CN114874986A - Method for amplifying NK cells by adopting K562 cells - Google Patents

Method for amplifying NK cells by adopting K562 cells Download PDF

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CN114874986A
CN114874986A CN202210682587.3A CN202210682587A CN114874986A CN 114874986 A CN114874986 A CN 114874986A CN 202210682587 A CN202210682587 A CN 202210682587A CN 114874986 A CN114874986 A CN 114874986A
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俞英豪
彭昉
王冶陶
王朝芳
洪茜茜
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Hangzhou Life Ark Biomedical Technology Co ltd
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Abstract

The invention discloses a method for amplifying NK cells by adopting K562 cells, which comprises the following steps: infecting K562 cells by recombinant lentivirus for 10-20h, and cloning by flow cytometry and limiting dilution method to obtain engineering cell strain; then, cobalt-60 irradiation sterilization is adopted to obtain trophoblasts; separating mononuclear cells by adopting peripheral blood, then adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts, uniformly mixing, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 2-6%; culturing to 3 days, centrifuging to remove supernatant, adding NK cell complete culture medium with the same volume, mixing, and culturing in incubator with carbon dioxide concentration of 2-6% at 37 deg.C; culturing to 7 days, and supplementing 1-2 μ g/mLIFN-gamma and autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.

Description

Method for amplifying NK cells by adopting K562 cells
Technical Field
The invention relates to the technical field of NK cell amplification, in particular to a method for amplifying NK cells by adopting K562 cells.
Background
Natural killer cells belong to human body mediated innate immune cells and are closely related to the resistance of the body to malignant tumors, viral infection and immunoregulation. Its killing of tumor cells or infected cells is independent of antibody participation and does not require antigen stimulation and sensitization; the recognition target cells have no major histocompatibility complex limitation, and can be directly contacted with the target cells to release perforin and granzyme to kill the target cells; can also release NK cytotoxic factor to combine with NK cytotoxic factor receptor of target cell, selectively kill and crack target cell; or killing of target cells by antibody-dependent cell-mediated cytotoxicity. The important role of NK cells in tumor immunity makes NK cell adoptive immunotherapy a new strategy for treating tumors.
NK cells account for about 5-10% of lymphocytes in blood, and sufficient cell number and cell purity are important factors for the NK tumor cell killing effect in cell therapy, but the proliferation of NK cells is weak, and a sufficient number of cells cannot be provided for administration, which is the biggest problem in clinical application. Therefore, it is still a hot topic of research of researchers to find an effective method for obtaining NK cells in sufficient quantity and purity in vitro.
At present, some researchers have reported proliferation of NK cells using tumor cell lines as trophoblast cells. The most commonly used trophoblasts are irradiated K562 cells expressing IL-21 and 4-1BBL, and for NK cells, IL-21 can enhance the secretion of IFN-gamma, up-regulate the secretion and expression of NK cell perforin, enhance the cytotoxic activity, and accelerate the NK cell maturation and the NK cell receptor expression; while 4-1BBL also regulates NK cell activation and signal transduction. However, this method can only expand cells to 100 times, and not only expands the difference of the number of times, but also has poor repeatability.
At present, how to adopt K562 cells to amplify NK cells to obtain high-purity, large-quantity and good-repeatability NK cells has great application prospect.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for amplifying NK cells by using K562 cells.
A method for expanding NK cells by using K562 cells, comprising the following steps:
s1, infecting K562 cells by adopting recombinant lentiviruses for 10-20h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then cobalt-60 irradiation sterilization is adopted to obtain trophoblasts;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 2-6%; culturing to 3 days, centrifuging to remove supernatant, adding NK cell complete culture medium with the same volume, mixing, and culturing in incubator with carbon dioxide concentration of 2-6% at 37 deg.C; culturing to 7 days, and supplementing 1-2 μ g/mL IFN- γ and autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Preferably, in S1, the irradiation dose of cobalt-60 is 20-50 Gy.
Preferably, in S1, the cell density of the obtained trophoblasts is 1-2X 10 7 one/mL.
Preferably, in S2, the trophoblasts are washed and recovered with PBS buffer before being added.
Preferably, in S2, the concentration of IFN- γ is 1.2-1.8 μ g/mL.
Preferably, the volume fraction of autologous plasma in S2 is 5-10%.
Preferably, the volume fraction of autologous plasma in S2 is 6-8%.
The technical effects of the invention are as follows:
(1) according to the invention, the K562 cells are infected by using specific recombinant lentiviruses and then used as trophoblasts to amplify the NK cells, explosive proliferation of the NK cells can be realized in a short time, the amplification multiple of the NK cells after in vitro amplification culture for 14 days is as high as 2500 times, the purity of the obtained NK cells reaches more than 95%, and the repeatability of the amplification multiple is good; the killing capacity of the obtained NK cells on tumor cells is more than 55%, and the exertion of the cytotoxicity effect of the NK cells is not reduced due to the increase of the passage number.
(2) The NK cells obtained by the method can be used for treating various diseases such as malignant tumors, virus infection, autoimmune diseases, rejection after transplantation, graft-versus-host disease and the like, the problems of high-purity NK cell standardization, scale amplification and the like are comprehensively solved, and the prepared NK cells are suitable for long-term cryopreservation, can be used after immediate recovery, and are safe and convenient.
Drawings
FIG. 1 is a graph comparing the amplification factor of example 5 and a comparative example.
FIG. 2 is a photograph showing the dilution of NK cells by 400 times after culturing for 14 days according to the amplification method of example 5 and the analysis result.
FIG. 3 is a cell flow scattergram of example 5.
FIG. 4 is a comparison of the purity of example 5 and comparative example.
FIG. 5 is a graph comparing the killing activity of example 5 and comparative examples on PANC-1 cells, U251 cells.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example 1
A method for expanding NK cells by using K562 cells, comprising the following steps:
s1, infecting K562 cells by adopting recombinant lentiviruses, wherein the infection time is 10h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then sterilizing by cobalt-60 irradiation to obtain the cell density of 1.027 × 10 7 one/mL of feeder cells; the irradiation dose of the cobalt-60 is 20 Gy;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by using peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts which are washed and revived by PBS buffer solution, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 2%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 2%; culturing to 7 days, and supplementing 1 μ g/Ml IFN-gamma and 5% volume fraction autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Example 2
A method for expanding NK cells by using K562 cells, comprising the following steps:
s1, infecting the K562 cells by adopting the recombinant lentivirus, wherein the infection time is 20h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then sterilizing by cobalt-60 irradiation to obtain cell density of 1.965 × 10 7 one/mL of feeder cells; the irradiation dose of the cobalt-60 is 50 Gy;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts which are washed and revived by PBS buffer solution, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 6%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 6%; culturing to 7 days, and supplementing 2 μ g/mL IFN- γ and 10% volume fraction autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Example 3
A method for expanding NK cells by using K562 cells, comprising the following steps:
s1, adopting heavy materialInfecting K562 cells by the lentivirus group for 12 hours, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then sterilizing by cobalt-60 irradiation to obtain the cell density of 1.71 × 10 7 one/mL of feeder cells; the irradiation dose of the cobalt-60 is 30 Gy;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts which are washed and revived by PBS buffer solution, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 5%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator with the carbon dioxide concentration of 3% at 37 ℃; culturing to 7 days, and supplementing 1.8 μ g/mL IFN-gamma and 6% volume fraction autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Example 4
A method for expanding NK cells using K562 cells, comprising the steps of:
s1, infecting the K562 cells by adopting recombinant lentiviruses, wherein the infection time is 18h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then sterilizing by cobalt-60 irradiation to obtain the cell density of 1.37 × 10 7 one/mL of feeder cells; the irradiation dose of the cobalt-60 is 40 Gy;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts which are washed and revived by PBS buffer solution, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 3%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 5%; culturing to 7 days, and supplementing 1.2 μ g/mL IFN-gamma and autologous plasma with volume fraction of 8%; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Example 5
A method for expanding NK cells by using K562 cells, comprising the following steps:
s1, infecting the K562 cells by adopting recombinant lentiviruses, wherein the infection time is 15h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then sterilizing by cobalt-60 irradiation to obtain the cell density of 1.53 × 10 7 one/mL of feeder cells; the irradiation dose of the cobalt-60 is 35 Gy;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts which are washed and revived by PBS buffer solution, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 4%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 4%; culturing to 7 days, and supplementing 1.5 μ g/mL IFN-gamma and 7% volume fraction autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
Comparative example
A method for expanding NK cells by using K562 cells, comprising the following steps:
separating mononuclear cells by adopting peripheral blood, then adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding K562 cells which are washed and revived by adopting a PBS buffer solution, uniformly mixing, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 4%; when the culture is carried out till the 3 rd day, centrifuging to remove supernatant, adding the same volume of NK cell complete culture medium, uniformly mixing, and continuously culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 4%; culturing to 7 days, and supplementing 1.5 μ g/mL IFN-gamma and 7% volume fraction autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
NK cells were expanded by the methods of example 5 and comparative example, a 0.4% trypan blue solution was prepared, the pH was adjusted to 7.0 to 7.2, and cells cultured on day 14 were collected. 50 mu L of cell suspension was taken, mixed with 50 mu L of trypan blue, stained for 4min, and the total number of cells was counted on a cell counting plate.
As shown in FIG. 1, the method of example 5 can greatly increase the amplification factor of NK cells. This is because K562 cells are infected with a specific recombinant lentivirus and then expanded as trophoblasts to thereby enable explosive proliferation of NK cells in a short time. The cells were cultured for 14 days by the amplification method of example 5, and the collected NK cells were diluted 400-fold and photographed and analyzed as shown in FIG. 2.
Taking the NK cells cultured by the methods of example 5 and comparative example respectively, and detecting the cell purity by using a flow detector, wherein the specific operations are as follows:
(1) preparation of single cell suspension: will be 1 × 10 5 -1×10 7 The cells are put into a 1.5mL centrifuge tube and centrifuged for 5 minutes at the rotating speed of 3000r/min, the supernatant is discarded, and 100 mu L of FACS buffer solution is added to suspend the cells;
(2) fluorescent antibody 20. mu.L, protected from light for 30 minutes;
(3) washing the cells to remove free fluorescent antibody; adding 350 mu L of FACS buffer solution, mixing gently, centrifuging for 5 minutes at 3000r/min, and discarding the supernatant;
(4) sample loading pretreatment: and (3) adding 100 mu L of instrument buffer solution into the cell sediment obtained in the step (3), gently mixing the suspended cells, transferring the cell suspension into a FACS special tube, and preparing for instrument detection and analysis.
As a result, as shown in FIGS. 3 and 4, the purity of NK cells amplified by the method of example 5 was much higher than that of the comparative example. According to the invention, the K562 cells are infected by using the specific recombinant lentivirus and then used as trophoblasts to amplify the NK cells, so that explosive proliferation of the NK cells can be realized in a short time, and the purity of the obtained NK cells reaches over 95%.
A96-well flat-bottom culture plate is adopted, and 3 experimental wells, simple effector cell wells and simple target cell wells are arranged respectively, and blank wells are arranged. PANC-1 cells or U251 cells were added to the experimental wells and the target cell wells alone. The NK cells cultured for 14 days in example 1 and comparative example were added to the experimental wells and the wells for single effector cells, respectively, and the ratio of effector cells to target cells was set to 1: 1. 3: 1. 10: 1 and 20: 1, four groups, and culturing for 24 h. Adding 20 mu LWST, continuing to culture for 6h, and detecting the OD value by an enzyme labeling instrument.
As a result, as shown in FIG. 5, NK cells amplified by the method of example 5 can significantly kill tumor cells. The death rate of PANC-1 cells and U251 cells is 20: 1, 55.91% and 66.35%, respectively, which shows that the NK cells induced and expanded by the special recombinant lentivirus infected K562 cells and serving as feeder cells can effectively kill tumor cells, and can be considered for immunotherapy of tumor patients.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A method for amplifying NK cells by using K562 cells, which comprises the following steps:
s1, infecting K562 cells by adopting recombinant lentiviruses for 10-20h, and cloning by flow cytometry and a limiting dilution method to obtain an engineering cell strain; then, cobalt-60 irradiation sterilization is adopted to obtain trophoblasts;
the recombinant lentivirus is formed by two plasmid systems of envelope plasmid pLP/VSVG and carrier plasmid, and the carrier plasmid carries Blasticidin screening gene Blasticidin;
s2, separating mononuclear cells by peripheral blood, adding the mononuclear cells into a complete NK cell culture medium for resuspension, adding trophoblasts, mixing uniformly, and culturing in an incubator at 37 ℃ and with the carbon dioxide concentration of 2-6%; culturing to 3 days, centrifuging to remove supernatant, adding NK cell complete culture medium with the same volume, mixing, and culturing in incubator with carbon dioxide concentration of 2-6% at 37 deg.C; culturing to 7 days, and supplementing 1-2 μ g/mL IFN- γ and autologous plasma; culturing is continued until day 14 to obtain NK cells expanded by K562 cells.
2. The method for amplifying NK cells using K562 cells according to claim 1, wherein the irradiation dose of cobalt-60 in S1 is 20-50 Gy.
3. The method for expanding NK cells using K562 cells according to claim 1, wherein the cell density of the obtained trophoblasts is 1-2X 10 in S1 7 one/mL.
4. The method for expanding NK cells using K562 cells according to claim 1, wherein the trophoblasts are washed and recovered with PBS before being added in S2.
5. The method for amplifying NK cells using K562 cells according to claim 1, wherein IFN-. gamma.is contained in S2 at a concentration of 1.2 to 1.8. mu.g/mL.
6. The method for expanding NK cells using K562 cells according to claim 1, wherein the volume fraction of autologous plasma in S2 is 5-10%.
7. The method for expanding NK cells using K562 cells according to claim 6, wherein the volume fraction of autologous plasma in S2 is 6-8%.
CN202210682587.3A 2022-06-16 2022-06-16 Method for amplifying NK cells by adopting K562 cells Pending CN114874986A (en)

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