CN115651903B - High-lethality immune cell population, and culture method, reagent composition and application thereof - Google Patents
High-lethality immune cell population, and culture method, reagent composition and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cell biology, and particularly relates to a high-lethality immune cell population, a culture method, a reagent composition and application thereof. The effector cells of this immune cell population consist of CD3+ CD56+ cells and CD3+ CD8+ cells. The invention takes Retronectin and CD16 monoclonal antibody as coating liquid, adopts an activation culture medium composed of a serum-free culture medium, CD3 monoclonal antibody, IL-15, IL-21 and IL-2, and adopts an amplification culture medium composed of the serum-free culture medium and the IL-2 to culture immune cell population. The culture method comprises the following steps: inoculating the cells into a cell culture bottle coated with a coating solution, activating and culturing for 14 days, and adding 10%, 5% and 2% serum substitutes respectively on the first day, the fourth day and the sixth day to obtain immune cell populations. The cell has high killing ability on chronic myelogenous leukemia cell K562, and can be used for treating chronic myelogenous leukemia.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a high-lethality immune cell population, a culture method, a reagent composition and application thereof.
Background
Chronic Myelocytic Leukemia (CML) is a clonal proliferative disease of hematopoietic stem cells, and the bone marrow is mainly characterized by myeloproliferation, peripheral blood leukocytosis and splenomegaly. The chronic myelocytic leukemia has a high morbidity rate in China, which accounts for about 15-20% of adult leukemia, and the existing treatment methods comprise chemotherapy, gene therapy, immunotherapy, bone marrow transplantation, targeted drug therapy and the like, but the treatment effect of the methods is not very ideal, and most of the methods have the problems of high cost, low success rate, large side effect and the like.
In recent years, cell therapy has been rapidly developed. The cell is used as an independent living body and has strong vitality, proliferation and differentiation capacity and functional plasticity capacity. The mechanism of cell therapy for treating diseases is mainly divided into two main categories, one is the direct action of cells, and the specific biological activity of the cells is directly applied to repair injured tissues and organs or play a role in specific/non-specific killing; the other is the indirect effect of the cells, such as the secretion of related factors or active molecules to regulate the proliferation and functional activities of the cells of the patient. Compared with the traditional malignant tumor treatment method, the cell therapy has the unique advantages of good curative effect, small side effect, individuation and the like. Known therapeutic cells include NK cells, γ δ T cells, CD3AK, CIK cells, and the like. The CIK population is a heterogeneous cell population composed of effector cells CD3+ CD56+ cells, CD3+ CD8+ cells and the like. At present, the CIK culture method needs to use serum, has the problems of poor proliferation efficiency, low cell purity, weak cytotoxicity to tumor cells and the like, and hinders the development of cellular immunotherapy to a certain extent.
The invention patent with publication number CN103173409B discloses a preparation method of CD3+ CD56+ cells derived from perinatal placental blood high-killing K562 cells, although the killing rate of the CD3+ CD56+ cells cultured by the invention to the K562 cells can reach more than 85%, the culture method is complicated, and the adopted blood sample is perinatal placental blood, which has difficulty in clinical popularization and is not beneficial to industrialized preparation.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a high-killing immune cell population, which has high ability to secrete factors associated with cytolytic toxicity, and has a very high killing ability on chronic myelogenous leukemia cell K562.
In order to achieve the purpose, the invention adopts the following technical scheme:
a population of immune cells with high lethality, the effector cells of the population of immune cells consisting of CD3+ CD56+ cells and CD3+ CD8+ cells; the proportion of cells secreting Granzyme B in the immune cell population is not less than 90%.
Further, the proportion of CD3+ CD56+ cells is not less than 40% of the total number of cells; the proportion of CD3+ CD8+ cells is not less than 65% of CD3+ cells.
The other purpose of the invention is to provide a method for culturing the immune cell population.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of culturing a population of immune cells comprising the steps of:
s1: coating a cell culture flask with Retronectin and CD16 monoclonal antibody;
s2: separating a blood sample by using a lymphocyte separation solution to obtain mononuclear cells;
s3: inoculating the cells obtained in the S2 into a cell culture flask coated with Retronectin and CD16 monoclonal antibody for activation culture;
on the first day, cells were cultured in an activated medium containing 10% by volume of serum replacement;
on the fourth day, the activated culture medium containing 5% by volume of serum substitute is supplemented for continuous culture;
on the sixth day, the amplification culture medium containing 2% by volume of serum substitute is supplemented for continuous culture;
s4: after 14 days of culture, the logarithmically growing immune cells were collected.
The culture method only needs to add serum substitutes with the volume percentage of 10 percent, 5 percent and 2 percent respectively on the first day, the fourth day and the sixth day, and does not need to add the serum substitutes, thereby well saving the cost.
Further, the blood sample is umbilical cord blood and/or peripheral blood.
Further, in S3, after the sixth day of cell activation culture, cell density is detected every two days, and the cell is continuously cultured by supplementing the amplification culture medium without serum substitute, and the cell density is kept to be not less than 1.0 multiplied by 10 6 one/mL.
Further, in S3, the activated medium containing 5% by volume of serum replacement is added in an amount of three times the volume of the original medium in the cell culture flask; the amount of amplification medium containing 2% by volume of serum replacement added was twice the volume of the original medium in the cell culture flask.
Furthermore, in the first day of the activation culture of the cells in S3, after adding 10% by volume of serum substitute into the activation culture medium, the content of the serum substitute is 0.5-1.5 multiplied by 10 6 Resuspending the cells at a density of one/ml, transferring the cells into a coating flaskAnd performing activation culture.
Further, there are 2 coating modes in S1: (1) coating the cells overnight at 4 ℃ one day before cell culture; (2) the cells were incubated at 37 ℃ for 2 hours.
It is another object of the present invention to provide a reagent composition for culturing the immune cell population, which can activate immune cells by using CD3 mAb in a free state to obtain a specific ratio of target cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a reagent composition for culturing the immune cell population, the reagent composition comprising a coating solution, an activation medium, and an amplification medium; the coating solution consists of Retronectin with the final concentration of 10-50ug/ml and CD16 monoclonal antibody with the final concentration of 50-100 ug/ml; the activation culture medium consists of a serum-free culture medium, a CD3 monoclonal antibody with the final concentration of 200-1000ng/ml, IL-15 with the final concentration of 20-100ng/ml, IL-21 with the final concentration of 20-100ng/ml and IL-2 with the final concentration of 200-1000U/ml; the amplification culture medium consists of a serum-free culture medium and IL-2 with the final concentration of 200-1000U/ml.
Further, the coating solution consists of Retronectin with a final concentration of 50ug/ml and CD16 monoclonal antibody with a final concentration of 50 ug/ml; or the coating solution consists of Retronectin with the final concentration of 50ug/ml and CD16 monoclonal antibody with the final concentration of 100 ug/ml.
Further, the activation medium consists of a serum-free medium, a CD3 monoclonal antibody with a final concentration of 400-800ng/ml, IL-15 with a final concentration of 30-90ng/ml, IL-21 with a final concentration of 30-90ng/ml and IL-2 with a final concentration of 500U/ml.
Further, the activation medium consists of a serum-free medium, a CD3 monoclonal antibody with a final concentration of 400ng/ml, IL-15 with a final concentration of 30ng/ml, IL-21 with a final concentration of 30ng/ml and IL-2 with a final concentration of 500U/ml.
Further, the activation medium consists of a serum-free medium, a CD3 monoclonal antibody with a final concentration of 800ng/ml, IL-15 with a final concentration of 90ng/ml, IL-21 with a final concentration of 90ng/ml and IL-2 with a final concentration of 500U/ml.
Further, the amplification medium consisted of serum-free medium and IL-2 at a final concentration of 500U/ml.
The fourth purpose of the invention is to provide an application of the immune cell population in preparing an agent for killing chronic myelogenous leukemia cells K562.
The fifth purpose of the invention is to provide the application of the immune cell population in preparing the medicine for treating chronic myelogenous leukemia.
The invention has the beneficial effects that:
1. the immune cell population claimed in this patent wherein the primary effector cells are CD3+ CD56+ cells and CD3+ CD8+ cells. The immune cell population has the capability of secreting relevant factors of cytolytic toxicity at high level, has extremely high killing capability on chronic myelogenous leukemia cells K562, and has extremely high clinical application potential in the aspect of treating chronic myelogenous leukemia;
2. compared with the conventional cell culture method, the culture method only needs to add 10 percent, 5 percent and 2 percent of serum substitutes respectively on the first day, the fourth day and the sixth day, and does not need to add the serum substitutes, so that the cost can be well saved;
3. in the prior art, CD3 monoclonal antibody is usually coated into a culture flask to activate immune cells, while in the present invention, CD3 monoclonal antibody in free state is selected to activate immune cells to obtain a specific ratio of target cells.
Drawings
Fig. 1 is a graph of the total cell expansion fold of CD3+ CD56+ cells and CD3+ CD8+ cells in expansion culture for 14 days, where there is a significant difference p <0.05 between control and experimental-2.
Fig. 2 is a graph of the proportion of cell subsets, with a significant difference p <0.01 between control and experimental-2 in CD3+ CD8+ cells.
FIGS. 3A and 3B are graphs showing the results of flow cytometry for the detection of cell subsets; FIG. 3A is a graph showing the ratio of CD3+ CD56+ cells, wherein the abscissa CD3 PC7-A represents the expression density of leukocyte differentiation antigen 3 of test cells, the ordinate CD56 PE-A represents the expression density of leukocyte differentiation antigen 56 of test cells, Q2-UL represents cells expressing leukocyte differentiation antigen 56 but not leukocyte differentiation antigen 3, Q2-UR represents cells expressing leukocyte differentiation antigen 56 and leukocyte differentiation antigen 3, Q2-LL represents cells not expressing leukocyte differentiation antigen 56 and leukocyte differentiation antigen 3, and Q2-LR represents cells expressing leukocyte differentiation antigen 3 but not expressing leukocyte differentiation antigen 56; FIG. 3B is se:Sub>A graph showing the ratio of CD3+ CD8+ cells, wherein the abscissse:Sub>A CD4 PC5.5-A represents the expression density of leukocyte differentiation antigen 4 of test cells, the ordinate CD8 FITC-A represents the expression density of leukocyte differentiation antigen 8 of test cells, Q1-UL represents cells expressing leukocyte differentiation antigen 8 but not leukocyte differentiation antigen 4, Q1-UR represents cells expressing leukocyte differentiation antigen 8 and leukocyte differentiation antigen 4, Q1-LL represents cells not expressing leukocyte differentiation antigen 8 and leukocyte differentiation antigen 4, and Q1-LR represents cells expressing leukocyte differentiation antigen 4 but not expressing leukocyte differentiation antigen 8; PE-A, PC7-A, FITC-A, PC5.5-A represent different fluorescence channels.
FIGS. 4A and 4B are graphs showing the results of the measurement of the killing ability of immunocytes; FIG. 4A is a statistical comparison of three killing experiments, where there is a significant difference p <0.001 between control and experimental-1; there was a significant difference p <0.0001 between control and experimental-2; significant difference p <0.01 exists between experimental group-1 and experimental group-2; FIG. 4B is se:Sub>A graph showing the results of se:Sub>A representative killing test, in which the abscissse:Sub>A 7AAD PC5.5-A indicates whether or not the test cell survives, i.e., whether or not the test cell survives within se:Sub>A range defined by P5, the left side of P5 is se:Sub>A live cell, the ordinate SSC-A indicates the refractive index of the cell membrane, cytoplasm, and nuclear membrane of the test cell and the properties of intracellular granules, and P5 indicates the 5 th "gate" set for the selected test cell during the test.
Fig. 5 is a graph of the results of the cytokine-secreting cell ratio assay, wherein a1, a2, and a3 represent the assay results of Perforin + cells, and the significant difference p between the control group and the experimental group-2 is less than 0.01; b1, B2 and B3 represent the detection result of the Granzyme B + cells, and the significant difference p between a control group and an experimental group-2 is less than 0.01; c1, c2 and c3 represent the detection result of IFN-gamma + cells, and the significant difference p between the control group and the experimental group-2 is more than 0.05.
Detailed Description
The technical solution of the present invention will be further clearly and completely described with reference to the following specific examples. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by those skilled in the art without inventive efforts shall fall within the scope of the present invention.
In the following examples:
activating a culture medium: for activating resting immune cells;
amplification medium: allowing the activated immune cells to proliferate and differentiate;
CD3+ CD56+ cells: immune cells with cytotoxic activity similar to NK cells and T cells;
CD3+ CD8+ cells: cytotoxic T lymphocytes;
the serum-free medium may be any one of the immune cell culture media commonly used in the art;
retronectin is a human fibronectin fragment, purchased from Takara;
the CD16 monoclonal antibody is a leukocyte differentiation antigen 16 monoclonal antibody and is purchased from American BD company;
the CD3 monoclonal antibody is a leukocyte differentiation antigen 3 monoclonal antibody and is purchased from ThermoFisher company;
IL-15 is interleukin-15, available from PeproTech, USA;
IL-21 is interleukin-21, available from PeproTech, USA;
IL-2 is interleukin-2, available from PeproTech, USA;
perforin + cells are Perforin positive cells;
granzyme B + cells are Granzyme B positive cells;
IFN-gamma is gamma interferon;
IFN-gamma + cells are gamma interferon positive cells.
Example 1 Experimental group
1. Reagent:
(1) Coating liquid: retronectin and CD16 monoclonal antibodies;
(2) Activating a culture medium: serum-free culture medium, CD3 monoclonal antibody, IL-15, IL-21 and IL-2;
(3) Amplification medium: serum-free medium, IL-2.
The CD3+ CD56+ cells and CD3+ CD8+ cells (referred to as experimental groups for short) are cultured by adopting the reagent, the experimental group comprises an experimental group-1 and an experimental group-2, and the difference between the experimental group-1 and the experimental group-2 lies in that the concentrations of partial culture reagents are different, and the detailed table is shown in table 1.
Table 1.
2. The culture method of the CD3+ CD56+ cells and the CD3+ CD8+ cells specifically comprises the following steps:
(1) Coating the cell culture flask with a coating solution one day before the culture (overnight coating at 4 ℃) or one day on the culture (coating for 2 hours at 37 ℃);
(2) Separating to obtain mononuclear cells by using a lymphocyte separating medium;
(3) Adding 10% serum substitute into activated culture medium according to the ratio of 0.5-1.5 × 10 6 Resuspending the cells at a density of one/ml, and transferring the cells into a coating bottle for activation culture;
(4) On the fourth day, the culture was continued by adding three times the volume of the activated medium containing 5% serum replacement;
(5) On the sixth day, the amplification culture medium containing 2 percent serum substitute with two times of volume is supplemented for continuous culture;
(6) Thereafter, the cell density was measured every two days, and the cell density was adjusted to not less than 1.0X 10 by adding an amplification medium containing no serum substitute 6 Per mL;
(7) After 14 days of culture, the logarithmically growing immune cells were collected.
Comparative example 1 control group
1. Reagent:
(1) Coating liquid: retronectin (50 ug/ml), CD3 mab (50 ug/ml);
(2) Activating a culture medium: serum-free medium, IFN-gamma (500U/ml), IL-2 (500U/ml);
(3) Amplification medium: serum-free medium, IL-2 (500U/ml).
CD3+ CD56+ cells and CD3+ CD8+ cells (control group for short) were cultured using the above-mentioned reagents.
2. The cell culture method comprises the following steps: same as in example 1.
Example 2
The cells obtained by culturing the cells of example 1 and comparative example 1 were examined as follows:
(1) Detecting the amplification multiple of the total cells;
(2) Detecting the cell subpopulation by flow cytometry;
(3) The ratio of immune cells to CFSE labeled K562 was 10:1 (effective target ratio) and incubating for 4 hours, and detecting the killing capacity of the immune cells obtained by culture;
(4) After intracellular staining of immune cells using conventional experimental methods, the proportion of cytokine-secreting cells was detected using flow cytometry.
The results of the detection are as follows. In the following results, cord blood samples from 3 different healthy volunteers were used for the control group and the experimental group-2, and cord blood samples from 2 healthy volunteers out of 3 volunteers were used for the experimental group-1, to thereby carry out comparative experiments.
(1) The results of the cell fold expansion measurements are shown in table 2 and fig. 1. The results show that the total cell amplification times detected by using the samples-1 to-3 in the control group are 285.0 times, 349.9 times and 411.5 times in sequence; the total cell amplification times detected by using the sample-1 and the sample-2 in the experimental group-1 are 385.2 times and 299.5 times in turn; the total cell amplification times detected by using samples-1 to-3 in the experimental group-2 were 525.3 times, 559.1 times and 417.6 times in this order. As can be seen, the average amplification fold of the experimental group-2 was the highest, the control group was lower than the experimental group-2, and the experimental group-1 was the lowest. Since the amplification factor of the experimental group-1 was lower than that of the control group and the experimental group-2 in the first two experiments, a subsequent third comparative experiment was not performed.
Table 2 fold expansion of cells.
(2) The results of flow cytometry for cell subpopulation detection are shown in table 3 and fig. 2, 3A and 3B. The results showed that, in the control group, the subpopulations of CD3+ CD56+ cells detected using sample-1 to sample-3 were 26.24%,41.25% and 16.43% in this order, and the subpopulations of CD3+ CD8+ cells detected using sample-1 to sample-3 were 29.12%,52.97% and 21.52% in this order; in the experimental group-1, the proportion of CD3+ CD56+ cell subsets detected using the sample-1 and the sample-2 was 27.59% and 32.38%, respectively, and the proportion of CD3+ CD8+ cell subsets detected using the sample-1 and the sample-2 was 53.05% and 39.70%, respectively; in the experimental group-2, the subpopulations of CD3+ CD56+ cells detected using the samples-1 to-3 were 40.89%,47.93% and 42.67%, respectively, and the subpopulations of CD3+ CD8+ cells detected using the samples-1 to-3 were 58.73%,82.82% and 69.48%, respectively. Fig. 3A and 3B are representative flow cytometry test results. FIG. 3A shows that the proportion of CD3+ CD56+ cells in the Q2-UR range can reach 42.67%, and the proportion of CD3+ cells in the Q2-LR range is 28.18%. Further, FIG. 3B shows that, among CD3+ cells, the proportion of CD3+ CD8+ cells expressed in the Q1-UL range was 69.48%, while the proportion of CD3+ CD4+ cells expressed in the Q1-LR range was 23.22%. It can be seen that the average ratio of the two cell populations in the control group was lower than that in the experimental group-2, and that in the experimental group-1, the ratio was in-between. The experimental group-2 can obtain a larger number of effector cells for clinical treatment under the condition of the same cell yield, thereby achieving better treatment effect.
TABLE 3 proportion of cell subsets.
(3) The results of the killing ability of the immune cells obtained by the culture of the experimental group and the control group are shown in table 4 and fig. 4A and 4B. The results show that in the control group, the ratios of the lysed cells detected by using samples-1 to-3 were 8.8%,14.5% and 9.5% in this order; experimental group-1 the ratios of lysed cells detected using sample-1 and sample-2 were 58.9% and 48.3% in this order; the ratios of lysed cells detected in the experimental group-2 using the samples-1 to-3 were 81.8%,80.0%, and 79.4% in this order. FIG. 4B is a graph showing representative results of detecting the killing ability of cultured immunocytes to the tumor cells K562 by flow cytometry. The proportion of K562 cells killed by the immune cells represented in the P5 gate range in the figure was 81.76% of the total K562 cells. As can be seen, the average proportion of lysed cells was lower in the control group than in the experimental group-2, with the experimental group-1 in between. The immune cells cultured by the experimental group-2 can kill more tumor cells and have higher anti-tumor capability.
TABLE 4 immune cell killing ability.
(4) The results of the cytokine secretion assay are shown in table 5 and fig. 5. As a result, in the control group, the proportion of Perforin + cells measured by using samples-1 to-3 was 16.3%,25.34% and 44.39% in this order, the proportion of Granzyme B + cells measured by using samples-1 to-3 was 58.08%,66.61% and 60.31% in this order, and the proportion of IFN-. Gamma. + cells measured by using samples-1 to-3 was 24.04%,37.47% and 48.94% in this order. In the experimental group-1, the Perforin + cell ratio measured using the sample-1 and the sample-2 was 27.45% and 46.44%, respectively; the proportion of Granzyme B + cells detected using sample-1 and sample-2 was 75.95% and 87.61% in order; the proportion of IFN-. Gamma. + cells detected using sample-1 and sample-2 was 18.31% and 43.35%, respectively. In the experimental group-2, the proportion of Perforin + cells measured using samples-1 to-3 was 70.55%,55.15% and 60.19% in this order, the proportion of Granzyme B + cells measured using samples-1 to-3 was 91.32%,95.7% and 93% in this order, and the proportion of IFN-. Gamma. + cells measured using samples-1 to-3 was 20.9%,48.58% and 35.33% in this order. Although the average proportion of IFN-gamma + cells secreting IFN-gamma which mainly plays a role in immune regulation in the control group is higher than that in the experimental group-1 and slightly higher than that in the experimental group-2, the average proportion of Perforin + cells secreting Perforin and Granzyme B which are related to cytotoxicity (namely the killing capacity on tumor cells) and the average proportion of Granzyme B + cells in the control group are lower than that in the experimental group-1 and the experimental group-2, and as a whole, the proportion of cytotoxic immune cells with the capacity of killing tumor cells in the experimental group-1 and the experimental group-2 is higher, and the results in Table 4 are indirectly verified.
TABLE 5 measurement of the cell ratio of cytokine secretion.
Claims (9)
1. A population of immune cells with high lethality, wherein the effector cells of said population of immune cells consist of CD3+ CD56+ cells and CD3+ CD8+ cells; the proportion of cells secreting Granzyme B in the immune cell population is not less than 90%;
the method for culturing the immune cell population comprises the following steps:
s1: coating a cell culture flask with Retronectin and CD16 monoclonal antibody;
s2: separating a blood sample by using a lymphocyte separation solution to obtain mononuclear cells;
s3: inoculating the cells obtained in the S2 into a cell culture flask coated with Retronectin and CD16 monoclonal antibody for activation culture;
on the first day, cells were cultured in an activated medium containing 10% by volume of serum replacement;
on the fourth day, the activated culture medium containing 5% by volume of serum substitute is supplemented for continuous culture;
on the sixth day, the amplification culture medium containing 2% serum substitute by volume percentage is supplemented for continuous culture;
s4: culturing for 14 days, and collecting logarithmically grown immune cells; the activation culture medium consists of a serum-free culture medium, a CD3 monoclonal antibody with the final concentration of 200-1000ng/ml, IL-15 with the final concentration of 20-100ng/ml, IL-21 with the final concentration of 20-100ng/ml and IL-2 with the final concentration of 200-1000U/ml; the amplification culture medium consists of a serum-free culture medium and IL-2 with the final concentration of 200-1000U/ml.
2. The immune cell population of claim 1, wherein the proportion of CD3+ CD56+ cells is not less than 40% of the total number of cells; the proportion of CD3+ CD8+ cells is not less than 65% of CD3+ cells.
3. The immune cell population of claim 1, wherein in S3, the cell density is measured every two days after the sixth day of cell activation culture, and the cells are continued to be cultured by supplementing the expansion medium without serum replacement while maintaining the cell density at not less than 1.0X 10 6 one/mL.
4. The immune cell population of claim 1, wherein in S3, the activated culture medium containing 5% by volume serum replacement is added in an amount of three times the volume of the original culture medium in the cell culture flask; the amount of amplification medium containing 2% by volume of serum replacement added was twice the volume of the original medium in the cell culture flask.
5. A reagent composition for culturing the immune cell population of claim 1, wherein the reagent composition comprises a coating solution, an activation medium, and an amplification medium; the coating solution consists of Retronectin with the final concentration of 10-50ug/ml and CD16 monoclonal antibody with the final concentration of 50-100 ug/ml; the activation culture medium consists of a serum-free culture medium, a CD3 monoclonal antibody with the final concentration of 200-1000ng/ml, IL-15 with the final concentration of 20-100ng/ml, IL-21 with the final concentration of 20-100ng/ml and IL-2 with the final concentration of 200-1000U/ml; the amplification culture medium consists of a serum-free culture medium and IL-2 with the final concentration of 200-1000U/ml.
6. Reagent composition according to claim 5, wherein the coating solution consists of Retronectin at a final concentration of 50ug/ml and CD16 mab at a final concentration of 50 ug/ml; or the coating solution consists of Retronectin with the final concentration of 50ug/ml and CD16 monoclonal antibody with the final concentration of 100 ug/ml.
7. The reagent composition of claim 5, wherein the activation medium consists of serum-free medium, CD3 monoclonal antibody at a final concentration of 400-800ng/ml, IL-15 at a final concentration of 30-90ng/ml, IL-21 at a final concentration of 30-90ng/ml, and IL-2 at a final concentration of 500U/ml; the amplification medium consists of a serum-free medium and IL-2 with a final concentration of 500U/ml.
8. Use of the immune cell population of claim 1 in the preparation of an agent for killing chronic myelogenous leukemia cells K562.
9. Use of the immune cell population of claim 1 in the preparation of a medicament for treating chronic myelogenous leukemia.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
| WO2022226522A1 (en) * | 2021-04-22 | 2022-10-27 | Baylor College Of Medicine | Methods of engineering immune cells having reduced fratricidal activity |
| CN115404215A (en) * | 2021-05-28 | 2022-11-29 | 任涛 | Method for efficiently activating and amplifying NK immune cells in vitro |
Family Cites Families (18)
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| KR101039843B1 (en) * | 2010-08-30 | 2011-06-09 | 주식회사 엔케이바이오 | A medium composition for cultivating self activated lymphocyte and the cultivation method using the same |
| CN103173409B (en) * | 2013-01-15 | 2014-12-31 | 江苏和泽生物科技有限公司 | Method for preparing CD3+CD56+cells through high-killing K562cells from perinatal placental blood |
| CN103627672B (en) * | 2013-12-17 | 2016-03-30 | 天津医科大学附属肿瘤医院 | NK cell injuring model method |
| CN103740643B (en) * | 2014-01-21 | 2015-09-30 | 山东省齐鲁干细胞工程有限公司 | The external evoked cultural method of the restricted killer T cell of a kind of MHC |
| CN105219708A (en) * | 2015-07-21 | 2016-01-06 | 中山大学 | Immunocyte cultivates test kit, immunocyte cultural method and application |
| CN106011061A (en) * | 2016-08-04 | 2016-10-12 | 广东省第二人民医院 | In-vitro large-scale amplification method of natural killer cells |
| KR101866827B1 (en) * | 2016-09-28 | 2018-07-19 | 한국원자력의학원 | Method for preparing Natural Killer cells using irradiated PBMCs, and Anti-cancer cellular immunotherapeutic agent comprising the NK cells |
| CN113061577A (en) * | 2017-09-05 | 2021-07-02 | 四川新生命干细胞科技股份有限公司 | Isolated culture method of high-purity NK cells |
| CN108676775B (en) * | 2018-06-08 | 2021-05-28 | 天晴干细胞股份有限公司 | Method for amplifying cord blood NK in vitro |
| US20200032211A1 (en) * | 2018-07-26 | 2020-01-30 | Oespedale Pediatrico Bambino Gesù (Opbg) | Therapeutic preparations of gamma-delta t cells and natural killer cells and methods for manufacture and use |
| CA3106056A1 (en) * | 2018-07-26 | 2020-01-30 | Ospedale Pediatrico Bambino Gesu (Opbg) | Therapeutic preparations of gamma-delta t cells and natural killer cells and methods for manufacture and use |
| CN109825473A (en) * | 2019-01-07 | 2019-05-31 | 山东博森医学工程技术有限公司 | A kind of cultural method of the autologous peripheral blood lymphocyte using the stimulation of TLR7 agonist |
| EP3982979A4 (en) * | 2019-06-12 | 2023-10-18 | The Regents Of The University Of California | Engineered off-the-shelf immune cells and methods of use thereof |
| CN113088490A (en) * | 2021-04-22 | 2021-07-09 | 河北森朗生物科技有限公司 | Mixed innate lymphocytes, methods of preparation and uses thereof |
| CN113817675A (en) * | 2021-09-26 | 2021-12-21 | 杭州原生生物科技有限公司 | Serum-free CIK amplification culture method |
| CN115074326A (en) * | 2022-07-07 | 2022-09-20 | 广州希灵生物科技有限公司 | Culture method for in-vitro amplification of NK cells by combining CD16 antibody with cytokine |
| CN115478051B (en) * | 2022-10-12 | 2023-04-18 | 北京奇迈永华生物科技有限公司 | Method for efficiently culturing human NK cells in serum-free manner |
| CN115558641B (en) * | 2022-11-14 | 2023-05-12 | 四川新生命干细胞科技股份有限公司 | High-purity effector immune cell population, culture method, reagent composition and application thereof |
-
2022
- 2022-11-14 CN CN202211417109.6A patent/CN115651903B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111573A1 (en) * | 2020-11-25 | 2022-06-02 | 上海君赛生物科技有限公司 | Pharmaceutical composition for enhancing cell killing, and use thereof |
| WO2022226522A1 (en) * | 2021-04-22 | 2022-10-27 | Baylor College Of Medicine | Methods of engineering immune cells having reduced fratricidal activity |
| CN115404215A (en) * | 2021-05-28 | 2022-11-29 | 任涛 | Method for efficiently activating and amplifying NK immune cells in vitro |
Non-Patent Citations (1)
| Title |
|---|
| Pittari G等.Revving up Natural Killer Cells and Cytokine-Induced Killer Cells Against Hematological Malignancies.2015,第6卷doi: 10.3389/fimmu. * |
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