CN113174369A - Establishment method of immune cell bank with pleiotropic immunoregulation function - Google Patents

本发明公开了一种多效免疫调节功能的免疫细胞库的建立方法，步骤如下：采集胎盘组织血；分离单个核细胞并其分成三份；刺激其中一份单个核细胞得到CD4⁺T细胞；分离另一份单个核细胞中的单核细胞；对分离单核细胞得到的上清液进行B淋巴细胞分选，得到B淋巴细胞、T淋巴细胞和NK细胞；将B淋巴细胞采用细胞因子激活得到LAK细胞；将最后一份单个核细胞采用细胞因子进行诱导，分离悬浮淋巴细胞和贴壁DC细胞；将贴壁DC细胞进行肿瘤特异性抗原负载，得到成熟DC细胞；将悬浮淋巴细胞分成两份，一份负载肿瘤特异性抗原得到CTL细胞；另一份采用细胞因子诱导产生NKT细胞；冻存上述细胞。本申请免疫细胞库具有较好的抗肿瘤效果。The invention discloses a method for establishing an immune cell bank with pleiotropic immune regulation functions. The steps are as follows: collecting placental tissue blood; separating mononuclear cells and dividing them into three parts; stimulating one of the mononuclear cells to obtain CD4 ⁺ T cells; Isolate the monocytes in another mononuclear cell; perform B lymphocyte sorting on the supernatant obtained by isolating the monocytes to obtain B lymphocytes, T lymphocytes and NK cells; activate the B lymphocytes with cytokines LAK cells were obtained; the last mononuclear cells were induced with cytokines, and the suspended lymphocytes and adherent DC cells were separated; the adherent DC cells were loaded with tumor-specific antigens to obtain mature DC cells; the suspended lymphocytes were divided into two parts. One was loaded with tumor-specific antigens to obtain CTL cells; the other was induced by cytokines to generate NKT cells; the above cells were cryopreserved. The immune cell bank of the present application has a good anti-tumor effect.

Establishment method of immune cell bank with pleiotropic immunoregulation function

Technical Field

The invention relates to the technical field of cell bank construction, in particular to a method for establishing an immune cell bank with a pleiotropic immune regulation function.

Background

Tumors are caused by the fact that the immune function of the body is insufficient to overcome various pathogenic factors, and the occurrence and development of the tumors are closely related to the immune state of a host. Traditional tumor treatment methods include surgery, chemotherapy and radiotherapy, which not only involve the risk of tumor migration and recurrence, but also have the serious side effects of damaging normal tissues and immune functions. Since the 80's of the 20 th century, cellular immunotherapy was classified by the national institutes of health as the fourth major tumor treatment modality in addition to the three approaches described above.

The cell immunotherapy mainly uses immune active cells as carriers, and can stimulate immune cells in vivo or in vitro, secrete cytokines to regulate the functions of other immune cells or directly kill tumor cells. At present, immune cells used for clinical treatment mainly include killer cells activated by lymphokines (LAK cells), cytokine-induced killer Cells (CIK), tumor-infiltrating lymphocytes (TIL cells), Dendritic Cells (DCs), natural killer cells (NK), and the like.

At present, in the process of clinically treating tumors by adopting immune cells, the immune cells mainly come from patients, but the immune function of the tumor patients is damaged, the capacity of killing tumor cells is low, and the capacity of killing the tumor cells by the immune cells is enhanced but still lower than the capacity of killing the tumor cells by healthy human immune cells even though the cells are activated by adopting cytokines. In order to overcome the defects, the method is a very good method for establishing an immune cell bank, storing high-quality immune cells and carrying out autoimmune cell treatment when needed in the future in a healthy state. However, the establishment of immune cell banks has the following problems, because the population base of China is large, and the establishment of an individual immune cell bank aiming at each healthy person requires a large amount of manpower, material resources and financial resources to realize the collection, separation and storage of immune cells, so that if a method for breaking through the bottleneck that the immune cells can only carry out self-treatment can be found, the establishment of the immune cell bank has important clinical significance.

Disclosure of Invention

The invention provides a method for establishing an immune cell bank with pleiotropic immunoregulation function, aiming at solving the problems in the prior art.

The invention relates to a method for establishing an immune cell bank with a pleiotropic immunoregulation function, which adopts the following technical scheme.

A method for establishing an immune cell bank with pleiotropic immunoregulation function comprises the following steps:

s1, collecting placenta tissue blood;

s2, separating mononuclear cells from placenta tissue blood; dividing the obtained mononuclear cells into three parts;

s3, stimulating one mononuclear cell obtained in the step S2 by using tumor specific antigen to obtain CD4⁺A T cell;

s4, separating the mononuclear cells in the other mononuclear cell obtained in the step S2;

s5, carrying out B lymphocyte sorting on the supernatant obtained by separating the mononuclear cells in the step S4 to obtain B lymphocytes and a mixture of T lymphocytes and NK cells;

s6, dividing the B lymphocytes obtained in the step S5 into two parts, wherein one part of the B lymphocytes is activated by adopting cell factors to obtain LAK cells;

s7, inducing the last mononuclear cell by adopting a cytokine, and separating suspended lymphocytes and adherent DC cells;

s8, carrying out tumor specific antigen loading on the adherent DC cells obtained in the step S7 to obtain mature DC cells;

s9, dividing the suspension lymphocytes obtained in the step S7 into two parts, and loading tumor specific antigens on one part to obtain CTL cells; the other part adopts cytokine to induce and produce NKT cells;

s10, adding the freezing liquid into the immune cells obtained in the steps S1-S9 for freezing, and coding and warehousing.

Through adopting above-mentioned technical scheme, this application adopts placenta tissue blood as the source of immunocyte, and the immunogenicity of placenta tissue blood is more weak, can tolerate HLA match type not to take to the at utmost, carries out the immunocyte treatment after the separation of placenta tissue blood is induced, can break through the bottleneck that the immunocyte can only carry out the autotherapy, satisfies the requirement of different crowds to the cell therapy, very big saving the required manpower, material resources and the financial resources that drop into of immune cell reservoir of establishing. Moreover, the placental tissue blood contains a large number of lymphocytes with strong proliferation potential, and the clinical treatment effect can be further improved. In addition, the immune cell bank constructed by the application is a collection of various immune cells, has a multi-effect immune regulation function, and can achieve a treatment effect by selecting proper immune cells for reinfusion in the treatment process, so that the time for waiting for cell culture is shortened, the delay of the state of an illness is avoided, and the clinical treatment effect is improved.

Optionally, in step S2, the method for separating mononuclear cells from the blood of the placenta tissue comprises: centrifuging the collected placenta tissue blood at 800-1500rpm/min for 10-20min, adding physiological saline with the volume 1-2 times that of the precipitate into the precipitate obtained by centrifugation, then adding 8% hydroxyethyl starch solution with the volume 0.2-0.4 time that of the precipitate, treating for 5-15min under the condition of 30-50rpm/min, then centrifuging at 600-800rpm/min for 6-8min, and collecting the supernatant to obtain the mononuclear cells.

By adopting the technical scheme, the 8% hydroxyethyl starch solution is adopted to separate mononuclear cells from the placenta tissue blood, the separation operation steps are simple, and the mononuclear cells in the placenta tissue blood can be separated to the maximum extent.

Optionally, in step S3, the tumor specific antigen is NY-ESO-1 antigen; the method for stimulating the mononuclear cells by the NY-ESO-1 antigen comprises the following steps: irradiating mononuclear cells with radiation of 2500-⁺T cells.

By miningBy adopting the technical scheme, the application adopts NY-ESO-1 antigen to stimulate mononuclear cells to obtain CD4⁺T cell, CD4⁺T cells can help activate CD8⁺T cells, which play an important role in the generation and maintenance of immune responses by memory cytotoxic T lymphocytes. Furthermore, CD4 was used⁺T cells can enhance the effectiveness of adoptive transfer immunotherapy, particularly against CD8⁺The T cells can not kill the tumor cells, and the function of killing the tumor cells is achieved.

Optionally, in step S4, the method for separating monocytes is: transferring the mononuclear cells into RPMI-1640 culture solution, wherein the density of the mononuclear cells is 10⁶-10⁷Standing for 2-4h, and sucking supernatant for later use; the adherent cells after the supernatant fluid is sucked are blown down and centrifuged at 600-800rpm/min for 6-8min, and then the mononuclear cells are obtained.

By adopting the technical scheme, the mononuclear cell is obtained by simple separation, has strong phagocytosis capacity and defense capacity, is an important component of body defense, is separated and frozen, and has important significance.

Optionally, in step S5, B lymphocyte sorting is performed on the supernatant obtained in step S4 by using a positive sorting immunomagnetic bead method, the antibody is a CD19 antibody, and the obtained cells are centrifuged at 600-800rpm/min for 6-8min to obtain B lymphocytes; the remaining cells in the supernatant after B lymphocyte sorting are the mixture of T lymphocytes and NK cells.

Through adopting above-mentioned technical scheme, the supernatant that this application will separate monocyte and obtain carries out B lymphocyte and selects separately, obtains the higher B lymphocyte of purity and T lymphocyte and NK cell's mixture, and this application also can adopt just selecting separately the immunomagnetic bead method to carry out T lymphocyte to the solution after B lymphocyte selects separately and select separately, obtains the higher T lymphocyte of purity and NK cell. T lymphocytes, B lymphocytes and NK cells are three most important immune cells of an immune system, and particularly, the NK cells have strong immunoregulation function besides a strong killing function, and have the effects of other various immune cells of an organism to regulate the immune state and the immune function of the organism.

Optionally, in step S6, the LAK cells are obtained by: transferring B lymphocyte into RPMI-1640 culture solution with lymphocyte density of 10⁶-10⁷Adding 80-100ng/ml IL-15 and 50-60ng/ml IL-15 alpha, and 5% CO at 37 deg.C₂Culturing for 24-48h under the condition to obtain LAK cells.

By adopting the technical scheme, the application combines IL-15 and IL-15 alpha to stimulate B lymphocytes to obtain LAK cells, the LAK cells have high anti-tumor activity, the killing activity is exerted without antigen sensitization, tumor cells insensitive to NK cells can be killed, but normal cells have no killing effect, and the LAK cells are a tumor adoptive cell treatment method which is wide in application range and definite in curative effect at present.

Optionally, in step S7, transferring the mononuclear cell into the RPMI-1640 culture solution, wherein the density of the mononuclear cell is 10⁶-10⁷Adding 80-100ng/ml rhGM-CSF to the mixture at 37 deg.C and 5% CO₂Culturing for 72-96h under the condition, and separating the suspended lymphocytes and the attached DC cells.

Optionally, in step S8, culturing the DC cells obtained in step S7 in RPMI-1640 culture medium containing 80-100ng/ml rhSCF and 80-100ng/ml rhIL-4; after 3-4 days of culture, monospecific tumor antigen was added followed by 5% CO at 37 deg.C₂Continuously culturing for 18-36h under the condition; then 80-100ng/ml rhTNF-alpha is added, and 5% CO is added at 37 DEG C₂And continuously culturing for 36-48h under the condition to obtain the mature DC cells.

By adopting the technical scheme, the mature DC cell is obtained through the two steps of operation, and the DC cell can promote the growth of the T cell, so that the T cell with the effect can grow and play a role in the tumor part; and simultaneously can secrete cytokines to regulate the functions of NK cells. The application adopts tumor antigen load with single specificity, enhances the targeting property of immune cell therapy, reduces the incidence rate of graft-versus-host disease and improves the safety of cellular immunotherapy.

Optionally, in step S9, transferring one of the suspension lymphocytes obtained in step S7 to RPMI-1640 for culturingIn liquid, the density of the suspension lymphocytes is 10⁶-10⁷Per ml, 30-60ng/ml alpha-GalCer is added, 5% CO at 37 deg.C₂Culturing for 18-36h under the condition to obtain NKT cells; transferring another part of the suspension lymphocytes obtained in the step S7 into RPMI-1640 culture medium, wherein the density of the suspension lymphocytes is 10⁶-10⁷Adding 80-100ng/ml rhIL-2 and 80-100ng/ml rhIL-15, and reacting at 37 deg.C with 5% CO₂Culturing for 72-96 hr, adding monospecific tumor antigen, and adding 5% CO at 37 deg.C₂Culturing for 18-36h under the condition to obtain CTL cell.

By adopting the technical scheme, the NKT cells and the CTL cells are obtained by respectively inducing the suspended immune cells. The NKT cell has the characteristics of both T cells and NK cells, and can show cytotoxic activity for various tumor NK samples; when the application adopts alpha-GalCer to stimulate the mononuclear cells, NKT cells in the mononuclear cells can generate a large amount of IFN-gamma and IL-4, activate or inhibit NK, DC and CD8⁺T, and the like. CTL cells are the most important effector cells in the anti-tumor reaction of an organism, and are activated by adopting tumor antigens with single specificity, so that the anti-tumor specificity is endowed on one hand, and the safety of cellular immunotherapy is improved on the other hand.

Optionally, in step S10, the immune cell is administered 10 times³-10⁸Freezing and storing at the concentration of/ml; the frozen stock solution comprises the following components: 3-5% of DMSO, 30-40% of placenta serum, 10-15% of human serum albumin and 40-57% of basal medium.

By adopting the technical scheme, the frozen concentration of the immune cells is optimized and strictly controlled by the components of the frozen liquid, so that the immune cells after being frozen can keep higher survival rate, and the treatment effect of cellular immunotherapy is effectively ensured.

The present invention obtains the following advantageous effects.

1. The placenta tissue blood is used as the source of the immune cells, so that the bottleneck that the immune cells can only carry out self-treatment is broken through, and the manpower, material resources and financial resources required for building an immune cell storage library are saved;

2. the immune cell bank constructed by the application is a collection of various immune cells, has a multi-effect immune regulation function, and can be used for enabling a patient to select proper immune cells to return in the treatment process, so that the treatment effect can be achieved, the time for waiting for cell culture is shortened, the delay of the state of an illness is avoided, and the clinical treatment effect is improved;

3. the components of the frozen stock solution and the proportion among the components are continuously optimized, so that the survival rate of immune cells after freezing is effectively guaranteed, and the treatment effect of cellular immunotherapy is guaranteed.

Detailed Description

The present invention will be further described with reference to examples.

Example 1

A method for establishing an immune cell bank with pleiotropic immunoregulation function comprises the following steps:

s1, collecting placenta tissue blood;

s2, centrifuging the collected placenta tissue blood at 800rpm/min for 20min, adding 1 time of physiological saline in volume of the precipitate into the precipitate obtained by centrifugation, adding 0.2 time of 8% hydroxyethyl starch solution in volume of the precipitate, treating for 15min under the condition of 30rpm/min, centrifuging at 800rpm/min for 6min, and collecting supernatant to obtain mononuclear cells; dividing the obtained mononuclear cells into three parts;

s3, radiating one part of mononuclear cells obtained in the step S2 by adopting 3500rad of radiation dose, then adding 8ug/ml NY-ESO-1 antigen, culturing and incubating for 40min in a serum-free culture medium, washing the incubated cells to obtain CD4⁺A T cell;

s4, transferring another single nucleus cell obtained in the step S2 into an RPMI-1640 culture solution, wherein the density of the single nucleus cell is 10⁶Standing for 2h, and sucking supernatant for later use; blowing off adherent cells after the supernatant is sucked, and centrifuging at 800rpm/min for 6min to obtain mononuclear cells;

s5, carrying out B lymphocyte sorting on the supernatant obtained in the step S4 by adopting a positive sorting immunomagnetic bead method, wherein the antibody is a CD19 antibody, and centrifuging the obtained cells at 800rpm/min for 6min to obtain lymphocyte B cells; the rest cells in the supernatant after B lymphocyte sorting are the mixture of T lymphocytes and NK cells;

s6, dividing the B lymphocytes obtained in the step S5 into two parts, transferring one part of the B lymphocytes into RPMI-1640 culture solution, wherein the density of the lymphocytes is 10⁶Adding 80ng/ml IL-15 and 60ng/ml IL-15 alpha, 5% CO at 37 ℃₂Culturing for 48h under the condition to obtain LAK cells;

s7, transferring the last single nucleus cell into RPMI-1640 culture solution, wherein the density of the single nucleus cell is 10⁷Adding 100ng/ml rhGM-CSF to the solution at 37 deg.C and 5% CO₂Culturing for 72h under the condition, and separating suspended lymphocytes and adherent DC cells;

s8, continuously culturing the DC cells obtained in the step S7 in an RPMI-1640 culture solution containing 80ng/ml of rhSCF and 100ng/ml of rhIL-4; after 4 days of culture, monospecific tumor antigen was added followed by 5% CO at 37 deg.C₂Continuously culturing for 18h under the condition; then 100ng/ml rhTNF-alpha is added, and 5% CO is added at 37 DEG C₂Continuously culturing for 48h under the condition to obtain mature DC cells;

s9, dividing the suspension lymphocytes obtained in the step S7 into two parts, transferring one part of the suspension lymphocytes obtained in the step S7 into RPMI-1640 culture solution, wherein the density of the suspension lymphocytes is 10⁷Per ml, 30ng/ml of alpha-GalCer was added, 5% CO at 37 ℃₂Culturing for 18h under the condition to obtain NKT cells; transferring another part of the suspension lymphocytes obtained in the step S7 into RPMI-1640 culture medium, wherein the density of the suspension lymphocytes is 10⁷Adding 100ng/ml rhIL-2 and 80ng/ml rhIL-15, and keeping the temperature at 37 ℃ with 5% CO₂Culturing for 72 hr, adding tumor antigen with single specificity, and culturing at 37 deg.C

5％CO₂Continuously culturing for 36h under the condition to obtain CTL cells;

s10, adding a freezing solution into the immune cells obtained in the steps S1-S9 for freezing, and coding and warehousing; the concentration of immune cells is 10³Per ml; the frozen stock solution comprises the following components: 3% DMSO, 40% placental serum, 10% human serum albumin, 47% basal medium.

Performing quality detection on the obtained immune cells before step S10, wherein the number of live cells before and after cryopreservation is detected by using a cell counter using a trypan blue staining method; the mycoplasma detection adopts a PCR method; the sterility test adopts a culture method; HBV, HIV and HCV adopt an ELISA method; bacterial endotoxin was limited by bacterial endotoxin. The results are shown in Table 1.

TABLE 1

As can be seen from Table 1, the immune cells prepared by the embodiment have high survival rate, the survival rate can still be more than 80% even after the frozen storage, and the immune cells have no virus or bacterial pollution and can meet the requirements of clinical use. The experimental results show that the immune cell bank with the pleiotropic immune regulation function is successfully constructed by adopting the method disclosed by the embodiment of the application.

Example 2

A method for establishing an immune cell bank with pleiotropic immunoregulation function comprises the following steps:

s1, collecting placenta tissue blood;

s2, centrifuging the collected placenta tissue blood at 1500rpm/min for 10min, adding physiological saline with 2 times of the volume of the precipitate into the precipitate obtained by centrifugation, adding 8% hydroxyethyl starch solution with 0.4 time of the volume of the precipitate, treating for 5min under the condition of 50rpm/min, centrifuging at 600rpm/min for 8min, and collecting supernatant to obtain mononuclear cells; dividing the obtained mononuclear cells into three parts;

s3, irradiating one part of mononuclear cells obtained in the step S2 by adopting 2500rad radiant quantity, then adding 12ug/ml NY-ESO-1 antigen, culturing and incubating for 90min in a serum-free culture medium, and washing the incubated cells to obtain CD4⁺A T cell;

s4, obtaining the step S2The obtained another single nucleus cell is transferred into RPMI-1640 culture solution, and the density of the single nucleus cell is 10⁷Standing for 4h, and sucking supernatant for later use; blowing off adherent cells after the supernatant is sucked, and centrifuging at 600rpm/min for 8min to obtain mononuclear cells;

s5, carrying out B lymphocyte sorting on the supernatant obtained in the step S4 by adopting a positive sorting immunomagnetic bead method, wherein the antibody is a CD19 antibody, and centrifuging the obtained cells at 600rpm/min for 8min to obtain lymphocyte B cells; the rest cells in the supernatant after B lymphocyte sorting are the mixture of T lymphocytes and NK cells;

s6, dividing the B lymphocytes obtained in the step S5 into two parts, transferring one part of the B lymphocytes into RPMI-1640 culture solution, wherein the density of the lymphocytes is 10⁷Per ml, 100ng/ml IL-15 and 50ng/ml IL-15 α were added, 5% CO at 37 ℃₂Culturing for 24h under the condition to obtain LAK cells;

s7, transferring the last single nucleus cell into RPMI-1640 culture solution, wherein the density of the single nucleus cell is 10⁶Adding 80ng/ml rhGM-CSF to the solution at 37 deg.C and 5% CO₂Culturing for 96h under the condition, and separating suspended lymphocytes and adherent DC cells;

s8, continuously culturing the DC cells obtained in the step S7 in an RPMI-1640 culture solution containing 100ng/ml of rhSCF and 80ng/ml of rhIL-4; after 3 days of culture, monospecific tumor antigen was added followed by 5% CO at 37 deg.C₂Continuously culturing for 36h under the condition; then 80ng/ml rhTNF-alpha is added, and 5% CO is added at 37 DEG C₂Continuously culturing for 36h under the condition to obtain mature DC cells;

s9, dividing the suspension lymphocytes obtained in the step S7 into two parts, transferring one part of the suspension lymphocytes obtained in the step S7 into RPMI-1640 culture solution, wherein the density of the suspension lymphocytes is 10⁶Per ml, 60ng/ml α -GalCer was added, 5% CO at 37 deg.C₂Culturing for 36h under the condition to obtain NKT cells; transferring another part of the suspension lymphocytes obtained in the step S7 into RPMI-1640 culture medium, wherein the density of the suspension lymphocytes is 10⁶Adding 80ng/ml rhIL-2 and 100ng/ml rhIL-15, and reacting at 37 deg.C with 5% CO₂Culturing for 96h under the condition, and adding monospecificSexual tumor antigen, 5% CO at 37 deg.C₂Continuously culturing for 18h under the condition to obtain CTL cells;

s10, adding a freezing solution into the immune cells obtained in the steps S1-S9 for freezing, and coding and warehousing; the concentration of immune cells is 10⁸Per ml; the frozen stock solution comprises the following components: 5% DMSO, 33% placental serum, 15% human serum albumin, 47% basal medium.

Performing quality detection on the obtained immune cells before step S10, wherein the number of live cells is detected by trypan blue staining using a cell counter; the mycoplasma detection adopts a PCR method; the sterility test adopts a culture method; HBV, HIV and HCV adopt an ELISA method; bacterial endotoxin was limited by bacterial endotoxin. The results are shown in Table 2.

TABLE 2

As can be seen from Table 2, the immune cells prepared by the embodiment have high survival rate, the survival rate can still be more than 80% even after the frozen storage, and the immune cells have no virus or bacterial pollution and can meet the requirements of clinical use. The experimental results show that the immune cell bank with the pleiotropic immune regulation function is successfully constructed by adopting the method disclosed by the embodiment of the application.

Example 3

A method for establishing an immune cell bank with pleiotropic immunoregulation function comprises the following steps:

s1, collecting placenta tissue blood;

s2, centrifuging the collected placenta tissue blood at 1000rpm/min for 15min, adding 1.5 times of physiological saline in volume of the precipitate into the precipitate obtained by centrifugation, then adding 0.3 times of 8% hydroxyethyl starch solution in volume of the precipitate, treating for 10min under the condition of 40rpm/min, then centrifuging at 700rpm/min for 7min, and collecting supernatant to obtain mononuclear cells; dividing the obtained mononuclear cells into three parts;

s3, radiating one part of mononuclear cells obtained in the step S2 by adopting 3000rad of radiation, then adding 10ug/ml of NY-ESO-1 antigen, culturing and incubating for 60min in a serum-free culture medium, and washing the incubated cells to obtain CD4⁺A T cell;

s4, transferring another single nucleus cell obtained in the step S2 into an RPMI-1640 culture solution, wherein the density of the single nucleus cell is 5 multiplied by 10⁶Standing for 3h, and sucking supernatant for later use; blowing off adherent cells after the supernatant is sucked, and centrifuging at 700rpm/min for 7min to obtain mononuclear cells;

s5, carrying out B lymphocyte sorting on the supernatant obtained in the step S4 by adopting a positive sorting immunomagnetic bead method, wherein the antibody is a CD19 antibody, and centrifuging the obtained cells at 700rpm/min for 7min to obtain lymphocyte B cells; the rest cells in the supernatant after B lymphocyte sorting are the mixture of T lymphocytes and NK cells;

s6, dividing the B lymphocytes obtained in the step S5 into two parts, transferring one part of the B lymphocytes into RPMI-1640 culture solution, wherein the density of the lymphocytes is 5 multiplied by 10⁶Adding 90ng/ml IL-15 and 55ng/ml IL-15 alpha, 5% CO at 37 ℃₂Culturing for 36h under the condition to obtain LAK cells;

s7, transferring the last single nucleus cell into RPMI-1640 culture solution, wherein the density of the single nucleus cell is 5 multiplied by 10⁶Adding 90ng/ml rhGM-CSF to the solution at 37 deg.C and 5% CO₂Culturing for 84h under the condition, and separating suspended lymphocytes and adherent DC cells;

s8, continuously culturing the DC cells obtained in the step S7 in an RPMI-1640 culture solution containing 90ng/ml of rhSCF and 90ng/ml of rhIL-4; after 3 days of culture, monospecific tumor antigen was added followed by 5% CO at 37 deg.C₂Continuously culturing for 24h under the condition; then adding 90ng/ml rhTNF-alpha and 5% CO at 37 DEG C₂Continuously culturing for 48h under the condition to obtain mature DC cells;

s9, dividing the suspension lymphocyte obtained in the step S7 into two parts, and transferring the suspension lymphocyte obtained in the step S7 to RPMI-1640 for cultureIn the nutrient solution, the density of suspension lymphocytes is 5 × 10⁶Per ml, 50ng/ml α -GalCer was added, 5% CO at 37 deg.C₂Culturing for 24h under the condition to obtain NKT cells; another portion of the suspension lymphocytes obtained in step S7 was transferred to RPMI-1640 medium at a density of 5X 10⁶Adding 90ng/ml rhIL-2 and 90ng/ml rhIL-15, and keeping the temperature at 37 ℃ with 5% CO₂Culturing for 84h under the condition, adding monospecific tumor antigen, and adding 5% CO at 37 deg.C₂Continuously culturing for 24h under the condition to obtain CTL cells;

s10, adding a freezing solution into the immune cells obtained in the steps S1-S9 for freezing, and coding and warehousing; the concentration of immune cells is 10⁵Per ml; the frozen stock solution comprises the following components: 5% DMSO, 35% placental serum, 10% human serum albumin, 50% basal medium.

Performing quality detection on the obtained immune cells before step S10, wherein the number of live cells is detected by trypan blue staining using a cell counter; the mycoplasma detection adopts a PCR method; the sterility test adopts a culture method; HBV, HIV and HCV adopt an ELISA method; bacterial endotoxin was limited by bacterial endotoxin. The results are shown in Table 3.

TABLE 3

As can be seen from Table 3, the immune cells prepared by the method have high survival rate, the survival rate can still be more than 80% even after the frozen storage, and the immune cells have no virus or bacterial pollution and can meet the requirements of clinical use. The experimental results show that the immune cell bank with the pleiotropic immune regulation function is successfully constructed by adopting the method disclosed by the embodiment of the application.

The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.