CN117487859A - Method for improving transfection efficiency of NK cells - Google Patents
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Abstract
The invention discloses a method for improving transfection efficiency of NK cells, and belongs to the technical field of NK cell transfection. The method utilizes lentivirus to transfect NK cells twice; during the first transfection, a first solution is firstly added into NK cell liquid, then slow virus liquid is added, and the first solution, a second solution and a third solution are simultaneously added to obtain a first cell-virus liquid; adding NK cell fluid to the first cell-virus fluid and adding the first solution; during the second transfection, adding the slow virus liquid and simultaneously adding the first solution, the second solution and the third solution to obtain a second cell-virus liquid; NK cell fluid is added to the second cell-virus fluid and the first solution is added. The method improves transfection efficiency of NK cells, reduces genotoxicity and risk of insertion mutation, and does not affect function and proliferation of NK cells. In addition, the transfection method is also applicable to transfection of human peripheral blood primary NK cells.
Description
Technical Field
The invention relates to the technical field of NK cell transfection, in particular to a method for improving the transfection efficiency of NK cells.
Background
NK cells (natural killer cells) belong to immune cells of an innate immune system, and the proportion of the NK cells in peripheral blood lymphocytes is 10% -15%, so that a first defense line of an organism immune system is formed. Because NK cells do not need to be sensitized in advance, the NK cells can directly kill tumor cells, respond rapidly, have no GvHD (foreign cells resist host reaction), basically do not generate cytokine storm, do not need histocompatibility matching, can be produced in a large scale, thereby greatly reducing the production cost and the like, and are attracting more attention and research. NK cells have wide application prospects in tumor biotherapy, and active exploration of NK cell immune killing mechanism is a foundation for effectively improving NK cell immune therapy. One of the challenges limiting the intensive exploration of NK cell immune killing mechanisms is the way in which genes are transfected.
At present, NK cells are transfected mainly by the following three ways: one is retroviral transfection, where the efficiency of the retrovirus in NK cells is relatively high, but where NK cells are prone to mutation after transfection and where the viability of the engineered NK cells is low. Another is Lentiviral (LV) transfection, which, while reducing the risk of genotoxicity and insertional mutagenesis, is less efficient than retroviral transfection. The third is electroporation, which is technically simpler and more strictly regulated, and avoids the risks associated with the use of viral vectors and oncogenes, as well as activation and insertional mutagenesis. However, there is only a short activity on the expression of proteins, which may negatively affect the efficacy of NK cell immunotherapy.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the following technical scheme.
The invention provides a method for improving transfection efficiency of NK cells, which comprises the following steps: transfecting NK cells twice with lentivirus;
during the first transfection, a first solution is firstly added into NK cell liquid, then slow virus liquid is added, and the first solution, a second solution and a third solution are added to obtain a first cell-virus liquid; adding NK cell fluid to the first cell-virus fluid and adding the first solution;
during the second transfection, adding the slow virus liquid and simultaneously adding the first solution, the second solution and the third solution to obtain a second cell-virus liquid; adding NK cell fluid to the second cell-virus fluid and adding the first solution;
wherein the first solution contains BX795 inhibitor, the second solution contains IL-2, and the third solution contains polybrene.
According to the NK cell transfection method provided by the invention, the first solution containing the BX795 inhibitor, the second solution containing the IL-2 and the third solution containing the polybrene are added in the two transfection processes, so that the NK cell transfection efficiency is effectively improved.
Wherein the polybrene is also known as hexadimethicone bromide. IL-2 is an English abbreviation for interleukin-2. BX795 is a potent and relatively specific inhibitor of TBK1 and ikkepsilon, which in turn affects IRF3 activation and IFN- β production. The use of the anti-virus pathway can inhibit NK cells during transfection, thereby improving the transfection efficiency of lentiviruses and not affecting the function of NK cells in killing tumors.
Preferably, the time interval between the first transfection and the second transfection may be 72h or 96h.
Preferably, the time of the first transfection is 2 to 3 days after NK cells are cultured in vitro.
Preferably, the slow virus liquid is prepared as follows: preparing solution A and solution B, adding solution B into solution A, mixing, standing at room temperature for 20min, and adding into 293T cell culture solution to obtain the slow virus solution;
the solution A is prepared by uniformly mixing 4 mu L of Vigo transfection reagent and 500 mu L of serum-free culture medium and standing for 5 min;
the solution B is prepared by uniformly mixing 3.33 mu g of psPAX2, 1.67 mu g of pMD2.0G, 5 mu g of shRNA and 500 mu L of serum-free medium (Opti-MEM) and standing for 5 min.
Preferably, the first solution is a 6mM BX795 inhibitor in water; the second solution is 1000U/mL, the solute of the second solution is IL-2, and the solvent of the second solution is PBS of BSA with the mass-volume ratio of 0.5%; the third solution is a 10mg/mL aqueous solution of polybrene.
Preferably, at the first transfection, the final concentration of BX795 inhibitor in the transfection system is 6. Mu.M, the final concentration of IL-2 in the transfection system is 100U/mL, and the final concentration of polybrene in the transfection system is 10. Mu.g/mL; in the second transfection, the final concentration of BX795 inhibitor in the transfection system was 6. Mu.M, the final concentration of IL-2 in the transfection system was 100U/mL, and the final concentration of polybrene in the transfection system was 10. Mu.g/mL.
In the invention, NK cells can be cultured by in vitro amplification by adopting the following steps:
NK cells can be NK92 cells, and the in-vitro amplification culture solution of the passaged NK cells is as follows: MEM alpha basal medium+0.2 mM Inositol (Inositol) +0.1mM beta-mercaptoethanol (. Beta. -mercaptoethanol) +0.02mM Folic acid (Folic acid) +100U/mL recombinant IL-2+12.5% Horse serum) +12.5% Fetal bovine serum (fetal bovine serum) +1% Penicillin/Steptomycin solution (Penicillin/streptomycin) solution; the culture conditions are as follows: at 37deg.C, with 5% CO 2 Is cultured in a cell culture incubator. According to 5X 10 5 ～1×10 6 The cell/mL passaging ratio was passaged.
The primary NK cell in-vitro amplification culture solution comprises the following components: RPMI-1640 basal medium +5mM HEPES (4-hydroxyethyl piperazine ethane sulfonic acid) +5mM NEAA (non-essential amino acid) +5mM Pyruvate) +50. Mu.M beta. -mercaptoethanol) +9% Fetal bovine serum (fetal bovine serum) +1% Human A/B serum+1% Penicillin/Steptomycin solution (Penicillin/streptomycin) solution +100U/mL IL-2. The culture conditions are as follows: at 37deg.C, with 5% CO 2 Is cultured in a cell culture incubator.
The beneficial effects of the invention are as follows: according to the method provided by the invention, NK cells are transfected by using lentivirus, and the first solution containing the BX795 inhibitor, the second solution containing IL-2 and the third solution containing polybrene are respectively used during the two times of transfection, so that the transfection efficiency of the NK cells is improved, the risks of genotoxicity and insertion mutation are reduced, the functions and proliferation of the NK cells are not influenced, and a new breakthrough in the aspect of NK cell transfection is realized.
Drawings
FIG. 1 is a schematic view of the effect of the first transfection according to the present invention;
FIG. 2 is a schematic diagram showing the effect of the second transfection according to the present invention;
FIG. 3 is a schematic view showing the effect of primary NK cell transfection in the present invention.
Detailed Description
In order to better understand the above technical solutions, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The embodiment of the invention provides a method for improving the transfection efficiency of NK cells, which comprises the following steps:
step 1, first transfection: after two days of passage of NK92 cell line, cell fluid is obtained, 6mL of cell fluid is taken and 6 mu L of first solution is added into the cell fluid; 30min later, NK92 cells were collected, counted, 1.5X10 5 Transferring the individual cells into an EP tube for centrifugation, discarding the supernatant after centrifugation, then re-suspending the cells by using 500 mu L of slow virus liquid, simultaneously adding 0.5 mu L of first solution and 0.5 mu L of second solution, and then adding 0.5 mu L of third solution to obtain first cell-virus liquid; after centrifugation of 1000g of the EP tube containing the first cell-virus solution at room temperature for 1h, 500. Mu.L of the first cell-virus solution was transferred to a 3cm dish, 1.5mL of the NK92 cell culture solution was filled, and 1. Mu.L of the first solution was added thereto. After 6-8 h, the solution was changed, cells were harvested in 1.5mL EP tubes, centrifuged at 300g for 5min at room temperature, the supernatant was discarded, the cell pellet was resuspended using 1.5mL fresh NK92 cell culture solution and transferred to a 3cm dish for cultivation. After 36-48 h, the transfection effect was examined by flow cytometry, as shown in FIG. 1.
Step 2, secondary transfection: collecting 300g of transfected cells for the first time, standing for 5min, removing the supernatant, adding 500 mu L of slow virus liquid, simultaneously adding 0.5 mu L of first solution and 0.5 mu L of second solution, and adding 0.5 mu L of third solution to obtain second cell-virus liquid; after centrifugation of 1000g of the EP tube containing the second cell-virus solution at room temperature for 1h, 500. Mu.L of the second cell-virus solution was transferred to a 3cm dish, 1.5mL of the NK92 cell culture solution was filled, and 1. Mu.L of the first solution was added thereto. After 6-8 h, the solution was changed, cells were harvested in 1.5mL EP tubes, centrifuged at 300g for 5min at room temperature, the supernatant was removed, the cell pellet was resuspended using 1.5mL fresh NK92 cell culture medium, and transferred to a 3cm dish for culture. After 36-48 h, the transfection effect was examined as shown in FIG. 2.
Step 3, performing transfection verification on the primary NK cells of the human peripheral blood, obtaining cell sap after stable culture of the primary NK cells for two days, taking 500 mu L of the cell sap, and adding 1 mu L of the first solution into the cell sap; after 30min, human peripheral blood primary NK cells were collected, and counted at 1.5X10 5 Transferring the individual cells into a 1.5mL EP tube for centrifugation, removing the supernatant after centrifugation, then re-suspending the cells by using 500 mu L of slow virus liquid, simultaneously adding 1 mu L of first solution and 0.5 mu L of second solution, and then adding 1 mu L of third solution to obtain first cell-virus liquid; after centrifugation of 1700g of the EP tube containing the first cell-virus solution at room temperature for 1h, 500. Mu.L of the first cell-virus solution was resuspended and transferred to a 24-well plate. After 6-8 h, the solution was changed, cells were harvested in 1.5mL EP tubes, centrifuged at 500g for 5min at room temperature, the supernatant was removed, the cell pellet was resuspended using 500. Mu.L fresh NK cell culture medium, and transferred to 24 well plates for cultivation. After 36-48 h, the transfection effect was examined as shown in FIG. 3.
In fig. 1 to 3, NEG represents a negative control tube (Negtive); SSC-A represents Side scatter arese:Sub>A (Side scatter-arese:Sub>A); GFP represents green fluorescent protein (Green fluorescent protein); 48h represents 48 hours; BX795-1 represents sample 1 to which BX795 was added; BX795-2 represents sample 2 to which BX795 was added; NONE-1 represents sample 1 to which BX795 was not added; NONE-2 represents sample 2 to which BX795 was not added; NK92 in section represents transfection of NK92 cells; first Round-1 represents the First lentivirus transfection performed on sample 1; first Round-2 represents the First lentivirus transfection performed on sample 2; second Round-1 represents a Second lentivirus transfection of sample 1; second Round-2 represents a Second lentiviral transfection of sample 2.
As can be seen from FIG. 1, samples 1 and 2 transfected without BX795 showed GFP fluorescence rates of 5.13 and 4.49, respectively, and samples 1 and 2 transfected with BX795 showed GFP fluorescence rates of 19.1 and 19.7, respectively, as compared to NEG control. It can be seen that the addition of BX795 can increase transfection efficiency. As can be seen from FIG. 2, after the secondary transfection, the GFP fluorescence rate after the transfection was 32.5, which was significantly higher than that of the primary transfection GFP fluorescence rates 19.1 and 19.7, and the secondary transfection was able to greatly increase the transfection efficiency on the basis of the primary transfection. As can be seen from fig. 3, the two samples of the primary NK cells of human peripheral blood were subjected to the primary transfection verification, respectively, and the GFP fluorescence rates after the transfection were 8.96 and 9.32, respectively, and the GFP fluorescence rates after the secondary transfection were 18.7 and 20.6, respectively, after the secondary transfection, respectively, and thus the transfection efficiency of the primary NK cells of human peripheral blood could be greatly improved. The method provided by the invention is also suitable for transfection of human peripheral blood primary NK cells.
Therefore, the method provided by the invention can improve the transfection efficiency by more than two times after the first transfection, and the second transfection can further improve the transfection efficiency on the basis of the first transfection result. Moreover, the transfection scheme provided by the invention is also suitable for transfection of human peripheral blood primary NK cells.
Packaging of lentiviruses involved in the transfection procedure of the above steps may be carried out as follows:
preparing solution A and solution B, adding solution B into solution A, mixing uniformly, standing at room temperature for 20min, adding into 293T cell culture solution (density 70% -80%), discarding supernatant after 6-8 h, rinsing with 2mL PBS, adding 10mL fresh 293T cell culture solution for liquid exchange, collecting supernatant in 50mL centrifuge tubes after 24h and 48h respectively, and storing in a refrigerator at 4 ℃; wherein, the solution A is prepared by uniformly mixing 4 mu L of Vigo transfection reagent and 500 mu L of serum-free culture medium and standing for 5 min; solution B was prepared by mixing 3.33. Mu.g of psPAX2, 1.67. Mu.g of pMD2.0G, 5. Mu.g of core plasmid shRNA (pLKO.1-puro-shSmalcb 1) and 500. Mu.L of serum-free medium, respectively, uniformly, and standing for 5 min.
The concentration of lentivirus involved in the transfection process in the above steps may be performed by PEG-8000 concentration, and specifically may include the steps of:
1.5 XPEG 8000 (polyethylene glycol 8000) and NaCl solution were prepared: weighing NaCl8.766g and PEG8000 50g, dissolving in 200mL of ultrapure water, performing wet heat sterilization at 121 ℃ for 30min, and preserving at 4 ℃;
2. filtering the supernatant collected during the packaging of the lentivirus by using a 0.45 mu m filter head to obtain a lentivirus initial solution;
3. adding 7.5mL of prepared 5 XPEG-8000 and NaCl solution into every 30mL of lentivirus initial solution to obtain lentivirus mixed solution, and placing the mixed solution in a refrigerator at 4 ℃ for overnight; centrifuging the lentivirus mixed solution at 4 ℃ and 4000g for 20min; discarding the supernatant, standing for 1-2 minutes, and sucking away residual liquid to obtain slow virus precipitate; adding proper amount of slow virus dissolving solution (solution for re-suspending and concentrating slow virus) to dissolve slow virus precipitate to obtain slow virus suspension;
4. executing the step 3 once every 20-30 min for 3-5 times;
5. the lentiviral suspension was concentrated and then split into 50. Mu.L portions (1X 10) 7 TU) is stored in the finished tube. Quick-freezing with crushed dry ice and storing at-80 ℃.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. A method of increasing NK cell transfection efficiency comprising: transfecting NK cells twice by using lentivirus, namely, carrying out secondary transfection on the basis of the primary transfection solution;
during the first transfection, a first solution is firstly added into NK cell liquid, then slow virus liquid is added, and the first solution, a second solution and a third solution are simultaneously added to obtain a first cell-virus liquid; adding NK cell fluid to the first cell-virus fluid and adding the first solution;
during the second transfection, adding the slow virus liquid and simultaneously adding the first solution, the second solution and the third solution to obtain a second cell-virus liquid; adding NK cell fluid to the second cell-virus fluid and adding the first solution;
wherein the first solution contains BX795 inhibitor, the second solution contains IL-2, and the third solution contains polybrene.
2. The method of claim 1, wherein the time interval between the first transfection and the second transfection is 72 hours or 96 hours.
3. The method of claim 1, wherein the first transfection is performed between days 2 and 3 after the in vitro expansion culture of NK cells.
4. The method of claim 1, wherein the lentiviral fluid is prepared according to the following method: preparing solution A and solution B, adding solution B into solution A, mixing, standing at room temperature for 20min, and adding into 293T cell culture solution to obtain the slow virus solution;
the solution A is prepared by uniformly mixing 4 mu L of Vigo transfection reagent and 500 mu L of serum-free culture medium and standing for 5 min;
the solution B is prepared by uniformly mixing 3.33 mu g of psPAX2, 1.67 mu g of pMD2.0G, 5 mu g of shRNA and 500 mu L of serum-free medium (Opti-MEM) and standing for 5 min.
5. The method of claim 1, wherein the first solution is a 6mM BX795 inhibitor in water; the second solution is 1000U/mL, the solute of the second solution is IL-2, and the solvent of the second solution is PBS of BSA with the mass-volume ratio of 0.5%; the third solution is a 10mg/mL aqueous solution of polybrene.
6. The method of claim 1, wherein the final concentration of BX795 inhibitor in the transfection system is 6 μm, the final concentration of IL-2 in the transfection system is 100U/mL, and the final concentration of polybrene in the transfection system is 10 μg/mL at the first transfection; in the second transfection, the final concentration of BX795 inhibitor in the transfection system was 6. Mu.M, the final concentration of IL-2 in the transfection system was 100U/mL, and the final concentration of polybrene in the transfection system was 10. Mu.g/mL.
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| CN114891742A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | Culture medium and in-vitro amplification method for NK (natural killer) cells with strong killing property |
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| CN114891742A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | Culture medium and in-vitro amplification method for NK (natural killer) cells with strong killing property |
| CN114891742B (en) * | 2022-06-23 | 2024-06-04 | 杭州中赢生物医疗科技有限公司 | Culture medium of NK cells with strong killing property and in-vitro amplification method |
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