CN108676775B - Method for amplifying cord blood NK in vitro - Google Patents
Method for amplifying cord blood NK in vitro Download PDFInfo
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Abstract
The invention discloses a method for amplifying cord blood NK (natural killer) in vitro, relates to a method for amplifying cord blood NK, and aims to solve the problems that the existing cord blood NK amplification method is complex and high in cost, and is low in safety, long in vitro amplification time and low in cell purity due to the introduction of animal-derived components. The invention has simple operation, low cost, no introduction of animal source components and high safety. The invention can harvest the cord blood NK cells after culturing for 15 days, the cell purity is 95.33%, the total number of cells is 120 hundred million/part of cord blood, and the killing effect on lung cancer A549 cells is good.
Description
Technical Field
The invention relates to a method for amplifying cord blood NK in vitro.
Background
Surgery and chemotherapy have been used as the primary methods of eliminating tumors for the last decade. However, the rate of tumor recurrence is significantly increased by the resistance of the body to anti-tumor drugs. Therefore, there is an urgent need to develop an effective method for resolving the problem of tumor recurrence. Recent studies have shown that the immune system plays an important role in suppressing the onset of malignant tumors. Natural killer cells (NK) are derived from hematopoietic precursor cells in the bone marrow and are widely distributed in different tissues, such as peripheral blood, spleen, lung, liver and uterus. NK cells account for 5% -10% of lymphocytes in normal human peripheral blood and 10% -20% of lymphocytes in cord blood, and a great deal of research has proved the role and the treatment potential of NK cells in cancer immunity, and simultaneously, the occurrence of solid tumors and hematologic malignant tumors is related to the function damage of the NK cells. The NK cell has an effective anti-tumor function, can selectively identify and kill a ligand of a high-expression NK cell activating receptor and a non-self tumor cell of a low-expression MHC I molecule, inhibits the growth and the metastasis of the tumor cell, and further effectively eliminates the tumor.
The immune system of tumor patients is worse than healthy people, and part of patients are already subjected to radiotherapy and chemotherapy in the early stage, so that the quality and the amplification capacity of immune cells are poorer, and meanwhile, a large number of clinical researches show that the self NK cell feedback treatment effect is poor, mainly because inhibitory KIR molecules exist on the surface of NK cells, can recognize and combine with self HLA-I molecules, and therefore activation of NK cells is inhibited. The proportion of NK cells in umbilical cord blood is obviously higher than that of NK cells in peripheral blood, and meanwhile, a large number of CD34+ stem cells exist in the umbilical cord blood, and can be differentiated into the NK cells through factor induction, and the source of the umbilical cord blood is rich, so that the umbilical cord blood can be used as an amplification material of the NK cells to perform in-vitro induced differentiation and amplification on the NK cells and the stem cells, thereby providing allogeneic NK cells for tumor treatment and providing an optimal method for immunotherapy.
Currently, various methods of expanding NK cells in vitro have been reported, thereby achieving higher therapeutic doses of cells and enhancing activity and in vivo proliferation potential. Such as the trophoblast method, which is the method of adding tumor cell lines, such as K562 cells, etc., and the factor method, the exosomes released by the trophoblast method can promote the transformation of normal hematopoietic stem cells, thereby stimulating the proliferation of NK cells, but can cause the risk of tumor cell generation during the NK back transfusion treatment process; the factor method is characterized in that related factors for promoting NK cell differentiation and proliferation, such as interleukin-2 (IL-2), interleukin-15 (IL-15) and the like are added, so that the activity of NK cells can be enhanced, the long-term growth of the NK cells can be maintained, no risk is introduced, and the safety is high. IL-15 plays an important role in the process of NK cell differentiation and development, and researches have proved that IL-15 can promote the directional differentiation of CD34+ cells separated from cord blood into NK cells, IL-21, namely interleukin-21, has a molecular structure similar to IL-15, participates in regulating B cell proliferation, and cooperates with IL-15 to promote the proliferation of bone marrow precursor cells and the proliferation, differentiation and cytotoxic activity of NK cells.
NK cells have an anti-tumor effect and are increasingly paid more attention to clinical application. In clinical application, NK cells need to reach a certain cell number and higher cell purity, so how to obtain higher cell treatment dose and higher purity when the cord blood NK cells are expanded in vitro becomes the key of tumor treatment.
Disclosure of Invention
The invention aims to solve the problems of complex and high cost of the existing cord blood NK amplification method, low safety, long in-vitro amplification time and low cell purity due to the introduction of animal-derived components, and provides the method for in-vitro amplification of the cord blood NK.
The invention relates to a method for amplifying cord blood NK in vitro, which comprises the following steps:
firstly, adding coating liquid and normal saline into a T75 culture bottle according to the volume ratio of 1: 4; 1mL of coating solution contains the following components: CD16 monoclonal antibody with the concentration of 3-7 mug/mL, IFN-gamma with the concentration of 800-1200 mug/mL and GM-CSF with the concentration of 400-600 IU/mL; shaking the cell bottle to make the coating solution spread on the bottom of the bottle, standing at room temperature for 1h, removing the coating solution, washing the bottom of the bottle with physiological saline once, removing the physiological saline, and placing in a T75 culture bottle for later use;
second, activation of mononuclear cells in cord blood
A. Isolation of mononuclear cells:
the method comprises the following steps of (1) subpackaging 80-100mL of cord blood in a centrifuge tube, centrifuging for 15min at room temperature at 650g, removing upper-layer light yellow plasma, and adding physiological saline with the same volume as the upper-layer light yellow plasma to obtain diluted cord blood; taking another centrifuge tube, adding lymphocyte separation liquid, adding diluted cord blood into the lymphocyte separation liquid, layering the diluted blood and the lymphocyte separation liquid, then centrifuging for 30min at room temperature at 650g, removing part of supernatant, absorbing the middle tunica albuginea layer into the centrifuge tube, then adding physiological saline with the same volume, centrifuging for 10min at room temperature of 260g, removing supernatant, washing for three times, and counting;
wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15: 45-50;
B. activation of monocytes:
removing supernatant from the precipitate in the previous step at a cell density of 2.0 × 106Inoculating the cells/mL into GT-T551H3 serum-free culture medium, and adding factors to form a mixed solution; placing the mixed solution in the T75 culture flask coated in the first step, and adding 5 vol% CO at 37 deg.C2Culturing for 1-5 days in an incubator with saturated humidity; and at 3d, the cell density is 0.8-1.0 x 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished;
wherein the factor is formed by mixing IL-2, IL-7, IL-15 and IL-21; the concentration of IL-2 in the mixed solution is 400-600 IU/mL, the concentration of IL-7 is 8-12 ng/mL, the concentration of IL-15 is 80-120 ng/mL, and the concentration of IL-21 is 150-200 ng/mL;
the fluid replacement in the step is a GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL;
third, in vitro amplification of cord blood NK cells
After 5 days of the previous step of culture, the cell density is 0.8-1.0 multiplied by 10 every 2 days6Supplementing each/mL of the solution, and completing the in-vitro amplification cord blood NK method after amplification;
the fluid replacement in the step is GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL.
The GT-T551H3 serum-free medium used in the present invention was purchased from TAKARA.
The invention has the following beneficial effects:
1. the method has the advantages of simple operation in both the activation process and the amplification process of the mononuclear cells, fewer steps, simple experimental process, avoidance of pollution caused by complicated operation process and high safety.
2. In the invention, the cord blood NK cells can be harvested after 15 days of culture, and the total number of the cells is up to 120 hundred million, thereby greatly shortening the in vitro amplification time; the culture time is 15 days, the cell density is high, the use of a culture medium is saved, the manpower and material resources are saved, and the cost is reduced.
3. The purity of the NK cells of the cord blood obtained by the invention is high and can reach 95.33%, and meanwhile, the NK cells have a good killing effect on tumor cells, and only the effective target ratio is 10: the killing efficiency can reach 91.65% at 1 hour.
4. According to the invention, the factor stimulation method is used for culturing the cord blood NK, the trophoblast cells are not added, the risk of inducing tumors in the NK reinfusion treatment process is avoided, animal-derived components are not introduced into the system, and the safety is higher.
5. The coating liquid contains various factors and can stimulate the activation of mononuclear cells, the cells of the NK cells of the cord blood are more agglomerated in the 5d, and compared with other coating liquids, the coating liquid has a more obvious activation effect.
6. In the invention, by adopting a differential culture mode, CD34+ cells in cord blood are differentiated and induced by utilizing high-concentration IL-15(100ng/mL) and IL-21(180ng/mL), and low-concentration IL-15(50ng/mL) and IL-21(100ng/mL) are adopted in the induction and amplification of NK cells and precursor cells thereof, so that the efficiency of NK differentiation into precursor cells is ensured, and mature cord blood NK cells with higher quantity, high purity and killing activity are amplified.
Drawings
FIG. 1: 0d cord blood NK cell map;
FIG. 2: cord blood NK cell morphology map 15 d;
FIG. 3: current methods No. 5d cord blood NK cell morphogram;
FIG. 4: in the method of the invention, the 5d cord blood NK cell morphology map;
FIG. 5: detecting the 0d cord blood NK cell map by a flow cytometer;
FIG. 6: detecting the purity map of the 15d cord blood NK cells by a flow cytometer;
FIG. 7: the ratio of the umbilical blood NK cells to the lung cancer A549 cells is 10:1 killing experiment result chart.
Detailed Description
The first embodiment is as follows: the method for amplifying the cord blood NK in vitro of the embodiment comprises the following specific steps:
firstly, adding coating liquid and normal saline into a T75 culture bottle according to the volume ratio of 1: 4; 1mL of coating solution contains the following components: CD16 monoclonal antibody with the concentration of 3-7 mug/mL, IFN-gamma with the concentration of 800-1200 mug/mL and GM-CSF with the concentration of 400-600 IU/mL; shaking the cell bottle to make the coating solution spread on the bottom of the bottle, standing at room temperature for 1h, removing the coating solution, washing the bottom of the bottle with physiological saline once, removing the physiological saline, and placing in a T75 culture bottle for later use;
second, activation of mononuclear cells in cord blood
A. Isolation of mononuclear cells:
the method comprises the following steps of (1) subpackaging 80-100mL of cord blood in a centrifuge tube, centrifuging for 15min at room temperature at 650g, removing upper-layer light yellow plasma, and adding physiological saline with the same volume as the upper-layer light yellow plasma to obtain diluted cord blood; taking another centrifuge tube, adding lymphocyte separation liquid, adding diluted cord blood into the lymphocyte separation liquid, layering the diluted blood and the lymphocyte separation liquid, then centrifuging for 30min at room temperature at 650g, removing part of supernatant, absorbing the middle tunica albuginea layer into the centrifuge tube, then adding physiological saline with the same volume, centrifuging for 10min at room temperature of 260g, removing supernatant, washing for three times, and counting;
wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15: 45-50;
B. activation of monocytes:
removing supernatant from the precipitate in the previous step at a cell density of 2.0 × 106Inoculating the cells/mL into GT-T551H3 serum-free culture medium, and adding factors to form a mixed solution; placing the mixed solution in the T75 culture flask coated in the first step, and adding 5 vol% CO at 37 deg.C2Culturing for 1-5 days in an incubator with saturated humidity; and at 3d, the cell density is 0.8-1.0 x 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished;
wherein the factor is formed by mixing IL-2, IL-7, IL-15 and IL-21; the concentration of IL-2 in the mixed solution is 400-600 IU/mL, the concentration of IL-7 is 8-12 ng/mL, the concentration of IL-15 is 80-120 ng/mL, and the concentration of IL-21 is 150-200 ng/mL;
the fluid replacement in the step is a GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL;
third, in vitro amplification of cord blood NK cells
After 5 days of the previous step of culture, the cell density is 0.8-1.0 multiplied by 10 every 2 days6Supplementing each/mL of the solution, and completing the in-vitro amplification cord blood NK method after amplification;
the fluid replacement in the step is GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: 1mL of coating solution contains the following components: CD16 monoclonal antibody with concentration of 5 mug/mL, IFN-gamma with concentration of 1000 mug/mL and GM-CSF with concentration of 500 IU/mL. The rest is the same as the first embodiment.
The third concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the volume ratio of the lymphocyte separation solution to the diluted cord blood was 15: 50. The rest is the same as the first embodiment.
The fourth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the factor is formed by mixing IL-2, IL-7, IL-15 and IL-21; and the concentration of IL-2 in the mixed solution is 500IU/mL, the concentration of IL-7 is 10ng/mL, the concentration of IL-15 is 100ng/mL, and the concentration of IL-21 is 180 ng/mL. The rest is the same as the first embodiment.
The fifth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the second-step fluid replacement is GT-T551H3 serum-free medium containing IL-2 with the concentration of 500IU/mL, IL-7 with the concentration of 10ng/mL, IL-15 with the concentration of 50ng/mL and IL-21 with the concentration of 100 ng/mL. The rest is the same as the first embodiment.
The sixth specific implementation mode: the first difference between the present embodiment and the specific embodiment is: the third-step fluid replacement is GT-T551H3 serum-free medium containing IL-2 with the concentration of 500IU/mL, IL-7 with the concentration of 10ng/mL, IL-15 with the concentration of 50ng/mL and IL-21 with the concentration of 100 ng/mL. The rest is the same as the first embodiment.
The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.
The beneficial effects of the present invention are demonstrated by the following examples:
example 1
The method for amplifying the cord blood NK in vitro comprises the following specific steps:
first, antibody coated cell bottle
1mL of coating solution and 4mL of physiological saline were added to a T75 culture flask, wherein the 1mL of coating solution contained the following components: CD16 monoclonal antibody (5. mu.g/mL), IFN-. gamma. (1000. mu.g/mL), GM-CSF (500 IU/mL); slowly shaking the cell bottle to enable the coating solution to be paved at the bottom of the bottle, standing for 1h at room temperature, removing the coating solution, and washing the bottom of the bottle once by using 10mL of physiological saline;
second, activation of mononuclear cells in cord blood
Isolation of mononuclear cells:
one part of cord blood is about 80-100mL, the cord blood is divided into 2 centrifuge tubes with 50mL, the centrifuge tube is centrifuged for 15min at room temperature under 650g, after removing the upper pale yellow plasma, physiological saline is used for supplementing the original volume of the plasma, 2 new centrifuge tubes with 50mL are taken, 15mL of lymphocyte separation liquid (Ficoll) is added, diluted blood is slowly added into the Ficoll, the diluted blood and the Ficoll have clear layering, the centrifuge tube is centrifuged for 30min at room temperature under 650g, obvious layering can be seen at this moment, the lower layer is red blood cells, the middle white membrane layer is mononuclear cells, after removing part of supernatant, the white membrane layer is carefully sucked into a new centrifuge tube with 50mL, the isometric physiological saline is added, the centrifuge tube is centrifuged for 10min at room temperature under 260g, the supernatant is removed, the supernatant is washed for three times, and the count is carried out;
activation of monocytes:
in 0-5d is the activation process of the mononuclear cells;
firstly, the cell density is 2.0 multiplied by 106one/mL the pellet from the previous step was inoculated into a coated T75 flask containing 20mL GT-T551H3 serum-free medium, and IL-2(500IU/mL), IL-7(10ng/mL), IL-15(5 ng/mL) were added0ng/mL) and placed at 37 ℃ in 5% CO2Culturing in an incubator with saturated humidity;
at 3d, the cell density is 0.8-1.0X 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished; the supplement solution is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL) and IL-15(50 ng/mL);
third, in vitro amplification of cord blood NK cells
Then every 2d according to the cell density of 0.8-1.0 multiplied by 106Per mL of the solution is replenished; the liquid supplementing culture medium is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL) and IL-15(50 ng/mL); count at 15d, check purity and kill experiment.
Example 2
The method for amplifying the cord blood NK in vitro comprises the following specific steps:
first, antibody coated cell bottle
1mL of coating solution and 4mL of physiological saline were added to a T75 culture flask, wherein the 1mL of coating solution contained the following components: CD16 monoclonal antibody (5. mu.g/mL), IFN-. gamma. (1000. mu.g/mL), GM-CSF (500 IU/mL); slowly shaking the cell bottle to enable the coating solution to be paved at the bottom of the bottle, standing for 1h at room temperature, removing the coating solution, and washing the bottom of the bottle once by using 10mL of physiological saline;
second, activation of mononuclear cells in cord blood
Isolation of mononuclear cells:
one part of cord blood is about 80-100mL, the cord blood is divided into 2 centrifuge tubes with 50mL, the centrifuge tube is centrifuged for 15min at room temperature under 650g, after removing the upper pale yellow plasma, physiological saline is used for supplementing the original volume of the plasma, 2 new centrifuge tubes with 50mL are taken, 15mL of lymphocyte separation liquid (Ficoll) is added, diluted blood is slowly added into the Ficoll, the diluted blood and the Ficoll have clear layering, the centrifuge tube is centrifuged for 30min at room temperature under 650g, obvious layering can be seen at this moment, the lower layer is red blood cells, the middle white membrane layer is mononuclear cells, after removing part of supernatant, the white membrane layer is carefully sucked into a new centrifuge tube with 50mL, the isometric physiological saline is added, the centrifuge tube is centrifuged for 10min at room temperature under 260g, the supernatant is removed, the supernatant is washed for three times, and the count is carried out;
activation of monocytes:
in 0-5d is the activation process of the mononuclear cells;
firstly, the cell density is 2.0 multiplied by 106one/mL was inoculated into a coated T75 flask containing 20mL of GT-T551H3 serum-free medium, and IL-2(500IU/mL), IL-7(10ng/mL), IL-15(50ng/mL), IL-21(100ng/mL) were added and placed in a medium at 37 ℃ with 5% CO2Culturing in an incubator with saturated humidity;
at 3d, the cell density is 0.8-1.0X 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished; the supplement solution is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL), IL-15(50ng/mL) and IL-21(100 ng/mL);
third, in vitro amplification of cord blood NK cells
Then every 2d according to the cell density of 0.8-1.0 multiplied by 106Per mL of the solution is replenished; the liquid supplementing culture medium is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL), IL-15(50ng/mL) and IL-21(100 ng/mL); count at 15d, check purity and kill experiment.
Example 3
The method for amplifying the cord blood NK in vitro comprises the following specific steps:
first, antibody coated cell bottle
1mL of coating solution and 4mL of physiological saline were added to a T75 culture flask, wherein the 1mL of coating solution contained the following components: CD16 monoclonal antibody (5. mu.g/mL), IFN-. gamma. (1000. mu.g/mL), GM-CSF (500 IU/mL); slowly shaking the cell bottle to enable the coating solution to be paved at the bottom of the bottle, standing for 1h at room temperature, removing the coating solution, and washing the bottom of the bottle once by using 10mL of physiological saline;
second, activation of mononuclear cells in cord blood
Isolation of mononuclear cells:
one part of cord blood is about 80-100mL, the cord blood is divided into 2 centrifuge tubes with 50mL, the centrifuge tube is centrifuged for 15min at room temperature under 650g, after removing the upper pale yellow plasma, physiological saline is used for supplementing the original volume of the plasma, 2 new centrifuge tubes with 50mL are taken, 15mL of lymphocyte separation liquid (Ficoll) is added, diluted blood is slowly added into the Ficoll, the diluted blood and the Ficoll have clear layering, the centrifuge tube is centrifuged for 30min at room temperature under 650g, obvious layering can be seen at this moment, the lower layer is red blood cells, the middle white membrane layer is mononuclear cells, after removing part of supernatant, the white membrane layer is carefully sucked into a new centrifuge tube with 50mL, the isometric physiological saline is added, the centrifuge tube is centrifuged for 10min at room temperature under 260g, the supernatant is removed, the supernatant is washed for three times, and the count is carried out;
activation of monocytes:
in 0-5d is the activation process of the mononuclear cells;
firstly, the cell density is 2.0 multiplied by 106one/mL was inoculated into a coated T75 flask containing 20mL of GT-T551H3 serum-free medium, and IL-2(500IU/mL), IL-7(10ng/mL), IL-15(100ng/mL), IL-21(180ng/mL) were added and placed in a medium at 37 ℃ with 5% CO2Culturing in an incubator with saturated humidity;
at 3d, the cell density is 0.8-1.0X 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished; the supplement solution is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL), IL-15(50ng/mL) and IL-21(100 ng/mL);
third, in vitro amplification of cord blood NK cells
Then every 2d according to the cell density of 0.8-1.0 multiplied by 106Per mL of the solution is replenished; the replacement fluid is GT-T551H3 serum-free culture medium containing IL-2(500IU/mL), IL-7(10ng/mL), IL-15(50ng/mL) and IL-21(100 ng/mL); count at 15d, check purity and kill experiment.
Validation of cord blood NK cells after expansion in examples 1 to 3:
1. cord blood NK cells trypan blue count:
mixing NK cells of cord blood, mixing 10 μ L and 10 μ L0.2% trypan blue, adding into counting plate, and counting with microscope.
After each counting, the number is 0.8-1.0 multiplied by 106The solution is replenished per mL. The total number of NK cells in cord blood at day 0 and day 15 are shown in Table 1, and the cell morphology is shown in FIG. 1 and FIG. 2The 5d cord blood NK cell morphology is shown in FIGS. 3 and 4;
2. detecting the purity of NK cells in cord blood by a flow cytometer:
collecting 5mL of the cord blood NK cells cultured for 0 day and the cord blood NK cells cultured for 15 days, centrifuging at 1300rpm for 2 min, removing supernatant, washing with PBS, and regulating cell number to 2 × 106each/mL, 500. mu.L of LPBS were resuspended and placed in a flow cytometry EP tube, 5. mu.L of each of CD3 and CD56 antibody were added, incubated at room temperature for 20min in the dark, isotype control was set, centrifuged at 1300rpm for 2 min, the supernatant was removed, 500. mu.L of PBS was added and centrifuged at 1300rpm for 2 min, the supernatant was removed, and finally resuspended with 500. mu.L of PBS and tested on the machine.
The results of day 15 cord blood NK cell flow assays are shown in table 2 and fig. 5 and 6.
3. Tumor cell killing experiment:
1) to the killer well plate, 50. mu.L of complete medium for culturing A549 cells (human lung cancer cells) was added, and the baseline was adjusted.
2) A549 cells with good growth state are used as target cells, the confluence degree of more than 90% can be digested by 0.25% pancreatin, trypan blue count is carried out after digestion is stopped, centrifugation is carried out at 1300rpm for 5min, the cells are suspended by culture medium after supernatant is removed, and the diluted cell density reaches 1 × 105Per mL, 100. mu.L of cell suspension was added to the kill well plate and incubated overnight in an incubator.
3) The NK cells of the cord blood are counted the next day, the NK cells of the cord blood are taken as effector cells, and the cell density reaches 2 multiplied by 106individual/mL, in an effective target ratio of 5: 1. 10: 1. 20:1, add to killer well plates, set up in 3 replicates per set, incubate overnight in incubator.
Tumor killing data analysis is shown in table 3 and fig. 7.
TABLE 1
| Example 1 | Example 2 | Example 3 | |
| NK cell number at |
4.0*107An | 4.0*107An | 4.0*107An |
| NK cell number at day 15 | 6.8*109An | 8.2*109An | 12.0*109An |
| Fold expansion of cells | 170 | 205 | 300 |
TABLE 2
TABLE 3
| Example 1 | Example 2 | Example 3 | |
| Killing rate at effective target ratio (5:1) | 24.63％ | 26.17％ | 30.57％ |
| Killing rate at effective target ratio (10:1) | 85.07％ | 87.56％ | 91.65％ |
| Killing rate at effective target ratio (20:1) | 81.71％ | 84.36％ | 88.29％ |
Through the analysis and comparison of different cytokines and concentrations in the above examples 1-3, it can be seen that the cytokine and concentration used in example 3 have the best effect, and the cord blood NK cells can be harvested after 15 days of culture in example 3, and the total number of cells is up to 120 hundred million, thereby greatly shortening the in vitro amplification time. The purity of the NK cells of the cord blood obtained in the embodiment 3 is high and can reach 95.33%, and meanwhile, the NK cells have a good killing effect on tumor cells, and only the effective target ratio is 10: the killing efficiency can reach 91.65% at 1 hour.
Claims (6)
1. A method for amplifying cord blood NK in vitro is characterized by comprising the following specific steps:
firstly, adding coating liquid and normal saline into a T75 culture bottle according to the volume ratio of 1: 4; 1mL of coating solution contains the following components: CD16 monoclonal antibody with the concentration of 3-7 mug/mL, IFN-gamma with the concentration of 800-1200 mug/mL and GM-CSF with the concentration of 400-600 IU/mL; shaking the cell bottle to make the coating solution spread on the bottom of the bottle, standing at room temperature for 1h, removing the coating solution, washing the bottom of the bottle with physiological saline once, removing the physiological saline, and placing in a T75 culture bottle for later use;
second, activation of mononuclear cells in cord blood
A. Isolation of mononuclear cells:
the method comprises the following steps of (1) subpackaging 80-100mL of cord blood in a centrifuge tube, centrifuging for 15min at room temperature at 650g, removing upper-layer light yellow plasma, and adding physiological saline with the same volume as the upper-layer light yellow plasma to obtain diluted cord blood; taking another centrifuge tube, adding lymphocyte separation liquid, adding diluted cord blood into the lymphocyte separation liquid, layering the diluted blood and the lymphocyte separation liquid, then centrifuging for 30min at room temperature at 650g, removing part of supernatant, absorbing the middle tunica albuginea layer into the centrifuge tube, then adding physiological saline with the same volume, centrifuging for 10min at room temperature of 260g, removing supernatant, washing for three times, and counting;
wherein the volume ratio of the lymphocyte separation liquid to the diluted cord blood is 15: 45-50;
B. activation of monocytes:
removing supernatant from the precipitate in the previous step at a cell density of 2.0 × 106Inoculating the cells/mL into GT-T551H3 serum-free culture medium, and adding factors to form a mixed solution; placing the mixed solution in the T75 culture flask coated in the first step, and adding 5 vol% CO at 37 deg.C2Culturing for 1-5 days in an incubator with saturated humidity; and at 3d, the cell density is 0.8-1.0 x 106Supplementing each/mL, turning to the bag at 5d, and packaging according to the cell density of 0.8-1.0 × 106Per mL of the solution is replenished;
wherein the factor is formed by mixing IL-2, IL-7, IL-15 and IL-21; the concentration of IL-2 in the mixed solution is 400-600 IU/mL, the concentration of IL-7 is 8-12 ng/mL, the concentration of IL-15 is 80-120 ng/mL, and the concentration of IL-21 is 150-200 ng/mL;
the fluid replacement in the step is a GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL;
third, in vitro amplification of cord blood NK cells
After 5 days of the previous step of culture, the cell density is 0.8-1.0 multiplied by 10 every 2 days6Supplementing each/mL of the solution, and completing the in-vitro amplification cord blood NK method after amplification;
the fluid replacement in the step is GT-T551H3 serum-free culture medium containing IL-2 with the concentration of 400-600 IU/mL, IL-7 with the concentration of 8-12 ng/mL, IL-15 with the concentration of 40-60 ng/mL and IL-21 with the concentration of 80-120 ng/mL.
2. The method for in vitro expansion of NK from cord blood according to claim 1, wherein 1mL of coating solution comprises the following components: CD16 monoclonal antibody with concentration of 5 mug/mL, IFN-gamma with concentration of 1000 mug/mL and GM-CSF with concentration of 500 IU/mL.
3. The method of claim 1, wherein the volume ratio of lymphocyte separation solution to diluted cord blood is 15: 50.
4. The method of claim 1, wherein the factor is a mixture of IL-2, IL-7, IL-15 and IL-21; and the concentration of IL-2 in the mixed solution is 500IU/mL, the concentration of IL-7 is 10ng/mL, the concentration of IL-15 is 100ng/mL, and the concentration of IL-21 is 180 ng/mL.
5. The method of claim 1, wherein the second fluid replacement step is GT-T551H3 serum-free medium containing IL-2 at a concentration of 500IU/mL, IL-7 at a concentration of 10ng/mL, IL-15 at a concentration of 50ng/mL, and IL-21 at a concentration of 100 ng/mL.
6. The method of claim 1, wherein the fluid replacement in step three is GT-T551H3 serum-free medium containing IL-2 at a concentration of 500IU/mL, IL-7 at a concentration of 10ng/mL, IL-15 at a concentration of 50ng/mL, and IL-21 at a concentration of 100 ng/mL.
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| CN109536455B (en) * | 2018-12-11 | 2020-02-21 | 武汉波睿达生物科技有限公司 | CAR-NK cell and preparation method and application thereof |
| CN109628397B (en) * | 2019-02-03 | 2023-01-10 | 福建省海西细胞生物工程有限公司 | Method for in-vitro amplification culture of NK (natural killer) cells |
| CN111073854A (en) * | 2020-01-23 | 2020-04-28 | 中科宝承生物医学科技有限公司 | Efficient amplification process suitable for NK cells from cord blood |
| CN113416700B (en) * | 2021-07-09 | 2023-04-11 | 天晴干细胞股份有限公司 | Efficient factor-secreting NK cell amplification method and application thereof |
| CN115651903B (en) * | 2022-11-14 | 2023-03-17 | 四川新生命干细胞科技股份有限公司 | High-lethality immune cell population, and culture method, reagent composition and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861435A (en) * | 2016-05-11 | 2016-08-17 | 南京华奥生物医药技术有限公司 | In-vitro amplification method of natural killer cells (NK) |
| CN107164323A (en) * | 2017-07-13 | 2017-09-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of method for the lymphocyte subgroup for obtaining tool High Fragmentation tumour cell ability |
| CN108103020A (en) * | 2018-02-01 | 2018-06-01 | 上海莱馥生命科学技术有限公司 | A kind of preparation method of efficient amplification natural killer cells |
-
2018
- 2018-06-08 CN CN201810589681.8A patent/CN108676775B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861435A (en) * | 2016-05-11 | 2016-08-17 | 南京华奥生物医药技术有限公司 | In-vitro amplification method of natural killer cells (NK) |
| CN107164323A (en) * | 2017-07-13 | 2017-09-15 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of method for the lymphocyte subgroup for obtaining tool High Fragmentation tumour cell ability |
| CN108103020A (en) * | 2018-02-01 | 2018-06-01 | 上海莱馥生命科学技术有限公司 | A kind of preparation method of efficient amplification natural killer cells |
Non-Patent Citations (2)
| Title |
|---|
| A Two-Phase Expansion Protocol Combining Interleukin(IL)-15 and IL-21 Improves Natural Killer Cell Proliferation and Cytotoxicity against Rhabdomyosarcoma.;Wagner et al.;《frontiers in Immunology》;20170612;第8卷(第676期);1-16 * |
| NK细胞的高效扩增及其对体外肿瘤模型的杀伤效果研究;刘春香 等;《中国免疫学杂志》;20171231;第33卷;1186-1190,1196 * |
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