (GlutaMAX) (item number 35050061), and sodium pyruvate solution (item number 11360070) were all purchased from Thermo Fisher Scientific (China) Co., Ltd.; fetal bovine serum (item number BC-SE-FBS07) was purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; streptomycin / penicillin solution (item number MA0110-1) was purchased from Meilun Biotechnology Co., Ltd.; flow cytometry antibody (PE anti-mouse CD4 Antibody (abbreviated as PE-CD4, Catalog No. 100512), APC / Cyanine7 anti-mouse CD8a Antibody (abbreviated as APC-CY7-CD8, Catalog No. 100713), APC anti-mouse CD45 Antibody (abbreviated as APC-CD45, Catalog No. 103112), Alexa AF700 anti-mouse CD90.2 (abbreviated as AF700-CD90 antibody, catalog number 105320) was purchased from Biolegend.

[0080] Example 1

[0081] In this example, mice were 12-week-old female BALB / c mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0082] This embodiment provides a method for CD8 + a medium for expanding and purifying T cells, comprising a first culture medium and a second culture medium;

[0083] The first culture medium includes the following components:

[0084] 5 ng / mL IL-2, 400 ng / mL anti-CD3 antibody, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0085] The second culture medium includes the following components:

[0086] 5 ng / mL IL-2, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% by volume of fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0087] This embodiment provides a method for amplifying and purifying CD8 + The T cell method comprises the following steps:

[0088] (1) Providing a treatment solution comprising the following components:

[0089] 10% fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the treatment solution), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 culture medium.

[0090] (2) According to ethical standards, mice were euthanized and then disinfected by immersing in 75% alcohol for 10 min.

[0091] (3) Place the sterilized mouse in a sterile tray and transfer it to a clean bench. First, use a set of scissors and tweezers to cut the fur. Use another pair of sterile tweezers to remove the mouse lymph nodes (i.e., 6 inguinal lymph nodes and axillary lymph nodes in total) and place them in the above-mentioned treatment solution.

[0092] (4) Place the mouse lymph nodes in the treatment solution on a 70 μm cell strainer and press and grind them with a 1 mL syringe piston handle until there is no tissue structure. Collect the cell suspension and filter it with a 40 μm cell strainer into a 15 mL centrifuge tube. Centrifuge at 500 g for 5 minutes at 4°C.

[0093] (5) Discard the supernatant, resuspend the lymph node cells in the first culture medium, and count them.

[0094] (6) Adjust the lymph node cell concentration to 5×10 5 cells / ml to obtain a cell suspension resuspended in the first culture medium.

[0095] (7) Pipette 1 mL of the cell suspension resuspended in the first culture medium into a 24-well plate, which is the initial state (i.e., culture day 0).

[0096] (8) Place the cell culture plate in a 37°C, 5% CO2 incubator for culture.

[0097] (9) At the end of the second day of culture (i.e., 2 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, and add all the cells to a new 24-well plate (cell concentration is 1.0 × 10 5 ~5.0×10 5 The cells were in the range of 10 cells / ml, so all were added to a new well plate. The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0098] (10) At the end of the third day of culture (i.e., 3 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count, take 500 μL of the cell suspension into a new 24-well plate, and add 500 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0099] (11) At the end of the fourth day of culture (i.e., 4 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, take 160 μL of the cell suspension into a new 24-well plate, and add 840 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0100] (12) At the end of the fifth day of culture (i.e., 5 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, take 100 μL of the cell suspension into a new 24-well plate, and add 900 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture. At the end of the sixth day of culture (i.e., after 6 days of culture), relevant tests were performed.

[0101] test:

[0102] At the end of culture day 0 (initial state) and day 6 (end state), 1 mL of cell suspension was drawn into a 1.5 mL EP tube. Centrifuged at 350 g for 5 minutes at 4°C. Discard the supernatant, add 100 μL of PBS (phosphate buffered saline), and 1 μL each of PE-CD4, APC-CY7-CD8, APC-CD45, and AF700-CD90 antibodies, and incubate at 4°C for 30 minutes. Add 1 mL of PBS and centrifuge at 350 g for 5 minutes at 4°C. Discard the supernatant, add 200 μL of PBS, and use flow cytometry to detect the ratio of CD45, CD90, CD4, and CD8. The results are as follows. Figure 1 As shown, and according to the detected T cells, CD4 + T cells, CD8 + T cell ratio (such as Figure 1 As shown) draw CD8 + The statistical chart of the proportion of T cells to total cells is as follows Figure 2 As shown. Figure 1 and Figure 2 It can be seen that after the lymph node cell culture is completed using the method provided by the present invention, CD8 + The proportion of T cells was 96.14%, indicating that the method provided by the present invention played a role in purifying CD8 + The role of T cells, and the acquisition of CD8 + T cell purity reached CD8 + T cell purity.

[0103] Example 2

[0104] In this example, the mice were 6-week-old C57 male mice (small black mice) purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0105] This embodiment provides a method for CD8 + a medium for expanding and purifying T cells, comprising a first culture medium and a second culture medium;

[0106] The first culture medium includes the following components:

[0107] 10 ng / mL IL-2, 100 ng / mL anti-CD3 antibody, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0108] The second culture medium includes the following components:

[0109] 10 ng / mL IL-2, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% by volume of fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0110] This embodiment provides a method for amplifying and purifying CD8 + The T cell method comprises the following steps:

[0111] (1) Provide a treatment solution, wherein the components and contents of the treatment solution are the same as those in Example 1.

[0112] (2) According to ethical standards, mice were euthanized and then disinfected by immersing in 75% alcohol for 10 min.

[0113] (3) Place the sterilized mouse in a sterile tray and transfer it to a clean bench. First, use a set of scissors and tweezers to cut the fur. Use another pair of sterile tweezers to remove the mouse lymph nodes (i.e., 6 inguinal lymph nodes and axillary lymph nodes in total) and place them in the above-mentioned treatment solution.

[0114] (4) Place the mouse lymph nodes in the treatment solution on a 70 μm cell strainer and grind them with a 1 mL syringe plunger until there is no tissue structure. Collect the cell suspension and filter it through a 40 μm cell strainer into a 15 mL centrifuge tube. Centrifuge at 500 g for 5 min at 4°C.

[0115] (5) Discard the supernatant, resuspend the lymph node cells in the first culture medium, and count them.

[0116] (6) Adjust the lymphocyte concentration to 1×10 5 cells / ml to obtain a cell suspension resuspended in the first culture medium.

[0117] (7) Pipette 1 mL of the cell suspension resuspended in the first culture medium into a 24-well plate, which is the initial state (i.e., culture day 0).

[0118] (8) Place the cell culture plate in a 37°C, 5% CO2 incubator for culture.

[0119] (9) At the end of the second day of culture (i.e., 2 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, and add all the cells to a new 24-well plate (cell concentration is 1.0 × 10 5 ~5.0×10 5The cells were in the range of 10 cells / ml, so all were added to a new well plate. The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0120] (10) At the end of the third day of culture (i.e., 3 days of culture), the cells were removed from the incubator and the cell suspension was pipetted into a 1.5 mL EP tube on a sterile operating table. The suspension was centrifuged at 160 g for 5 min at 4°C. The supernatant was discarded, 1 mL of the second culture medium was added, the cells were counted, and all the cells were added to a new 24-well plate (cell concentration was 1.0 × 10 5 ~5.0×10 5 The cells were in the range of 10 cells / ml, so all were added to a new well plate. The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0121] (11) At the end of the fourth day of culture (i.e., 4 days of culture), the cells were removed from the incubator and the cell suspension was pipetted into a 1.5 mL EP tube on a sterile operating table. The suspension was centrifuged at 160 g for 5 min at 4°C. The supernatant was discarded, 1 mL of the second culture medium was added, the cells were counted, and all the cells were added to a new 24-well plate (cell concentration was 1.0 × 10 5 ~5.0×10 5 The cells were in the range of 10 cells / ml, so all were added to a new well plate. The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0122] (12) At the end of the fifth day of culture (i.e., 5 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube on a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count, take 150 μL of the cell suspension into a new 24-well plate, and then add 850 μL of T cell culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture. At the end of the sixth day of culture (i.e., after 6 days of culture), relevant tests were performed.

[0123] To test:

[0124] At the end of culture day 0 (initial state) and day 6 (end state), draw 1 mL of cell suspension into a 1.5 mL EP tube. Centrifuge at 350 g for 5 minutes at 4°C. Discard the supernatant, add 100 μL of PBS, and 1 μL each of PE-CD4, APC-CY7-CD8, APC-CD45, and AF700-CD90 antibodies, and incubate at 4°C for 30 minutes. Add 1 mL of PBS, centrifuge at 350 g for 5 minutes at 4°C. Discard the supernatant, add 200 μL of PBS, and use flow cytometry to detect the ratio of CD45, CD90, CD4, and CD8. The results are as follows. Figure 3 As shown, and according to the detected T cells, CD4 + T cells, CD8 + T cell ratio (such as Figure 3 As shown), draw CD8 + The statistical chart of the proportion of T cells to total cells is as follows Figure 4 As shown. Figure 3 and Figure 4 It can be seen that after the lymph node cell culture is completed using the method of the present invention, CD8 + The proportion of T cells was 96.50%, indicating that the method provided by the present invention played a role in purifying CD8 + The role of T cells, and the acquisition of CD8 + T cell purity reached CD8 + T cell purity.

[0125] Example 3

[0126] In this example, mice were 10-week-old female C57 mice (small black mice) purchased from Guangdong Yaokang Biotechnology Co., Ltd.; Annexin V binding buffer was purchased from Biolegend, and FITC Annexin V Apoptosis Detection Kit with 7-AAD was purchased from Biolegend (Cat. No. 640922).

[0127] This embodiment provides a method for CD8 + a medium for expanding and purifying T cells, comprising a first culture medium and a second culture medium;

[0128] The first culture medium includes the following components:

[0129] 10 ng / mL IL-2, 200 ng / mL anti-CD3 antibody, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0130] The second culture medium includes the following components:

[0131] 10 ng / mL IL-2, 55 μM 2-mercaptoethanol, 1 mM pyruvate, 2 mM GlutaMAX, 10% by volume of fetal bovine serum (the volume of fetal bovine serum accounts for 10% of the total volume of the first culture medium), 100 U / mL penicillin, 0.1 mg / mL streptomycin and RPMI-1640 medium.

[0132] This embodiment provides a method for amplifying and purifying CD8 + The T cell method comprises the following steps:

[0133] (1) Provide a treatment solution, wherein the components and contents of the treatment solution are the same as those in Example 1.

[0134] (2) According to ethical standards, mice were euthanized and then disinfected by immersing in 75% alcohol for 10 min.

[0135] (3) Place the sterilized mice in a sterile tray and transfer them to a clean bench. First, use a set of scissors and tweezers to cut the fur. Use another pair of sterile tweezers to remove the inguinal lymph nodes and axillary lymph nodes and place them in the treatment solution.

[0136] (4) Place the mouse lymph nodes in the treatment solution on a 70 μM cell strainer and grind them with a 1 mL syringe plunger until there is no tissue structure. Collect the cell suspension and filter it through a 40 μm cell strainer into a 15 mL centrifuge tube. Centrifuge at 500 g for 5 min at 4°C.

[0137] (5) Discard the supernatant, resuspend the lymph node cells in the first culture medium, and count them.

[0138] (6) Adjust the lymphocyte concentration to 1×10 6 cells / ml to obtain a cell suspension resuspended in the first culture medium.

[0139] (7) Pipette 1 mL of the cell suspension resuspended in the first culture medium into a 24-well plate, which is the initial state (i.e., culture day 0).

[0140] (8) Place the cell culture plate in a 37°C, 5% CO2 incubator for culture.

[0141] (9) At the end of the second day of culture (i.e., 2 days of culture), the cells were removed from the incubator and the cell suspension was pipetted into a 1.5 mL EP tube in a sterile operating table. The cell count was 1.06 × 10 6 cells / ml. Centrifuge at 160g for 5 min at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count, take 500 μL of the cell suspension and add it to a new 24-well plate, then add 500 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0142] (10) At the end of the third day of culture (i.e., 3 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube in a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, take 200 μL of the cell suspension and add it to a new 24-well plate, and then add 800 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0143] (11) At the end of the fourth day of culture (i.e., 4 days of culture), the cells were removed from the incubator and the cell suspension was pipetted into a 1.5 mL EP tube in a sterile operating table. The cell count was 1.7 × 10 6 Cells / ml were centrifuged at 160g for 5 min at 4°C. The supernatant was discarded, 1 mL of the second culture medium was added, and the cells were counted. 100 μL of the cell suspension was added to a new 24-well plate, and 900 μL of the second culture medium was added (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5 The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0144] (12) At the end of the fifth day of culture (i.e., 5 days of culture), remove the cells from the incubator and use a pipette to remove the cell suspension into a 1.5 mL EP tube in a sterile operating table. Centrifuge at 160 g for 5 minutes at 4°C. Discard the supernatant, add 1 mL of the second culture medium, count the cells, take 100 μL of the cell suspension and add it to a new 24-well plate, and then add 900 μL of the second culture medium (this ensures that the cell concentration is 1.0×10 5 ~5.0×10 5The cell culture plate was placed in a 37°C, 5% CO2 incubator for culture. At the end of the sixth day of culture (i.e., after 6 days of culture), the cell count was 3.84×10 5 cells / ml and perform relevant tests.

[0145] To test:

[0146] (1) At the end of culture day 0 (initial state) and day 6 (end state), draw 1 mL of cell suspension into a 1.5 mL EP tube. Centrifuge at 350 g for 5 min at 4 °C. Discard the supernatant, add 100 μL of PBS, and 1 μL each of PE-CD4, APC-CY7-CD8, APC-CD45, and AF700-CD90 antibodies, and incubate at 4 °C for 30 min. Add 1 mL of PBS, centrifuge at 350 g for 5 min at 4 °C. Discard the supernatant, add 200 μL of PBS, and use flow cytometry to detect the ratio of CD45, CD90, CD4, and CD8. The results are as follows. Figure 5 As shown, and based on T cells, CD4 + T cells, CD8 + T cell ratio (such as Figure 5 As shown), draw CD8 + The statistical chart of the proportion of T cells to total cells is as follows Figure 6 As shown. Figure 5 and 6 It can be seen that after the lymph node cell culture is completed using the method provided by the present invention, CD8 + The proportion of T cells was 95.19%, indicating that the method provided by the present invention played a role in purifying CD8 + The role of T cells, and the acquisition of CD8 + T cell purity reached CD8 + T cell purity.

[0147] (2) According to the cell counts at different days, the cell proliferation times were plotted. The results are as follows Figure 7 As shown, CD8 + T cells are able to proliferate efficiently.

[0148] (3) At the end of the 6th day, the cells were removed from the incubator and the cell suspension was removed by pipette into a 1.5 mL EP tube in a sterile operating table. Centrifuge at 350 g for 5 min at 4 °C. Discard the supernatant, add 1 mL of PBS, and centrifuge at 350 g for 5 min at 4 °C. Discard the supernatant, add 100 μL of Annexin V binding buffer, and 1 μL and 2 μL of FITC-Annexin V and percep-7AAD antibodies respectively, incubate at room temperature for 15 min, add 400 μL of Annexin V binding buffer, and detect apoptosis on the instrument. The results are as follows: Figure 8 As shown, CD8 + T cells are in good condition.

[0149] In summary, the present invention provides a method for amplifying and purifying CD8 + T cell method, culture medium and application thereof, the culture medium provided by the present invention can make CD8 + T cells are effectively activated and proliferated, but CD4 + Proliferation and activation of T cells and other cells, thereby purifying CD8 + The present invention realizes the function of CD8 T cells for the first time through the culture method. + The purification of T cells is simple to operate, low-cost, and has application prospects in scientific research.

[0150] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. For CD8 + A culture medium for T cell expansion and purification, characterized in that The use of CD8 + The T cell expansion and purification medium contains the following components: 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, basic culture medium, serum and antibiotics.

2. For CD8 + A culture medium for T cell expansion and purification, characterized in that The use of CD8 + The T cell expansion and purification medium contains the following components: 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

3. For CD8 + A culture medium for T cell expansion and purification, characterized in that The use of CD8 + The culture medium for expanding and purifying T cells includes a first culture medium and a second culture medium; The first culture medium includes the following components: 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, 50-400 ng / mL anti-CD3 antibody, and basal culture medium, serum, and antibiotics; The second culture medium includes the following components: 5-25 ng / mL interleukin-2, 1 mM sodium pyruvate, 2 mM L-alanyl-L-glutamine dipeptide, 55 μM 2-mercaptoethanol, as well as basic culture medium, serum and antibiotics.

4. The method for use in CD8 according to any one of claims 1 to 3. + A culture medium for T cell expansion and purification, characterized in that The basic culture medium is RPMI-1640 culture medium.

5. The method for use in CD8 according to any one of claims 1 to 3. + A culture medium for T cell expansion and purification, characterized in that The antibiotics include penicillin and streptomycin, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 0.1 mg / mL; and / or, The volume of serum is the same as that used for CD8 + 9% to 12% of the volume of the culture medium for T cell expansion and purification.

6. A method for CD8 according to any one of claims 1 to 5. + T cell expansion and purification medium in purified CD8 + Application in T cells.

7. Amplification and purification of CD8 + T cell method, characterized in that The steps include: provided mouse lymph node cells; The mouse lymph node cells were incubated with culture medium to obtain CD8 + T cells; The culture medium is the one for CD8 + T cell expansion and purification medium.

8. The method according to claim 7, characterized in that The mouse lymph node cells include mouse inguinal lymph node cells and / or mouse axillary lymph node cells.

9. The method according to claim 7, characterized in that When the mouse lymph node cells were incubated with the culture medium, the initial concentration of the mouse lymph node cells was 1×10 5 ~1×10 6 cells / ml.

10. The method according to claim 7, characterized in that The culture medium is the one for CD8 + The culture medium for T cell expansion and purification was used to incubate the mouse lymph node cells to obtain CD8 + The T cell steps specifically include: The mouse lymph node cells were incubated in the first culture medium for several days, and the initial concentration of the mouse lymph node cells was 1×10 5 ~1×10 6 cells / ml; Then, the first culture medium was replaced with the second culture medium, and the incubation was continued for several days. During the incubation process, the second culture medium was replaced every day, and the initial concentration of mouse lymph node cells after the replacement of the second culture medium was 1.0×10 5 ~5.0×10 5 cells / ml, CD8 + T cells.

Images