CN115109749A - Induced culture method of CIK cells - Google Patents

Induced culture method of CIK cells Download PDF

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CN115109749A
CN115109749A CN202210972485.5A CN202210972485A CN115109749A CN 115109749 A CN115109749 A CN 115109749A CN 202210972485 A CN202210972485 A CN 202210972485A CN 115109749 A CN115109749 A CN 115109749A
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解静
陈博
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Nanjing Sijijing Technology Co ltd
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Abstract

The invention discloses an induced culture method of CIK cells, which comprises the following steps: (1) isolating mononuclear cells from peripheral blood; (2) suspending the mononuclear cells with X-VIVO 15 medium, culturing, supplementing IL-2, IL-18, green algae growth factor, glutamine, and CD3 monoclonal antibody into the culture medium on the 2 nd day of culture, and supplementing solution every 2-3 days to the 7 th day; (3) supplementing liquid every 2-3 days from day 8, and performing induction culture for 14 days; the nutrient medium of the liquid supplementing in the step (2) is prepared by adding the following components into an X-VIVO 15 nutrient medium: IL-2, IL-18, green algae growth factor; the liquid supplementing culture medium in the step (3) is prepared by adding the following components into an X-VIVO 15 culture medium: IL-2, vitexin, methimazole. The induction culture method provided by the invention promotes the proliferation of the CIK cells, effectively improves the proliferation efficiency of the CIK cells, obtains the CIK cells with high purity and good tumor killing activity, and ensures the application effect of the CIK cells in the immunotherapy.

Description

Induced culture method of CIK cells
Technical Field
The invention relates to the field of immune cells, in particular to an induction culture method of CIK cells.
Background
CIK cells are a group of cells obtained by co-culturing human mononuclear cells with various cytokines in vitro for a period of time, are also called NK cell-like T lymphocytes, have T cell and NK cell-like phenotypic characteristics, and have the advantages of strong anti-tumor activity of the T lymphocytes and non-MHC (major histocompatibility complex) tumor killing of the NK cells. The CIK cell has the characteristics of strong proliferation capacity, strong tumor killing activity, wide tumor killing spectrum, small toxicity to normal bone marrow hematopoietic precursor cells and the like, and is widely used in cellular immunotherapy.
CIK cells have various tumor killing mechanisms, can directly kill tumors, induce tumor cell apoptosis through Fas/FasL signal channels, generate the tumor killing inhibition effect of a large amount of cell factors after CIK activation, activate the immune system of an organism and improve the immune function of the organism. The CIK cell can specifically identify tumor cells and cells infected by viruses without reacting to healthy cells, and has application in treating malignant melanoma, gastric cancer, renal cancer, breast cancer, liver cancer, lung cancer and the like.
Therapeutic CIK cells are usually derived from patient peripheral blood mononuclear cells, but the number of killer cells in peripheral blood is limited, so that a large amount of demands for CIK cells in a therapeutic process cannot be met, and thus, the CIK cells need to be expanded in vitro. The existing CIK culture method needs to use serum, has the problems of slow growth, poor proliferation effect, weak cytotoxicity to tumor cells and the like of the CIK cells, also has the problem of complex preparation process, and the proliferation activity and the lethality of the CIK cells are very important for the effect of the CIK cell-based tumor immunotherapy. Therefore, it is necessary to research a culture method capable of improving the in vitro proliferation capacity and the tumor killing capacity of the CIK cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an induction culture method of CIK cells, which improves the efficiency of in vitro induction culture of CIK cells.
The purpose of the invention is realized by adopting the following technical scheme:
an induction culture method of CIK cells comprises the following steps:
(1) isolating mononuclear cells from peripheral blood;
(2) suspending mononuclear cells with X-VIVO 15 medium, culturing, supplementing IL-2, IL-18, green algae growth factor, glutamine, and CD3 monoclonal antibody to the culture medium on day 2, and culturing once every 2-3 days to day 7;
(3) supplementing liquid once every 2-3 days from day 8, and performing induction culture for 14 days;
the nutrient medium of the liquid supplementing in the step (2) is prepared by adding the following components into an X-VIVO 15 nutrient medium: IL-2, IL-18, green algae growth factor;
the nutrient medium of the liquid supplementing in the step (3) is prepared by adding the following components into an X-VIVO 15 nutrient medium: IL-2, vitexin, methimazole.
Further, the concentration of IL-2, IL-18, chlorella growth factor, glutamine, CD3 monoclonal antibody in the culture medium supplemented on the 2 nd day of culture in step (2) is: IL-21000 IU/mL, IL-18100 ng/mL, green algae growth factor 20-30ng/mL, glutamine 5-10 μ g/mL, CD3 monoclonal antibody 1-5 μ g/mL.
Further, the concentrations of IL-2, IL-18 and green alga growth factor in the X-VIVO 15 culture medium supplemented in the step (2) are as follows: IL-21000 IU/mL, IL-18100 ng/mL, and chlorella growth factor 20-30 ng/mL.
Further, the concentrations of IL-2, vitexin and methimazole in the X-VIVO 15 culture medium supplemented in the step (3) are as follows: IL-21000 IU/mL, vitexin 2-6 mu g/mL, methimazole 10-20 ng/mL.
Further, the seeding density of the mononuclear cells in the X-VIVO 15 culture medium in the step (2) is 1.5-4X 10 6 One per mL.
Further, the cell density in step (2) and step (3) is maintained at 1.5-4X 10 by replenishing the fluid 6 one/mL.
Further, the cell culture process was carried out at 37 ℃ with 5% CO 2 Is carried out in an incubator.
Further, the process for separating single nuclear cells in the step (1) is as follows: collecting peripheral blood, adding PBS buffer solution for dilution, adding lymphocyte separation solution, centrifuging, removing supernatant, and washing with PBS to obtain mononuclear cells.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an induced culture method of CIK cells, which comprises the steps of firstly adding components such as IL-2, IL-18, green alga growth factors and the like into a culture medium of mononuclear cells, then supplementing liquid in time in the culture process, adjusting the added components in the supplemented culture medium after culturing for 7 days, and better promoting the proliferation of the CIK cells by adding IL-2, vitexin and methimazole, thereby effectively improving the proliferation efficiency of the CIK cells.
Drawings
FIG. 1 shows the ratio of CD3+ CD56+ cells in CIK cells cultured in example 1 and comparative examples 1 to 5 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
An induction culture method of CIK cells comprises the following steps:
(1) isolation of mononuclear cells from peripheral blood: collecting peripheral blood, adding PBS buffer solution to dilute according to the volume ratio of 1:2, adding Ficoll lymphocyte separation solution, subpackaging in a centrifuge tube, centrifuging, discarding supernatant, and washing with PBS to obtain mononuclear cells;
(2) the mononuclear cells were resuspended in X-VIVO 15 medium and then transferred to flasks at 37 ℃ with 5% CO 2 Cultured in an incubator with a cell seeding density of 1.5X 10 6 Adding the culture medium with IL-21000 IU/mL, IL-18100 ng/mL, chlorella growth factor 20ng/mL, glutamine 5 μ g/mL and CD3 monoclonal antibody 1 μ g/mL on the 2 nd day of culture, continuing culture, and supplementing solution once every 2 th day to the 7 th day, wherein the cell density is maintained at 1.5 × 10 after each solution supplementation 6 Per mL; the nutrient solution is prepared by adding the following components into a supplemented X-VIVO 15 culture medium: IL-2, IL-18 and green algae growth factor, wherein the concentration of each component in a liquid supplementing culture medium is as follows: IL-21000 IU/mL, IL-18100 ng/mL, green algae growth factor 20 ng/mL;
(3) the fluid infusion is performed every 2 days from day 8, and the cell density is maintained at 1.5 × 10 after each fluid infusion 6 The following were added to each mL of the supplemented X-VIVO 15 mediumThe concentration of each component is as follows: IL-21000 IU/mL, vitexin 2 mug/mL and methimazole 10ng/mL, and the induction culture is carried out for 14 days.
Example 2
An induction culture method of CIK cells comprises the following steps:
(1) isolation of mononuclear cells from peripheral blood: collecting peripheral blood, adding PBS buffer solution to dilute according to the volume ratio of 1:2, adding Ficoll lymphocyte separation solution, subpackaging in a centrifuge tube, centrifuging, discarding supernatant, and washing with PBS to obtain mononuclear cells;
(2) the mononuclear cells were resuspended in X-VIVO 15 medium and then placed in culture flasks at 37 ℃ with 5% CO 2 Cultured in an incubator with a cell seeding density of 3X 10 6 Adding the culture medium with IL-21500 IU/mL, IL-18110 ng/mL, chlorella growth factor 25ng/mL, glutamine 8 μ g/mL, CD3 monoclonal antibody 3 μ g/mL on 2 days of culture, continuing culture, and supplementing solution once every 3 days to 7 days, wherein the cell density is maintained at 3 × 10 after each solution supplementation 6 Per mL; the nutrient solution is prepared by adding the following components into a supplemented X-VIVO 15 culture medium: IL-2, IL-18 and green algae growth factor, wherein the concentration of each component in a liquid supplementing culture medium is as follows: IL-21500 IU/mL, IL-18110 ng/mL, chlorella growth factor 25 ng/mL;
(3) replenishing every 2-3 days from day 8, and maintaining cell density at 3 × 10 after each fluid replenishment 6 Adding the following components in the following concentration into a supplemented X-VIVO 15 culture medium per mL: IL-21500 IU/mL, vitexin 3. mu.g/mL, methimazole 15ng/mL, induced culture for 14 days.
Example 3
An induction culture method of CIK cells comprises the following steps:
(1) isolation of mononuclear cells from peripheral blood: collecting peripheral blood, adding PBS buffer solution, diluting according to a volume ratio of 1:2, adding Ficoll lymphocyte separation solution, subpackaging in a centrifuge tube, centrifuging, removing supernatant, washing with PBS to obtain mononuclear cells;
(2) the mononuclear cells were resuspended in X-VIVO 15 medium and then transferred to flasks at 37 ℃ with 5% CO 2 Cultured in an incubator to a cell seeding densityIs 4 x 10 6 Adding IL-22000 IU/mL, IL-18120 ng/mL, chlorella growth factor 30ng/mL, glutamine 10 ug/mL, CD3 monoclonal antibody 5 ug/mL into the culture medium on the 2 nd day of culture, continuing culture, and supplementing solution once every 2 th day to the 7 th day, wherein the cell density is maintained at 4 × 10 after each solution supplementation 6 Per mL; the nutrient solution is prepared by adding the following components into a supplemented X-VIVO 15 culture medium: IL-2, IL-18 and green algae growth factor, wherein the concentration of each component in a liquid supplementing culture medium is as follows: IL-22000 IU/mL, IL-18120 ng/mL, green algae growth factor 30 ng/mL;
(3) the fluid infusion is performed every 2 days from day 8, and the cell density is maintained at 4 × 10 after each fluid infusion 6 Adding the following components in the following concentration into a supplemented X-VIVO 15 culture medium per mL: IL-22000 IU/mL, vitexin 6 mu g/mL and methimazole 20ng/mL, and the induction culture is carried out for 14 days.
Comparative example 1
Comparative example 1 provides a culture method for inducing CIK cells, which is different from example 1 in that: the green alga growth factor added in the step (2) is omitted.
Comparative example 2
Comparative example 2 provides a method for induction culture of CIK cells, which is different from example 1 in that: the vitexin added in the step (3) is omitted.
Comparative example 3
Comparative example 3 provides a method for induction culture of CIK cells, which is different from example 1 in that: the methimazole added in the step (3) is omitted.
Comparative example 4
Comparative example 4 provides a culture method for inducing CIK cells, which is different from example 1 in that: the vitexin and methimazole added in the step (3) are saved.
Comparative example 5
Comparative example 5 provides a culture method for inducing CIK cells, which is different from example 1 in that: the vitexin and methimazole added in the step (3) are omitted, and the vitexin and methimazole are replaced by IL-18100 ng/mL and green alga growth factor of 20 ng/mL.
The fold amplifications of the cells of examples 1 to 3 and comparative examples 1 to 5 were counted, respectively, and calculated as follows: the number of harvested cells was divided by the number of inoculated mononuclear cells after 14 days of culture, which was the fold expansion of the cells, and the cells were counted using a full-automatic cell counter by Invitrogen, and the cell viability of each group was counted using trypan blue staining method, 3 times for each group, and the results were averaged as shown in table 1.
The killing activity of the CIK cells is detected by adopting an MTT method: using K562 cells as target cells, adjusting the concentration of effector cells to 1 × 10 6 And (2) paving holes in a 96-well plate according to an effective target ratio of 20:1, after co-culturing for 4h, centrifuging, removing supernatant, adding DMSO, shaking for dissolution, detecting an absorbance value (A) at 570nm by using a microplate reader, and meanwhile, setting a target cell control hole, an effector cell control hole and a blank control hole, wherein the results are shown in Table 1.
Killing activity ═ target cell control well a value- (experimental well a value-effector cell control well a) ]/target cell control well a value × 100%.
TABLE 1
Group of Amplification factor Cell survival rate (%) Killing activity (%)
Example 1 186.6 97.1 85.7
Example 2 195.3 97.5 86.1
Example 3 189.2 97.2 85.4
Comparative example 1 167.8 92.4 59.4
Comparative example 2 146.4 84.9 73.8
Comparative example 3 131.7 83.3 76.5
Comparative example 4 95.3 79.5 70.7
Comparative example 5 117.5 88.7 80.2
As can be seen from Table 1, the cells in examples 1 to 3 had higher fold expansion and higher survival rates. In comparative examples 1 to 5, the culture process was adjusted, and neither the proliferating activity nor the killing activity of the cells was equal to that in examples 1 to 3.
Taking example 1, comparative examples 1 to 5Centrifuging CIK cell suspension after culturing for 14 days, discarding supernatant, adding PBS to resuspend cells, and adjusting cell density to 2 × 10 6 Adding the monoclonal antibody CD3+ CD56+ into the cells per mL, incubating the cells for 15min in a dark place, and detecting the proportion of CD3+ CD56+ cells in the CIK cells by using a flow cytometer, wherein the result is shown in a figure 1.
As can be seen from FIG. 1, the CIK cells of example 1 have a higher proportion of CD3+ CD56+ cells and the CIK cells of comparative example 1 have the lowest proportion, which indicates that the green alga growth factors have a better effect of increasing the proportion of CD3+ CD56+ cells in the CIK cells.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims (8)

1. The induction culture method of the CIK cells is characterized by comprising the following steps:
(1) isolating mononuclear cells from peripheral blood;
(2) suspending the mononuclear cells with X-VIVO 15 medium, culturing, supplementing IL-2, IL-18, green algae growth factor, glutamine, and CD3 monoclonal antibody into the culture medium on the 2 nd day of culture, and supplementing solution every 2-3 days to the 7 th day;
(3) supplementing liquid every 2-3 days from day 8, and performing induction culture for 14 days;
the nutrient medium of the liquid supplementing in the step (2) is prepared by adding the following components into an X-VIVO 15 nutrient medium: IL-2, IL-18, green algae growth factor;
the nutrient medium of the liquid supplementing in the step (3) is prepared by adding the following components into an X-VIVO 15 nutrient medium: IL-2, vitexin, methimazole.
2. The method for inducing and culturing CIK cells according to claim 1, wherein the concentrations of IL-2, IL-18, chlorella growth factor, glutamine, and CD3 mab in the culture medium supplemented at day 2 of the culturing in step (2) are: IL-21000-2000IU/mL, IL-18100-120 ng/mL, green algae growth factor 20-30ng/mL, glutamine 5-10 μ g/mL, CD3 monoclonal antibody 1-5 μ g/mL.
3. The method for inducing and culturing CIK cells according to claim 1, wherein the concentrations of IL-2, IL-18 and chlorella growth factor in the supplemented X-VIVO 15 medium in step (2) are as follows: IL-21000 IU/mL, IL-18100 ng/mL, and chlorella growth factor 20-30 ng/mL.
4. The method for inducing and culturing CIK cells according to claim 1, wherein the concentrations of IL-2, vitexin and methimazole in the X-VIVO 15 medium supplemented in step (3) are as follows: IL-21000-2000IU/mL, vitexin 2-6 mu g/mL, methimazole 10-20 ng/mL.
5. The method for inducing and culturing CIK cells according to claim 1, wherein the mononuclear cells are seeded in the X-VIVO 15 medium at a density of 1.5-4X 10 in step (2) 6 one/mL.
6. The method for inducing and culturing CIK cells, according to claim 1, wherein the cell density of the cells in step (2) and step (3) is maintained at 1.5-4X 10 by replenishing the cells with a cell density of 1.5 6 one/mL.
7. The method for inducing and culturing CIK cells according to claim 1, wherein the cells are cultured at 37 ℃ and 5% CO 2 Is carried out in an incubator.
8. The method for inducing and culturing CIK cells according to claim 1, wherein the single nuclear cells are isolated in step (1) as follows: collecting peripheral blood, adding PBS buffer solution for dilution, adding lymphocyte separation solution, centrifuging, removing supernatant, and washing with PBS to obtain mononuclear cells.
CN202210972485.5A 2022-08-15 2022-08-15 Induced culture method of CIK cells Withdrawn CN115109749A (en)

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