CN113699107A - Peripheral blood NKT cell culture solution and culture method - Google Patents
Peripheral blood NKT cell culture solution and culture method Download PDFInfo
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Abstract
The invention discloses a peripheral blood NKT cell culture solution and a culture method, wherein the culture method of NKT cells comprises the following steps: adding peripheral blood mononuclear cells into a lymphocyte serum-free culture medium containing autologous serum of cell factors to culture for about 14 days; during the culture period, a culture solution containing the cell factors is required to be added every 2-3 days; after the culture is finished, performing centrifugal treatment and washing by 0.9% sodium chloride solution to obtain peripheral blood NKT cells; human serum albumin was added to peripheral blood NKT cells, and the volume was fixed with 0.9% sodium chloride solution to obtain peripheral blood NKT cells. According to the culture method, the peripheral blood single check has no immune rejection reaction to the autologous serum, the cells are purer, and the safety is higher; NKT cells grow fast and have high amplification rate; NKT cells have high killing activity and stronger tumor cell killing effect.
Description
Technical Field
The invention relates to the field of cytology, in particular to a peripheral blood NKT cell culture solution and a culture method.
Background
NKT cells (natural killer T cells), a class of killer cells induced by multiple cytokines, are immune cells mediating the strongest cytotoxic activity, and have both the high tumoricidal activity of T lymphocytes and the restricted tumoricidal effect of NK Cell non-Major Histocompatibility Complexes (MHC). The NKT cell has the advantages of high proliferation speed, high tumor killing activity and the like.
In the present stage, NKT cell culture is to treat isolated Peripheral Blood Mononuclear Cells (PBMCs) with IFN- γ for 24 hours, and then add CD3 monoclonal antibodies, IL-1 α and IL-2 factors to stimulate and induce them to obtain a certain number of NKT cells, but the final obtained NKT cells have low purity of effective cell content, weak cell killing activity, and relatively low cell expansion rate.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide a high-purity peripheral blood NKT cell culture solution with a high expansion ratio and a culture method.
The first technical scheme of the invention is as follows:
a peripheral blood NKT cell culture solution comprises a lymphocyte serum-free culture medium containing 1-20v/v% of autologous serum, wherein cytokines are added into the culture medium: 10-3000 IU/ml of IL-2, 5-500 ng/ml of IL-15, 5-500 ng/ml of IL-18, 5-1000 ng/ml of CD3 monoclonal antibody, 0.01-5 ng/ml of dasatinib and 5-500 IU/ml of POM.
Preferably, in one embodiment, the NKT cell culture fluid comprises lymphocyte serum-free medium containing 10v/v% of autologous serum, and cytokines are added into the lymphocyte serum-free medium: 1000IU/ml IL-2, 50ng/ml IL-15, 50ng/ml IL-18, 200ng/ml CD3 monoclonal antibody, 2ng/ml dasatinib and 100IU/ml POM.
The invention also provides a culture method of peripheral blood NKT cells, which comprises the following steps:
s1, separating mononuclear cells from peripheral blood on day 0, adding the mononuclear cells into a lymphocyte serum-free culture medium containing 1-20v/v% of autologous serum, and adding cytokines into the culture medium, wherein the cytokines comprise IL-2 with the concentration of 10-3000 IU/ml, IL-15 with the concentration of 5-500 ng/ml, IL-18 with the concentration of 5-500 ng/ml, CD3 monoclonal antibody with the concentration of 5-1000 ng/ml, dasatinib with the concentration of 0.01-5 ng/ml and POM with the concentration of 5-500 IU/ml; standing and culturing for 14 days;
s2, adding a lymphocyte serum-free culture medium and cytokines on days 3 and 5 respectively, wherein the cytokines comprise IL-2 with the concentration of 10-3000 IU/ml, IL-15 with the concentration of 5-500 ng/ml, IL-18 with the concentration of 5-500 ng/ml, CD3 monoclonal antibody with the concentration of 5-1000 ng/ml, dasatinib with the concentration of 0.01-5 ng/ml and POM with the concentration of 5-500 IU/ml; (ii) a
S3, on 7 th day, 9 th day and 11 th day, adding a lymphocyte serum-free culture medium and IL-2 cytokines with the concentration of 10-3000 IU/ml respectively;
s4, after cell culture is finished, transferring the cultured peripheral blood NKT cells into a centrifuge tube for centrifugal treatment, collecting the peripheral blood NKT cells, and repeatedly washing the peripheral blood NKT cells for 2-3 times by using a 0.9% sodium chloride solution;
s5, adding human serum albumin to the peripheral blood NKT cells obtained after the washing in step S5, and fixing the volume with 0.9% sodium chloride solution to obtain the peripheral blood NKT cells.
Preferably, in step S1 of the culturing method, the autologous serum is inactivated at 56 ℃ and has a concentration of 1-20 v/v%.
Preferably, in steps S1 and S2 of the culture method, the cytokine has a concentration of IL-2 of 1000IU/ml, a concentration of IL-15 of 50ng/ml, a concentration of IL-18 of 50ng/ml, a concentration of CD3 monoclonal antibody of 200ng/ml, a concentration of dasatinib of 2ng/ml and a concentration of POM of 100 IU/ml.
Preferably, in step S3 of the culture method, the concentration of the IL-2 is 1000 IU/ml.
Preferably, in the culture method, the lymphocyte serum-free medium includes any one of GT-T551H3 medium, Corning KBM581 lymphocyte serum-free medium and RPMI-1640 medium
Preferably, in the culture method, the cell concentration is controlled to 1X 10 during the cell culture72 x 10 units/ml7One per ml.
Preferably, in step S5 of the culture method, 10ml of human serum albumin with a volume percentage of 10% is added to peripheral blood NKT cells, and the volume is adjusted to 200ml with 0.9 wt% sodium chloride solution.
Compared with the conventional culture method, the beneficial effects of the invention are shown in the following aspects:
(1) The mononuclear cells are separated from peripheral blood, the peripheral blood mononuclear cells have no immune rejection reaction to autologous serum, the cells are purer, and the safety is higher;
(2) The cultured NKT cells have high purity, fast growth and high amplification rate;
(3) The cultured NKT cells have high killing activity rate and stronger tumor cell killing effect;
therefore, the cytokines dasatinib and POM can promote the activation of NKT cells during the continuous addition of cell culture, and have a great promoting effect on the CD3+ CD56+ flow content of the NKT cells, so that the tumor killing activity of the NKT cells is effectively improved.
Drawings
FIG. 1 is a graph showing the growth of NKT in peripheral blood immunocytes in examples 1, 2 and 3 and comparative examples of the present invention;
FIG. 2 is a graph showing the NKT amplification rate of peripheral blood immunocytes in examples 1, 2 and 3 and a comparative example of the present invention;
FIG. 3 is a graph showing the killing of tumor cells K562 by peripheral blood immune cells NKT according to examples 1, 2, 3 and comparative examples of the present invention;
FIGS. 4a, 4b, 4c and 4d are the phenotypic test of CD3+ CD56+ after 14 days of culture of NKT in peripheral blood immunocytes of examples 1, 2 and 3 and comparative examples, respectively.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
Some commonly used terms in the present invention are described below:
IL-2: interleukin 2;
IL-15: interleukin 15;
IL-18: interleukin 18;
CD3 monoclonal antibody: cell surface molecule 3 monoclonal antibody.
Wherein:
IL-2, IL-15, IL-18, CD3 monoclonal antibodies and the like are essential for the culture of NK and NKT cells as basic cytokines for the culture of NKT cells;
IL-2: promoting the growth and proliferation of NKT cells;
IL-15 and IL-18: the NKT cells are stimulated to differentiate and proliferate under the synergistic effect;
CD3 monoclonal antibody: stimulation induces the formation of NKT cells and cytokine production;
dasatinib: inhibiting the proliferation of T cells, improving the purity of NKT cells, enhancing the killing activity of NKT cells and generating cytokines;
POM: the cell proliferation is promoted, the cell growth and the cell agglomeration are prevented, and the cell survival number and the expansion multiple are improved.
In the present invention, a method for culturing peripheral blood NKT cells is used, and the following procedure is specifically performed.
1. Isolation of peripheral blood mononuclear cells
(1) Crude separation
The blood was dispensed into two 50ml centrifuge tubes (labeled No. 1 and No. 2) each containing 30ml and the blood volume was 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, about 8X 10 cells were taken out from the cell fluid7The number of mononuclear cells were individually loaded into a number 8 centrifuge tube and resuspended to 50 ml. And then, centrifuging the No. 8 centrifuge tube, wherein the centrifugal force is 300g, the centrifugation time is 10min, and the centrifugal speed is increased by 8 steps and decreased by 8 steps during centrifugation. After centrifugation, the supernatant was discarded, and the pellet was the desired mononuclear cell, which also became Peripheral Blood Mononuclear Cells (PBMC).
2. Culture of peripheral blood NKT cells
1) Day 0, 5X 106Transferring the peripheral blood mononuclear cells/ml into a T175 culture bottle, placing the culture bottle into an incubator for standing culture for 3 days, and adding an autologous serum lymphocyte serum-free culture medium containing cytokines such as IL-2, IL-15, IL-18, CD3 monoclonal antibody, dasatinib and POM (2- (thiazole-2-ylamino) ethylene-1, 1-bis (tetrapentyloxymethyl) diphosphonate, pivaloyloxymethyl ester for short POM) and the like into the culture bottle.
In the step 1), the concentration of IL-2 in the cell factor is 10-3000 IU/ml, the concentration of IL-15 is 5-500 ng/ml, the concentration of IL-18 is 5-500 ng/ml, the concentration of CD3 monoclonal antibody is 5-1000 ng/ml, the concentration of dasatinib is 0.01-5 ng/ml and the concentration of POM is 5-500 IU/ml; the autologous serum is inactivated at 56 ℃, and the concentration of the autologous serum is 1-20 v/v%.
The lymphocyte serum-free medium includes any one of GT-T551H3 medium, Corning KBM581 lymphocyte serum-free medium and RPMI-1640 medium.
Meanwhile, during the static culture of the cells, the cell concentration is controlled to be 1 multiplied by 1072 x 10 units/ml7In the range of one/ml.
The conditions of static culture in the incubator are as follows: CO with the ambient temperature of 37 ℃, the volume percentage of 5 percent and the relative saturation humidity of 100 percent2。
2) Adding lymphocyte serum-free culture medium containing cell factors respectively on the 3 rd and 5 th days; from day 7 onwards, the cell culture was transferred to culture bags.
In the step, the concentration of IL-2 in the cell factor is 10-3000 IU/ml, the concentration of IL-15 is 5-500 ng/ml, the concentration of IL-18 is 5-500 ng/ml, the concentration of CD3 monoclonal antibody is 5-1000 ng/ml, the concentration of Dasatinib is 0.01-5 ng/ml, and the concentration of POM is 5-500 IU/ml.
Meanwhile, during the static culture of the cells, the cell concentration is controlled to be 1 multiplied by 1072 x 10 units/ml7In the range of one/ml.
3) And on the 7 th day, the 9 th day and the 11 th day, a lymphocyte serum-free culture medium and IL-2 cytokines with the concentration of 10-3000 IU/ml are respectively added.
4) After the 14 th day of cell culture, transferring the peripheral blood NKT cells and the culture solution in the culture bag into a centrifuge tube together, performing centrifugation treatment and removing the supernatant, and repeatedly centrifuging and washing the peripheral blood NKT cells on the lower layer for 2-3 times by adopting 0.9% sodium chloride solution; finally, collecting the precipitate on the lower layer of the centrifuge tube by using a volume fixing device, adding human serum albumin into the precipitate, and fixing the volume to 200ml by using 0.9% sodium chloride solution to obtain peripheral blood NKT cells; wherein the concentration of 0.9% sodium chloride solution is 0.9 wt%, the volume percentage concentration of human serum albumin is 10%, and 10ml of human serum albumin is added; the human serum albumin can prevent cell agglomeration and maintain cell viability.
Compared with the conventional culture method, the beneficial effects of the invention are shown in the following aspects:
(1) The mononuclear cells are separated from peripheral blood, the peripheral blood mononuclear cells have no immune rejection reaction to autologous serum, the cells are purer, and the safety is higher;
(2) The cultured NKT cells have high purity, fast growth and high amplification rate;
(3) The cultured NKT cells have high killing activity rate and stronger tumor cell killing effect.
First, culture of peripheral blood NKT cells
Example 1
On day 0, Corning KBM581 lymphocyte serum-free medium containing cytokines at a final concentration of 10% autologous serum was added to a T175 cell culture flask at a final volume of medium25 ml; then 5X 106The peripheral blood mononuclear cells obtained as described above were added to a Corning KBM581 lymphocyte serum-free medium containing 10v/v% of autologous serum, and the mixture was left to stand for 3 days in an incubator at 37 ℃ in the atmosphere of 5% by volume of CO2And CO with a relative saturation humidity of 100%2(ii) a Wherein, the cell factors comprise: IL-2 at a final concentration of 1000IU/ml, IL-15 at a final concentration of 50ng/ml, IL-18 at a final concentration of 50ng/ml, CD3 monoclonal antibody at a final concentration of 200ng/ml, dasatinib at a final concentration of 2ng/ml, and POM at a final concentration of 100 IU/ml.
On day 3 and day 5, 50ml of Corning KBM 581-containing serum-free lymphocyte culture medium and cytokines were added to each cell culture flask to culture cells at a cell concentration of 1X 1072 x 10 units/ml7Within a unit/ml range; wherein the cytokines include: the concentration of IL-2 is 1000IU/ml, the concentration of IL-15 is 300ng/ml, the concentration of IL-18 is 300ng/ml, the concentration of CD3 monoclonal antibody is 500ng/ml, the concentration of dasatinib is 2ng/ml and the concentration of POM is 300 IU/ml.
On day 7, the cells were transferred to two 1000ml standard cell culture bags together with the culture medium, and 250ml of Corning KBM581 lymphocyte serum-free medium containing IL-2 at a final concentration of 1000IU/ml was added to the cell culture bags; the cell culture was continued in the incubator.
On day 9, 500ml of Corning KBM581 lymphocyte serum-free medium containing IL-2 at a final concentration of 1000IU/ml was again added to the cell culture bag.
On day 11, 500ml of Corning KBM581 lymphocyte serum-free medium containing IL-2 at a final concentration of 1000IU/ml was further added to the cell culture bag.
On day 14, the culture is stopped, the cultured peripheral blood NKT cells are collected by a centrifuge tube, centrifuged, the supernatant is removed, and repeatedly centrifuged and washed for 3 times by 0.9% sodium chloride solution; and finally, collecting the precipitate at the lower layer of the centrifuge tube by using a volume fixing device, adding 10ml of 10% human serum albumin into the precipitate, and then fixing the volume to 200ml by using 0.9% sodium chloride solution to obtain the peripheral blood NKT cell product.
Example 2
On day 0, GT-T551H3 lymphocyte serum-free medium containing final concentration of 1% autologous serum was added to the T175 cell culture flask, and the final volume of the medium was 25 ml; then 5X 106Adding the obtained peripheral blood mononuclear cells into culture medium, and standing for 3 days in incubator with environment temperature of 37 deg.C and atmosphere of 5% CO2And CO with a relative saturation humidity of 100%2(ii) a Wherein, the cell factors comprise: IL-2 at a final concentration of 10IU/ml, IL-15 at a final concentration of 5ng/ml, IL-18 at a final concentration of 5ng/ml, CD3 monoclonal antibody at a final concentration of 5ng/ml, dasatinib at a final concentration of 0.01ng/ml, and POM at a final concentration of 5 IU/ml.
On day 3 and day 5, 50ml of the GT-T551H3 lymphocyte serum-free medium and cytokine were added to the cell culture flask, respectively, to culture the cells at a cell concentration of 1X 1072 x 10 units/ml7Within a unit/ml range; wherein the cytokines include: the concentration of IL-2 is 10IU/ml, the concentration of IL-15 is 5ng/ml, the concentration of IL-18 is 5ng/ml, the concentration of CD3 monoclonal antibody is 5ng/ml, the concentration of dasatinib is 0.01ng/ml and the concentration of POM is 5 IU/ml.
On day 7, the cells were transferred to two 1000ml standard cell culture bags together with the culture medium, and 250ml of GT-T551H3 lymphocyte serum-free medium containing IL-2 at a final concentration of 5IU/ml was added to the cell culture bags at the same time; the cell culture was continued in the incubator.
On day 9, 500ml of GT-T551H3 lymphocyte serum-free medium containing IL-2 at a final concentration of 5IU/ml was added to the cell culture bag again.
On day 11, 500ml of GT-T551H3 lymphocyte serum-free medium containing IL-2 at a final concentration of 5IU/ml was further added to the cell culture bag.
On day 14, the culture is stopped, the cultured peripheral blood NKT cells are collected by a centrifuge tube, centrifuged, the supernatant is removed, and repeatedly centrifuged and washed for 3 times by 0.9% sodium chloride solution; and finally, collecting the precipitate at the lower layer of the centrifuge tube by using a volume fixing device, adding 10ml of 10% human serum albumin into the precipitate, and then fixing the volume to 200ml by using 0.9% sodium chloride solution to obtain the peripheral blood NKT cell product.
Example 3
On day 0, RPMI-1640 medium containing a final concentration of 20% autologous serum was added to the T175 cell culture flask, and the final volume of the medium was 25 ml; then 5X 106Adding the obtained peripheral blood mononuclear cells into culture medium, and standing for 3 days in incubator with environment temperature of 37 deg.C and atmosphere of 5% CO2And CO with a relative saturation humidity of 100%2(ii) a Wherein, the cell factors comprise: IL-2 at a final concentration of 1000IU/ml, IL-15 at a final concentration of 500ng/ml, IL-18 at a final concentration of 500ng/ml, CD3 monoclonal antibody at a final concentration of 1000ng/ml, dasatinib at a final concentration of 5ng/ml, and POM at a final concentration of 500 IU/ml.
On day 3 and day 5, 50ml of medium containing RPMI-1640 and cytokine were added to the cell culture flask, respectively, to culture cells at a cell concentration of 1X 1072 x 10 units/ml7Within a unit/ml range; wherein the cytokines include: the concentration of IL-2 is 1000IU/ml, the concentration of IL-15 is 500ng/ml, the concentration of IL-18 is 500ng/ml, the concentration of CD3 monoclonal antibody is 1000ng/ml, the concentration of dasatinib is 5ng/ml and the concentration of POM is 500 IU/ml.
On day 7, the cells were transferred to two 1000ml standard cell culture bags together with the culture medium, and 250ml of GT-T551H3 lymphocyte serum-free medium containing IL-2 at a final concentration of 1000IU/ml was added to the cell culture bags at the same time; the cell culture was continued in the incubator.
On day 9, 500ml of RPMI-1640 medium containing IL-2 at a final concentration of 1000IU/ml was added again to the cell culture bags.
On day 11, 500ml of RPMI-1640 medium containing IL-2 at a final concentration of 1000IU/ml was added to the cell culture bags.
On day 14, the culture is stopped, the cultured peripheral blood NKT cells are collected by a centrifuge tube, centrifuged, the supernatant is removed, and repeatedly centrifuged and washed for 3 times by 0.9% sodium chloride solution; and finally, collecting the precipitate at the lower layer of the centrifuge tube by using a volume fixing device, adding 10ml of 10% human serum albumin into the precipitate, and then fixing the volume to 200ml by using 0.9% sodium chloride solution to obtain the peripheral blood NKT cell product.
Comparative example (conventional NKT cell culture method)
Take 5X 106Adding each/ml of the peripheral blood mononuclear cells prepared in the above into a culture bottle of RPMI-1640 culture medium containing 10% fetal bovine serum, adding a cytokine IFN-gamma into 50ml of the culture medium, wherein the final concentration of the cytokine IFN-gamma in the culture medium is 1200ng/ml respectively; placing the culture flask into CO with the ambient temperature of 37 ℃ and the ambient atmosphere of 5 percent by volume2And CO with a relative saturation humidity of 100%2The culture box of (2) was subjected to static culture for 3 days.
On the 2 nd day, factors such as IL-1 beta, IL-2, CD 3-monoclonal antibody and the like are added into the cell culture bottle for cell culture; wherein the final concentrations of IFN-gamma, IL-2 and CD 3-monoclonal antibody protein in the culture medium are 1200ng/ml, 120ng/ml and 200U/ml respectively.
On days 3 to 11, 400ml of RPMI-1640 medium containing IL-2 is added into the cell culture bottle every two days, and cell culture is continued; wherein the final concentration of IL-2 in the medium was 2000ng/ml, respectively.
On day 14, the culture is stopped, the cultured peripheral blood NKT cells are collected by a centrifuge tube, centrifuged, the supernatant is removed, and repeatedly centrifuged and washed for 3 times by 0.9% sodium chloride solution; and finally, collecting the precipitate at the lower layer of the centrifuge tube by using a volume fixing device, adding 10ml of 10% human serum albumin into the precipitate, and then fixing the volume to 200ml by using 0.9% sodium chloride solution to obtain the peripheral blood NKT cell product.
Second, characterization of experimental test results
(I) measurement of NKT cell growth
FIG. 1 shows the culture growth profiles of peripheral blood NKT cells of examples 1, 2, 3 and comparative examples. FIG. 1 shows the stimulating effect of Dasatinib cytokines in the culture method of the present invention after 14 days of culture proliferation of cellsThe proliferation of T cells is inhibited, and the purity of NKT cells is improved; under the stimulation of POM cell cytokines, NKT cells can be prevented from being adhered to form clusters, and the survival number of the cells is increased; in a word, the culture solution contains dasatinib and POM cytokines, and can effectively improve the proliferation multiple of NKT cells, so that the number of the NKT cells reaches nearly 1.12 multiplied by 1010The number of NKT cells cultured in the comparative example is 0.65X 1010As shown in table 1. In table 1, the cell growth numbers of peripheral blood NKT cells measured on days 0, 6, 8, 10, 12, and 14 in example 1, example 2, example 3, and comparative example, respectively; wherein, after 14 days of culture of NK cells, example 1, example 2, and example 3 are 1.95 times, 1.12 times, and 1.37 times, respectively, as compared with the comparative example.
TABLE 1 peripheral blood NKT cell growth number Scale (. times.10)7One)
(II) measurement of the amount of peripheral blood NKT cell proliferation
NKT cells cultured in example 1, example 2, example 3 and comparative example were stained with trypan blue and counted by a cell counter, and the current total number of cells was divided by the number of mononuclear cells before culture, which was the fold expansion of the cells. The proliferation status of cells can be dynamically observed by this method, as shown in FIG. 2. As shown in FIG. 2, after the cells are cultured and proliferated for 14 days, in the culturing method of the present invention, in the growth and proliferation process of NKT cells, dasatinib can inhibit the proliferation of peripheral blood T cells through continuous stimulation, so as to achieve the effect of improving the growth and proliferation of NKT cells; the POM can prevent the NKT cells from being adhered to each other to form a cluster, so that the survival number of the single NKT cells is increased, and further the effective proliferation multiple of the NKT cells is reached, so that the proliferation multiple of the NKT cells is far larger than that of the NKT cells in a comparative example, as shown in table 2, the cell proliferation multiples in examples 1 to 3 are 224.6, 143.6 and 158.2 respectively; table 2 shows the values of the amplification factors of peripheral blood NKT cells measured on days 0, 6, 8, 10, 12, and 14 in example 1, example 2, example 3, and comparative example, respectively; among them, examples 1, 2, and 3 are 1.95 times, 1.25 times, and 1.37 times as large as those of comparative examples.
TABLE 2 peripheral blood NKT cell expansion fold table
(III) detection of viability of NKT cells before and after cryopreservation
After trypan blue staining of the cells of example 1, example 2, example 3 and comparative example, the number of cells and the cell viability were calculated by counting the cells using a cell counting plate, as shown in table 3.
TABLE 3 cell number and cell viability Table
As can be seen from Table 3, the cell numbers and cell viability rates of examples 1, 2 and 3 are much higher than those of the comparative examples, which means that the NKT cell expansion rate of the culture solution containing the combination of the cytokines dasatinib and POM in examples 1, 2 and 3 is much higher than that of the comparative examples.
(IV) peripheral blood NKT cell killing Activity assay
Peripheral blood NKT cells were collected and the number of the cells was the same after 14 days of culture in each of example 1, example 2, example 3 and comparative example. Transferring to 3 sets of centrifuge tube sets (4 centrifuge tubes in each set, each set corresponding to NKT cells cultured in examples 1, 2, 3, and comparative examples), the concentration of each of the 3 sets was adjusted to 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 NKT cells and target cells K562 were plated in an effective target ratio of 1:1, 5:1, 10:1, 20:1, and 3 wells were plated for each group. Each well of NKT cells and K562 target cells was 500. mu.l. Each well was filled with 500. mu.l of the medium corresponding to each of the examples and comparative examples.
Effector cell NKT cell natural release group: 1.0X 106/ml、2.0×106/ml、4.0×106NKT 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 4 and fig. 3.
TABLE 4 detection of killing Activity of NKT cells against K562 target cells
As can be seen from Table 4, when K562 cells are killed, NKT cells induced by the method have better killing effect than NKT cells induced by a proportion, and the difference is obvious, which shows that when NKT cells are cultured by adopting the combination of the cell factor containing dasatinib and POM in the culture solution, the activity of killing tumors can be enhanced.
As shown in fig. 3, the killing profile of peripheral blood NKT cells against tumor cell K562; the curves of killing activity against peripheral blood NKT cells in example 1, example 2, example 3 and comparative example are shown.
(V) immunophenotyping of peripheral blood NKT cells
After 14 days, peripheral blood NKT cells cultured in example 1, example 2, example 3 and comparative example were each collected to prepare a cell suspension, and the cell concentration was adjusted to 1X 105Adding labeled CD3+ CD56+ monoclonal antibody into the mixture per ml, incubating the mixture for 20 minutes at room temperature in the dark, washing off excessive antibody, detecting the excessive antibody by an up-flow cytometer, and testing the result as shown in a peripheral blood NKT cell phenotype analysis chart of figures 4a, 4b and 4 c. Fig. 4a, 4b, 4c, and 4d show that the content of CD3+ CD56+ cells in peripheral blood NKT cells of examples 1, 2, and 3 was 55.34%, 40.99%, 43.63%, and 18.30%, respectively. Therefore, the combination of the cytokines dasatinib and POM added to the culture medium makes the content of CD3+ CD56+ cells as high as 55.34% and more than 3 times higher than the content of the comparative example 18.30%, thereby reflecting that the peripheral blood NKT cell antitumor activity of examples 1, 2 and 3 is strong.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (9)
1. A peripheral blood NKT cell culture solution, which is characterized by comprising a lymphocyte serum-free culture medium containing 1-20v/v% of autologous serum, wherein the culture medium is added with cytokines: 10-3000 IU/ml of IL-2, 5-500 ng/ml of IL-15, 5-500 ng/ml of IL-18, 5-1000 ng/ml of CD3 monoclonal antibody, 0.01-5 ng/ml of dasatinib and 5-500 IU/ml of POM.
2. The NKT cell culture fluid according to claim 1, wherein the culture fluid comprises a lymphocyte serum-free medium containing 10v/v% of autologous serum, and wherein a cytokine: 1000IU/ml IL-2, 50ng/ml IL-15, 50ng/ml IL-18, 200ng/ml CD3 monoclonal antibody, 2ng/ml dasatinib and 100IU/ml POM.
3. A method for culturing peripheral blood NKT cells, comprising the steps of:
s1, separating mononuclear cells from peripheral blood on day 0, adding the mononuclear cells into a lymphocyte serum-free culture medium containing 1-20v/v% of autologous serum, and adding cytokines into the culture medium, wherein the cytokines comprise IL-2 with the concentration of 10-3000 IU/ml, IL-15 with the concentration of 5-500 ng/ml, IL-18 with the concentration of 5-500 ng/ml, CD3 monoclonal antibody with the concentration of 5-1000 ng/ml, dasatinib with the concentration of 0.01-5 ng/ml and POM with the concentration of 5-500 IU/ml; standing and culturing for 14 days;
s2, adding a lymphocyte serum-free culture medium and cytokines on days 3 and 5 respectively, wherein the cytokines comprise IL-2 with the concentration of 10-3000 IU/ml, IL-15 with the concentration of 5-500 ng/ml, IL-18 with the concentration of 5-500 ng/ml, CD3 monoclonal antibody with the concentration of 5-1000 ng/ml, dasatinib with the concentration of 0.01-5 ng/ml and POM with the concentration of 5-500 IU/ml;
s3, on 7 th day, 9 th day and 11 th day, adding a lymphocyte serum-free culture medium and IL-2 cytokines with the concentration of 10-3000 IU/ml respectively;
s4, after cell culture is finished, transferring the cultured NKT cells into a centrifuge tube for centrifugal treatment, collecting the NKT cells, and repeatedly washing the NKT cells for 2-3 times by using 0.9% sodium chloride solution;
s5, adding human serum albumin to the NKT cells obtained after the washing in step S4, and diluting the mixture with 0.9% sodium chloride solution to a constant volume to obtain the peripheral blood NKT cells.
4. The culture method according to claim 3, wherein the autologous serum is inactivated at 56 ℃ at a concentration of 1-20v/v% in step S1.
5. The method according to claim 3, wherein the cytokine has a concentration of IL-2 of 1000IU/ml, IL-15 of 50ng/ml, IL-18 of 50ng/ml, CD3 monoclonal antibody of 200ng/ml, dasatinib of 2ng/ml and POM of 100IU/ml in steps S1 and S2.
6. The culture method according to claim 3, wherein the IL-2 concentration in the step S3 is 1000 IU/ml.
7. The culture method according to claim 3, wherein the lymphocyte serum-free medium comprises any one of GT-T551H3 medium, Corning KBM581 lymphocyte serum-free medium, and RPMI-1640 medium.
8. The culture method according to claim 3, wherein the cell concentration is controlled to 1X 10 during the cell culture72 x 10 units/ml7One per ml.
9. The culture method according to claim 3, wherein 10ml of human serum albumin is added to peripheral blood NKT cells in a volume percentage of 10% in step S5, and the volume is adjusted to 200ml with 0.9% sodium chloride solution.
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