CN114134111B - Immune cell activator and preparation method and application thereof - Google Patents

Immune cell activator and preparation method and application thereof Download PDF

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CN114134111B
CN114134111B CN202111487820.4A CN202111487820A CN114134111B CN 114134111 B CN114134111 B CN 114134111B CN 202111487820 A CN202111487820 A CN 202111487820A CN 114134111 B CN114134111 B CN 114134111B
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activator
cells
immune
vitamin
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CN114134111A (en
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孔伟圣
蓝欣
黄海娟
陈智妍
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Zhuhai Basso Cell Science And Technology Co ltd
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Abstract

The invention provides an immune cell activator and a preparation method and application thereof, belonging to the technical field of cell products. Immune cell activating agent includes immunomodulator, cephalosporin antibiotics, monoclonal antibody medicine, vitamin, heparin sodium and recombinant human protein. The invention uses the superior cell factor, medical recombinant protein and reagent, does not introduce exogenous animal-derived protein, has definite components, can efficiently induce and activate immune cells, improves the purity of the immune cells and the cell number, and has higher market competitiveness.

Description

Immune cell activator and preparation method and application thereof
Technical Field
The invention belongs to the technical field of cell products, and particularly relates to an immune cell activator and a preparation method and application thereof.
Background
Immune cell therapy has been growing in a debate as a post-treatment for four major cancers. As nobel medical physiology awards are issued to two immunotherapeutic scientists and immunotherapeutic drugs continue to enter the market, the public is beginning to pay more attention to immunotherapy.
Currently, cellular immunotherapy achieves significant efficacy in chronic myelogenous leukemia, acute myelogenous leukemia, non-hodgkin lymphoma, and multiple myeloma. China also follows the global steps in the field of cellular immunotherapy, more and more domestic pharmaceutical enterprises are actively put into research and development lines, and related enterprises are expected to meet the long-term development opportunity along with the gradual increase of the number of medicines on the market and the increasing abundance of batch-obtaining indications.
Cellular immunotherapy refers to the acquisition of immune cells from a patient, followed by in vitro culture and expansion, and then reinfusion into the patient to stimulate and enhance the autoimmune function of the body to treat tumors, and adoptive cellular immunotherapy includes Tumor Infiltrating Lymphocyte (TIL) therapy, TCR-T cell therapy, CAR-T cell therapy, lymphokine Activated Killer (LAK) therapy, cytokine Induced Killer (CIK) therapy, dendritic cell therapy, natural Killer (NK) therapy, and the like.
The number, purity and activity of immune cells are key elements of immune cell therapy, and these key factors are, in addition to the various factors added, also an important factor is the carrier-cell culture medium in which immune cells are cultured in vitro.
The cell culture medium used for the in vitro culture of the immune cells is a serum-free culture medium, and the serum-free culture medium is a synthetic culture medium which can maintain the growth and propagation of the cells in vitro for a long time without adding serum. Compared with the cells of the traditional cell culture medium, the serum-free medium does not contain serum, so that the repeatability, accuracy and stability of the experiment are improved; the problems of blood-borne pollution and the like caused by serum are avoided, and the damage of unknown components of the serum to cells is reduced; the protein in the serum-free culture medium is derived from recombinant protein or protein hydrolysate, so that the experimental efficiency is improved; the serum-free culture medium has relatively definite components and content, can improve the quality of cell products and is favorable for separating and purifying the products. However, the serum-free cell culture medium has the defects of low expansion times, poor induced differentiation capability and weak tumor killing activity of the existing serum-free cell culture medium for expanding immune cells. Therefore, development of a high-efficiency immune cell activator which is applied to a serum-containing or serum-free cell culture medium can not only meet the requirement of high-efficiency induction of cell activation, but also improve the expansion times and enhance the tumor killing activity, and meet the requirement of clinical application of immune cells.
Disclosure of Invention
Accordingly, the present invention is directed to an immune cell activator, a preparation method and an application thereof, which can efficiently induce immune cell activation and proliferation.
The invention provides an immune cell activator, which comprises the following components in parts by mass and 100-100 IU of heparin sodium: 102 to 450 parts of immunomodulator, 250 to 900 parts of cephalosporin antibiotics, 1.5 to 20 parts of monoclonal antibody medicine, 3000 to 110000 vitamins and 52 to 140 parts of recombinant human protein.
Preferably, the immunomodulator comprises Mycobacterium phlei F.U.36 injection, levamisole and ribonucleic acid.
Preferably, the mass ratio of the mycobacterium phlei F.U.36 injection, levamisole and ribonucleic acid is (1-100): (1-50): (100-300).
Preferably, the cephalosporin antibiotics include cefaclor, cefodizime and ceftazidime.
Preferably, the mass ratio of the cefaclor, the cefdezine and the ceftazidime is (1-4): (2-8): (2-6).
Preferably, the monoclonal antibody drug includes a CD50 monoclonal antibody and a CD52 monoclonal antibody;
the mass ratio of the CD50 monoclonal antibody to the CD52 monoclonal antibody is (0.5-10): (1-10).
Preferably, the vitamins include vitamin a, vitamin C, and vitamin E; the mass ratio of the vitamin A, the vitamin C and the vitamin E is (1-50): (1-50): (1-50).
Preferably, the recombinant human protein comprises recombinant human albumin, immunoglobulin, transferrin; the mass ratio of the recombinant human albumin to the immunoglobulin to the transferrin is (1-20): (1-20): (50-100).
Preferably, the immune cell activator comprises the following concentrations of components: mycobacterium phlei F.36 injection 1-100 μg/ml, levamisole 1-50 μg/ml, ribonucleic acid 100-300 μg/ml, cefaclor 50-200 μg/ml, cefodizime 100-400 μg/ml, ceftazidime 100-300 μg/ml, CD50 monoclonal antibody 0.5-10 μg/ml, CD52 monoclonal antibody 1-10 μg/ml, vitamin A1-10 mg/ml, vitamin C1-50 mg/ml, vitamin E1-10 mg/ml, heparin sodium 100-1000 IU/ml, recombinant human albumin 1-20 μg/ml, recombinant human immunoglobulin 1-20 μg/ml, recombinant human transferrin 50-100 μg/ml.
The invention provides a serum-free cell culture medium, which comprises the immune cell activator and a cell nutrition component.
The invention provides an application of the immune cell activator or the serum-free cell culture medium in immune cell culture.
Preferably, the immune cells comprise human peripheral blood immune cells;
the human peripheral blood immune cells comprise one or more of the following: cytokine-induced killer cells, LAK cells, natural killer cells, NKT cells, CTLs, and γδ T cells.
The immune cell activator provided by the invention comprises the following components in parts by mass and 100-100 IU of heparin sodium: 102 to 450 parts of immunomodulator, 250 to 900 parts of cephalosporin antibiotics, 1.5 to 20 parts of monoclonal antibody medicine, 3000 to 110000 vitamins and 52 to 140 parts of recombinant human protein. The immunomodulator of the activator component is an immunopotentiator, can promote the activation of immune cells, promote the secretion of specific antibodies and various cytokines, and enhance the functions of the immune cells. The cephalosporin antibiotics can stimulate lymphocyte proliferation and enhance cell activity; monoclonal antibody medicine promotes lymphocyte differentiation and proliferation; vitamins are bioactive substances for maintaining cell growth, play a role in regulating and controlling in cell metabolism, can increase cell membrane stability, improve cell viability and can maximally preserve cell viability; recombinant human proteins have a variety of physiological functions in cell culture, including heavy metal chelators, free radical scavengers, modulators of osmotic pressure and pH, stabilizers, carriers for growth factors, surfactants, nutrients, and the like; heparin sodium and growth factors act synergistically to promote cell proliferation. The components of the activator are mutually cooperated, so that the activating proliferation of cells can be effectively induced, and the tumor killing activity of immune cells can be improved. When the immune cells are cultured, the activator is added into the culture solution, so that the purity, activity and cell number of the immune cells can be effectively improved under the same condition, the in-vitro induced culture cells are simpler and more efficient, and better guarantee is provided for immune cell treatment. The high-efficiency immune cell activator uses the superior cell factor, the medical recombinant protein and the reagent, does not introduce exogenous animal source protein, has definite components, can efficiently induce and activate immune cells, and improves the purity and the cell number of the immune cells.
Drawings
FIG. 1 is a graph showing the proliferation number of NK cells in vitro in example 1;
FIG. 2 is a graph showing the proliferation amount of CIK culture in vitro in example 2;
FIG. 3 is a graph showing the proliferation amount of γδT culture in example 3.
Detailed Description
The invention provides an immune cell activator, which comprises the following components in parts by mass and 100-100 IU of heparin sodium: 102 to 450 parts of immunomodulator, 250 to 900 parts of cephalosporin antibiotics, 1.5 to 20 parts of monoclonal antibody medicine, 3000 to 110000 vitamins and 52 to 140 parts of recombinant human protein.
In the present invention, the immunomodulator preferably includes Mycobacterium phlei F.U.36 injection, levamisole and ribonucleic acid. The mass ratio of the mycobacterium phlei F.U.36 injection to the levamisole to the ribonucleic acid is preferably (1-100): (1-50): (100 to 300), more preferably (2 to 8): (1-4): (15-25), more preferably 1:1:4. Mycobacterium phlei F.U.36 injection, levamisole and ribonucleic acid are used as immunopotentiator, which can promote the activation of immune cells, promote the secretion of specific antibodies and various cytokines and enhance the functions of the immune cells.
In the examples of the present invention, mycobacterium phlei F.U.36 injection was purchased from Chengdu Jinxing health pharmaceutical Co. The levamisole was purchased from Guangdong North China pharmaceutical Co. Ribonucleic acid was purchased from Henan Tianzhi pharmaceutical Co.
In the present invention, the cephalosporin antibiotics preferably include cefaclor, cefditoren and ceftazidime. The mass ratio of the cefaclor, the cefdezine and the ceftazidime is preferably (1-4): (2-8): (2 to 6), more preferably (8 to 15): (10-30): (10-20), most preferably 1:1:1. Cefaclor, cefdezine and ceftazidime stimulate lymphocyte proliferation and enhance cell activity. In the present example, cefaclor, cefditoren and ceftazidime are all purchased from li-bead pharmaceutical factory, li-bead group.
In the present invention, the monoclonal antibody drug preferably includes a CD50 monoclonal antibody and a CD52 monoclonal antibody. The mass ratio of the CD50 monoclonal antibody to the CD52 monoclonal antibody is preferably (0.5-10): (1 to 10), more preferably (3 to 8): (3-7), more preferably 1:1.CD50 monoclonal antibodies and CD52 monoclonal antibodies promote lymphocyte differentiation and proliferation. In an embodiment of the invention, the CD50 monoclonal antibody and CD52 monoclonal antibody were purchased from bio-technology limited, san francisco, beijing.
In the present invention, the vitamins preferably include vitamin a, vitamin C and vitamin E. The mass ratio of the vitamin A, the vitamin C and the vitamin E is preferably (1-50): (1-50): (1 to 50), more preferably (1 to 4): (1-4): (1 to 4), most preferably 1:5:1. vitamin A, vitamin C and vitamin E are bioactive substances for maintaining cell growth, play a role in regulating and controlling cell metabolism, can increase cell membrane stability, improve cell activity, and can maximally preserve cell activity.
In the present invention, the recombinant human protein preferably includes recombinant human albumin, immunoglobulin, transferrin. The mass ratio of the recombinant human albumin, the immunoglobulin and the transferrin is preferably (1-20): (1-20): (50 to 100), more preferably (5 to 15): (1-20): (70-90), most preferably 1:1:5. Recombinant human albumin, immunoglobulins, transferrin have a variety of physiological functions in cell culture including heavy metal chelators, free radical scavengers, modulators of osmotic pressure and pH, stabilizers, carriers for growth factors, surfactants, nutrients and the like. In the examples of the present invention, recombinant human albumin, immunoglobulin, transferrin were purchased from Shanghai JieTeberlin Bio-pharmaceutical Co., ltd, shanghai Laishi blood products Co., ltd, shanghai derived leaf Biotechnology Co., respectively.
In the present invention, the immune cell activator preferably comprises the following components in the following concentrations: mycobacterium phlei F.36 injection 1-100 μg/ml, levamisole 1-50 μg/ml, ribonucleic acid 100-300 μg/ml, cefaclor 50-200 μg/ml, cefradine 100-400 μg/ml, ceftazidime 100-300 μg/ml, CD50 monoclonal antibody 0.5-10 μg/ml, CD52 monoclonal antibody 1-10 μg/ml, vitamin A1-10 mg/ml, vitamin C1-50 mg/ml, vitamin E1-10 mg/ml, heparin sodium 100-1000 IU/ml, recombinant human albumin 1-20 μg/ml, recombinant human immunoglobulin 1-20 μg/ml, recombinant human transferrin 50-100 μg/ml, more preferably Mycobacterium phlei F.36 injection 50 μg/ml, levamisole 50 μg/ml, ribose nucleic acid 200 μg/ml, cefradine 100 μg/ml, vitamin A1-10 mg/ml, recombinant human albumin 10 IU, recombinant human albumin 50 μg/ml, heparin sodium 100 μg/ml, recombinant human albumin 50 μg/100 μg/ml. The components of the activator are mutually cooperated, so that the activating proliferation of cells can be effectively induced, and the tumor killing activity of immune cells can be improved.
The invention provides a serum-free cell culture medium, which comprises the immune cell activator and a cell nutrition component.
In the invention, the immune cell activator is used for preparing a serum-free cell culture medium, and the serum-free cell culture medium is obtained by diluting 100 times of cell nutrition components. The composition and content of the cell nutrient components are not particularly limited in the present invention, and components of serum-free cell culture media well known in the art are employed.
The invention provides an application of the immune cell activator or the serum-free cell culture medium in immune cell culture.
In the present invention, the immune cells preferably include human peripheral blood immune cells. The human peripheral blood immune cells preferably comprise one or more of the following: cytokine-induced killer cells, LAK cells, natural killer cells, NKT cells, CTLs, and γδ T cells. When the serum-free cell culture medium prepared by the invention is used for culturing immune cells, the activator is added into the culture solution, so that the purity, activity and cell number of the immune cells can be effectively improved under the same condition, the in-vitro induced culture cells are simpler and more efficient, and better guarantee is provided for immune cell treatment. The high-efficiency immune cell activator uses the superior cell factor, the medical recombinant protein and the reagent, does not introduce exogenous animal source protein, has definite components, can efficiently induce and activate immune cells, and improves the purity and the cell number of the immune cells.
The following examples are provided to illustrate an immune cell activator, a method for preparing the same and an application thereof in detail, but they should not be construed as limiting the scope of the invention.
The experimental environment, experimental materials and instruments are as follows:
1. experimental environment: operating in a clean bench in a laboratory in a GMP environment.
2. Reagent: phosphate buffer PBS (Martensitic Soxhlet Biotechnology Co., ltd.), ALyS505N-0, ALyS505NK-AC, ALyS505NK-EX serum-free medium, lynectin-NK, lynectin- γδT (CSTI from Co., ltd.), human lymphocyte isolate, sodium heparin solution, CD3 monoclonal antibody, IL-1α, IL-2, IL-21, IL-15.
3. Instrument and apparatus: centrifuges (Thermo, U.S.), T75 suspension flasks, T175 suspension flasks, NIPRO cell culture bags (japan NIPRO corporation), CO2 incubators (sanyang, china), ultra clean benches (zhijing, china).
Example 1
The preparation of immune cell activator and its application in NK cell culture includes the following steps:
(1) The immune cell activator formulation is as follows: mycobacterium phlei F.36 injection 50 μg/ml, levamisole 50 μg/ml, ribonucleic acid 200 μg/ml, cefaclor 100 μg/ml, cefradine 100 μg/ml, CD50 monoclonal antibody 10 μg/ml, CD52 monoclonal antibody 10 μg/ml, vitamin A1mg/ml, vitamin C5 mg/ml, vitamin E1mg/ml, heparin sodium 500IU/ml, recombinant human albumin 10 μg/ml, recombinant human immunoglobulin 10 μg/ml, recombinant human transferrin 50 μg/ml. After the preparation, the mixture was added to ALyS505N-AC medium at a ratio of 1:100.
(2) A heparin sodium anticoagulation blood collection tube is used for collecting 40ml of peripheral blood of a healthy person, 20ml of fresh blood is slowly poured onto 15ml of lymph separation liquid, and 800g is centrifuged for 20min, and the blood is slowly lifted and lowered. The upper plasma is collected by a sterile straw and is collected to a centrifuge tube, the mononuclear cell layer is not required to be sucked by mistake, the plasma is heated at 56 ℃ for 30min, and is centrifuged at room temperature for 10min by 1200g, the supernatant is collected to a new centrifuge tube by the straw, and the centrifuge tube is preserved at 4 ℃ until the supernatant is taken out before use. The white film layer was collected into a new 50ml centrifuge tube by a pipette, and the cells were washed 3 times with DPBS for further use.
(3) Preparation of an activation medium: IL-2, IL-12 and IL-15 were added to 200ml ALyS505N-AC culture at a final concentration of IL-2 500IU/ml, IL-12 50IU/ml and IL-15 20IU/ml; preparation of amplification medium: IL-2 was added to ALyS505N-EX at a concentration of 500IU/ml.
(4) Adding 1ml of Lymantin-NK and 4ml of DPBS into a non-TC-treated T75 culture flask, fully and uniformly mixing, incubating for 1h at room temperature, discarding the coating liquid, adding 10ml of DPBS, washing the culture flask, adding 18ml of activation culture medium and 2ml of autologous plasma, adding PBMC prepared in the step (2), and adjusting the cell density to 1.3X10 6 Each ml, at 37℃and 5% CO 2 Culturing in an incubator; day3 and Day5 were added to 40ml and 140ml of the activation medium, respectively, and the culture was continued. Culturing until Day7 is transferred to 2 cell culture bags for expansion culture, and adding 400, 700 and 900ml (equally dividing two culture bags) of amplification culture medium every two days (namely Day7, day9 and Day 11) to maintain the cell density at 5-7×10 5 Every ml, stand at 37 ℃ 5% CO 2 Culturing in an incubator.
(5) The cells were cultured to Day14, counted, washed by centrifugation, and collected, and the number and the viability of the cells were measured. Fluorescent antibody is added to flow cytometry for detecting CD3 by flow cytometry - CD56 + Is a ratio of (2).
(6) Determination of cell killing Activity: taking K562 cells in logarithmic growth phase as target cells, NK cells as effector cells, setting experimental groups with the effective target ratios of 2:1, 4:1 and 5:1 respectively, and setting three compound holes in each group. The density of K562 cells is adjusted to 20 ten thousand/ml; regulating NK cell density according to the effective target ratio, and adding 50 μl of effector cells and target cell suspension into each well; simultaneously taking 50 μl of effector cell suspension, and adding 50 μl of 1640 culture medium to each well; mu.l of the target cell suspension was taken and 50. Mu.l of 1640 medium was added to each well as a natural release group, each group being provided with three duplicate wells. Placing in 37 ℃ and 5% CO 2 Culturing in incubator for 6 hr, taking out 96-well plate, adding 20 μl of CCK-8 solution into each well, adding 37 ℃ and 5% CO 2 After incubation for 90min in the incubator, detection was performed with a microplate reader at a wavelength of 450 nm. And calculating the killing rate according to a formula I.
Killing rate (%) = (1- (experimental group-control group)/natural release group) formula I
The following control groups were also set up in this example:
control group 1
The difference from example 1 is that no activator is added, and the rest is the same as example 1.
Control group 2
The components of the activator were different from those of example 1, and Mycobacterium phlei F.U.36 injection, levamisole and ribonucleic acid in the activator were omitted, and the rest was the same as in example 1.
Control group 3
Unlike example 1, the components of the activator were different from those of the activator, and cefaclor, cefdezine and ceftazidime in the activator were omitted, and the rest was the same as in example 1.
Control group 4
The difference from example 1 is that the components of the activator are different, and the CD50 mab and CD52 mab in the activator are omitted, and the rest is the same as example 1.
Control group 5
The components of the activator were different from those of example 1, except that vitamin A, vitamin C and vitamin E in the activator were omitted, and the rest was the same as in example 1.
Control group 6
Except for the difference in the composition of the activator from example 1, heparin sodium in the activator was omitted, and the rest was the same as in example 1.
Control group 7
The recombinant human proteins in the activator were omitted as albumin, immunoglobulin and transferrin, except that the components of the activator were different from those of example 1, and the rest was the same as in example 1.
The results of the detection of the immune cell activators prepared in example 1 and control groups 1 to 7 are shown in Table 1.
TABLE 1 cell culture results for immune cell activators prepared in example 1 and control groups 1 to 7
As shown in Table 1, the NK cells in example 1 had the highest cell number and flow phenotype, and the strongest killing function, and the NK cells had decreased cell number and NK cell purity after the control groups 1 to 7 did not add additives or reduced the components of the activator, because Mycobacterium phlei F.U.36 injection, levamisole, and ribonucleic acid in the present activator had the effects of promoting activation of immune cells, promoting secretion of specific antibodies and various cytokines, and enhancing functions of immune cells. Cefaclor, cefdezine and ceftazidime have the functions of regulating immune cell function and stimulating lymphocyte proliferation. CD50 and CD52 monoclonal antibodies act primarily to induce lymphocyte activation; vitamin A, vitamin C and vitamin E are bioactive substances for maintaining cell growth, play a role in regulating and controlling in cell metabolism, can increase cell membrane stability, improve cell viability, and can maximally preserve cell viability; heparin sodium and growth factors cooperate to promote cell proliferation, recombinant human proteins such as albumin, immunoglobulin and transferrin have various physiological functions in cell culture, including heavy metal chelating agents, free radical scavengers, osmotic pressure and pH value regulators, stabilizers, growth factor carriers, surfactants, nutrients and the like, and as can be seen from control groups 2-7, the NK cell culture effect of the recombinant human proteins is inferior to that of example 1 (see figure 1) of the invention after no activator is added or any component of the activator is omitted, and the synergistic effect of the components is further illustrated, so that the number and purity of immune cells are improved and the killing function of the immune cells is enhanced.
Example 2
(1) The activator is applied to the culture of CIK cells, and comprises the following steps:
the concentration of the main components of the cell immune activator is 50 mu g/ml of mycobacterium phlei F.36 injection, 50 mu g/ml of levamisole, 200 mu g/ml of ribonucleic acid, 100 mu g/ml of cefaclor, 100 mu g/ml of cefdezine, 100 mu g/ml of ceftazidime, 10 mu g/ml of CD50 monoclonal antibody, 10 mu g/ml of CD52 monoclonal antibody, 1mg/ml of vitamin A, 5mg/ml of vitamin C, 1mg/ml of vitamin E, 500IU/ml of heparin sodium, 10 mu g/ml of recombinant human albumin, 10 mu g/ml of recombinant human immunoglobulin and 50 mu g/ml of recombinant human transferrin. After the preparation is completed, the mixture is added into ALyS505N-0 culture medium according to the proportion of 1:100.
(2) 40ml of peripheral blood of a healthy person is collected by using a heparin sodium anticoagulation blood collection tube, 20ml of fresh blood is slowly poured onto 15ml of lymph separation liquid, and the blood is slowly lifted and lowered by centrifugation at 800xg for 20 min. The upper plasma is collected by a sterile straw and is collected to a centrifuge tube, the mononuclear cell layer is not required to be sucked by mistake, the plasma is heated at 56 ℃ for 30min, and is centrifuged at room temperature for 10min by 1200g, the supernatant is collected to a new centrifuge tube by the straw, and the centrifuge tube is preserved at 4 ℃ until the supernatant is taken out before use. The buffy coat was collected into another 50ml centrifuge tube with a pipette and the cells were washed 3 times with DPBS for further use.
(3) Preparation of ALyS505N-500 medium: IL-2 was added to 1000ml of ALyS505N-0 culture at a concentration of 500IU/ml.
(4) Adding 18ml of ALyS505N-500 and 2ml of autologous plasma into a non-TC-treated T75 culture flask, respectively adding CD3 1 μg/ml and IL-1α100IU/ml, adding PBMC prepared in step 2, and adjusting cell density to 1.5X10 6 Each ml, at 37℃and 5% CO 2 Culturing in an incubator; day3, day5 and Day7 were continuously cultured by adding 20ml, 60ml and 200ml of ALyS505N-500 medium, respectively. Culturing until Day7 is transferred to 2 cell culture bags for expansion culture, and adding 700 ml of amplification culture medium and 1000ml of amplification culture medium (equally dividing the two culture bags) every two days (namely Day9 and Day 11) to maintain the cell density to be 5-7 multiplied by 10 5 Every ml, stand at 37 ℃ and 5% CO 2 Culturing in an incubator.
(5) The cells were cultured to Day14, counted, washed by centrifugation, and collected, and the number and the viability of the cells were measured. Fluorescent antibody is added to flow cytometry for detecting CD3 by flow cytometry - CD56 + Is a ratio of (2).
(6) Determination of cell killing Activity: taking K562 cells in logarithmic growth phase as target cells, taking CIK cells as effector cells, setting experimental groups with the effective target ratios of 2:1, 4:1 and 5:1 respectively, and setting three compound holes in each group. The density of K562 cells is adjusted to 20 ten thousand/ml; adjusting CIK cell density according to the effective target ratio, and adding 50 μl of effector cells and target cell suspension into each well; simultaneous effector cell extraction50 μl of suspension was added to each well with 50 μl of 1640 medium as a control; mu.l of the target cell suspension was taken and 50. Mu.l of 1640 medium was added to each well as a natural release group, each group being provided with three duplicate wells. Placing in 37 ℃ and 5% CO 2 Culturing in incubator for 6 hr, taking out 96-well plate, adding 20 μl of CCK-8 solution into each well, adding 37 ℃ and 5% CO 2 After incubation for 90min in the incubator, detection was performed with a microplate reader at a wavelength of 450 nm. And calculating the killing rate according to a formula I.
Killing rate (%) = (1- (experimental group-control group)/natural release group) formula I
The present embodiment also provides a control group:
control 1, which is different from example 2 in that no activator was added, was the same as example 1.
Control group 2 was different from example 2 in that the components of the activator were different, mycobacterium phlei F.U.36 injection, levamisole, and ribonucleic acid were omitted from the activator, and the rest was the same as in example 2.
Control group 3 was different from example 2 in that the components of the activator were different, and cefaclor, cefdezine, and ceftazidime in the activator were omitted, and the rest was the same as in example 2.
Control group 4 was identical to example 2 except that the activator was different in composition, CD50 and CD52 in the activator were omitted, and the remainder was identical to example 2.
Control group 5 was different from example 2 in that the components of the activator were different, and vitamin A, vitamin C and vitamin E in the activator were omitted, and the rest was the same as example 2.
Control 6, which differs from example 2 in the composition of the activator, omits heparin sodium from the activator, and is otherwise identical to example 2.
Control 7, which differs from example 1 in the composition of the activator, omits the recombinant human proteins in the activator as albumin, immunoglobulin and transferrin, and is otherwise identical to example 1.
TABLE 2 Effect of culturing immune cell activators prepared in example 2 and control groups 1 to 7
As is clear from Table 2, the immune cell activator prepared in example 2 has the highest cell count and flow phenotype and the highest killing function, and the immune cells have decreased cell count and cell purity after no additive or reduced components of the activator are added to the control groups 1 to 7. As can be seen from the control groups 1-7, the immune cell culture effect is inferior to that of the embodiment of the invention (see FIG. 2) without adding the activator or omitting any component of the activator, further explaining the synergistic effect of the components, improving the number and purity of immune cells and enhancing the killing function of immune cells.
Example 3
The activator is applied to culturing gamma delta T cells, and comprises the following steps:
(1) Immune cell activators were as follows: mycobacterium phlei F.36 injection 50 μg/ml, levamisole 50 μg/ml, ribonucleic acid 200 μg/ml, cefaclor 100 μg/ml, cefodizime 100 μg/ml, ceftazidime 100 μg/ml, CD50 monoclonal antibody 10 μg/ml, CD52 monoclonal antibody 10 μg/ml, vitamin A1mg/ml, vitamin C5 mg/ml, vitamin E1mg/ml, heparin sodium 500IU/ml, recombinant human albumin 10 μg/ml, recombinant human immunoglobulin 10 μg/ml, recombinant human transferrin 50 μg/ml. After the preparation, the mixture was added to ALyS505N-0 medium at a ratio of 1:100.
(2) 40ml of peripheral blood of a healthy person is collected by using a heparin sodium anticoagulation blood collection tube, 20ml of fresh blood is slowly poured onto 15ml of lymph chaos, and the blood is slowly lifted and lowered by centrifugation at 800xg for 20 min. The upper plasma is collected by a sterile straw and is collected to a centrifuge tube, the mononuclear cell layer is not required to be sucked by mistake, the plasma is heated at 56 ℃ for 30min, and is centrifuged at room temperature for 10min at 1200Xg, the supernatant is collected to a new centrifuge tube by the straw, and the supernatant is stored at 4 ℃ until the supernatant is taken out before use. The buffy coat was collected into another 50ml centrifuge tube with a pipette and the cells were washed 3 times with DPBS for further use.
(3) Preparation of ALyS505N-500 medium: IL-2 was added to 1000ml of ALyS505N-0 culture at a concentration of 500IU/ml for the experimental group at a ratio of 1:100.
(4) 18ml of ALyS505N-500 and 2ml of autologous plasma, and 1ml of Lymantin- γδT were placed in a non-TC-treated T75 flask, and PBMC prepared in step 2 were added to adjust the cell density to 1.5X10 6 Individual/ml, 5% CO at 37 DEG C 2 Culturing in an incubator; day3, day5 and Day7 were continuously cultured by adding 20ml, 60ml and 200ml of ALyS505N-500 medium, respectively. Culturing until Day7 is transferred to 2 cell culture bags for expansion culture, and adding 700 and 1000ml (average of two culture bags) of amplification culture medium every two days (namely Day9 and Day 11) to maintain cell density at 5-7X10 5 Every ml, stand at 37 ℃ 5% CO 2 Culturing in an incubator.
(5) The cells were cultured to Day14, counted, washed by centrifugation, and collected, and the number and the viability of the cells were measured. Fluorescent antibody is added to flow cytometry for detecting CD3 by flow cytometry - CD56 + Is a ratio of (2). Detection method is the same as that of CD3 in example three - CD56 + NK cell phenotype detection step.
(6) Determination of cell killing Activity: taking K562 cells in logarithmic growth phase as target cells, taking gamma delta T cells as effector cells, setting experimental groups with the effective target ratios of 2:1, 4:1 and 5:1 respectively, and setting three compound holes in each group. The density of K562 cells is adjusted to 20 ten thousand/ml; adjusting the gamma delta T cell density according to the effective target ratio, and adding 50ul of effector cells and target cell suspensions into each hole; simultaneously taking 50 μl of effector cell suspension, and adding 50 μl of 1640 culture medium to each well; mu.l of the target cell suspension was taken and 50. Mu.l of 1640 medium was added to each well as a natural release group, each group being provided with three duplicate wells. Placing in 37 ℃ and 5% CO 2 Culturing in incubator for 6 hr, taking out 96-well plate, adding 20 μl of CCK-8 solution into each well, adding 37 ℃ and 5% CO 2 After incubation for 90min in the incubator, detection was performed with a microplate reader at a wavelength of 450 nm. And calculating the killing rate according to a formula I.
Killing rate = (1- (experimental group-control group)/natural release group) formula I
The following control groups were also set up in this example:
control 1, which is different from example 1 in that no activator was added, was the same as example 1.
Control group 2 was different from example 1 in that the components of the activator were different, mycobacterium phlei F.U.36 injection, levamisole, and ribonucleic acid were omitted from the activator, and the rest was the same as in example 1.
Control group 3 was different from example 1 in that the components of the activator were different, and cefaclor, cefdezine, and ceftazidime in the activator were omitted, and the rest was the same as in example 1.
Control group 4 was identical to example 1 except that the activator was different in composition, and CD50 and CD52 in the activator were omitted, and the remainder was identical to example 1.
Control group 5 was different from example 1 in that the components of the activator were different, and vitamin A, vitamin C and vitamin E in the activator were omitted, and the rest was the same as in example 1.
Control 6, which differs from example 1 in the composition of the activator, was prepared by omitting heparin sodium from the activator, and the remainder was the same as in example 1.
Control 7, which differs from example 1 in the composition of the activator, omits the recombinant human proteins in the activator as albumin, immunoglobulin and transferrin, and is otherwise identical to example 1.
The results are shown in Table 3
TABLE 3 results of cell culture with immunocyte activators prepared in example 3 and control groups 1-7
As is clear from Table 3, the immune cell activator prepared in example 3 has the highest cell count and flow phenotype and the highest killing function, and the immune cells have decreased cell count and cell purity after no additive or reduced components of the activator are added to the control groups 1 to 7. As can be seen from the control groups 1-7, the immune cell culture effect is inferior to that of the embodiment of the invention (see FIG. 3) without adding the activator or omitting any component of the activator, further explaining the synergistic effect of the components, improving the number and purity of immune cells and enhancing the killing function of immune cells.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (3)

1. An immune cell activator characterized by the following concentration of components: mycobacterium phlei F.U.36 injection 50 μg/ml, levamisole 50 μg/ml, ribonucleic acid 200 μg/ml, cefaclor 100 μg/ml, cefodizime 100 μg/ml, ceftazidime 100 μg/ml, CD50 monoclonal antibody 10 μg/ml, CD52 monoclonal antibody 10 μg/ml, vitamin A1mg/ml, vitamin C5 mg/ml, vitamin E1mg/ml, heparin sodium 500IU/ml, recombinant human albumin 10 μg/ml, recombinant human immunoglobulin 10 μg/ml, recombinant human transferrin 50 μg/ml.
2. A serum-free cell culture medium comprising the immune cell activator of claim 1 and a cell nutrient component;
the immune cell activator is used for preparing serum-free cell culture medium, and the serum-free cell culture medium is diluted by 100 times by cell nutrition components.
3. Use of the immune cell activator of claim 1 or the serum-free cell culture medium of claim 2 in immune cell culture, wherein the immune cells are NK cells, CIK cells and γδ T cells.
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