CN114736859A - Culture solution and culture method for cord blood NK cells - Google Patents
Culture solution and culture method for cord blood NK cells Download PDFInfo
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Abstract
The invention discloses a culture solution and a culture method of NK cells of umbilical cord blood; the culture solution comprises the following components: the culture medium is characterized in that a first culture medium consisting of cell factors IL-2, IL-15, IL-12, SCF, TGF, PDGF, OK432, StemRegenin 1 and halcinonide is added to a basal culture medium, a second culture medium consisting of cell factors IL-2, IL-21, SCF, soybean phospholipid and thymopentin is added to the basal culture medium, and a third culture medium consisting of cell factors IL-2, SCF, soybean phospholipid and thymopentin is added to the basal culture medium. The cell factor is added into the culture solution, so that the cell culture period is shortened by 37.5%, and the amplification multiple, purity and tumor killing effect of the NK cells are improved.
Description
Technical Field
The invention relates to a preparation method and a culture method of a cell culture solution, in particular to a culture solution and a culture method of cord blood NK cells.
Background
Natural Killer (NK) cells are one of the important components of the human immune system, are effector cells of the natural immune system, are derived from lymphoid stem cells of the bone marrow, are differentiated and matured in the bone marrow and thymus, and are distributed in the blood, lymph nodes, bone marrow, etc. NK cells, unlike T, B cells, are capable of non-specifically recognizing and killing tumor cells as well as virally infected cells, without antigen sensitization and without MHC restriction. Because of this important function, the research and application of NK cells in the field of tumor immunotherapy is gradually being emphasized.
In clinical application, NK cells have high requirements on quantity and purity, particularly purity, and cannot contain excessive lymphocytes such as T cells, B cells and the like. Because the lymphocyte proportion in peripheral blood has large individual difference, NK cells cultured by PBMC extracted from the peripheral blood by the same culture method have different purity, the immature T cells contained in umbilical cord blood are not easy to cause graft-versus-host disease (GVHD), the lymphocyte proportion in the umbilical cord blood is relatively stable, and the quality level of the NK cells cultured by the same culture method is stable, so the umbilical cord blood is an ideal source for preparing the NK cells. However, in the prior art, the amplification culture period of the cord blood NK cells is long, the proportion of effector cells is low (the purity is low), the operation is complex, the price of the cord blood NK cell culture kit is high, and the production cost is high.
Therefore, the exploration of a production process for culturing high-purity, high-quantity, stable, safe and strong-operability cord blood NK cells is a challenge at present.
Disclosure of Invention
In view of the above problems, the present invention provides a culture solution and a culture method for umbilical cord blood NK cells with high purity, high activity, and the like.
One of the technical schemes of the invention is as follows:
a culture solution of cord blood NK cells comprises the following components:
first medium: adding IL-2 with a final concentration of 1500-3000 IU/ml, IL-15 with a final concentration of 30-80 ng/ml, IL-12 with a final concentration of 50-100 ng/ml, IL-3 with a final concentration of 20-50 ng/ml, OK432 with a final concentration of 0.1-2 ug/ml, SCF (dry cell factor) with a final concentration of 20-40 ng/ml, TGF (transforming growth factor) with a final concentration of 20-40 ng/ml, PDGF (platelet-derived growth factor) with a final concentration of 5-20 ng/ml, StemRegenin 1 (SR1, antagonist) with a final concentration of 2-5 mu M and halcinonide with a final concentration of 0.05-1ng/ml into a basal medium;
a second medium: IL-2 with the final concentration of 1500-2000 IU/ml, IL-21 with the final concentration of 10-30 ng/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-800ug/ml and thymopentin with the final concentration of 1-5ug/ml are added into a basal culture medium;
a third medium: IL-2 with the final concentration of 1000-1500 IU/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-.
In one embodiment, the culture solution comprises the following components:
a first medium: IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 muM and Hacinonide with a final concentration of 0.15ng/ml are added to the basal medium;
a second medium: adding IL-2 with a final concentration of 1800IU/ml, IL-21 with a final concentration of 22ng/ml, SCF with a final concentration of 16ng/ml, soybean phospholipid with a final concentration of 550ug/ml and thymopentin with a final concentration of 3ug/ml into a basic culture medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at 550ug/ml and thymopentin at 3 ug/ml.
In one embodiment, the culture solution comprises the following components:
a first medium: IL-2 with a final concentration of 1500IU/ml, IL-15 with a final concentration of 80ng/ml, IL-12 with a final concentration of 50ng/ml, IL-3 with a final concentration of 20ng/ml, OK432 with a final concentration of 0.1ug/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml, StemRegenin 1 with a final concentration of 2 μ M and Hacinonide with a final concentration of 0.05ng/ml are added to a basal medium;
a second medium: IL-2 with the final concentration of 1500IU/ml, IL-21 with the final concentration of 10ng/ml, SCF with the final concentration of 10ng/ml, soybean phospholipid with the final concentration of 300ug/ml and thymopentin with the final concentration of 1ug/ml are added into a basal culture medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1000IU/ml, SCF at a final concentration of 10ng/ml, soybean phospholipids at a final concentration of 300ug/ml, and thymopentin at a final concentration of 1 ug/ml.
In one embodiment, the culture solution comprises the following components:
first medium: adding IL-2 with a final concentration of 3000IU/ml, IL-15 with a final concentration of 30ng/ml, IL-12 with a final concentration of 100ng/ml, IL-3 with a final concentration of 50ng/ml, OK432 with a final concentration of 2ug/ml, SCF with a final concentration of 40ng/ml, TGF with a final concentration of 20ng/ml, PDGF with a final concentration of 20ng/ml, StemRegenin 1 with a final concentration of 5 mu M and halcinonide with a final concentration of 1ng/ml to a basal medium;
a second medium: adding IL-2 with final concentration of 2000IU/ml, IL-21 with final concentration of 30ng/ml, SCF with final concentration of 20ng/ml, soybean phospholipid with final concentration of 800ug/ml and thymopentin with final concentration of 5ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1500IU/ml, SCF at a final concentration of 20ng/ml, soybean phospholipids at 800ug/ml, and thymopentin at 5 ug/ml.
The second technical scheme of the invention is as follows:
a culture method of cord blood NK cells comprises the following steps:
1) preparing a first culture medium, a second culture medium and a third culture medium; wherein the content of the first and second substances,
a first medium: adding IL-2 with a final concentration of 1500-3000 IU/ml, IL-15 with a final concentration of 30-80 ng/ml, IL-12 with a final concentration of 50-100 ng/ml, IL-3 with a final concentration of 20-50 ng/ml, OK432 with a final concentration of 0.1-2 ug/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml, StemRegenin 1 with a final concentration of 2-5 mu M and halcinonide with a final concentration of 0.05-1ng/ml into a basic culture medium;
a second medium: adding IL-2 with a final concentration of 1500-2000 IU/ml, IL-21 with a final concentration of 10-30 ng/ml, SCF with a final concentration of 10-20 ng/ml, soybean phospholipid with a final concentration of 300-800ug/ml and thymopentin with a final concentration of 1-5ug/ml into a basic culture medium;
third medium: IL-2 with the final concentration of 1000-1500 IU/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-800ug/ml and thymopentin with the final concentration of 1-5ug/ml are added into a basic culture medium;
2) inoculating PBMC of umbilical cord blood and culturing;
day 0: using a suspension cell culture flask 500, 285ml of the first medium and 15ml of 5v/v% blood were added; then, inoculating 5000 ten thousand cells, standing and culturing for 5 days, and after the 3 rd day, shaking the culture bottle for 1-2 times to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
In one embodiment, the cultivation method comprises, in step 1):
a first medium: IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 muM and Hacinonide with a final concentration of 0.15ng/ml are added to the basal medium;
a second medium: IL-2 with a final concentration of 1800IU/ml, IL-21 with a final concentration of 22ng/ml, SCF with a final concentration of 16ng/ml, soybean phospholipid with a final concentration of 550ug/ml and thymopentin with a final concentration of 3ug/ml are added into a basal culture medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at 550ug/ml and thymopentin at 3 ug/ml.
In one embodiment, the culturing method comprises step 1):
a first medium: adding IL-2 with a final concentration of 1500IU/ml, IL-15 with a final concentration of 80ng/ml, IL-12 with a final concentration of 50ng/ml, IL-3 with a final concentration of 20ng/ml, OK432 with a final concentration of 0.1ug/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml, StemRegenin 1 with a final concentration of 2 mu M and Hacinonide with a final concentration of 0.05ng/ml to a basal medium;
a second medium: adding IL-2 with final concentration of 1500IU/ml, IL-21 with final concentration of 10ng/ml, SCF with final concentration of 10ng/ml, soybean phospholipid with final concentration of 300ug/ml and thymopentin with final concentration of 1ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1000IU/ml, SCF at a final concentration of 10ng/ml, soybean phospholipids at a final concentration of 300ug/ml, and thymopentin at a final concentration of 1 ug/ml.
In one embodiment, the culturing method comprises step 1):
a first medium: adding IL-2 with a final concentration of 3000IU/ml, IL-15 with a final concentration of 30ng/ml, IL-12 with a final concentration of 100ng/ml, IL-3 with a final concentration of 50ng/ml, OK432 with a final concentration of 2ug/ml, SCF with a final concentration of 40ng/ml, TGF with a final concentration of 20ng/ml, PDGF with a final concentration of 20ng/ml, StemRegenin 1 with a final concentration of 5 mu M and halcinonide with a final concentration of 1ng/ml to a basal medium;
a second medium: adding IL-2 with final concentration of 2000IU/ml, IL-21 with final concentration of 30ng/ml, SCF with final concentration of 20ng/ml, soybean phospholipid with final concentration of 800ug/ml and thymopentin with final concentration of 5ug/ml into basal medium;
third medium: IL-2 was added to the basal medium at a final concentration of 1500IU/ml, SCF at a final concentration of 20ng/ml, soybean phospholipids at 800ug/ml, and thymopentin at 5 ug/ml.
The basic culture medium is KBM581, GT-T551, X-VIVO 15 or AIM-V.
Compared with the prior art, the invention has the following advantages:
1. preparing three culture matrixes with different nutrient components in the NK cell culture solution, wherein each culture matrix contains different cell factors, and different culture matrixes are added in different culture times according to the growth activity cycle of the NK cells, so that the cell culture cycle is shortened from 16 days to about 10 days;
2. in the three culture matrixes, the halcinonide component is added into the first culture matrix and is used for inhibiting the growth of T cells and mononuclear cells in the umbilical cord blood, and the purity of NK cells in the umbilical cord blood is effectively improved during early culture; the method has a great promoting effect on the CD3-CD56+ flow content of the NK cells, and improves the purity of the NK cells;
3. soybean phospholipid and thymopentin components are added into the second culture medium and the third culture medium, suspended cells are further evacuated through hydrophobicity and solubilization of the soybean phospholipid, substances generated by high-density cell metabolism can be fully dissolved through the solubilization, and massive death caused by local cell aggregation and agglomeration is reduced, so that high-density rapid amplification growth of the cells is maintained; meanwhile, the thymopentin has the functions of promoting NK cell proliferation, strengthening NK cell physiological activity and immunologic function, eliminating the immunosuppressive action of halcinonide in the first culture medium and realizing the later-stage rapid proliferation of the NK cells in the umbilical cord blood.
4. In the process of culturing the NK cells, the culture matrixes with different components are added in different culture times, so that the high killing activity rate of the NK cells is improved, and the effect of killing tumor cells is stronger.
Drawings
FIG. 1 is a graph of cord blood NK cell growth curves for examples 1, 2, 3, 4, 5 and 1; among them, the cord blood NK cells of examples 1 to 4 were cultured for 10 days, and the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days.
FIG. 2 is a graph showing the killing of tumor cells K562 by cord blood NK cells of examples 1 to 5 and comparative example 1; wherein, the umbilical cord blood NK cells of example 1, example 2, example 3, example 4 were cultured for 10 days; the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days.
FIGS. 3, 4, 5, 6, 7 and 8 are graphs showing the phenotype of CD3-CD56+ after culture of cord blood NK cells of examples 1 to 5 and comparative example 1, respectively; among them, the cord blood NK cells of examples 1 to 4 were cultured for 10 days, and the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
In the present invention, culture of NK cells in peripheral blood, i.e., umbilical cord blood, is employed. First, it is necessary to isolate PBMC from umbilical cord blood and then culture NK cells from the PBMC.
The specific operation of separating the mononuclear cells from the umbilical cord blood is as follows:
(1) crude separation
In the invention, the blood is subpackaged into two centrifuge tubes (marked as No. 1 and No. 2 centrifuge tubes) with the specification of 50ml, each centrifuge tube is subpackaged with 30ml, and the blood volume is calculated.
The blood sample was aspirated with a 3ml format pasteur pipette, 2 drops were added to the EP tube, and the cell number was measured with a hematology analyzer.
Centrifuging the blood in the centrifuge tube; in the centrifugation process, the centrifugal force is 650g, the centrifugation time is 10min, and the speed is firstly increased by 8 and then decreased by 8.
Two additional tubes of the same size (labeled as No. 3 and No. 4) were prepared, and 15ml of lymphocyte separation medium was added to each tube.
(2) Plasma preparation
After centrifugation of two centrifuge tubes filled with whole blood is finished, transferring the centrifuge tubes to a table board of a safety cabinet, and sucking upper plasma into a new centrifuge tube (marked as No. 5) by using a 25ml pipette; taking 1ml of plasma as a reserved sample, marking and storing in a refrigerator at-20 ℃; sealing the No. 5 centrifuge tube with residual blood plasma with sealing membrane, placing in 56 deg.C water bath, and inactivating for 30 min.
Immediately after the inactivation, the No. 5 centrifugal tube filled with the plasma is placed in a refrigerator at-20 ℃ for 15min, and then the No. 5 centrifugal tube is taken out for centrifugal treatment, wherein the centrifugal force is 3000g and the centrifugal time is 10 min.
And finally, pouring the plasma supernatant in the centrifuge tube No. 5 into another new centrifuge tube (marked as No. 6), sealing, and placing in a refrigerator at 2-8 ℃ for freezing and preservation for later use.
(3) Mononuclear cell preparation
Diluting the remaining lower layer blood cells in the No. 1 and No. 2 centrifuge tubes by using normal saline 1:1 and uniformly mixing; and adding the corresponding hemocyte diluent into No. 3 and No. 4 centrifuge tubes containing the lymphocyte separating medium respectively, and carrying out the following treatment.
Namely, a small amount of blood cell suspension is added to the position 1cm above the separation liquid by an electric pipettor respectively by tilting No. 3 and No. 4 centrifuge tubes which are filled with the lymph separation liquid by 45 degrees, and the blood cell suspension is spread; the purpose of tilting the centrifuge tube is to bring the suspension into contact with the separation liquid.
The remaining cell suspension was then added slowly and continuously along the walls of the No. 3 and No. 4 centrifuge tubes. During operation, the liquid level interface is not disturbed, and the maximum volume of each tube is 45 ml. After the completion, the centrifuge tubes No. 3 and No. 4 are kept vertically, and carefully transferred to a centrifuge for centrifugation, wherein the centrifugal force is 650g, the centrifugation time is 30 minutes, and the centrifuge tube is raised to 1 gear and lowered to 0 gear (namely, the slowest gear).
The centrifuge tubes 3 and 4 were centrifuged and carefully removed to clearly visualize the cell separation. The centrifuge tube is kept vertically to avoid damaging cell stratification. The cells were slowly transferred to a safety cabinet, and 2ml of residual plasma was removed from the upper layer using a 10ml pipette, and then about 10ml of the second layer of leukocytes were aspirated from centrifuge tubes # 3 and # 4, respectively, and added to the corresponding additional new centrifuge tubes (labeled # 7 and # 8).
Adding physiological saline into No. 7 and No. 8 centrifuge tubes to 50ml scale, and mixing. Centrifuging No. 7 and No. 8 centrifuge tubes, wherein the centrifugal force is 650g, the centrifugation time is 10min, and the centrifugal force is increased by 8 steps and is changed by 8 steps. After centrifugation is finished, 5-10 ml of normal saline is added into No. 7 and No. 8 centrifuge tubes respectively to resuspend cells, the cells in each centrifuge tube are precipitated, combined and collected into No. 7 centrifuge tubes, the normal saline is added to complement to 50ml of scales, and after the cells are uniformly blown by a 10ml pipette, 2 drops of the normal saline are added into an EP tube for counting.
After the counting, the mononuclear cells were individually loaded into a multi-branch centrifuge tube and resuspended to 50ml from the cell sap in 5000 ten thousand number. The centrifugal tubes are then centrifuged, the centrifugal force is 300g, the centrifugation time is 10min, and the centrifugal force is increased by 8 steps and decreased by 8 steps. After the centrifugation is finished, the supernatant is discarded, and the precipitate in each centrifuge tube is the required mononuclear cells and also becomes cord blood mononuclear cells (PBMC).
The invention provides a culture solution of cord blood NK cells, which comprises the following components in percentage by weight:
a first medium: adding IL-2 with a final concentration of 1500-3000 IU/ml, IL-15 with a final concentration of 30-80 ng/ml, IL-12 with a final concentration of 50-100 ng/ml, IL-3 with a final concentration of 20-50 ng/ml, OK432 with a final concentration of 0.1-2 ug/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml, StemRegenin 1 with a final concentration of 2-5 mu M and halcinonide with a final concentration of 0.05-1ng/ml into a basic culture medium;
a second medium: adding IL-2 with a final concentration of 1500-2000 IU/ml, IL-21 with a final concentration of 10-30 ng/ml, SCF with a final concentration of 10-20 ng/ml, soybean phospholipid with a final concentration of 300-800ug/ml and thymopentin with a final concentration of 1-5ug/ml into a basic culture medium;
a third medium: IL-2 with the final concentration of 1000-1500 IU/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-.
Preferably, the basal medium is any one of 581, GT-T551, X-VIVO 15 or AIM-V.
The invention also provides a method for culturing the cord blood NK cells by using the culture solution, which comprises the following culture process steps:
day 0: using a suspension cell culture flask 500, 285ml of the above first medium and 15ml of 5v/v% hemoreplacement were added; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days; after day 3, the culture flask is shaken for 1-2 times to disperse the aggregated cells; wherein, the shaking operation can be manual shaking operation if the number of the culture bottles is small; if the number of culture bottles is large, the culture bottles can be shaken by using a rocking bed.
Day 5: removing part of the culture solution, replacing and adding 400ml of the second culture medium, shaking up the cells, and standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of the third culture medium, shaking up the cells, and standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Among the three kinds of culture media, the basic culture medium and the added cytokines IL-2, IL-15, IL-12, IL-3, SCF, TGF, PDGF and the like are commonly used basic culture factors, and at present, NK culture media contain these cytokines or some cytokines.
The culture of cord blood NK cells is illustrated below by some specific examples.
First, NK cell culture
Example 1
1) Preparing a first culture medium, a second culture medium and a third culture medium
A first medium: IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 mu M and Hacinonide with a final concentration of 0.15ng/ml are added to an X-VIVO 15 basal medium;
a second medium: IL-2 with the final concentration of 1800IU/ml, IL-21 with the final concentration of 22ng/ml, SCF with the final concentration of 16ng/ml, soybean phospholipid with the final concentration of 550ug/ml and thymopentin with the final concentration of 3ug/ml are added into an X-VIVO 15 basal medium;
third medium: IL-2 was added to the basic X-VIVO 15 medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at a final concentration of 550ug/ml, and thymopentin at a final concentration of 3 ug/ml.
2) Inoculation of cord blood PBMC and culture
Day 0: adding 285ml of first culture medium and 15ml of 5v/v% blood into a suspension cell culture bottle with the specification of 500 ml; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days, and shaking the culture bottle 1-2 times after 3 days to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Example 2
1) Preparing a first culture medium, a second culture medium and a third culture medium
A first medium: IL-2 with a final concentration of 1500IU/ml, IL-15 with a final concentration of 80ng/ml, IL-12 with a final concentration of 50ng/ml, IL-3 with a final concentration of 20ng/ml, OK432 with a final concentration of 0.1ug/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml, StemRegenin 1 with a final concentration of 2 μ M and Hacinonide with a final concentration of 0.05ng/ml are added to KBM581 basal medium;
a second medium: IL-2 with a final concentration of 1500IU/ml, IL-21 with a final concentration of 10ng/ml, SCF with a final concentration of 10ng/ml, soybean phospholipid with a final concentration of 300ug/ml and thymopentin with a final concentration of 1ug/ml are added into a KBM581 basal medium;
third medium: IL-2 was added to KBM581 basal medium at a final concentration of 1000IU/ml, SCF at a final concentration of 10ng/ml, soybean phospholipids at 300ug/ml, and thymopentin at 1 ug/ml.
2) Inoculation of cord blood PBMC and culture
Day 0: adding 285ml of first culture medium and 15ml of 5v/v% blood into a suspension cell culture bottle with the specification of 500 ml; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days, and shaking the culture bottle 1-2 times after 3 days to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Example 3
1) Preparing a first culture medium, a second culture medium and a third culture medium
First medium: IL-2 at a final concentration of 3000IU/ml, IL-15 at a final concentration of 30ng/ml, IL-12 at a final concentration of 100ng/ml, IL-3 at a final concentration of 50ng/ml, OK432 at a final concentration of 2ug/ml, SCF at a final concentration of 40ng/ml, TGF at a final concentration of 20ng/ml, PDGF at a final concentration of 20ng/ml, StemRegenin 1 at a final concentration of 5. mu.M, and halcinonide at a final concentration of 1ng/ml were added to the AIM-V basal medium;
a second medium: IL-2 with final concentration of 2000IU/ml, IL-21 with final concentration of 30ng/ml, SCF with final concentration of 20ng/ml, soybean phospholipid with final concentration of 800ug/ml and thymopentin with final concentration of 5ug/ml are added into the AIM-V basal medium;
a third medium: IL-2 was added to the AIM-V basal medium at a final concentration of 1500IU/ml, SCF at a final concentration of 20ng/ml, soybean phospholipids at a final concentration of 800ug/ml, and thymopentin at a final concentration of 5 ug/ml.
2) Inoculation of cord blood PBMC and culture
Day 0: adding 285ml of first culture medium and 15ml of 5v/v% blood into a suspension cell culture bottle with the specification of 500 ml; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days, and shaking the culture bottle 1-2 times after 3 days to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Example 4 (second and third Medium cytokine-free Soybean Phospholipids and Thymopentin)
1) Preparing a first culture medium, a second culture medium and a third culture medium
A first medium: adding IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 mu M and halcinonide with a final concentration of 0.15ng/ml to X-VIVO 15 basal medium;
a second medium: IL-2 with a final concentration of 1800IU/ml, IL-21 with a final concentration of 22ng/ml and SCF with a final concentration of 16ng/ml are added into an X-VIVO 15 basic culture medium;
a third medium: IL-2 was added to the basic X-VIVO 15 medium at a final concentration of 1150IU/ml and SCF at a final concentration of 16 ng/ml.
2) Inoculation of cord blood PBMC and culture
Day 0: adding 285ml of the first culture medium and 15ml of 5v/v% blood by using a 500ml gauge suspension cell culture bottle; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days, and shaking the culture bottle 1-2 times after 3 days to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Example 5 (first Medium cytokine-free halcinonide)
1) Preparing a first culture medium, a second culture medium and a third culture medium
A first medium: IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml and StemRegenin 1 with a final concentration of 3 mu M are added into an X-VIVO 15 basal medium;
a second medium: IL-2 with the final concentration of 1800IU/ml, IL-21 with the final concentration of 22ng/ml, SCF with the final concentration of 16ng/ml, soybean phospholipid with the final concentration of 550ug/ml and thymopentin with the final concentration of 3ug/ml are added into an X-VIVO 15 basal medium;
third medium: IL-2 was added to the basic X-VIVO 15 medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at a final concentration of 550ug/ml, and thymopentin at a final concentration of 3 ug/ml.
2) Inoculation of cord blood PBMC and culture
Day 0: adding 285ml of the first culture medium and 15ml of 5v/v% hemocyte into a suspension cell culture bottle with the specification of 500 ml; then, 5000 ten thousand of the NK cells separated from the cord blood were inoculated and left to stand at 37 ℃ with 5% CO2Culturing in an incubator in a saturated humidity environment for 5 days, and shaking the culture bottle 1-2 times after 3 days to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping cell culture, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
Comparative example 1
1) Preparing an activation medium and a proliferation medium
Initial culture medium: X-VIVO 15 medium containing 1000U/ml IL-2, 50ng/ml IL-15 and 10. mu.M StemRegenin 1 (SR 1);
amplification medium: X-VIVO 15 medium containing 1000U/ml IL-2 and 50ng/ml IL-21;
2) and NK cell culture
Obtaining cord blood PBMC by adopting the normal blood separation;
inoculation on day 0: by 1X 106Inoculating T175 culture flask per mL, performing static culture with 45mL of activated culture medium +5mL of serum in a total volume of 50mL for 5 days, standing at 37 deg.C, and 5% CO2Saturated humidityCulturing in an incubator in the environment for 5 days;
transfer cells were collected on day 5: centrifuging to remove supernatant, resuspending cells with 180ml proliferation medium and 20ml serum, and inoculating into culture bag with total volume of 200 ml;
and (4) fluid infusion on the 7 th day: adding 200ml of proliferation culture medium, and making the total volume be 400 ml;
supplementing liquid on day 9, adding 400ml proliferation culture medium, and making total volume 800 ml;
supplementing liquid on day 11, adding 800ml proliferation culture medium, and making total volume be 1600 ml;
supplementing liquid on day 13, adding 800ml proliferation culture medium, and making total volume 2400 ml;
and on the 16 th day, stopping culturing, transferring the peripheral blood NK cells and the culture solution in the culture bag into a centrifuge tube together, carrying out centrifugation treatment, removing the supernatant, and repeatedly centrifuging and washing the umbilical cord blood NK cells on the lower layer for 2-3 times by adopting a 0.9% sodium chloride solution to obtain the umbilical cord blood NK cells.
Second, characterization of test results
(I), NK cell growth number and amplification times
FIG. 1 is a graph of cord blood NK cell growth curves for examples 1, 2, 3, 4, 5 and 1; among them, the cord blood NK cells of examples 1 to 4 were cultured for 10 days, and the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days. Table 1 is a table of statistics corresponding to the number of NK cell growth counts in cord blood in FIG. 1.
FIG. 1 and Table 1 show that, after proliferation of NK cells by culture for 10 days or 16 days, the culture methods of examples 1 to 4 of the present invention showed rapid proliferation of NK cells by culture, and the number of NK cells reached 121.1X 10 after culture for about 10 days8~133.8×108The cell per ml and the cell amplification multiple reach 242.8-267.6 times; in example 5 and comparative example 1, the proliferation rate of NK cells in culture is relatively slow, and the number of NK cells can reach 134.2X 10 after about 16 days of culture8129.4X 10 pieces/ml8The amplification times of cells can reach 268.4 times and 258.8 times per ml; that is, examples 1 to 4, compared to example 5 and comparative example 1, can shorten the culture time by 6 days by culturing the same number of NK cells,the time efficiency can be improved by 37.5%. In examples 1 to 5, the NK cell growth rate was superior to that in comparative example 1.
The detection result shows that: 1) the added halcinonide component in the first culture medium can inhibit the growth of T cells and mononuclear cells in umbilical cord blood, and is beneficial to the early rapid growth of NK cells; 2) adding cell factors soybean phospholipid and thymopentin into the second culture medium and the third culture medium; the soybean phospholipids have good hydrophobicity and solubilization, have an evacuation effect on suspended NK cells, can fully dissolve substances generated by high-density cell metabolism, and reduce mass death caused by local cell aggregation and agglomeration, so that high-density rapid amplification growth of the cells is maintained; the thymopentin has the functions of promoting NK cell proliferation, strengthening NK cell physiological activity and immunologic function, eliminating the immunosuppressive effect of halcinonide in the first culture medium and realizing the later-stage rapid proliferation of the NK cells in the umbilical cord blood.
TABLE 1 counting table of NK cell growth amounts of umbilical cord blood (. times.10)8One/ml)
(II) peripheral blood NK cell killing Activity test
Taking the NK cells of the umbilical cord blood after the respective culture of examples 1 to 5 and comparative example 1, wherein the number of the cells is the same; wherein, the umbilical cord blood NK cells of example 1, example 2, example 3, example 4 were cultured for 10 days; the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days.
Transferring to 3 sets of centrifuge tubes (6 centrifuge tubes in each set, each set corresponding to NK cells cultured in examples 1 to 5 and comparative example 1), the 3 sets of centrifuge tubes were individually adjusted to have a concentration of about 1.0X 106/ml、2.0×106/ml、4.0×106Cell suspension in/ml, as 3 groups of effector cells.
K562 cells were used as target cells, effector NK cells and target K562 cells were plated in an effective-to-target ratio of 1:1, 5:1, 10:1, 20:1, 3 duplicate wells per group. Each well of NK cells and K562 target cells was 500. mu.l. Each well was filled with 500. mu.l of the medium corresponding to each of examples 1 to 5 and comparative example 1.
Effector NK cell natural release group: 1.0X 106/ml、2.0×106/ml、4.0×106NK cells/ml, 500. mu.l per well, 3 replicates per group, 500. mu.l per well.
K562 target cell maximal release group: 1.0X 105Perml of target cells, 500. mu.l per well, 3 replicates per group, 500. mu.l per well of medium, 5% CO at 37 ℃2Incubate in incubator for 40 min.
K562 target cell spontaneous release group: 1.0X 105Perml of target cells, 500. mu.l per well, 3 replicates per group, 500. mu.l per well.
Culture solution blank control test group: mu.l per well, 3 replicates per group, 500. mu.l per well.
After plating, the plates were centrifuged at 250g for 4 minutes and placed at 37 ℃ in 5% CO2The culture was carried out in an incubator for 4 hours. 45 minutes before the end of the culture, the culture plate was removed, the thawing solution was added to the maximum release group of the target cells, the mixture was centrifuged at 250g for 4 minutes, the cells were gently mixed, the mixture was centrifuged at 300g for 5 minutes, the supernatant was collected, and the absorbance at 492nm was measured using LDH (lactate dehydrogenase).
The killing activity was calculated according to the following formula:
killing activity = (A-B-C)/(D-C). times.100%;
wherein:
a-represents the corrected value of the absorbance value of the test group;
b-represents effector cell natural release group;
c-represents a target cell spontaneous release group;
d-represents the maximum release group of target cells;
the test results are shown in table 2 and fig. 2; table 2 shows the specific arrangement of the corresponding data in fig. 2, and the two data are consistent with each other. As shown in fig. 2, the killing profile of NK cells against tumor cell K562; corresponding curves of the NK cell killing activity of cord blood in example 1, example 2, example 3, example 4, example 5 and comparative example 1 are shown.
TABLE 2 detection of killing activity of NK cells on K562 target cells
As can be seen from Table 2 and FIG. 2, when killing K562 cells, the NK cells induced in examples 1 to 5 of the present invention have better killing effect and more obvious difference than the NK cells induced in comparative example 1. It is demonstrated that the combination of cytokines in the culture medium of the present invention can enhance the activity of killing tumor cells when NK cells are cultured.
(III) flow-type phenotype detection of NK cells of umbilical cord blood
FIGS. 3, 4, 5, 6, 7 and 8 are graphs showing the phenotype of CD3-CD56+ after culture of cord blood NK cells of examples 1 to 5 and comparative example 1, respectively; among them, the cord blood NK cells of examples 1 to 4 were cultured for 10 days, and the cord blood NK cells of example 5 and comparative example 1 were cultured for 16 days.
NK cells cultured in examples 1 to 5 and comparative example 1 were each prepared into a cell suspension and adjusted to a cell concentration of 1X 105Adding a labeled CD3-CD56+ monoclonal antibody into the cells per ml, incubating the cells for 20 minutes at room temperature in the dark, washing off excess antibody, and detecting the antibody by using an up-flow cytometer, wherein the test results are shown in figures 3, 4, 5, 6, 7 and 8.
As is clear from FIGS. 3, 4, 5, 6, 7 and 8, the contents of CD3-CD56+ in the cord blood NK cells cultured in examples 1 to 5 and comparative example 1 were 96.34%, 95.81%, 94.87%, 86.71%, 64.51% and 60.82%, respectively.
Therefore, the combination of the components such as the cytokines halcinonide, the soybean phospholipid and the thymopentin, which are added into the culture medium, enables the content of CD3-CD56+ (examples 1 to 4) to reach 86.51-96.34%, which is far higher than that of CD3-CD56+ of comparative example 1 by 60.82%; in addition, in examples 1 to 5, since the cytokine halcinonide is not added to the first culture medium in example 5, the content of CD3-CD56+ in example 5 is relatively low, 64.51%, which is much less than the content of CD3-CD56+ in examples 1 to 4.
Therefore, from the above-described flow detection results, it is reflected that: in the culture solutions of examples 1 to 4, the cytokine halcinonide can inhibit the growth of T cells and monocytes in umbilical cord blood, effectively improve the purity of NK cells in umbilical cord blood during early culture, and the thymopentin can promote NK cell proliferation, enhance the physiological activity and immune function of NK cells, eliminate the immunosuppressive effect of halcinonide in the first culture medium, and realize the later-stage rapid proliferation of NK cells in umbilical cord blood.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be considered as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A culture solution of cord blood NK cells is characterized by comprising the following components:
a first medium: adding IL-2 with a final concentration of 1500-3000 IU/ml, IL-15 with a final concentration of 30-80 ng/ml, IL-12 with a final concentration of 50-100 ng/ml, IL-3 with a final concentration of 20-50 ng/ml, OK432 with a final concentration of 0.1-2 ug/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml, StemRegenin 1 with a final concentration of 2-5 mu M and halcinonide with a final concentration of 0.05-1ng/ml into a basic culture medium;
a second medium: adding IL-2 with a final concentration of 1500-2000 IU/ml, IL-21 with a final concentration of 10-30 ng/ml, SCF with a final concentration of 10-20 ng/ml, soybean phospholipid with a final concentration of 300-800ug/ml and thymopentin with a final concentration of 1-5ug/ml into a basic culture medium;
a third medium: IL-2 with the final concentration of 1000-1500 IU/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-.
2. The culture solution according to claim 1, comprising the following components:
a first medium: IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 muM and Hacinonide with a final concentration of 0.15ng/ml are added to the basal medium;
a second medium: adding IL-2 with a final concentration of 1800IU/ml, IL-21 with a final concentration of 22ng/ml, SCF with a final concentration of 16ng/ml, soybean phospholipid with a final concentration of 550ug/ml and thymopentin with a final concentration of 3ug/ml into a basic culture medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at 550ug/ml and thymopentin at 3 ug/ml.
3. The culture solution according to claim 1, comprising the following components:
a first medium: IL-2 with a final concentration of 1500IU/ml, IL-15 with a final concentration of 80ng/ml, IL-12 with a final concentration of 50ng/ml, IL-3 with a final concentration of 20ng/ml, OK432 with a final concentration of 0.1ug/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml, StemRegenin 1 with a final concentration of 2 μ M and Hacinonide with a final concentration of 0.05ng/ml are added to a basal medium;
a second medium: adding IL-2 with final concentration of 1500IU/ml, IL-21 with final concentration of 10ng/ml, SCF with final concentration of 10ng/ml, soybean phospholipid with final concentration of 300ug/ml and thymopentin with final concentration of 1ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1000IU/ml, SCF at a final concentration of 10ng/ml, soybean phospholipids at a final concentration of 300ug/ml, and thymopentin at a final concentration of 1 ug/ml.
4. The culture solution according to claim 1, comprising the following components:
a first medium: adding IL-2 with a final concentration of 3000IU/ml, IL-15 with a final concentration of 30ng/ml, IL-12 with a final concentration of 100ng/ml, IL-3 with a final concentration of 50ng/ml, OK432 with a final concentration of 2ug/ml, SCF with a final concentration of 40ng/ml, TGF with a final concentration of 20ng/ml, PDGF with a final concentration of 20ng/ml, StemRegenin 1 with a final concentration of 5 mu M and halcinonide with a final concentration of 1ng/ml to a basal medium;
a second medium: adding IL-2 with final concentration of 2000IU/ml, IL-21 with final concentration of 30ng/ml, SCF with final concentration of 20ng/ml, soybean phospholipid with final concentration of 800ug/ml and thymopentin with final concentration of 5ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1500IU/ml, SCF at a final concentration of 20ng/ml, soybean phospholipids at 800ug/ml, and thymopentin at 5 ug/ml.
5. The culture solution according to any one of claims 1 to 4, wherein the basal medium is KBM581, GT-T551, X-VIVO 15 or AIM-V.
6. A culture method of cord blood NK cells is characterized by comprising the following steps:
1) preparing a first culture medium, a second culture medium and a third culture medium; wherein the content of the first and second substances,
a first medium: adding IL-2 with a final concentration of 1500-3000 IU/ml, IL-15 with a final concentration of 30-80 ng/ml, IL-12 with a final concentration of 50-100 ng/ml, IL-3 with a final concentration of 20-50 ng/ml, OK432 with a final concentration of 0.1-2 ug/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml, StemRegenin 1 with a final concentration of 2-5 mu M and halcinonide with a final concentration of 0.05-1ng/ml into a basic culture medium;
a second medium: adding IL-2 with a final concentration of 1500-2000 IU/ml, IL-21 with a final concentration of 10-30 ng/ml, SCF with a final concentration of 10-20 ng/ml, soybean phospholipid with a final concentration of 300-800ug/ml and thymopentin with a final concentration of 1-5ug/ml into a basic culture medium;
a third medium: IL-2 with the final concentration of 1000-1500 IU/ml, SCF with the final concentration of 10-20 ng/ml, soybean phospholipid with the final concentration of 300-;
2) inoculating umbilical cord blood PBMC and culturing;
day 0: adding 285ml of first culture medium and 15ml of 5v/v% blood into a suspension cell culture bottle with the specification of 500 ml; then, inoculating 5000 ten thousand cells, standing and culturing for 5 days, and after 3 days, shaking the culture bottle for 1-2 times to disperse the aggregated cells;
day 5: removing part of the culture solution, replacing and adding 400ml of a second culture medium, shaking up the cells, and then standing and culturing for 3 days;
day 8: removing part of the culture solution, replacing and adding 500ml of a third culture medium, shaking up the cells, and then standing and culturing for 2 days;
day 10: stopping culturing, collecting cells, centrifuging to remove supernatant, collecting precipitate, and repeatedly washing the precipitate with normal saline for multiple times to obtain high-purity and high-activity cord blood NK cells.
7. The culture method according to claim 6, wherein in the step 1),
a first medium: adding IL-2 with a final concentration of 2000IU/ml, IL-15 with a final concentration of 50ng/ml, IL-12 with a final concentration of 75ng/ml, IL-3 with a final concentration of 42ng/ml, OK432 with a final concentration of 1.2ug/ml, SCF with a final concentration of 30ng/ml, TGF with a final concentration of 28ng/ml, PDGF with a final concentration of 16ng/ml, StemRegenin 1 with a final concentration of 3 mu M and halcinonide with a final concentration of 0.15ng/ml to a basal medium;
a second medium: adding IL-2 with a final concentration of 1800IU/ml, IL-21 with a final concentration of 22ng/ml, SCF with a final concentration of 16ng/ml, soybean phospholipid with a final concentration of 550ug/ml and thymopentin with a final concentration of 3ug/ml into a basic culture medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1150IU/ml, SCF at a final concentration of 16ng/ml, soybean phospholipids at 550ug/ml and thymopentin at 3 ug/ml.
8. The culture method according to claim 6, wherein in the step 1),
a first medium: IL-2 with a final concentration of 1500IU/ml, IL-15 with a final concentration of 80ng/ml, IL-12 with a final concentration of 50ng/ml, IL-3 with a final concentration of 20ng/ml, OK432 with a final concentration of 0.1ug/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml, StemRegenin 1 with a final concentration of 2 μ M and Hacinonide with a final concentration of 0.05ng/ml are added to a basal medium;
a second medium: adding IL-2 with final concentration of 1500IU/ml, IL-21 with final concentration of 10ng/ml, SCF with final concentration of 10ng/ml, soybean phospholipid with final concentration of 300ug/ml and thymopentin with final concentration of 1ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1000IU/ml, SCF at a final concentration of 10ng/ml, soybean phospholipids at a final concentration of 300ug/ml, and thymopentin at a final concentration of 1 ug/ml.
9. The culture method according to claim 6, wherein in the step 1),
a first medium: adding IL-2 with a final concentration of 3000IU/ml, IL-15 with a final concentration of 30ng/ml, IL-12 with a final concentration of 100ng/ml, IL-3 with a final concentration of 50ng/ml, OK432 with a final concentration of 2ug/ml, SCF with a final concentration of 40ng/ml, TGF with a final concentration of 20ng/ml, PDGF with a final concentration of 20ng/ml, StemRegenin 1 with a final concentration of 5 mu M and halcinonide with a final concentration of 1ng/ml to a basal medium;
a second medium: adding IL-2 with final concentration of 2000IU/ml, IL-21 with final concentration of 30ng/ml, SCF with final concentration of 20ng/ml, soybean phospholipid with final concentration of 800ug/ml and thymopentin with final concentration of 5ug/ml into basal medium;
a third medium: IL-2 was added to the basal medium at a final concentration of 1500IU/ml, SCF at a final concentration of 20ng/ml, soybean phospholipids at 800ug/ml, and thymopentin at 5 ug/ml.
10. The method according to any one of claims 6 to 9, wherein the basal medium is KBM581, GT-T551, X-VIVO 15 or AIM-V.
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