CN114438129B - Kit for assisting lentivirus with CAR to transfect primary NK cells and application of kit - Google Patents
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Abstract
The invention provides a kit for assisting lentivirus with CAR in transfecting primary NK cells and application thereof. The kit comprises a first infection-assisting agent, a second infection-assisting agent, a third infection-assisting agent and a fourth infection-assisting agent; the first infection-assisting agent is: the final concentration of polybrene is 8-100 mg/mL; the second infection-promoting agent is: interleukin-2 with a final concentration of 500000-5000000U/mL and a solvent of 0.3-0.7 vol% human serum albumin; the third infection-promoting agent is: interleukin-12 with final concentration of 20000-200000U/mL and solvent of 0.3-0.7 vol% human serum albumin solution; the fourth infection-promoting agent is: the final concentration of the phytohemagglutinin is 1-20 mg/mL. The kit is applied to the preparation of CAR-NK cells, and meanwhile, the transfection efficiency of the NK cells can be effectively improved by controlling the time point of adding the lentiviral vector and the dosage of the infection assisting agent and carrying out centrifugal treatment after the infection assisting agent is added for the first time.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a kit for assisting lentivirus with CAR in transfecting primary NK cells and application thereof.
Background
Natural Killer (NK) cells belong to granular lymphocytes, are important immune cells of the body and are closely related to nonspecific immunity. As a broad spectrum immune cell, NK cells can secrete various cytokines to regulate immune response, can directly kill certain tumor cells and virus infected cells, and play an important role in resisting tumor and virus infection and regulating immunity. It has been shown that both NK cells are more immunogenic and have higher lethality than T cells.
A Chimeric Antigen Receptor (CAR) is a cell surface receptor that can recognize a specific protein (antigen), which is mainly composed of two domains: an extracellular domain and an intracellular signaling region. When the CAR structure and the tumor-associated antigen antibody are fused and expressed on the surface of a cell membrane of an NK cell, the NK cell can be endowed with the capacity of specifically recognizing the corresponding tumor cell, and the NK cell is the CAR-NK cell which is usually called.
At present, CAR-T cells are mainly adopted for cellular immunotherapy of tumors, and CAR-NK cells are relatively rarely applied, mainly because NK cells are high in transfection difficulty, low in virus titer and difficult to infect, so that the infection rate of the NK cells is low, the number of obtained total CAR-NK cells is small, and the application of the CAR-NK cells in tumor therapy is limited.
Disclosure of Invention
The invention aims to provide a kit for assisting lentivirus with CAR in transfecting primary NK cells and application thereof, so as to improve the transfection efficiency of the lentivirus with CAR on the primary NK cells.
According to a first aspect of the invention, there is provided a kit for assisting lentivirus bearing a CAR in transfecting primary NK cells, comprising a first, a second, a third, a fourth co-infectious agent;
the first infection-assisting agent is: the final concentration of polybrene is 8-100 mg/mL;
the second infection-promoting agent is: interleukin-2 (IL-2) with a final concentration of 500000-5000000U/mL and a solvent of 0.3-0.7 vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 (IL-12) with final concentration of 20000-200000U/mL and solvent of 0.3-0.7 vol% human serum albumin solution;
the fourth infection-promoting agent is: the final concentration of Phytohaemagglutinin (PHA) is 1-20 mg/mL.
The kit for assisting the lentivirus with the CAR to transfect the primary NK cells contains four infection aids, wherein the four infection aids respectively contain polybrene, IL-2, IL-12 and PHA, and the transfection efficiency of the lentivirus with the CAR on the primary NK cells can be improved through the synergistic effect of the four infection aids, so that more CAR-NK cells can be obtained.
Preferably, the kit for assisting lentivirus with CAR in transfecting primary NK cells comprises a first infection adjuvant, a second infection adjuvant, a third infection adjuvant and a fourth infection adjuvant;
the first infection-promoting agent is: an aqueous solution with a final polybrene concentration of 80 mg/mL;
the second infection-promoting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/mL and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
According to a second aspect of the invention, there is provided the use of a kit as described above to assist lentiviruses carrying a CAR to transfect primary NK cells in the preparation of CAR-NK cells.
According to a third aspect of the present invention, there is provided a method for preparing CAR-NK cells, comprising the steps of:
(1) Adding a CAR-bearing lentivirus into primary NK cells according to MOI = 2.5-20, then adding a first co-infection agent, culturing for 5-7 h, and then adding the CAR-bearing lentivirus into a transfection system according to MOI = 0-10;
(2) Adding a second infection adjuvant, a third infection adjuvant and a fourth infection adjuvant into the transfection system, and continuously culturing for 24-48 h to obtain CAR-NK cells;
the first infection-promoting agent is: the final concentration of polybrene is 8-100 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 500000-5000000U/mL and a solvent of 0.3-0.7 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with final concentration 20000-200000U/ml and solvent 0.3-0.7 vol% human serum albumin solution;
the fourth infection-promoting agent is: the final concentration of the phytohemagglutinin is 1-20 mg/mL.
Preferably, in the above method for preparing CAR-NK cells, the first infection adjuvant is: an aqueous solution with a final polybrene concentration of 80 mg/mL;
the second infection-promoting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/mL and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
Preferably, in the above method for preparing CAR-NK cells, step (1) is: CAR-bearing lentivirus was added to primary NK cells at MOI = 2.5-20, followed by the addition of a first co-infectious agent, centrifuged, cultured for 5-7 h, and then CAR-bearing lentivirus was added to the transfection system at MOI = 0-10.
According to the scheme, after lentivirus with CAR is added into primary NK cells, a first infection assisting reagent is added, and centrifugation is performed before culture, so that the first infection assisting reagent can be fully mixed with lentivirus with CAR and the primary NK cells, transfection of lentivirus with CAR on the primary NK cells is facilitated, and infection positive rate of the NK cells is effectively improved.
Preferably, the conditions of centrifugation are: 800-1200g at 30-37 ℃ for 0.5-5 h.
Preferably, in the above method for preparing CAR-NK cells, step (1) is: adding CAR-bearing lentivirus to primary NK cells according to MOI = 2.5-10, then adding a first co-infecting agent, culturing for 5-7 h, then adding CAR-bearing lentivirus to the transfection system according to MOI = 2.5-10.
Preferably, in the above method for preparing CAR-NK cells, step (1) is: CAR-bearing lentivirus was added to primary NK cells at MOI = 2.5-10, followed by the addition of the first co-infectious agent, centrifuged, cultured for 5-7 h, and then CAR-bearing lentivirus was added to the transfection system at MOI = 2.5-10.
The scheme adopts a mode of adding lentiviruses with CAR step by step to prepare CAR-NK cells, after adding lentiviruses with CAR and a first auxiliary infectious agent into primary NK cells according to MOI = 2.5-10, carrying out centrifugal treatment, then culturing for 5-7 h, and then adding lentiviruses with CAR into a transfection system according to MOI = 2.5-10, so that the transfection efficiency of the NK cells is higher and more CAR-NK cells are obtained by the mode of adding the lentiviruses with CAR step by step.
Preferably, the conditions of centrifugation are: 30-37 ℃, 800-1200g for 0.5-5 h.
Preferably, in the step (1), the final concentration of polybrene in the transfection system is 6-10 mug/mL; in the step (2), the final concentration of interleukin 2 in the transfection system is 460-540U/mL, the final concentration of interleukin 12 in the transfection system is 16-24U/mL, and the final concentration of phytohemagglutinin in the transfection system is 0.6-1.4 mug/mL.
The beneficial effects of the invention are as follows: the invention provides a kit for assisting lentivirus with CAR to transfect primary NK cells, which contains four infection assisting agents, and the infection assisting agents can act synergistically to improve the transfection efficiency of the NK cells. The kit is applied to the preparation of CAR-NK cells, and the transfection efficiency of the NK cells can be effectively improved by controlling the time point of adding the lentiviral vector and the dosage of the infection assisting agent and simultaneously performing centrifugation after the infection assisting agent is added for the first time.
Detailed Description
Technical features in the technical solutions provided by the present invention are further clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =20, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 10 mug/mL), and putting the 12-well plate into an incubator for culturing for 7h;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36h to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a polybrene final concentration of 80 mg/mL;
the second infection-promoting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution having a final phytohemagglutinin concentration of 10 mg/mL.
Example 2
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =10, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 10 mug/mL), and putting the 12-well plate into an incubator for culturing for 7h;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36h to obtain CAR-NK cells;
the first infection assisting agent, the second infection assisting agent, the third infection assisting agent and the fourth infection assisting agent are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-assisting agent is: an aqueous solution with a polybrene final concentration of 80 mg/mL;
the second infection-promoting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
Example 3
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =5, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 8 mug/mL), and putting the 12-well plate into an incubator for culturing for 6h;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 460U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 24U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.0 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 24 hours to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: a polybrene final concentration of 8 mg/mL;
the second infection-promoting agent is: a solution of interleukin-2 with a final concentration of 300000U/mL and a solvent of 0.7vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 100000U/ml and a solvent of 0.7vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 1 mg/mL.
Example 4
A method for producing CAR-NK cells comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =2.5, adding a first infection adjuvant (the final concentration of the first infection adjuvant in a transfection system is 6 mug/mL), and putting the 12-well plate into an incubator for culturing for 5h;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 500U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 16U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 0.6 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 48 hours to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a final polybrene concentration of 100 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 500000U/mL and a solvent of 0.3vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 20000U/ml and a solvent of 0.3vol% human serum albumin;
the fourth infection-assisting agent is: an aqueous solution having a final phytohemagglutinin concentration of 20 mg/mL.
Example 5
A method for producing CAR-NK cells comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =10, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 10 mug/mL), putting the 12-well plate into an incubator for culturing for 7h, and then adding the lentivirus with the CAR into the transfection system according to MOI = 10;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36 hours to obtain CAR-NK cells;
the first infection assisting agent, the second infection assisting agent, the third infection assisting agent and the fourth infection assisting agent are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a final polybrene concentration of 80 mg/mL;
the second infection-promoting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-assisting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
Example 6
Example 6 provides a method for preparing CAR-NK cells, which differs from example 5 in that: in step (2), the MOI values were 5 for both additions of the CAR-bearing lentivirus.
Example 7
Example 7 provides a method of preparing CAR-NK cells, which differs from example 5 in that: in step (2), the MOI values were 2.5 for both additions of the CAR-bearing lentivirus.
Example 8
A method for producing CAR-NK cells comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the proportion of 10-100 ten thousand per well (no more than 1 mL).
(2) To 12-well plates were added lentiviruses with the corresponding CAR at MOI =20, followed by the addition of the first co-infective agent (final concentration of the first co-infective agent in the transfection system was 10 μ g/mL); adjusting a constant temperature centrifuge to idle and preheat for 10min at 33 ℃ and 1000g, quickly placing a 12-hole plate in the centrifuge, balancing, and centrifuging for 3h at 33 ℃ and 1000 g; putting the 12-hole plate into an incubator for culturing for 7 hours;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36h to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a polybrene final concentration of 80 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution having a final phytohemagglutinin concentration of 10 mg/mL.
Example 9
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) To 12-well plates were added lentiviruses with the corresponding CAR at MOI =20, followed by the addition of the first co-infective agent (final concentration of the first co-infective agent in the transfection system was 10 μ g/mL); adjusting a constant temperature centrifuge to idle and preheat for 10min at 37 ℃ and 800g, quickly placing a 12-hole plate in the centrifuge, balancing, and centrifuging for 0.5h at 37 ℃ and 800 g; putting the 12-hole plate into an incubator for culturing for 7 hours;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36h to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a polybrene final concentration of 80 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-assisting agent is: an aqueous solution having a final phytohemagglutinin concentration of 10 mg/mL.
Example 10
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) To 12-well plates were added lentiviruses with the corresponding CAR at MOI =10, followed by the addition of the first co-infective agent (final concentration of the first co-infective agent in the transfection system was 10 μ g/mL); adjusting a constant temperature centrifuge to idle and preheat for 10min at 30 ℃ and 1200g, quickly placing a 12-hole plate in the centrifuge, balancing, and centrifuging for 5h at 30 ℃ and 1200 g; placing the 12-well plate into an incubator for 7h, and then adding the CAR-bearing lentivirus into a transfection system according to MOI = 10;
(3) Adding a second auxiliary infectious agent, a third auxiliary infectious agent and a fourth auxiliary infectious agent into the transfection system, wherein the final concentration of the second auxiliary infectious agent in the transfection system is 540U/ml, the final concentration of the third auxiliary infectious agent in the infection system is 20U/ml, and the final concentration of the fourth auxiliary infectious agent in the infection system is 1.4 mu g/ml, and putting the 12-hole plate into an incubator for culturing for 36h to obtain CAR-NK cells;
the first infection adjuvant, the second infection adjuvant, the third infection adjuvant and the fourth infection adjuvant are all components in a kit for assisting lentivirus with CAR to transfect primary NK cells;
the first infection-promoting agent is: an aqueous solution with a polybrene final concentration of 80 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
Comparative example 1
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Lentiviruses with the corresponding CAR were added to 12-well plates at MOI =20, and the 12-well plates were placed in an incubator and cultured for 36h to obtain CAR-NK cells.
Comparative example 2
A method for producing CAR-NK cells, comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the proportion of 10-100 ten thousand per well (no more than 1 mL).
(2) Adding lentivirus with corresponding CAR into a 12-well plate according to MOI =10, putting the 12-well plate into an incubator for culturing for 7h, then adding lentivirus with CAR into a transfection system according to MOI =10, putting the 12-well plate into the incubator for culturing for 36h, and obtaining CAR-NK cells;
comparative example 3
A method for producing CAR-NK cells comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentiviruses with the corresponding CAR to 12-well plates at MOI = 20; adjusting a constant temperature centrifuge to idle and preheat for 10min at 30 ℃ and 1200g, quickly placing a 12-hole plate in the centrifuge, balancing, and centrifuging for 5h at 30 ℃ and 1200 g; and putting the 12-hole plate into an incubator to culture for 36h to obtain the CAR-NK cells.
Comparative example 4
A method for producing CAR-NK cells comprising the steps of:
(1) Taking a proper amount of primary NK cell suspension with a good growth state, centrifuging for 5min at 300g, removing supernatant, collecting primary NK cells, re-suspending the cells by using a fresh serum-free culture medium, and then inoculating the primary NK cells into a 12-well plate according to the proportion of 10-100 ten thousand per well (no more than 1 mL).
(2) Adding lentiviruses with the corresponding CAR to 12-well plates at MOI = 10; adjusting a constant temperature centrifuge to idle and preheat for 10min at 30 ℃ and 1200g, quickly placing a 12-hole plate in the centrifuge, balancing, and centrifuging for 5h at 30 ℃ and 1200 g; the 12-well plate was placed in an incubator for 7h, then lentivirus with CAR was added to the transfection system according to MOI =10, and the 12-well plate was placed in an incubator for 36h to obtain CAR-NK cells.
Comparative example 5
This comparative example provides a method for preparing CAR-NK cells, which differs from example 5 in that: this comparative example contained no fourth infection aid.
Test example
1. Construction mode of experiment
The CAR-NK cells prepared in examples 1 to 10 and comparative examples 1 to 5 (transfection system containing lentivirus and infection promoter) were tested by flow cytometry after their virus removal and sampling, and the GFP-positive cells were successfully transfected in the flow assay because lentivirus was labeled with GFP (green fluorescent protein), and the cells that were successfully transfected (CAR-NK cells) and negative control (untreated) were counted to obtain the infection positivity, i.e., the lentivirus transfection efficiency of NK cells, by calculating the ratio of CAR-NK cells to untreated cells. The calculation formula is as follows: lentiviral transfection efficiency of NK cells = CAR-NK cells/total NK cells.
2. Results of the experiment
TABLE 1 Lentiviral transfection efficiency of NK cells
The slow virus transfection efficiency results of NK cells obtained by transfecting primary NK cells by using the CAR-NK cell preparation methods of examples 1 to 10 and comparative examples 1 to 5 are shown in Table 1. As can be seen from table 1, the transfection efficiency of primary NK cells with four infection promoters to assist the CAR-bearing lentiviruses in examples 1 to 10 was significantly improved, with a transfection efficiency of > 73%, compared to comparative examples 1 to 5. In addition, the transfection efficiencies of examples 8 to 10 were 77.15%, 76.44% and 78.96%, respectively, which are higher than those of examples 1 to 7, indicating that the lentiviral transfection efficiency of NK cells can be improved by adding the CAR-bearing lentivirus in two steps and centrifuging after adding the first co-infecting agent. The result shows that the kit for assisting the lentivirus with the CAR to transfect the primary NK cells contains four infection assisting agents, the four infection assisting agents have good synergistic effect and can improve the lentivirus transfection efficiency of the primary NK cells, and when the kit is used for preparing the CAR-NK cells, the transfection efficiency of the NK cells can be effectively improved, and more CAR-NK cells can be obtained.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (3)
1. A method for producing CAR-NK cells, comprising the steps of:
(1) Adding a CAR-bearing lentivirus into primary NK cells according to MOI = 2.5-10, then adding a first infection adjuvant, centrifuging, culturing for 5-7 h, and then adding the CAR-bearing lentivirus into a transfection system according to MOI = 2.5-10;
(2) Adding a second infection assisting agent, a third infection assisting agent and a fourth infection assisting agent into the transfection system, and continuously culturing for 24-48 h to obtain the CAR-NK cells;
the first infection assistant is: the final concentration of polybrene is 8-100 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 500000-5000000U/mL and a solvent of 0.3-0.7 vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with final concentration of 20000-200000U/mL and solvent of 0.3-0.7 vol% human serum albumin solution;
the fourth infection-assisting agent is: the final concentration of the phytohemagglutinin is 1-20 mg/mL.
2. The method of claim 1 for producing CAR-NK cells, wherein: the centrifugation conditions were: 30-37 ℃, 800-1200g for 0.5-5 h.
3. The method of claim 1 for producing CAR-NK cells, wherein: in the step (1), the final concentration of polybrene in a transfection system is 6-10 mug/mL; in the step (2), the final concentration of the interleukin-2 in the transfection system is 460-540U/mL, the final concentration of the interleukin-12 in the transfection system is 16-24U/mL, and the final concentration of the phytohemagglutinin in the transfection system is 0.6-1.4 mu g/mL.
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| CN108893493A (en) * | 2018-07-26 | 2018-11-27 | 北京呈诺医学科技有限公司 | A method of promoting NK cell transfecting efficiency |
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| CN108893493A (en) * | 2018-07-26 | 2018-11-27 | 北京呈诺医学科技有限公司 | A method of promoting NK cell transfecting efficiency |
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