CN108004213B - Method and kit for rapid amplification of CIK cells - Google Patents
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Abstract
The invention discloses a method and a kit for rapidly amplifying CIK cells, which adopt the technical scheme that the kit comprises an activator, a CIK cell activator and a CIK cell proliferation agent, wherein the activator is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the CIK cell activator is prepared by dissolving IL-2, IL-1 α and IFN-gamma in the PBS buffer solution, and the CIK cell proliferation agent is prepared by dissolving IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin10And (4) respectively.
Description
Technical Field
The invention relates to the crossing technical field of biotechnology, life science and medicine, in particular to a method and a kit for rapidly amplifying CIK cells.
Background
CIK cells, cytokine-induced killer cells (cytokine-induced killer cells), are a heterogeneous population of cells obtained by co-inducing human peripheral blood mononuclear cells with multiple cytokines in vitro. Is a new generation of cytokine-activated lymphocytes developed on the basis of LAK cells (Lymphokine-activated killer cells). LAK cells are first reported by Grimm et al in 1982, and a non-specific killer cell induced by adding various cytokines into peripheral blood mononuclear cells and culturing the peripheral blood mononuclear cells in vitro for 4 to 6 days can kill various tumor cells. The Rosenberg group used IL-2 and LAK for the first time in 1984 to treat 25 patients with renal cell carcinoma, melanoma, lung cancer, colon cancer, etc., of which 11 tumors were 50% smaller and 1 had completely resolved melanoma. In 1988 the study group summarized 222 tumor patients with synergistic treatment, 16 of them had complete tumor metastasis regression and 26 had more than 50% tumor regression, and the treatment was effective on metastatic renal cell carcinoma, melanoma, colon cancer and non-hodgkin's lymphoma.
CD3 in CIK cells+CD56+The double positive cells are main effector cells and express two membrane protein molecules of CD3 and CD56, so the double positive cells are also called natural killer cell-like T lymphocytes, have strong anti-tumor activity of the T lymphocytes and strong recognition capability on the tumor cells, can accurately recognize and kill the tumor cells without damaging normal cells. Especially has remarkable effect on patients after operation or radiotherapy and chemotherapy, can eliminate residual tiny metastasis focus, prevent cancer cell diffusion and recurrence, and improve organism immunity. China is a country with more clinical tests of CIK cells, relevant people make statistics, and the clinical tests of CIK cells in China relate to multiple aspects, such as various malignant tumors of liver cancer, gastric cancer, breast cancer, kidney cancer, leukemia, nasopharyngeal carcinoma and the like, and the total effective rate is 51.7%. A large number of researches show that the CIK cells have higher proliferation capacity and killing activity than LAK, CTL (cytotoxic T lymphocytes) and TIL (tumor infiltrating lymphocytes), are novel and efficient immune effector cells with broad-spectrum tumor killing activity, and have no Major Histocompatibility Complex (MHC) limitation, so the CIK cells are considered as a first choice scheme of a new generation of tumor adoptive cell immunotherapy, and show great application value in the tumor immunotherapy.
At present, the common activation method of the CIK cells is to activate the peripheral blood mononuclear cells obtained by separation by using factors such as CD3 monoclonal antibody, interleukin-1 (IL-1 α), interleukin-2 (IL-2) and gamma-interferon (IFN- γ), and further to amplify the cells by using a static and intermittent liquid changing method in a long-term culture method, however, the common phenomena of low activation efficiency, long culture period and limitation of cell growth due to nutrient depletion or toxic byproduct accumulation exist, and the cell amplification effect is difficult to meet clinical requirements.
Disclosure of Invention
The invention aims to provide a method and a kit for rapidly amplifying CIK cells, which are used for solving the technical problems of low amplification efficiency, long culture time consumption, large using amount of culture solution and difficult operation of the conventional CIK cells.
In order to achieve the above purpose, the first technical solution of the present invention is: a method for rapid expansion of CIK cells, comprising the step of isolating peripheral blood mononuclear cells, the method further comprising the steps of:
activating a culture flask: adding an activating agent into the culture bottle until the bottom of the culture bottle is covered, and incubating to obtain an activated culture bottle, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution;
preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium to obtain an activation medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution;
③ inoculation of mononuclear cells were suspended in activated medium and 10% by volume plasma was added and inoculated in an activated flask at 37 ℃ with 5% CO2Culturing for 48-96h in an incubator to obtain CIK cells;
④ expanding CIK cells by removing the activation medium, adding the amplification medium, 37 deg.C, 5% CO2Culturing in an incubator to a required amount, wherein the amplification culture medium is a basal culture medium containing IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin.
Preferably, the concentration of the CD3 monoclonal antibody in the activator in the step (i) is 30-100 mu g/mL, and the concentration of IFN-gamma is 1000-3000U/mL; the concentration of IL-2 in the CIK cell activator of the step (II) is 20000-100000U/mL, the concentration of IL-1 alpha is 20000-100000U/mL, and the concentration of IFN-gamma is 50000-150000U/mL; the concentration of IL-2 in the amplification culture medium in the step (IV) is 500-1000U/L, the concentration of ascorbic acid is 100-400mg/L, the concentration of vitamin E is 300-800mg/L, the concentration of human plasma albumin is 3-8g/L, and the concentration of insulin is 5-20 ng/L.
More preferably, the concentration of the CD3 monoclonal antibody in the activator in the step (I) is 50 mug/mL, and the concentration of IFN-gamma is 2000U/mL; the concentration of IL-2 in the CIK cell activator in the step II is 50000U/mL, the concentration of IL-1 alpha is 50000U/mL, and the concentration of IFN-gamma is 100000U/mL; the concentration of IL-2 in the amplification culture medium in the step (IV) is 700U/L, the concentration of ascorbic acid is 200mg/L, the concentration of vitamin E is 500mg/L, the concentration of human plasma albumin is 5g/L, and the concentration of insulin is 10 ng/L.
Preferably, the basic culture medium is RPMI-1640 basic culture medium; in the step III, the plasma is inactivated autologous plasma, and the plasma content is 10% of the volume of the activation culture medium.
Preferably, the incubation condition in the first step is 4-37 ℃, and the incubation time is 1-18 h.
Preferably, when the CIK cells are amplified in the step (IV), the amplification culture medium is replaced every 1 to 3 days.
The second technical scheme is as follows: a kit for rapid amplification of CIK cells comprises an activating agent, a CIK cell activating agent and a CIK cell proliferating agent, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS (phosphate buffer solution); the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution; the CIK cell proliferation agent comprises IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin.
Preferably, the concentration of the CD3 monoclonal antibody in the activator is 30-100 mu g/mL, and the concentration of IFN-gamma is 1000-3000U/mL; the concentration of IL-2 in the CIK cell activator is 20000-100000U/mL, the concentration of IL-1 alpha is 20000-100000U/mL, the concentration of IFN-gamma is 50000-150000U/mL, and the CIK cell activator is diluted by 50 times when in use; the CIK cell proliferation agent contains 500-1000U of IL-2, 100-400mg of ascorbic acid, 800mg of vitamin E, 3-8g of human plasma albumin and 5-20ng of insulin, and is diluted to 1000mL by a basic culture medium when in use.
More preferably, the CD3 monoclonal antibody concentration in the activator is 50 mug/mL, and the IFN-gamma concentration is 2000U/mL; IL-2 concentration in CIK cell activator is 50000U/mL, IL-1 alpha concentration is 50000U/mL, IFN-gamma concentration is 100000U/mL, and the CIK cell activator is diluted by 50 times when in use; the CIK cell proliferation agent contains 700U of IL-2, 200mg of ascorbic acid, 500mg of vitamin E, 5g of human plasma albumin and 10ng of insulin, and is diluted to 1000mL by a basal medium when in use.
Preferably, the basic culture medium is RPMI-1640 basic culture medium.
In the technical scheme, the method for rapidly amplifying the CIK cells comprises the steps of firstly coating an incubation culture bottle with an activating agent containing a CD3 monoclonal antibody and IFN-gamma, wherein the CD3 monoclonal antibody has the mitogen activity effect and can be crosslinked with CD3 on the surface of the T cells to induce the activation of the mitogen monoclonal antibody, and the IFN-gamma can induce the synthesis of factors such as interleukin-1 and the like; stimulating mononuclear cells to induce and form the CIK cells by using a CIK cell activating agent containing IL-2, IL-1 alpha and IFN-gamma and an activation culture medium of autologous plasma, wherein the IL-1 alpha mainly can mediate and up-regulate the expression of IL-2R on the surface of peripheral blood lymphocytes, and further enhancing the growth, proliferation and differentiation of the lymphocytes by the IL-2; on the other hand, the cells adopt IL-1 alpha and IFN-gamma under the excitation of the CD3 monoclonal antibody, so that the killing effect of the CIK cells is obviously improved; finally, the CIK cells are cultured by an amplification culture medium containing IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin, and the IL-2 can promote the proliferation and activation of the CIK cells and keep the cells to divide and proliferate at a higher speed; the human plasma albumin is mainly used as a serum substitute to provide required nutrition for cell growth, and in the further improved technical scheme, autologous plasma is adopted to reduce the incidence rate of immune reaction to the maximum extent; the ascorbic acid and the vitamin E have antioxidant effect, and the vitamin E is fat-soluble, can effectively prevent the oxidation reaction of cell membranes and has the effects of promoting cell proliferation and activity; ascorbic acid is an antioxidant of extracellular fluid, is combined with free radicals to prevent cell membranes from being damaged by lipid peroxidation, and also has the effects of remarkably improving the function of DC cells (dendritic cells) and further promoting the proliferation of T cells and the secretion of cytokines. The scientific addition of the nutrient substances ensures the efficient and rapid proliferation of the CIK cells, particularly, the addition of the plasma albumin further clarifies the components of the culture medium, and reduces the influence of exogenous substances on the clinical application of the CIK cells.
The method and the kit for rapidly amplifying the CIK cells have the advantages that the method and the kit are convenient to use and simple to operate, the CIK cells can be efficiently and rapidly amplified, the culture period is greatly shortened, the amplification efficiency is improved, and the method and the kit can be used for inducing and amplifying the CIK cells separated from 50-10 mL of peripheral blood to reach 2 × 10 within 7-15 days without a trophoblast10～8×1010The clinical CIK cell sample is far higher than the general demand, and a small amount of peripheral blood samples can meet the clinical application requirement of CIK cells, so that the clinical CIK cell sample provides hope for clinical CIK cell treatment.
Drawings
FIG. 1 is a graph showing the results of the killing rate of CIK cells against cancer cells obtained by the conventional amplification method in example 1 and comparative example.
FIG. 2 is a graph showing the results of the killing rate of CIK cells on cancer cells obtained by the conventional amplification method in example 2 and comparative example.
Detailed Description
The following examples are given to illustrate the present invention but are not intended to limit the scope of the present invention, and the procedures and methods involved in the examples are all conventional ones, and the reagents involved in the examples, such as PBS buffer, CD3 monoclonal antibody, IFN-. gamma.and IL-2, are all conventional ones unless otherwise specified, and the cell density is 4 × 105The concentration of 4 × 10 per mL of cell suspension5And (4) cells.
Example 1 method for Rapid expansion of CIK cells
This example illustrates in detail the method for rapid expansion of CIK cells, comprising the steps of:
① activating the flask by adding an activator to the flask to cover the bottom of the flask, in this example, to 75cm2Adding 10mL of activating agent into the cell culture bottle to fill the bottom of the bottle, and incubating at 4 ℃ for 12h to obtain an activated culture bottle, wherein the activating agent is CD3 monoThe monoclonal antibody and IFN-gamma are dissolved in PBS buffer solution (pH7.35-7.45) to prepare the antibody, the concentration of the CD3 monoclonal antibody is 50 mug/mL, and the concentration of the IFN-gamma is 2000U/mL;
preparing an activation culture medium: adding 5mL of autologous plasma and 1mL of CIK cell activator into 44mL of basal medium to obtain an activation medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution, the concentration of IL-2 is 50000U/mL, the concentration of IL-1 alpha is 50000U/mL, and the concentration of IFN-gamma is 100000U/mL, and the autologous plasma is obtained by collecting anticoagulated peripheral blood, centrifuging at 2000rpm/min for 20min, separating and collecting the plasma, inactivating at 56 ℃ for 30min, and centrifuging at 1500rpm/min for 10 min;
③ separation and inoculation of mononuclear cells
First, peripheral blood mononuclear cells were isolated: taking 50-100 mL of anticoagulated peripheral blood, centrifuging at 2000rpm/min for 20min, separating and collecting plasma, inactivating at 56 ℃ for 30min, centrifuging at 1500rpm/min for 10min, and obtaining plasma from a subject for preparing an activation culture medium;
in addition, sterile PBS (1-1.5 times the volume of plasma) is added to the blood from which the plasma is removed to dilute the blood, and Peripheral Blood Mononuclear Cells (PBMCs) are separated by Ficoll-Paque (product number BTN131136, available from Beijing Baiolai Botech Co., Ltd.) to obtain mononuclear cells.
The mononuclear cells were then resuspended in activated medium at a controlled seeding density of 1 × 10, formulated at step ②6seed/mL, inoculated in the activated culture flask treated in step ①, at 37 ℃ with 5% CO2Culturing for 72h in an incubator to obtain CIK cells, and gently washing the activated culture bottle with PBS (phosphate buffer solution) for 1-2 times before inoculation;
④ expanding CIK cells by centrifuging to remove the activated culture medium, adding the amplified culture medium, and standing at 37 deg.C under 5% CO2Culturing in an incubator, continuously using CIK cell amplification culture medium to dilute the cell density to 4 × 10 every 48h5The culture was carried out in/mL until the number of CIK cells reached the desired number. The amplification culture medium is a basic culture medium containing IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin, and the concentration of each component is as follows: IL-2 content of 700U/L, ascorbic acid 200mg/L, vitamin E500mgL, 5g/L human plasma albumin and 10ng/L insulin.
In the embodiment, after 15 days of culture, the amplification quantity of CIK cells reaches 2 × 1010～8×1010Is far greater than the conventional demand 7 × 109Therefore, the method and the provided reagent can efficiently amplify the CIK cells.
In order to compare with the conventional CIK cell expansion method, the present example also provides a comparative example during the culture, which is cultured by the conventional culture method, specifically as follows:
taking 50-100 mL of anticoagulated peripheral blood, centrifuging at 2000rpm/min for 20min, separating and collecting plasma, inactivating at 56 ℃ for 30min, centrifuging at 1500rpm/min for 10min, and obtaining plasma from a subject for preparing an activation culture medium;
in addition, sterile PBS (1-1.5 times of the volume of the plasma) is added to the blood from which the plasma is removed to dilute the blood, and Peripheral Blood Mononuclear Cells (PBMCs) are separated by Ficoll-Paque to obtain mononuclear cells.
Cell density was adjusted to 1 × 10 using RPMI1640 medium containing 10% volume fetal bovine serum6Adding IFN-gamma to a final concentration of 2000U/mL, adding CD3 monoclonal antibody 24 hours later, wherein the final concentration of CD3 is 50 mug/mL, human interleukin-2 (IL-2), the final concentration of IL-2 is 500U/mL, the final concentration of IL-1 α is 50000U/mL, counting every 48 hours later, and counting according to 4 × 105Density of/mL was passaged while supplemented with IL-2, IL-1 α and IFN- γ.
The following two indexes (one) and (two) are adopted for comparison:
(I) comparison of proliferation fold of CIK cells
The CIK cells cultured by the two different culture methods are counted respectively on the 3 rd day, the 5 th day, the 10 th day and the 15 th day of the amplification stage, the amplification multiple is calculated, and statistical analysis is carried out. The results are as follows:
CIK proliferation multiple comparison statistical table
| Grouping | 3d | 5d | 10d | 15d |
| Comparative example | 2.2±0.4 | 7.5±2.1 | 98.6±13.9 | 784.2±43.7 |
| The invention | 3.1±0.3* | 12.8±3.4* | 164.3±21.7* | 1031.5±56.8* |
*: compared with the common culture method, the difference is that (P is less than 0.05)
As can be seen from the above table, the amplification times of the amplification method of the present invention during the culture period are much higher than those of the common culture method, and have significant differences, which are 1.41 times, 1.71 times, 1.67 times and 1.32 times of the amplification times of the conventional methods on days 3, 5, 10 and 15, respectively.
(II) measurement of cell killing Activity of CIK
The cells at the 9 th day of the expansion period of this culture method and the conventional culture method were collected, washed three times with PBS, and adjusted to a concentration of 2 × 10 using RPMI1640 culture medium6A cell suspension in/ml as effector cells; dividing A549 and K562 cells in logarithmic growth phase into fractionsRespectively 2 × 105A target cell suspension per mL; respectively taking 500 mu L of effector cells cultured by the method of the invention and 500 mu L of comparative effector cells, respectively acting on 500 mu L of target cell suspension, and uniformly mixing to obtain an experimental group. Setting effector cell natural release group (adding only CIK cell 500uL and RPMI1640 culture solution 500uL), target cell natural release group (adding only target cell 500uL and RPMI1640 culture medium 500uL), target cell maximum release group (adding only target cell 500uL and RPMI1640 culture medium 500uL and adding 10 uL/mL Triton-100 40min before detection), placing in an incubator for culturing for 6h, then gently mixing the cells, centrifuging at 1000rpm for 10min, collecting supernatant, measuring absorbance value A of 492nm wavelength by using lactate dehydrogenase method, setting 3 multiple wells for each group, and calculating killing activity average value according to the following formula.
The results are shown in fig. 1, the killing rate of the CIK cells cultured by the amplification method and the traditional amplification method to the cancer cells K562 and A549 is over 60%, and the killing effect of the CIK cells obtained by the method of the invention to the K562 cells is obviously higher than that of the traditional method, and the killing rate to the A549 is not lower than 70% at all.
Example 2 method for Rapid expansion of CIK cells
The method for rapidly amplifying the CIK cells in the embodiment is as follows:
① activating the culture flask to 75cm2Adding 13mL of activating agent into the cell culture bottle to cover the bottom of the bottle, and incubating for 1h at 37 ℃ to obtain an activated culture bottle, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody is 70 mu g/mL, and the concentration of the IFN-gamma is 1500U/mL;
preparing an activation culture medium: adding 5mL of autologous plasma and 1mL of CIK cell activator into 44mL of basal medium to obtain an activation medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution, the concentration of IL-2 is 70000U/mL, the concentration of IL-1 alpha is 70000U/mL, and the concentration of IFN-gamma is 80000U/mL, and the autologous plasma is obtained by taking anticoagulated peripheral blood, centrifuging at 2000rpm/min for 20min, separating and collecting the plasma, inactivating at 56 ℃ for 30min, and centrifuging at 1500rpm/min for 10 min;
③ separation and inoculation of mononuclear cells
First, peripheral blood mononuclear cells were isolated: taking 80ml of anticoagulated peripheral blood, centrifuging at 2000rpm/min for 20min, separating and collecting plasma, inactivating at 56 ℃ for 30min, centrifuging at 1500rpm/min for 10min, and obtaining plasma from a subject for preparing an activation culture medium;
the blood was diluted with 1-fold volume of sterile PBS buffer solution to the blood from which the plasma was removed, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll-Paque (available from Beijing Baiolai Boke technologies, Ltd., product No. BTN131136) to obtain mononuclear cells.
The mononuclear cells were then resuspended in activated medium at a controlled seeding density of 2 × 10, formulated at step ②6/mL, inoculated in the activated flask treated in step ①, 37 ℃ with 5% CO2Culturing for 72h in an incubator to obtain CIK cells, and gently washing the activated culture bottle with PBS (phosphate buffer solution) for 1-2 times before inoculation;
④ expanding CIK cells by centrifuging to remove the activated culture medium, adding the amplified culture medium, and standing at 37 deg.C under 5% CO2Culturing in an incubator, continuously using CIK cell amplification culture medium to dilute the cell density to 4 × 10 every 48h5The culture was carried out in/mL until the number of CIK cells reached the desired number. The amplification culture medium is a basic culture medium containing IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin, and the concentration of each component is as follows: the content of IL-2 is 600U/L, the content of ascorbic acid is 300mg/L, the content of vitamin E is 400mg/L, the content of human plasma albumin is 3g/L and the content of insulin is 15 ng/L.
In the embodiment, after 15 days of culture, the amplification quantity of CIK cells reaches 7 × 1010And (4) respectively.
Similar to example 1, during the culture period, the comparative example was cultured in a conventional culture method, specifically, a mononuclear cell was isolated in the same manner, except that the cell density was adjusted to 2 × 10 using RPMI1640 medium containing 10% volume of fetal bovine serum6/mL, adding IFN-gamma, and the final concentration is 1500U/mL; after 24 hours, addCD3 monoclonal antibody, CD3 final concentration is 70 μ g/mL, interleukin-2 (IL-2), IL-2 final concentration is 600U/mL, IL-1 α final concentration is 1400U/mL, count every 48h thereafter, according to 4 × 105Density of/mL was passaged while supplemented with IL-2, IL-1 α and IFN- γ.
The following two indexes (one) and (two) are adopted for comparison:
(I) comparison of proliferation fold of CIK cells
The CIK cells cultured by the two different culture methods are counted respectively on the 3 rd day, the 5 th day, the 10 th day and the 15 th day of the amplification stage, the amplification multiple is calculated, and statistical analysis is carried out. The results are as follows:
CIK proliferation multiple comparison statistical table
| Grouping | 3d | 5d | 10d | 15d |
| Comparative example | 2.5±0.3 | 11.4±2.8 | 129.3±14.4 | 980.6±42.7 |
| The invention | 3.6±0.2* | 16.1±3.1* | 193.8±16.1* | 1268.4±60.5* |
*: compared with the common culture method, the difference is that (P is less than 0.05)
As can be seen from the above table, the amplification times of the amplification method of the present invention during the culture period are much higher than those of the common culture method, and have significant differences, which are 1.44 times, 1.41 times, 1.50 times and 1.29 times of the amplification times of the conventional methods on days 3, 5, 10 and 15, respectively.
(II) measurement of cell killing Activity of CIK
The killing activity of CIK cells was measured in the same manner as in example 1, and the average value of the killing activity was calculated.
The results are shown in fig. 2, the killing rates of the CIK cells cultured by the amplification method and the traditional amplification method on the cancer cells K562 and A549 are both over 60%, and the killing effect of the CIK cells obtained by the method on the K562 cells is obviously higher than that of the traditional method, and the killing rate on the A549 is not lower than 70% at all.
Example 3
Based on the exploration of the method, the invention also provides a kit for rapid amplification of CIK cells, and the following contents and concentrations are only an example, and the concentration and the contents can be expanded by a person skilled in the art according to the conditions and requirements of the preparation, such as 10 times (10 times of final concentration in use), 50 times (50 times of final concentration in use) and the like so as to facilitate packaging, storage and application.
The kit comprises an activating agent, a CIK cell activating agent and a CIK cell proliferating agent.
The activator is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody is 30-100 mu g/mL, preferably 50 mu g/mL, and the concentration of the IFN-gamma is 1000-3000U/mL, preferably 2000U/mL;
the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution; the concentration of IL-2 in the CIK cell activator is 20000-100000U/mL, the concentration of IL-1 alpha is 20000-100000U/mL, the concentration of IFN-gamma is 50000-150000U/mL, preferably, the concentration of IL-2 is 50000U/mL, the concentration of IL-1 alpha is 50000U/mL, the concentration of IFN-gamma is 100000U/mL, and the dilution is 50 times when in use;
the CIK cell proliferation agent comprises IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin. The CIK cell proliferation agent contains 500-1000U of IL-2, 100-400mg of ascorbic acid, 800mg of vitamin E300, 3-8g of human plasma albumin and 5-20ng of insulin, preferably contains 700U of IL-2, 200mg of ascorbic acid, 500mg of vitamin E, 5g of human plasma albumin and 10ng of insulin, and is diluted by 1000mL of basal medium when in use, namely the CIK cell proliferation agent is used at the final concentration of 700U/L of IL-2, 200mg/L of ascorbic acid, 500mg/L of vitamin E, 5g/L of human plasma albumin and 10ng/L of insulin.
Example 4
According to the method steps described in example 1, this example mainly examines the amplification efficiency of CIK cells by different components of activation medium and amplification medium, and the experimental group setting and processing methods are shown in the following table:
the experiment was performed with reference to the method described in example 1, except that, in the step of amplifying CIK cells, the following experimental group and comparative group were set to examine the influence of the amplification medium components on the amplification efficiency.
*: compared with the experimental group, the compound has significant difference that P is less than 0.05.
Experiments prove that when the components in the amplification medium are matched, the cell proliferation promoting effect is most obvious and is respectively higher than that of other groups by about 41.5%, 16.7%, 61.4% and 21.2%.
Example 5
This example examines the effect of activated flasks on the phenotype of CIK cells, the experimental method referring to the method of example 1, except that the experimental and comparative groups were operated as follows during the activated flask step:
*: compared with the experimental group, the compound has significant difference that P is less than 0.05.
The above data demonstrate that in the method of the present invention, activated flasks are responsible for CD3, the primary effector cell of the CIK cells obtained+CD56+The proportion of double positive cells was more affected, compared to the blank control (comparative example 3) and the single component control (comparative example 1 and comparative example 2), in which CD3 was present in the experimental group+CD56+The proportion of double positive cells is obviously increased even higher than the sum of the three groups of the comparative examples, which shows that the activating reagent provided by the invention has scientific and reasonable proportion of the two components, has synergistic effect and obviously improves the CD3 of the main effect cell of CIK+CD56+Proportion of double positive cells.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for rapidly expanding CIK cells, which comprises the step of separating peripheral blood mononuclear cells, and is characterized by further comprising the following steps:
activating a culture flask: adding an activating agent into the culture bottle until the bottom of the culture bottle is covered, and incubating to obtain an activated culture bottle, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution;
preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium containing plasma to obtain an activation medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution;
③ inoculation of mononuclear cellsResuspending the mononuclear cells in the medium, inoculating in an activated flask, 5% CO at 37 ℃2Culturing for 48-96h in an incubator to obtain CIK cells;
④ expanding CIK cells by removing the activation medium, adding the amplification medium, 37 deg.C, 5% CO2Culturing in an incubator to a required amount, wherein the amplification culture medium is a basal culture medium containing IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin;
the concentration of the CD3 monoclonal antibody in the activator of the step I is 30-100 mu g/mL, and the concentration of IFN-gamma is 1000-3000U/mL; the concentration of IL-2 in the CIK cell activator of the step (II) is 20000-100000U/mL, the concentration of IL-1 alpha is 20000-100000U/mL, and the concentration of IFN-gamma is 50000-150000U/mL; the concentration of IL-2 in the amplification culture medium in the step (IV) is 500-1000U/L, the concentration of ascorbic acid is 100-400mg/L, the concentration of vitamin E is 300-800mg/L, the concentration of human plasma albumin is 3-8g/L, and the concentration of insulin is 5-20 ng/L.
2. The method as claimed in claim 1, wherein the CD3 monoclonal antibody concentration in the activator of step (i) is 50 μ g/mL, and the IFN- γ concentration is 2000U/mL; the concentration of IL-2 in the CIK cell activator in the step II is 50000U/mL, the concentration of IL-1 alpha is 50000U/mL, and the concentration of IFN-gamma is 100000U/mL; the concentration of IL-2 in the amplification culture medium in the step (IV) is 700U/L, the concentration of ascorbic acid is 200mg/L, the concentration of vitamin E is 500mg/L, the concentration of human plasma albumin is 5g/L, and the concentration of insulin is 10 ng/L.
3. The method of claim 1, wherein the basal medium is RPMI-1640 basal medium; in the step III, the plasma is inactivated autologous plasma, and the plasma content is 10% of the volume of the activation culture medium.
4. The method as claimed in claim 1, wherein the incubation in step (i) is carried out at a temperature of 4 ℃ to 37 ℃ for 1 to 18 hours.
5. The method according to claim 1, wherein the amplification medium is replaced every 1 to 3 days when the CIK cells are amplified in the step (iv).
6. The kit for rapid amplification of CIK cells is characterized by comprising an activating agent, a CIK cell activating agent and a CIK cell proliferating agent, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS (phosphate buffer solution); the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution; the CIK cell proliferation agent comprises IL-2, ascorbic acid, vitamin E, human plasma albumin and insulin.
7. The kit as claimed in claim 6, wherein the activator contains CD3 monoclonal antibody at a concentration of 30-100 μ g/mL, and IFN- γ at a concentration of 1000-3000U/mL; the concentration of IL-2 in the CIK cell activator is 20000-100000U/mL, the concentration of IL-1 alpha is 20000-100000U/mL, the concentration of IFN-gamma is 50000-150000U/mL, and the CIK cell activator is diluted by 50 times when in use; the CIK cell proliferation agent contains 500-1000U of IL-2, 100-400mg of ascorbic acid, 800mg of vitamin E, 3-8g of human plasma albumin and 5-20ng of insulin, and is diluted to 1000mL by a basic culture medium when in use.
8. The kit of claim 6, wherein the CD3 monoclonal antibody concentration in the activator is 50 μ g/mL, the IFN- γ concentration is 2000U/mL; IL-2 concentration in CIK cell activator is 50000U/mL, IL-1 alpha concentration is 50000U/mL, IFN-gamma concentration is 100000U/mL, and the CIK cell activator is diluted by 50 times when in use; the CIK cell proliferation agent contains 700U of IL-2, 200mg of ascorbic acid, 500mg of vitamin E, 5g of human plasma albumin and 10ng of insulin, and is diluted to 1000mL by a basal medium when in use.
9. The kit of claim 6 or 7, wherein the basal medium is RPMI-1640 basal medium.
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