Culture method for inducing and amplifying central memory T cells

Abstract

The invention provides a culture method for inducing and amplifying central memory T cells with high expression of CD62L typing, which comprises the following steps: collecting peripheral blood mononuclear cells, adding the peripheral blood mononuclear cells into a culture medium containing stimulant CD3 / CD28 antibody coupling magnetic beads, amplifying for 7-12 days to obtain a cell population with TCM, and the culture medium further comprises IL-2, IL-7, IL-15 and syringaresinol. The method can effectively improve the TCM cell content and purity of the high-expression CD62L marker, and can be effectively used for treating tumors, infection or other immune related diseases.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and in particular to an immune cell culture method. Background Art

[0002] There are two main types of specific T cells: CD4 + T cells and CD8 + T cells. In cell adoptive immunotherapy, CD4 + In addition to their own killing ability, T cells can also assist the body's own immune cells and co-adopted immune cells. They are highly versatile and can interact with CD8 + T cells complement each other. + T lymphocytes can be divided into three subgroups: initial T lymphocytes, effector T lymphocytes, and memory T lymphocytes. Memory T cells can be divided into three subgroups based on their phenotypic characteristics and functions, namely stem cell-like T cells (T SCM ), central memory T cells (central memory T cells, T CM ) and effector memory T cells (T EM ). Among them, central memory T cells have the ability to self-renew, and have a short response time, a long action time, and few side effects on the human body. In recent years, multiple research groups have reported that after T cells with memory function are infused back into tumor patients, the therapeutic effect is obvious and the treatment time is long, showing strong anti-tumor effects and clinical effects, which can reduce the pain of patients and reduce the biosafety risks of excessive cell in vitro culture times, and have good application prospects.

[0003] Central memory T cells express high levels of molecules such as CD45RO, CD62L, CD44, and CCR7 on their surface, which play an important role in maintaining the stability and function of central memory T cells. + CD45RA + CCR7 + ) to stem cell-like memory T cells (T SCM , CD3 + CD45RA + CCR7 + ), central memory T cells (T CM , CD3 + CD45RO + CCR7 + ), effector memory T cells (T EM , CD3 + CD45RO+ CCR7 - ) and effector T cells (T E , CD3 + CD45RO - CCR7 - ) is a gradual differentiation process. In clinical practice, T cells need to be amplified in vitro to meet the therapeutic dose. Although some T cell amplification methods have been developed in the prior art, these T cell amplification methods often lead to the terminal differentiation of T cells into effector T cells. After being infused back into the patient's body, the proliferation and amplification capacity of the cells are low, which seriously affects the efficacy of adoptive T cell therapy. Therefore, how to obtain a large number of memory T cells in vitro and reduce the terminal differentiation of T cells, so that more T cells can differentiate into T cells? CM , is a difficult problem in cell immunotherapy.

[0004] Syringaresinol (Syr), an important pharmacologically active ingredient from the traditional Chinese medicine Sargentodoxae, is a natural Chinese medicine monomer with multiple pharmacological effects, especially in anti-inflammatory and anti-oxidation. In recent years, some studies have found that syringaresinol has a certain promoting effect on the cultivation of immune cells. For example, WO2016140485A1 discloses a composition for regulating T cells. Syringaresinol can enhance the efficacy of vaccines and increase the number of T cells that are reduced due to aging; CN117987361B discloses a T cell culture medium, including IL-2, IL-15, CD3 monoclonal antibody, sodium polystyrene sulfonate, dulaglutide, syringaresinol, etc. The synergistic effect of dulaglutide and syringaresinol added to the culture medium can enhance the proliferation capacity of T cells. However, the use of syringaresinol in T cell differentiation has not been reported, and the existing technology does not disclose the concentration of syringaresinol in the differentiation of central memory T cells and the combination of other components. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a magnetic bead method for culturing T CM Cell (central memory T cell, Central Memory T cell) method.

[0006] The present invention relates to a method for inducing and expanding central memory T cells (T CM ) cultivation method.

[0007] Peripheral blood mononuclear cells (PBMCs) were collected and added to a culture medium containing stimulatory CD3 / CD28 antibody-coupled magnetic beads. After 7-12 days of expansion, T cells were obtained. CM of cell populations.

[0008] Preferably, the T cells are harvested after 8-11 days of expansion. CMCells are preferably expanded to harvest T cells on day 10-11. CM cell.

[0009] Specifically, induction and expansion of central memory T cells (T CM ) in the culture method, after the magnetic beads are added, the magnetic beads are removed twice; Specifically, induction and expansion of central memory T cells (T CM ) in the culture method, after the magnetic beads are added, a further step of removing the magnetic beads is required, and the number of times the magnetic beads are removed is preferably 2-3 times; preferably, the magnetic beads contact the cells for 1-3 days.

[0010] Preferably, a special culture medium for central memory T cells needs to be added during the culture process, and preferably the culture medium includes a basal culture medium and a supplementary culture medium; The basic culture medium can be a self-prepared common basic nutrient culture medium, which contains basic amino acids, inorganic salts, vitamins, etc. required by T cells.

[0011] The amino acids include, but are not limited to, L-Asp, L-Glu, L-Ile, L-Ser, L-Gly, L-Pro, L-His hydrochloride monohydrate, L-Arg hydrochloride, L-Thr, L-Ala, L-Tyr disodium salt dihydrate, L-Val, L-Met, L-Leu, L-cysteine, L-Phe, L-Lys hydrochloride, L-Trp, L-Asn monohydrate, L-Cys hydrochloride monohydrate, and L-hydroxyproline; The inorganic salts include, but are not limited to, NaCl, KCl, Na2HPO4, MgSO4, NaHCO3, CaCl2, etc.; The microorganisms include but are not limited to folic acid, VB12, vitamin C, inositol, biotin and the like.

[0012] The basal culture medium can also be a commercial basal culture medium with a defined composition, such as CTS OpTmizer T cell expansion SFM culture medium + CTS OpTmizer T cell expansion auxiliary concentrated culture medium (referred to as CTS), IMDM, DMEM, DMEM / F12, aMEM, RPMI1640, M199 medium basal culture medium can be one or a mixture of several thereof, preferably CTS culture medium, DMEM culture medium, RPMI1640 culture medium.

[0013] Supplementary culture medium is further added to the basal culture medium, including but not limited to sufficient supplementary nutrition and inducers of adoptive cell immunotherapy. The inducers of adoptive cell immunotherapy may be interleukins, syringaresinol, and further may be IL-2, IL-7, IL-15. The supplementary nutrition may be albumin or glutamine.

[0014] Further, the supplemented culture medium may contain glutamine 20-30 μg / mL, albumin 20-50 g / L, IL-2 100-200 IU / mL, IL-7 100-200 IU / mL, IL-15 100-500 IU / mL, and syringaresinol 5-35 μg / L; Further preferably, glutamine 22-27 μg / mL, albumin 20-40 g / L, IL-2 100-200 IU / mL, IL-7 150-200 IU / mL, IL-15 100-400 IU / mL, syringaresinol 10-30 μg / L; More preferably, glutamine 22-27 μg / mL, albumin 20-40 g / L, IL-2 150 IU / mL, IL-7 150 IU / mL, IL-15 400 IU / mL, syringaresinol 10-20 μg / L.

[0015] Furthermore, the culture medium may be as shown in Table 1.

[0016] The present invention further provides the use of memory T cells that highly express CD62L in treating tumors, infections or other immune-related diseases, as well as the use in preparing drugs for treating tumors, infections or other immune-related diseases. The tumor may be melanoma, breast cancer, prostate cancer, skin cancer, non-small cell lung cancer, gastric cancer, cervical cancer, etc.

[0017] The present invention further provides the use of syringaresinol in the culture of central memory T cells, specifically, for improving T cells in the separation process. CM The content and purity of CD62L typing in it, and its use in promoting T cell differentiation.

[0018] Beneficial Effects The present invention provides a method for inducing and amplifying central memory T cells with high expression of CD62L typing, which innovatively uses CD3 / CD28 antibody-coupled magnetic beads for sorting, and uses a basal culture medium and a supplementary culture medium for mixing, and further contains albumin, glutamine, IL-2, IL-7, IL-15, and syringaresinol, which has a synergistic effect and can improve T CMThe cells have high purity and can be further effectively used to treat tumors, infections or other immune-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Control group 1 T CM Cell ratio analysis flow cytometry detection chart Figure 2 Control group 2 T CM Cell ratio analysis flow cytometry detection chart Figure 3 Control group 3 T CM Cell ratio analysis flow cytometry detection chart Figure 4 Control group 4 T CM Cell ratio analysis flow cytometry detection chart Figure 5 Control group 5 T CM Cell ratio analysis flow cytometry detection chart Figure 6 Experimental group AT CM Cell ratio analysis flow cytometry detection chart Figure 7 Experimental group BT CM Cell ratio analysis flow cytometry detection chart Figure 8 Experimental group CT CM Cell ratio analysis flow cytometry detection chart Fig. 9 Experimental group DT CM Cell ratio analysis flow cytometry detection chart Fig.10 Experimental group ET CM Cell ratio analysis flow cytometry detection chart Fig.11 Experimental group FT CM Cell ratio analysis flow cytometry detection chart Fig.12 Experimental group GT CM Cell ratio analysis flow cytometry detection chart Fig.13 Experimental group HT CM Cell ratio analysis flow cytometry detection chart Fig.14 Experimental Group IT CM Cell ratio analysis flow cytometry detection chart Fig.15 Experimental group JT CM Cell ratio analysis flow cytometry detection chart Fig.16 Experimental group KT CMCell ratio analysis flow cytometry detection chart Fig.17 Experimental group LT CM Cell ratio analysis flow cytometry detection chart Fig.18 Experimental group MT CM Cell ratio analysis flow cytometry detection chart Fig.19 Experimental group C uses CD3-PerCP to sort out T lymphocytes, showing the flow cytometry detection of T lymphocytes in lymphocytes Figure 20-22 Experimental group C on culture days 0, 5, and 10 CM Cell ratio analysis flow cytometry detection chart DETAILED DESCRIPTION

[0020] 1. Experimental consumables and experimental groups (1) Reagents: Human peripheral blood components were donated by healthy volunteers, sodium chloride injection, lymphocyte separation solution, human serum albumin, CD3 / CD28 antibody-coupled magnetic beads (purchased from Gibco, Catalog No.: SUPF-31), trypan blue; CTS OpTmizer T cell expansion SFM medium (purchased from Gibco, Catalog No. A10221-01) + CTS OpTmizer T cell expansion auxiliary concentrated medium (purchased from Gibco, Catalog No. A1048402), referred to as CTS, 1640 and DMEM were purchased from Gibco culture medium, IL-2, IL-7, IL-15 (recombinant human interleukin, purchased from Jiangsu Dongkang), L-glutamine (purchased from Gibco); (2) Consumables: T75 culture flasks, cell culture bags, 200 mL disposable plastic blood bags, pipettes (5 mL, 10 mL, 25 mL, 50 mL), centrifuge tubes (50 mL, 250 mL), disposable cell counting chambers, syringes (10 mL, 50 mL), gun tips (10 μL, 200 μL, 1000 μL), 2 mL cryovials, EP tubes (0.65 mL / 1.5 mL); 75% alcohol gauze, sealing film.

[0021] (3) Instruments: BD Accuri TM C6 Plus flow cytometer, clean bench, Eppendorf centrifuge, CO2 incubator (37°C, 5%), refrigerator, electric pipette, single-channel micropipette (10-100μL, 20-200μL, 100-1000μL), sealer, heat sealer, microscope, 50mL / 1.5mL magnetic rack. Clean bench / biosafety cabinet, electric pipette, liquid level visible safety alcohol lamp, elbow hemostatic forceps.

[0022] Preparation method of culture medium: 1000mL of T cell expansion SFM medium + 26mL of T cell expansion auxiliary concentrated medium (a set of culture medium is referred to as CTS), or the same volume of 1640 and DMEM medium as the basic culture medium, and further add glutamine, IL-2, IL-7, IL-15, and syringaresinol in proportion. The concentration is prepared according to the groups in Table 1 (NA means not added).

[0023] Table 1

[0024] Second, the cultivation process (1) Isolation of PBMCs Turn on the ultraviolet light of the clean bench. After 30 minutes of ultraviolet disinfection, turn on the fan and lighting switch of the clean bench, and wipe the table with 75% alcohol or disinfected towels; take 8 50mL centrifuge tubes, add 20mL lymphocyte separation solution to each, mark for later use; divide the anticoagulated blood equally into 4 50mL centrifuge tubes (about 20mL / tube), use a pipette to take 2mL of whole blood into 2 2mL cryopreservation tubes, seal, mark, and freeze in a -80℃ refrigerator; add physiological saline in a 1:1 ratio, use a 25mL pipette to fully mix each tube, and use the cocktail addition method to slowly add the diluted anticoagulated blood along the tube wall to the lymphocyte separation solution layer; 610g Centrifuge for 20 minutes; discard the top layer of serum (about 10 mL) with a 25 mL pipette. If there is too much serum left, use a 5 mL pipette to remove some serum before absorbing the white film layer; carefully absorb the white film layer (about 10 mL) of each tube with a 5 mL pipette, transfer it to 8 new 50 mL centrifuge tubes, add sodium chloride injection to 45 mL, use a 10 mL pipette to mix the cell suspension (about 3 times), and mark it; centrifuge at 250 g for 10 minutes; discard the supernatant after centrifugation, and gently loosen the cells at the bottom of each tube; suspend the cells in each tube in turn with 5-8 mL of sodium chloride injection and merge them into 2 new 50 mL centrifuge tubes, wash it twice in this way, add sodium chloride injection to 40 mL, use a 10 mL pipette to mix the cell suspension (about 3 times), and mark it; centrifuge at 210 g for 10 minutes; (2) Remove adherent cells Discard the supernatant after centrifugation, gently loosen the cells at the bottom of the tube, and according to the thickness of the cells at the bottom of the tube, use 20mL of culture medium to mix the cells in the tube three times and merge them into another centrifuge tube; move the mixed cells to a new T75 culture flask, rinse the cell centrifuge tube once with 10mL of culture medium and move it to the T75 culture flask, shake gently to mix, mark it, and culture it in a CO2 incubator.

[0025] Cell culture day 1 (1) PBMC count Take the cells out of the CO2 incubator, take about 500 μL of cells from the well-mixed tube and put them into a 1.5 mL EP tube, then put the cells into the CO2 incubator for culture; count the cells by 10-fold dilution with trypan blue staining; 10× dilution count = 1:9, that is, "10 μL of well-mixed cell suspension" is added to "90 μL of trypan blue staining solution"; the total number of cells in the four grids = the total number of live cells in the four grids (translucent and round, not stained) + the total number of dead cells in the four grids (stained, excluding lysed cells); the viability rate = the total number of live cells in the four grids / the total number of dead cells in the four grids × 100%; the total number of cells = (the total number of cells in the four grids / 4) × 10 × 10 4 × Total volume of cell suspension; if there are more red blood cells, the total number of cells is converted to 1 / 2-2 / 3 of the total number of counts; inoculation volume (mL) = total number of cells / cell inoculation density (cell inoculation density is about 2M / mL); magnetic bead suspension volume (mL) = inoculation volume (mL) × 0.04; (add 40μL of magnetic bead suspension to each mL of cell suspension) (2) Rinse the magnetic beads Place 4 autoclaved 1.5mL EP tubes on a small magnetic rack, mix the magnetic bead suspension with a pipette and divide it equally into 1.5mL EP tubes, discard the suspension carefully to avoid bringing out the magnetic beads, add 1000μL culture solution to each tube in the air (avoid the tip of the pipette from contacting the magnetic beads to cause contamination), and cover the EP tube cap; rotate each 1.5mL EP tube for two weeks with a hemostatic forceps, open the EP tube cap, discard the suspension with a pipette carefully to avoid bringing out the magnetic beads, change the tip of the pipette, add 1000μL culture solution to each tube in the air, cover the EP tube cap, and remove the 1.5mL EP tube from the small magnetic rack for use; (3) T CM Vaccination Divide the magnetic beads equally into a new T75 culture flask (15-45mL / flask); mix the cells in the original T75 culture flask and divide them equally into the new T75 culture flask; rinse the original T75 culture flask with the remaining culture medium and divide them equally into the new T75 culture flask; use a 10mL pipette to gently blow and mix the cells, mark the current time, subject information, and preparation date on the T75 culture flask, and after observation, place it flat with the "8" method to mix, and culture it in a CO2 incubator.

[0026] Cell culture day 2 Use a 10 mL pipette to gently mix the cell suspension.

[0027] Cell culture day 3 First time removal of magnetic beads In T CM48 hours after cell inoculation, use a 10mL pipette to gently blow up the cells on the bottom and wall of the bottle and mix the cell suspension, then transfer it to a 50mL centrifuge tube. Each tube should not exceed 45mL. Cover the centrifuge tube lid but do not tighten it. Place the centrifuge tube containing the cell suspension on a 50mL magnetic rack for 10 minutes. Gently unscrew the centrifuge tube lid to prevent the centrifuge tube from rotating, and carefully transfer the cell suspension to two new T75 culture flasks to avoid touching the wall of the centrifuge tube. Mark them. After observation under a microscope, place it flat using the "8" method to mix, and culture it in a CO2 incubator.

[0028] Cell culture day 4 Take out the cells from the CO2 incubator and observe under a microscope. If the cells become larger and fall off and suspend, it means they are fully stimulated and meet the bag transfer standard. (1) Second removal of magnetic beads Use a 10mL pipette to gently blow up the cells at the bottom and wall of the bottle and mix the cell suspension, then transfer it to a 50mL centrifuge tube. Each tube should not exceed 45mL. Cover the centrifuge tube lid and do not tighten it. Place the centrifuge tube containing the cell suspension on a 50mL magnetic rack, rotate it slowly once, and leave it for 5 minutes. After 5 minutes, use a 10mL pipette to transfer the suspension to a new 50mL centrifuge tube in mid-air, avoiding touching the wall of the centrifuge tube. Observe under the sampling microscope to see if the magnetic beads are completely removed (if not completely removed, remove the magnetic beads again). (2) Bag transfer Place a 50mL disposable syringe on the fluid infusion rack and connect it to the cell culture bag; use a 10mL pipette to mix the cell suspension and transfer it to the culture bag; rinse the 50mL centrifuge tube with 5-10mL culture solution and transfer the cell suspension to the culture bag; exhaust the air in the bag; add an appropriate amount of culture solution according to the color of the cell suspension and the colony situation; pat gently to mix and place in a CO2 incubator for culture; Replenishment of fluid and microbial testing on day 5-6 of cell culture Place a 50mL disposable syringe on the rehydration rack and connect it to the cell culture bag; exhaust the air in the bag; add an appropriate amount of culture medium according to the color of the cell suspension and the colony situation; pat and mix well, and then place in a CO2 incubator for culture. Sampling: pat and mix the cell suspension, and use a 10mL disposable syringe to take out 4mL of cell suspension from the sampling port; divide it equally into 4 2mL cryopreservation tubes, 1mL in each tube for bacteria, fungi, and mycoplasma detection, seal it, label it, and give it to QC personnel for external inspection; pat and mix well, and then place in a CO2 incubator for culture.

[0029] Replenish the fluid on the 7th to 12th day of cell culture and harvest and test the cells as needed. Extract cells: Take out cells from CO2 incubator and pour the cell suspension into a labeled 250mL centrifuge tube; Count: Use a 10mL pipette to fully mix the cell suspension, take about 0.5mL of cell suspension and stain with 5-fold trypan blue for dilution counting; Place cells in an Eppendorf centrifuge at 1700rpm for 8min; Rinse cells: discard the supernatant after centrifugation, gently loosen the cells at the bottom of each tube, add about 10mL of sodium chloride injection to the original centrifuge tube, blow up gently with a 10mL pipette, and transfer the cell suspension evenly into 2 new 50mL centrifuge tubes (if the total number of cells is less than 1200×10 6 cells, transfer them to a 50mL centrifuge tube) and rinse twice in the same way, dilute to 45mL / tube with sodium chloride injection solution, gently pipette up and down to mix for about 3 times with a 10mL pipette, mark the cells; place the cells in an Eppendorf centrifuge, centrifuge at 1400rpm for 8min; discard the supernatant after centrifugation, gently loosen the cells at the bottom of each tube, wash the cells in a 50mL centrifuge tube with sodium chloride injection solution for 3 times, merge into another 50mL centrifuge tube, dilute to 40mL with sodium chloride injection solution, gently pipette up and down to mix for about 3 times with a 10mL pipette; place the cells in an Eppendorf centrifuge, centrifuge at 1400rpm for 8min; Microbiological testing After centrifugation, use a pipette to take about 4 mL of cell supernatant and divide it equally into 4 2 mL cryotubes (1 mL / tube), 1 mL for bacterial detection, 1 mL for mycoplasma detection, and 2 mL for fungal detection. Seal the tubes and affix corresponding labels for microbial detection.

[0030] Collect cells Discard the supernatant after centrifugation and gently loosen the cells at the bottom of each tube; connect a 200mL disposable plastic blood bag to a 50mL disposable syringe and mark it; add about 10mL of sodium chloride injection to the centrifuge tube, gently mix with a 10mL pipette, filter once with a 70μm cell sieve, pass into a 200mL disposable plastic blood bag, repeat 3 times, and discard the used 70μm cell sieve; use a pipette to draw 1mL of albumin and add it to a disposable empty blood collection bag through a 50mL disposable syringe (if the number of cells exceeds 3×10 9 , add 2 mL of albumin); add sodium chloride injection to 150 mL and mix the cell suspension; Endotoxin testing sampling and cryopreservation Use a pipette to take about 1 mL of the final cell product into a 2 mL cryopreservation tube and affix a corresponding label; use a pipette to take about 2 mL of the final cell product into two 2 mL cryopreservation tubes (1 mL / tube), affix corresponding labels, and keep samples; heat seal the tubes, leaving about 1 cm of sealed tube containing the final cell product, affix corresponding labels, and keep samples; check all the information, and after confirming that they are correct, affix corresponding labels on the final cell product bag and packaging box, put them into a self-sealing bag, and then put them into the corresponding packaging box.

[0031] 3. Persister T cell phenotype detection method Take samples at specific times as needed, tap the sampled cell suspension to mix, and use a 10mL disposable syringe to take out 1mL of cell suspension from the sampling port; perform Persister T cell phenotype detection (flow cytometry). Mix the sample cell suspension upside down, add 100μL of cell suspension to each tube 1-6, centrifuge at 300g for 5min, discard the supernatant, and flick the cells at the bottom of the tube, and add fluorescent labeled antibodies according to the table below. (When there are more than one sample, increase the number of sample tubes.) As shown in Table 2 Table 2

[0032] Mix thoroughly, incubate at room temperature in the dark for 30 minutes, then add 2 mL of 1× PBS to each of tubes 2-6 and vortex to mix. Centrifuge at 300 g for 5 minutes, discard the supernatant, flick the cells in the tube, add 1 mL of 1× PBS to resuspend, and test on the analyzer.

[0033] 4. Test results and analysis 1 T CM Cell ratio analysis The lymphocytes in the cell population were preliminarily screened out from cell debris, monocytes, neutrophils, senescent cells, apoptotic cells, etc. by using FSC and SSC values. The cells were stained and labeled with fluorescent-conjugated antibodies according to different cell surface markers to analyze the cell types and subpopulations. There are three subpopulations of memory T cells: (1) effective memory T cells (T EM (2) tissue-resident memory T cells (T cells) RM (3) Central memory T cells (T CM ), through surface marker analysis, CD62L-PE and CD45RO-APC screening can be used to determine the T cells in lymphocytes. CM Cell percentage, T EM +T RM Cell percentage, T NFurthermore, the total T cells can be obtained by using CD4-FITC and CD8-PerCP dyes. CM Cell subsets, i.e. CD4 + Helper T cells and CD8 + Cytotoxic T cell subsets were sorted and further screened using CD62L-PE and CD45RO-APC to further obtain CD4 + Helper T cells—T CM Proportion and CD8 + Killer T cells - T CM For details, see Table 3 below, and the flow sorting diagram is shown in Figure 1-18 As shown, the cells were sequentially compared with the control group 1 to the experimental group M (the following data were obtained by sampling and testing on the 11th day of cell culture).

[0034] Table 3

[0035] Control groups 1-3 are mainly used to verify the effects of different types of basal culture media on T. CM The experimental results show that when 1640, DMEM, and CTS are used as basic culture medium without adding induction components, the CM The total amount of the culture medium had a relatively low impact, and CTS was slightly better than 1640 and DMEM culture medium. In the control groups 4 and 5, albumin and interleukin were added to the basal culture medium, respectively. The test results showed that albumin had a significant effect on T CM The total amount had little effect, but after adding interleukin, T CM The total amount of the experiment group AE was significantly improved, reaching 73.6%. CM The total amount can reach more than 90%. Taking experimental group C as an example, after adding syringaresinol to the culture medium, T CM The total amount can reach 96.8%, and CD4 + Helper T cells, CD8 + The results of cytotoxic T cell typing show that 77.6% belong to CD8 + Cytotoxic T cells, and 87.5% of them are T CM cells, compared with the control group 5, both T CM The total amount and the proportion of cytotoxic cells have been significantly improved, so the addition of syringaresinol and IL-2, IL-7, and IL-15 can play a synergistic role. CM The total amount can reach more than 86%, less than 90%; in experimental groups L and M, syringaresinol was fixed, that is, compared with experimental group C, the basic culture medium was changed to 1640 and DMEM, T CMThe total amount can still be maintained at more than 85% even if it decreases to a certain extent. It can be seen that the optimized supplementary induction medium components of the present invention have a certain synergistic effect with the basic medium.

[0036] Analysis of lymphocyte proportion Using CD3-PerCP dye for labeling and screening, the inventors detected the percentage of T lymphocytes in the control group and the experimental group AM, and found no significant difference, both reaching more than 95%. Fig.19 It shows the proportion of T lymphocytes in lymphocytes. Taking experimental group C as the target group, it is verified that the proportion of T lymphocytes can reach 96.40%.

[0037] Cultivation time analysis: The culture timeline of experimental group C is shown in Table 4. Tests were performed on days 0, 5, 10, and 11. The test results are shown in Table 4. Figure 20-22 , Figure 8 shown.

[0038] Table 4

[0039] On the 12th to 14th day of culture, T CM The total amount did not increase but decreased to a certain extent, so the comprehensive cultivation time and T CM The total amount of cells, D11 was selected as the optimal harvest time.

[0040] T CM Stimulation of cell proliferation in vitro Take experimental group C as the target group to harvest T CM T CM In vitro cell proliferation stimulation assay T CM The cells were co-cultured with dendritic cells loaded with E6 and E7 proteins of human papillomavirus (HPV) using conventional basal culture medium. After a certain period of culture, the number of cells was detected by cell counter and the metabolic activity of cells was detected by CCK-8 method. CM The number of cells is 1×10 8 After 72 hours of co-culture, the cell number increased to 2.5×10 8 The metabolic activity of cells increased by 2 times, indicating that T CM Cells proliferate in response to specific antigen stimulation.

[0041] Cytokine stimulation validation The experimental group C was used as the target group for cytokine stimulation verification experiments. CMThe cells were resuscitated and cultured, and IL-7 and IL-5 were added at 150 IU / mL, and the cell number and cell cycle distribution were detected at different time points. The test results showed that the proportion of cells in the G0 / G1 phase was 70% when not stimulated. After 48 hours of stimulation, the proportion of cells in the G0 / G1 phase decreased to 50%, the proportion of cells in the S phase and G2 / M phase increased, and the cell number increased by 1.8 times.

[0042] T CM Verification of long-term cell culture and proliferation effect Taking experimental group C as the target group, a long-term culture proliferation experiment was carried out. CM The cells were cultured for a long time in a culture system containing IL-2 and basal medium. The cell number and activity were regularly tested and a cell growth curve was drawn. The results showed that the number of cells on the first day of culture was 2×10 8 On day 7, the number of cells was 5×10 8 On day 14, the number of cells was 1×10 9 , showing T CM The cells have good long-term proliferation potential.

[0043] In vitro target cell killing assay Experimental group C was used as the target group, and the tumor cell line non-small cell lung cancer cell line A549 was used as the target cell and T CM The target cells were co-cultured and the target cell death was detected by LDH release method. Experimental results: The LDH activity of target cells cultured alone was 100 U / L, which was similar to that of T cells. CM After 48 hours of co-culture, the LDH activity increased to 500 U / L, and the target cell death rate was calculated to be 40%, indicating that T CM The cells have a certain killing ability in vitro.

[0044] In vivo killing experiment Take experimental group C as the target group and set T CM The cells were transplanted into tumor-bearing mice, and the changes in tumor volume and the survival time of the mice were measured. The tumor-bearing mice were purchased from Saiye Biotechnology. This in vivo killing experiment verified A375 human melanoma tumor-bearing mice. The results showed that the untransplanted T CM The tumor volume of mice transplanted with T cells increased from 100 mm³ to 500 mm³ within 2 weeks, while CM The tumor volume of mice in the experimental group with T cells only increased to 300 mm³ in the same period of time. The average survival time of mice in the experimental group was 40 days, while that in the control group was 30 days, indicating that T CM The cells have a killing effect on tumor cells in the body.

[0045] 9. Immune memory function test Taking experimental group C as the target group, a secondary stimulation reaction experiment was conducted. CM The cells were transplanted into mice by tail vein injection and quickly entered the blood circulation. The cells were stimulated with SARS-CoV-2 spike protein at a concentration of 50 μg / mL and an injection volume of 0.1 mL to observe the immune response and T CM Activation of cells. After the first stimulation, T CM The cell ratio is 10%. After the second stimulation, T CM The proportion of cells increased to 20%, and the antibody level in mouse serum also increased significantly, indicating that T CM The cells have immune memory function in animals.

Claims

1. A method for inducing the expansion of central memory T cells (T CM The method comprises collecting peripheral blood mononuclear cells, adding the cells to a culture medium containing stimulatory CD3 / CD28 antibody-coupled magnetic beads, and amplifying the cells for 7-12 days to obtain T cells. CM A cell population characterized in that The culture medium further contained IL-2, IL-7, IL-15, and syringaresinol.

2. The method according to claim 1, characterized in that: After 8-11 days of expansion, harvest T CM Cells are preferably harvested on day 10-11. CM cell.

3. The culture method according to claim 1, characterized in that After the magnetic beads are added, the magnetic beads are removed twice; the number of times of removing the magnetic beads is preferably 2-3 times; the time for the magnetic beads to contact the cells is 1-3 days.

4. The culture method according to claim 1, characterized in that The culture medium includes a basic culture medium and a supplementary culture medium; the basic amino acids include L-Asp, L-Glu, L-Ile, L-Ser, L-Gly, L-Pro, L-His hydrochloride monohydrate, L-Arg hydrochloride, L-Thr, L-Ala, L-Tyr disodium salt dihydrate, L-Val, L-Met, L-Leu, L-cysteine, L-Phe, L-Lys hydrochloride, L-Trp, L-Asn monohydrate, L-Cys hydrochloride monohydrate, L-hydroxyproline; NaCl, KCl, Na2HPO4, MgSO4, NaHCO3, CaCl2; folic acid, VB12, vitamin C, inositol, biotin.

5. The culture method according to claim 4, characterized in that The basal culture medium can be a mixture of one or more of CTS OpTmizer T cell expansion SFM culture medium and CTS OpTmizer T cell expansion auxiliary concentrated culture medium (CTS), IMDM, DMEM, DMEM / F12, aMEM, RPMI1640, and M199; further, CTS culture medium, DMEM culture medium, and RPMI1640 culture medium are preferred.

6. The culture method according to claim 4, characterized in that The supplemented culture medium includes, but is not limited to, albumin, glutamine, interleukin, and syringaresinol.

7. The culture method according to claim 6, characterized in that: The supplemented culture medium contains glutamine 20-30 μg / mL, albumin 20-50 g / L, IL-2 100-200 IU / mL, IL-7 100-200 IU / mL, IL-15 100-500 IU / mL, and syringaresinol 5-35 μg / L; or, Glutamine 22-27 μg / mL, albumin 20-40 g / L, IL-2 100-200 IU / mL, IL-7 150-200 IU / mL, IL-15 100-400 IU / mL, syringaresinol 10-30 μg / L; or, Glutamine 22-27μg / mL, albumin 20-40 g / L, IL-2 150IU / mL, IL-7 150IU / mL, IL-15 400IU / mL, syringaresinol 10-20μg / L.

8. Use of the central memory T cells obtained by the culture method according to claims 1-7 in the preparation of drugs for treating tumors, infections or other immune-related diseases.

9. Use of syringaresinol in the isolation and culture of central memory T cells, characterized in that: Used to increase T during separation CM The content and purity of CD62L typing in the cell can promote T cell differentiation.

10. Use of syringaresinol, IL-2, IL-7 and IL-15 in the isolation and culture of central memory T cells, characterized in that: Used to increase T during separation CM The content and purity of CD62L typing in the cell can promote T cell differentiation.

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