CD4 + T cell culture solution and culture method thereof

Abstract

The invention discloses a CD4 + T cell culture solution and a culture method of CD4 + T cells. The culture solution comprises a complete culture medium, and human AB serum with the final concentration of 0.1-10v / v%, Rh IL-2 with the final concentration of 50-1000IU / mL and fish oil with the final concentration of 10-50mu M are added into the complete culture medium. Omega-3 polyunsaturated fatty acids (EPA and DHA) contained in the fish oil component of the culture solution can inhibit excessive activation of cells, promote rapid amplification and growth of CD4 + T cells and increase production of anti-inflammatory factors (such as IL-4 and IL-10), so that the proinflammatory microenvironment is reduced, and immune balance is maintained; meanwhile, the CD4 + T cells cultured by the CD4 + T cell culture fluid are high in activity, and the anti-tumor immunity can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell culture technology, in particular to a CD4+ T cell culture medium and a method for culturing CD4+ T cells using the culture medium. BACKGROUND

[0002] T cells are key lymphocytes in the human immune system, which mature in the thymus and are responsible for recognizing and eliminating cells infected by pathogens or abnormal cells. According to the function, they can be divided into helper T cells (Thh), cytotoxic T cells (CTL), regulatory T cells (Treg), etc., which maintain immune balance by secreting cytokines or directly killing target cells.

[0003] T cell-mediated immunity is a self-adaptive process that develops antigen (Ag)-specific T lymphocytes to eliminate viral, bacterial, parasitic infections or malignant tumors.

[0004] CD4+ T cells (helper T cells) play an important coordinating role in the immune system, such as enhancing regulatory T cells (Treg) to maintain immune tolerance, releasing pro-inflammatory cytokines, and improving anti-tumor immunity. However, during the culture of CD4+ T cells, there are problems such as excessive proliferation of cells, resistance to apoptosis, and low cell activity. SUMMARY

[0005] Based on the above problems, the present application aims to provide a culture medium that can reduce CD4+ T cell apoptosis, release a large amount of pro-inflammatory cytokines, and has high cell activity.

[0006] The technical solution of the present application is as follows:

[0007] A CD4+ T cell culture medium, comprising a complete culture medium, wherein human AB serum with a final concentration of 0.1-10 v / v%, rhIL-2 with a final concentration of 50-1000 IU / mL, and fish oil with a final concentration of 10-50 μM are added to the complete culture medium.

[0008] Preferably, in an embodiment, the complete culture medium in the CD4+ T cell culture medium is X-Vivo 10 medium, RPMI-1640 medium, and GIBCO OpTmizer TM CTS TM T medium.

[0009] The present application also provides a culture method of CD4+ T cells, comprising the following steps:

[0010] On day 0, 1.0E+07 PBMCs are sorted, and the sorted cells are activated with magnetic beads for 2 days to obtain activated CD4+ T cells;

[0011] After the culture, the activated CD4+ T cells are collected and resuspended in the culture solution, and the cell suspension is transferred to a 24-well plate, and the CD4+ T cell culture solution is added, and the cell density is controlled to be 1.0E+06 cells / mL, and the plate is cultured in a 37°C, 5% CO2 incubator;

[0012] From day 5 to day 9, the CD4+ T cell culture solution is added every two days, and the culture is continued;

[0013] On day 11, the culture is completed, the cells and the culture solution are centrifuged, the supernatant is removed, and the cells are washed with PBS for 2-3 times to obtain high-purity CD4+ T cells.

[0014] Preferably, in the culture method, on day 0, the PBMC sorting and cell activation steps specifically include the following treatments:

[0015] First, the centrifuge tube is added with the sorting reagent antibody MIX, 1.0E+07 PBMCs, and 10 mL of the CD4+ T cell culture solution, and is placed in a 4°C refrigerator for 20 min;

[0016] After the freezing is completed, the centrifuge tube is taken out and CD4 negative selection magnetic beads are added, and the tube is placed at room temperature for 30 min

[0017] Then, the centrifuge tube is placed in a magnetic stand for 1 min, and the supernatant is aspirated to obtain a CD4+ T cell suspension;

[0018] After the standing is completed, the CD4+ T cell suspension is collected, CD3 / CD28 magnetic beads are added, and the mixture is activated and cultured at 37°C in a 5% CO2 incubator for 2 days to obtain activated CD4+ T cells.

[0019] Preferably, in the culture method, on day 3, the activated CD4+ T cells are collected before being transferred to a 24-well plate, and the following treatments are further included:

[0020] After the activation culture is completed, the activated CD4+ T cells are collected and transferred to a new centrifuge tube;

[0021] The new centrifuge tube is placed in a magnetic stand for 1 min, the supernatant is aspirated, and the cell precipitate is collected after centrifugation to obtain activated CD4+ T cells.

[0022] Preferably, in the culture method, the cell culture steps from day 5 to day 9 are as follows:

[0023] On day 5, the CD4+ T cell culture solution is supplemented and the cell density is controlled at 0.5E+06 cells / mL, and the culture is continued;

[0024] On day 7, the CD4+ T cell culture solution is supplemented and the cell density is controlled at 0.8E+06 cells / mL, and the culture is continued;

[0025] On day 9, the CD4+ T cell culture solution is supplemented and the cell density is controlled at 1.0E+06 cells / mL, and the culture is continued.

[0026] Preferably, in the culture method, on day 11, the centrifugation is 500g for 30 min.

[0027] The CD4+ T cell culture solution and culture method have the following advantages:

[0028] 1) In the fish oil component of the culture solution, the Omega-3 polyunsaturated fatty acids (EPA and DHA) can inhibit the overactivation of cells and promote the rapid expansion and growth of CD4+ T cells;

[0029] 2) Omega-3 polyunsaturated fatty acids (EPA and DHA) can reduce the production of pro-inflammatory factors (such as IFN-γ) and increase the production of anti-inflammatory factors (such as IL-4 and IL-10), thereby reducing the pro-inflammatory microenvironment and maintaining immune balance;

[0030] 3) The CD4+ T cells cultured using the CD4+ T cell culture solution have strong activity and can improve the anti-tumor immunity. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The CD4+ T cell growth curve on day 11 of the culture in Examples 1 to 6 is shown in the figure.

[0032] Figure 2 The CD4+ T cell purity flow cytometry graph on day 11 of the culture in Example 1 is shown in the figure.

[0033] Figure 3 The CD4+ T cell purity flow cytometry graph on day 11 of the culture in Example 2 is shown in the figure.

[0034] Figure 4 The CD4+ T cell purity flow cytometry graph on day 11 of the culture in Example 3 is shown in the figure.

[0035] Figure 5 The CD4+ T cell purity flow cytometry graph on day 11 of the culture in Example 4 is shown in the figure.

[0036] Figure 6Flow cytometry plot of CD4+ purity for day 11 CD4+ T cells cultured in Example 5;

[0037] Figure 7 Flow cytometry plot of CD4+ purity for day 11 CD4+ T cells cultured in Example 6;

[0038] Figure 8 Column chart of CD4+ T cell cytokine secretion concentration for day 11 cultured in Examples 1-6;

[0039] Figure 9 Graph of CD4+ T cell specific killing rate for day 11 cultured in Examples 1-6. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present application are further described in detail below with reference to the accompanying drawings.

[0041] The CD4+ T cell culture solution provided by the present application has the immune regulatory effect of Omega-3 polyunsaturated fatty acids (EPA and DHA) in the fish oil component on CD4+ T cells, specifically as follows:

[0042] 1) Promoting Th1 / Th2 balance

[0043] CD4+ T cells, also known as helper T cells, can be differentiated into different subgroups (such as Th1, Th2, Th17, Treg, etc.), and Omega-3 polyunsaturated fatty acids can regulate the differentiation direction of CD4+ T cells and affect immune responses, such as EPA and DHA can inhibit the overactivation of Th1 cells, reduce pro-inflammatory factors such as IFN-γ, while promoting the differentiation of Th2 cells and increasing anti-inflammatory factors (such as IL-4, IL-10), thereby maintaining immune balance;

[0044] 2) Enhancing Treg cell function

[0045] Omega-3 can promote the proliferation and functional expression of Treg cells, such as increasing IL-10 secretion, inhibiting autoimmune reactions and chronic inflammation;

[0046] 3) Anti-inflammatory effect and immunosuppression

[0047] Inhibiting the release of pro-inflammatory factors: excessive activation of CD4+ T cells can release a large amount of pro-inflammatory factors (such as TNF-α, IL-6), and Omega-3 can reduce the production of these pro-inflammatory factors by inhibiting the NF-κB signaling pathway, thereby alleviating inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease;

[0048] Reduce immune cell overreaction: In the infection or tumor microenvironment, Omega-3 can reduce the overproliferation and apoptosis resistance of CD4+ T cells, avoid damage to normal tissues by the immune system;

[0049] 4), tumor immunotherapy assistance

[0050] After the cancer patients supplement fish oil, the Omega-3 in fish oil can enhance CD4+ T cell activity, promote the generation of CD8+ T cells, improve CD4+ T cell-mediated immune homeostasis by restoring colonic microbiota and metabolic profile, thereby improving anti-tumor immunity.

[0051] Specifically, the CD4+ T cell culture solution of the present application is prepared as follows:

[0052] In the CD4+ T cell culture solution, based on the complete culture medium, 0.1-10 v / v% human AB serum, 50-1000 IU / mL rhIL-2 and 10-50 μM fish oil are added to the complete culture medium with a final concentration; wherein the complete culture medium is any one of X-Vivo 10, X-Vivo 15, RPMI-1640 and GIBCO OpTmizer TM CTS TM T medium.

[0053] In the present application, the complete culture medium is preferably X-Vivo 10, and the complete culture medium used in the present application is commercially available.

[0054] Preferably, in the CD4+ T cell culture solution, the complete culture medium contains 0.5-8 v / v% human AB serum, 100-800 IU / mL rhIL-2 and 215-40 μM fish oil with a final concentration; or, the complete culture medium contains 1-5 v / v% human AB serum, 200-600 IU / mL rhIL-2 and 20-35 μM fish oil with a final concentration, etc.

[0055] When the CD4+ T cell culture solution prepared as described above is used to culture CD4+ T cells, the obtained CD4+ T cells have the performance of reducing pro-inflammatory microenvironment, maintaining immune balance, and thus improving cellular immunity.

[0056] Therefore, the present application also provides a method for culturing CD4+ T cells using the above-mentioned CD4+ T cell culture solution, and the specific steps are as follows:

[0057] On day 0, first, a 50 mL size No. 1 centrifuge tube (the number of No. 1 centrifuge tubes can be 1 or more than 2 according to the culture requirement) was taken, and the antibody MIX in the commercially available kit, 10 mL of the prepared CD4+T cell culture solution and 1.0E+07 PBMCs were added, and the No. 1 centrifuge tube was placed in a 4℃ refrigerator for 20 min; then, after the freezing was completed, the No. 1 centrifuge tube was taken out and CD4 negative selection magnetic beads were added, and all cells except CD4+T cells were adsorbed and removed, and then the No. 1 centrifuge tube and the cell culture supernatant were placed in a magnetic stand for 1 min, and the CD4 negative selection magnetic beads were adsorbed using the magnetic stand, and the supernatant was reserved, and thus a CD4+T cell suspension was obtained; finally, a No. 2 centrifuge tube (the number of No. 2 centrifuge tubes can also be 1 or more than 2 according to the specific situation) was used to collect the CD4+T cell suspension, and CD3 / CD28 magnetic beads were added, and the No. 2 centrifuge tube was placed in a 37℃, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0058] On day 3, after the activation culture was completed, the No. 2 centrifuge tube was placed in a magnetic stand for 1 min, and the supernatant was sucked and centrifuged at 300g for 5 min, and the cell precipitate was collected, and thus activated CD4+T cells were obtained; then, the activated CD4+T cells were resuspended with the above-mentioned CD4+T cell culture solution, and the cell suspension was transferred into a 24-well plate, and the CD4+T cell culture solution was added and the cell density in each well was controlled to be 1.0E+06 cells / mL, and the culture was carried out in a 37℃, 5% CO2 constant temperature incubator;

[0059] On day 5, the CD4+T cell culture solution was added and the cell density in each well was controlled to be 0.5E+06 cells / mL, and the culture was continued;

[0060] On day 7, the CD4+T cell culture solution was added and the cell density in each well was controlled to be 0.8E+06 cells / mL, and the culture was continued;

[0061] On day 9, the CD4+T cell culture solution was added and the cell density in each well was controlled to be 1.0E+06 cells / mL, and the culture was continued;

[0062] On day 11, the culture was completed, and the cells and the culture solution were transferred into a No. 3 centrifuge tube (the number of No. 3 centrifuge tubes can also be 1 or more than 2 according to the specific situation), and the No. 3 centrifuge tube was centrifuged at 500g for 5 min, and the supernatant was removed, and the cell precipitate was washed with PBS for 2 to 3 times, and thus high-purity CD4+T cells were obtained, and the cells were stored for later use.

[0063] In the culture of CD4+T cells, the seed cells can be PBMCs (peripheral blood mononuclear cells) or CBMCs (cord blood mononuclear cells), and in the present application, the PBMCs separated from peripheral blood are preferred.

[0064] The specific steps of isolating PBMC from peripheral blood are as follows:

[0065] 1) Extract 100 mL of peripheral blood from a healthy person and place it in a sterile heparin sodium blood collection tube. Then, transfer the blood into three 50 mL 11# centrifuge tubes. After centrifugation, obtain blood plasma and whole blood cells.

[0066] 2) After the blood plasma is combined, transfer it to a new 12# centrifuge tube and place it in a 56°C water bath for inactivation for 20 min. Then, place it in a -20°C environment for rapid freezing for 15 min. Centrifuge at 1500 g for 30 min to obtain clear blood plasma, which is stored in a 2-8°C environment.

[0067] 3) Transfer the whole blood cells to a 13# centrifuge tube, dilute them with physiological saline at a ratio of 1:1, and then slowly add them to the lymph separation liquid at a ratio of 1:2. Try to ensure clear layering, with an increase of 1 and a decrease of 0, and centrifuge at 650 g for 30 min.

[0068] 4) After centrifugation, transfer the white membrane layer in the middle of the 13# centrifuge tube to a 14# centrifuge tube. Then, wash and resuspend it with physiological saline twice to collect the mononuclear cells PBMC, which are then divided into cryogenic tubes and stored for later use.

[0069] The technical solutions of the present application will be described in further detail through specific examples. In the following examples, the PBMC used are obtained from the peripheral blood described above and belong to the same batch of peripheral blood.

[0070] Example 1

[0071] 1.1 Preparation of CD4+ T cell culture solution

[0072] Take 2000 mL of X-Vivo10 culture medium and add human AB serum with a final concentration of 0.1 v / v%, rhIL-2 with a final concentration of 50 IU / mL, and fish oil with a final concentration of 10 μM to the culture medium.

[0073] 1.2 CD4+ T cell culture

[0074] Day 0, first, take 50 mL centrifuge tube, add the antibody MIX in the commercially available kit, 10 mL CD4+T cell culture solution prepared in step 1.1 of the embodiment and 1.0E+07 PBMC, put into 4℃ refrigerator, 20 min; then, after the freezing is completed, take out the centrifuge tube and add CD4 negative selection magnetic beads, adsorb all cells except CD4+T cells, then place the centrifuge tube and cell culture supernatant on the magnetic stand for 1 min, use the magnetic stand to adsorb the CD4 negative selection magnetic beads, and retain the supernatant suspension, that is, the CD4+T cell suspension is obtained; finally, collect the CD4+T cell suspension with the centrifuge tube, add CD3 / CD28 magnetic beads, and place in a 37℃, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0075] Day 3, first, after the activation culture is completed, place the centrifuge tube containing the CD3 / CD28 magnetic bead cell suspension on the magnetic stand for 1 min, remove the CD3 / CD28 magnetic beads, retain the supernatant and centrifuge at 300g for 5 min; collect the cell precipitate, that is, the activated CD4+T cells are obtained; then, resuspend the activated CD4+T cells with the CD4+T cell culture solution prepared in step 1.1 of the embodiment, and spread the cell suspension into 10 holes of a 24-hole plate, and add 1 mL of CD4+T cell culture solution prepared in step 1.1 of the embodiment in the corresponding holes, and control the cell density in each hole to be 0.5E+06 cells / ml, and place in a 37℃, 5% CO2 constant temperature incubator for 2 days;

[0076] Days 5 to 11, CD4+T cell culture solution prepared in step 1.1 of the embodiment needs to be added every two days, and the cell density in each hole is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL and 1.0E+06 cells / mL respectively after the addition of the solution on day 6, day 8 and day 10;

[0077] Day 11, after the culture is completed, collect the cultured cells and culture solution and transfer to a new centrifuge tube, centrifuge at 500g for 5 min, remove the supernatant, wash with PBS for 2 to 3 times, and obtain high-purity CD4+T cells for storage.

[0078] Example 2

[0079] 2.1, preparation of CD4+T cell culture solution

[0080] Take 2000 mL RPMI-1640 medium, and add human AB serum with a final concentration of 2%, rhIL-2 with a final concentration of 400 IU / mL and fish oil with a final concentration of 25 μM to the medium.

[0081] 2.2, CD4+T cell culture

[0082] Day 0, first, take 50 mL centrifuge tube, add the antibody MIX in commercially available kit, 10 mL CD4+T cell culture solution prepared in step 2.1 of the embodiment and 1.0E+07 PBMC, put into 4℃ refrigerator, 20 min; then, after freezing, take out the centrifuge tube and add CD4 negative selection magnetic beads, adsorb all cells except CD4+T cells, then put the centrifuge tube and cell culture supernatant in a magnetic stand for 1 min, use the magnetic stand to adsorb CD4 negative selection magnetic beads, and reserve the supernatant suspension, that is, the CD4+T cell suspension; finally, collect the CD4+T cell suspension with the centrifuge tube, add CD3 / CD28 magnetic beads, and put in a 37℃, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0083] Day 3, first, after the activation culture, put the centrifuge tube containing the cell suspension with CD3 / CD28 magnetic beads in a magnetic stand for 1 min, remove the CD3 / CD28 magnetic beads, reserve the supernatant and centrifuge the supernatant at 300g for 5 min; collect the cell precipitate, that is, the activated CD4+T cells; then, resuspend the activated CD4+T cells with the CD4+T cell culture solution prepared in step 2.1 of the embodiment, and spread the cell suspension into 10 holes of a 24-hole plate, and add 1 mL of CD4+T cell culture solution prepared in step 2.1 of the embodiment in the corresponding holes, and control the cell density of each hole to be 0.5E+06 cells / ml, in a 37℃, 5% CO2 constant temperature incubator for 2 days;

[0084] Days 5 to 11, every two days, the CD4+T cell culture solution prepared in step 2.1 of the embodiment needs to be added, and the cell density of each hole is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL and 1.0E+06 cells / mL respectively after the addition of the solution on the 6th day, the 8th day and the 10th day;

[0085] Day 11, after the culture, collect the cultured cells and culture solution and transfer to a new centrifuge tube, centrifuge at 500g for 5 min, remove the supernatant, wash with PBS for 2 to 3 times, and obtain high-purity CD4+T cells for storage.

[0086] Example 3

[0087] 3.1, preparation of CD4+T cell culture solution

[0088] Take 2000 mL GIBCO OpTmizer TM CTS TM T medium, and add human AB serum containing a final concentration of 10 v / v%, rhIL-2 containing a final concentration of 1000 IU / mL and fish oil containing a final concentration of 50 μM in the medium.

[0089] 3.2, CD4+T cell culture

[0090] On day 0, first, take 50 mL centrifuge tube, add antibody MIX in commercially available kit, 10 mL CD4+ T cell culture solution prepared in step 3.1 of the embodiment, and 1.0E+07 PBMC, and place in 4°C refrigerator for 20 min; then, after the freezing is completed, take out the centrifuge tube and add CD4 negative selection magnetic beads, adsorb all cells except CD4+ T cells, and then place the centrifuge tube and cell culture supernatant on a magnetic stand for 1 min, use the magnetic stand to adsorb the CD4 negative selection magnetic beads, and retain the supernatant suspension, that is, the CD4+ T cell suspension is obtained; finally, collect the CD4+ T cell suspension with the centrifuge tube, and then add CD3 / CD28 magnetic beads, and place in a 37°C, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0091] On day 3, first, after the activation culture is completed, place the centrifuge tube containing the cell suspension with CD3 / CD28 magnetic beads on a magnetic stand for 1 min, remove the CD3 / CD28 magnetic beads, retain the supernatant, and centrifuge the supernatant at 300g for 5 min; collect the cell precipitate, that is, the activated CD4+ T cells are obtained; then, resuspend the activated CD4+ T cells with the CD4+ T cell culture solution prepared in step 3.1 of the embodiment, and spread the cell suspension into 10 holes of a 24-hole plate, and add 1 mL of the CD4+ T cell culture solution prepared in step 3.1 of the embodiment into the corresponding holes, and control the cell density in each hole to be 0.5E+06 cells / ml, and place in a 37°C, 5% CO2 constant temperature incubator for 2 days;

[0092] On day 6 to day 10, the CD4+ T cell culture solution prepared in step 2.1 of the embodiment needs to be added every two days, and the cell density in each hole is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL, and 1.0E+06 cells / mL respectively after the solution is added on day 6, day 8, and day 10;

[0093] On day 11, the culture is completed, the cultured cells and culture solution are transferred to a new centrifuge tube, centrifuged at 500g for 5 min, the supernatant is removed, and the cells are washed 2 to 3 times with PBS to obtain high-purity CD4+ T cells, which are stored for later use.

[0094] Example 4 (comparative example 1)

[0095] 4.1, preparation of CD4+ T cell culture solution

[0096] Take 2000 mL X-Vivo 10 culture medium, and add human AB serum containing a final concentration of 10 v / v%, rhIL-2 containing a final concentration of 1000 IU / mL, and fish oil containing a final concentration of 25 μM to the culture medium.

[0097] 4.2, CD4+ T cell culture

[0098] On day 0, first, take a 50 mL centrifuge tube, add the antibody MIX in the commercially available kit, 10 mL of the CD4+ T cell culture solution prepared in step 4.1 of this example, and 1.0E+07 PBMCs, and place it in a 4°C refrigerator for 20 min; then, after the freezing is completed, take out the centrifuge tube and add CD4 negative selection magnetic beads to adsorb all cells except CD4+ T cells, and then place the centrifuge tube and cell culture supernatant on a magnetic stand for 1 min, use the magnetic stand to adsorb the CD4 negative selection magnetic beads, and retain the supernatant suspension, i.e., obtain the CD4+ T cell suspension; finally, collect the CD4+ T cell suspension with the centrifuge tube, add CD3 / CD28 magnetic beads, and place it in a 37°C, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0099] On day 3, first, after the activation culture is completed, place the centrifuge tube containing the cell suspension with CD3 / CD28 magnetic beads on a magnetic stand for 1 min to remove the CD3 / CD28 magnetic beads, retain the supernatant, and centrifuge the supernatant at 300g for 5 min; collect the cell precipitate, i.e., obtain the activated CD4+ T cells; then, resuspend the activated CD4+ T cells with the CD4+ T cell culture solution prepared in step 4.1 of this example, and spread the cell suspension into 10 wells of a 24-well plate, and add 1 mL of the CD4+ T cell culture solution prepared in step 4.1 of this example to the corresponding wells, and control the cell density in each well to be 0.5E+06 cells / mL, and place it in a 37°C, 5% CO2 constant temperature incubator for 2 days;

[0100] On days 5 to 11, the CD4+ T cell culture solution prepared in step 4.1 of this example needs to be added every two days, and the cell density in each well is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL, and 1.0E+06 cells / mL on days 6, 8, and 10, respectively, after the addition of the solution;

[0101] On day 11, the culture is completed, the cultured cells and culture solution are transferred to a new centrifuge tube, centrifuged at 500g for 5 min, the supernatant is removed, washed with PBS for 2 to 3 times, and high-purity CD4+ T cells are obtained, which are stored for later use.

[0102] Example 5 (Comparative Example 2)

[0103] 5.1, Preparation of CD4+ T cell culture solution

[0104] Take 2000 mL of X-Vivo 10 culture medium, and add human AB serum containing a final concentration of 2v / v%, rhIL-2 containing a final concentration of 400 IU / mL, and fish oil containing a final concentration of 1 μM to the culture medium.

[0105] 5.2, CD4+ T cell culture

[0106] On day 0, first, take 50 mL centrifuge tube, add antibody MIX in commercially available kit, 10 mL CD4+ T cell culture solution prepared in step 5.1 of the present embodiment and 1.0E+07 PBMC, and place in 4°C refrigerator for 20 min; then, after the freezing is completed, take out the centrifuge tube and add CD4 negative selection magnetic beads, adsorb all cells except CD4+ T cells, and then place the centrifuge tube and cell culture supernatant on a magnetic stand for 1 min, use the magnetic stand to adsorb the CD4 negative selection magnetic beads, and retain the supernatant suspension, i.e. obtain the CD4+ T cell suspension; finally, collect the CD4+ T cell suspension with the centrifuge tube, add CD3 / CD28 magnetic beads, and place in a 37°C, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0107] On day 3, first, after the activation culture is completed, place the centrifuge tube containing the cell suspension with CD3 / CD28 magnetic beads on a magnetic stand for 1 min, remove the CD3 / CD28 magnetic beads, retain the supernatant and centrifuge at 300g for 5 min; collect the cell precipitate, i.e. obtain the activated CD4+ T cells; then, resuspend the activated CD4+ T cells with the CD4+ T cell culture solution prepared in step 5.1 of the present embodiment, and spread the cell suspension into 10 holes of a 24-well plate, and add 1 mL of the CD4+ T cell culture solution prepared in step 5.1 of the present embodiment to the corresponding holes, and control the cell density in each hole to be 0.5E+06 cells / ml, and place in a 37°C, 5% CO2 constant temperature incubator for 2 days;

[0108] On days 5 to 11, the CD4+ T cell culture solution prepared in step 5.1 of the present embodiment needs to be added every two days, and the cell density in each hole is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL and 1.0E+06 cells / mL respectively on days 6, 8 and 10 after the replenishment of the solution;

[0109] On day 11, after the culture is completed, collect the cultured cells and culture solution and transfer to a new centrifuge tube, centrifuge at 500g for 5 min, remove the supernatant, wash with PBS for 2 to 3 times, and obtain high-purity CD4+ T cells, which are stored for later use.

[0110] Example 6 (Comparative Example 3)

[0111] 6.1, Preparation of CD4+ T cell culture solution

[0112] Take 2000 mL X-Vivo 10 culture medium, and add human AB serum containing a final concentration of 2 v / v% and rhIL-2 containing a final concentration of 400 IU / mL to the culture medium.

[0113] 6.2, CD4+ T cell culture

[0114] Day 0, first, take 50 mL centrifuge tube, add the antibody MIX in the commercially available kit, 10 mL CD4+T cell culture solution prepared in step 6.1 of this example and 1.0E+07 PBMC, put into 4℃ refrigerator, 20 min; then, after freezing, take out the centrifuge tube and add CD4 negative selection magnetic beads, adsorb all cells except CD4+T cells, then put the centrifuge tube and cell culture supernatant in the magnetic stand for 1 min, use the magnetic stand to adsorb CD4 negative selection magnetic beads, and reserve the supernatant suspension, that is, the CD4+T cell suspension is obtained; finally, collect the CD4+T cell suspension with the centrifuge tube, add CD3 / CD28 magnetic beads, and put it in a 37℃, 5% CO2 constant temperature incubator for 2 days of activation culture;

[0115] Day 3, first, after the activation culture is completed, the centrifuge tube containing the cell suspension with CD3 / CD28 magnetic beads is placed in the magnetic stand for 1 min, the CD3 / CD28 magnetic beads are removed, the supernatant is reserved and centrifuged at 300g for 5 min; collect the cell precipitate, that is, the activated CD4+T cells are obtained; then, resuspend the activated CD4+T cells with the CD4+T cell culture solution prepared in step 1.1 of this example, and spread the cell suspension into 10 holes of a 24-hole plate, and add 1 mL of CD4+T cell culture solution prepared in step 6.1 of this example in the corresponding holes, and control the cell density in each hole to be 0.5E+06 cells / ml, in a 37℃, 5% CO2 constant temperature incubator for 2 days;

[0116] Days 5 to 11, CD4+T cell culture solution prepared in step 6.1 of this example needs to be added every two days, and the cell density in each hole is controlled to be 0.5E+06 cells / mL, 0.8E+06 cells / mL and 1.0E+06 cells / mL respectively after the addition of the solution on the 6th day, the 8th day and the 10th day;

[0117] Day 11, the culture is completed, the cultured cells and culture solution are transferred to a new centrifuge tube, centrifuged at 500g for 5 min, the supernatant is removed, washed with PBS for 2 to 3 times, and high-purity CD4+T cells are obtained for storage.

[0118] Example 7

[0119] 7.1, cell growth amplification multiple detection test

[0120] The CD4+T cells cultured in Examples 1 to 6 are sampled on the 0th day, the 4th day, the 6th day, the 8th day, the 10th day and the 11th day, and counted with a cell counter; the detection results are shown in Table 1 and Figure 1 Figure 1 ​Based on the data in Table 1. In Table 1, the cells detected by counting on day 0 are PBMCs, and the number is 1.0E+07. After day 4, the cells detected by sampling are CD4+T cells.

[0121] Table 1: CD4+T cell expansion quantity

[0122]

[0123] From Table 1 and Figure 1 It can be seen that: 1) from Examples 1 to 3 and Example 5, the fish oil component in the CD4+T cell culture solution can promote the rapid expansion and growth of CD4+T cells; 2) from Examples 1 to 5, the fish oil component in the CD4+T cell culture solution can promote the rapid expansion and growth of CD4+T cells, but too high or too low fish oil concentration has an inhibitory effect on the growth of CD4+T cells.

[0124] Therefore, the concentration of the fish oil component in the CD4+T cell culture solution is in the range of 10 to 50 μM, and the Omega-3 polyunsaturated fatty acids (EPA and DHA) contained therein can inhibit the overactivation of cells and promote the rapid expansion and growth of CD4+T cells.

[0125] 7.2: CD4+positive rate detection test of cells

[0126] On the 11th day of cell culture in Examples 1 to 6, each sample was taken, and the number of CD4+T cells was 1.0E+06. The cells were washed twice with 2% FBS+DPBS, then centrifuged at 500g for 2 min, the supernatant was discarded, and the CD4+T cell precipitate was stained with CD4 antibody, followed by flow sampling detection. The detection results are shown in Figures 2 to 7 .

[0127] From Figures 2 to 7 It can be seen that the flow detection CD4+positive rates of the CD4+T cells cultured in Examples 1 to 6 are 76.59%, 92.56%, 75.07%, 65.92%, 61.18%, and 28.37%, respectively. Therefore, it is shown that the fish oil with a concentration of 25 μM provided by the application has the best effect on the expansion and functional expression of CD4+T cells. The CD4+T cells obtained by culturing in Example 6 without fish oil component have the lowest CD4+positive rate, which is much lower than the CD4+positive rates of the CD4+T cells obtained by culturing in Examples 1 to 5 with fish oil component.

[0128] 7.3: IFN-γ, IL-4, IL-10, and other factor concentration detection test

[0129] The cells of Example 1 to 6 were sampled on the 11th day of cell culture, and the concentration detection test of IFN-γ, IL-4, IL-10 and other factors was carried out. The cytokine detection was carried out using an ELISA kit, and the specific process was as follows:

[0130] IL-4 concentration detection: Human IL-4 ELISA Kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., product number PI 618);

[0131] IL-10 concentration detection: Human IL-10 ELISA Kit (purchased from Yixing Biotechnology (Shanghai) Co., Ltd., product number 97063ES96);

[0132] IFN-γ concentration detection: Human IFN-γ Valukine ELISA Kit (R&D Systems VAL104C).

[0133] The concentration detection results of IL-4, IL-10, IFN-γ and other cytokines are shown in Table 2 and Figure 8 ; wherein, Figure 8 is a graph plotted based on the data in Table 2.

[0134] Table 2 Cytokine secretion concentration

[0135]

[0136] From Table 2 and Figure 8 , it can be seen that: 1) in Example 1 to 3 and Example 6, the Omega-3 polyunsaturated fatty acids (EPA and DHA) in the fish oil component of the CD4+ T cell culture solution can reduce the production of pro-inflammatory factors (such as IFN-γ) and increase the production of anti-inflammatory factors (such as IL-4 and IL-10); 2) in Example 1 to 5, the concentration of the fish oil component in the CD4+ T cell culture solution is too high or too low, which is not conducive to the production of cytokines IFN-γ, IL-4 and IL-10.

[0137] Therefore, in the CD4+ T cell culture solution, the concentration of the fish oil component is in the range of 10-50 μM, and the Omega-3 polyunsaturated fatty acids (EPA and DHA) contained therein can reduce the production of pro-inflammatory factor IFN-γ and increase the secretion of anti-inflammatory factors IL-4 and IL-10, thereby improving the anti-inflammatory ability of CD4+ T cells.

[0138] 7.4, Specific killing rate test of CD4+ T cells

[0139] The CD4+ T cells in the 11th day of the cell culture in the examples 1 to 6 were sampled, and co-cultured with Raji cells (i.e. Raji target cells) for 132 hours, and the co-culture ratio was CD4+ T cells: Raji cells = 10:1, and the tumor killing efficiency was detected by using a real time xCELLigence analysis system (RTCA), and the sampling detection results are shown in Table 3. Figure 9 Based on the data in Table 3, the following figure was obtained.

[0140] As shown in Table 3 and Figure 9 , the killing curve of CD4+ T cells on Raji target cells; the 6 curves respectively represent the killing activity rate curves of the corresponding CD4+ T cells in the example 1, the example 2, the example 3, the example 4 (comparative example 1), the example 5 (comparative example 2), and the example 6 (comparative example 3) at the effector target ratio E:T = 10:1.

[0141] Table 3 CD4+ T cell specific killing rate

[0142]

[0143] As shown in Table 3 and Figure 9 , the killing curve of CD4+ T cells on Raji target cells; the 6 curves respectively represent the killing activity rate curves of the corresponding CD4+ T cells in the example 1, the example 2, the example 3, the example 4 (comparative example 1), the example 5 (comparative example 2), and the example 6 (comparative example 3) at the effector target ratio E:T = 10:1.

[0144] Therefore, it is illustrated that the Omega-3 in the fish oil component in the CD4+ T cell culture solution can enhance the activity of the CD4+ T cells, promote the generation of the CD8+ T cells, improve the immune homeostasis mediated by the CD4+ T cells, and thus improve the anti-tumor immunity; however, the concentration of the fish oil component in the CD4+ T cell culture solution is too high or too low, which affects the killing rate of the CD4+ T cells, and when the concentration of the fish oil is 10-50 μM, the killing rate of the CD4+ T cells cultured in the CD4+ T cell culture solution is better.

[0145] It should be understood that the above description of the preferred embodiments of the present application is more detailed, and therefore should not be considered as a limitation on the patent protection scope of the present application, and the patent protection scope of the present application should be subject to the appended claims.

Claims

1. A CD4+ T cell culture medium, characterized in that, The culture medium contains a complete culture medium supplemented with and containing human AB serum at a final concentration of 0.1–10 v / v%, rhIL-2 at a final concentration of 50–1000 IU / mL, and fish oil at a final concentration of 10–50 μM.

2. The CD4+ T cell culture medium according to claim 1, characterized in that, The complete culture medium consisted of X-Vivo 10 medium, RPMI-1640 medium, and GIBCO OpTmizer. TM CTS TM Any of the T-mediums.

3. A method for culturing CD4+ T cells, characterized in that, Includes the following steps: On day 0, 1.0E+07 PBMCs were sorted, and the sorted T cells were cultured together with the activation magnetic beads for 2 days. On day 3, after activation culture, the activated CD4+ T cells were collected and resuspended in the culture medium as described in claim 1. The cell suspension was transferred into a 24-well plate, and the CD4+ T cell culture medium as described in claim 1 was added. The cell density per well was controlled at 1.0E+06 cells / mL, and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2. From day 5 to day 9, the CD4+ T cell culture medium was added every two days, and the cells were kept in static culture. On day 11, the culture was completed. The cells and culture medium were collected, centrifuged, the supernatant was removed, and the cells were washed 2 to 3 times with PBS to obtain high-purity CD4+ T cells.

4. The cultivation method according to claim 3, characterized in that, Day 0, the PBMC sorting and cell activation steps specifically include the following treatments: First, take a centrifuge tube, add the sorting reagent antibody MIX, 1.0E+07 PBMCs and 10mL of CD4+ T cell culture medium as described in claim 1, and freeze it at 4°C for 20min. After freezing, remove the centrifuge tubes and add CD4 cathode beads, then let stand at room temperature for 30 minutes. Subsequently, the centrifuge tube was placed on a magnetic rack and left to stand for 1 minute. The supernatant suspension was then aspirated to obtain a CD4+ T cell suspension. Collect CD4+ T cell suspension, add CD3 / CD28 magnetic beads, and incubate at 37℃ in a 5% CO2 constant temperature incubator for 2 days to activate the cells.

5. The cultivation method according to claim 1, characterized in that, On day 3, activated CD4+ T cells were collected and subjected to the following treatments before being transfected into 24-well plates: After activation culture is complete, the activated CD4+ T cells are collected and transferred to new centrifuge tubes; Place the new centrifuge tube on a magnetic rack and let it stand for 1 minute. Aspirate the supernatant, centrifuge, and collect the cell pellet to obtain activated CD4+ T cells.

6. The cultivation method according to claim 1, characterized in that, The cell culture steps from day 5 to day 9 are as follows: On day 5, the CD4+ T cell culture medium was added again, and the cell density per well was controlled at 0.5E+06 cells / mL, and the cells were kept in static culture for a longer period. On day 7, the CD4+ T cell culture medium was added again, and the cell density per well was controlled at 0.8E+06 cells / mL. The cells were then allowed to continue to be cultured statically. On day 9, the CD4+ T cell culture medium was added again, and the cell density per well was controlled at 1.0E+06 cells / mL. The cells were then allowed to continue to be cultured statically.

7. The cultivation method according to claim 3, characterized in that, On day 11, the centrifugation was performed at 500g for 30 minutes.

Images