CN114438129A - 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-promoting agent is: a polybrene final concentration of 8-100 mg/mL; the second infection-promoting agent is: a solution of human serum albumin with a final concentration of 500000-5000000U/mL interleukin-2 and a solvent of 0.3-0.7 vol%; the third infection-promoting agent is: a solution of human serum albumin with a final concentration of interleukin-12 of 20000 to 200000U/mL and a solvent of 0.3 to 0.7 vol%; 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 adjuvant and performing centrifugal treatment after the infection adjuvant 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 non-specific 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 specific proteins (antigens) and 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 in the transfection of primary NK cells with a CAR-bearing lentivirus, comprising a first, a second, a third, a fourth co-infectious agent;
the first infection-promoting agent is: a polybrene final concentration of 8-100 mg/mL;
the second infection-promoting agent is: a solution of 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-promoting agent is: a solution of interleukin-12 (IL-12) with a final concentration of 20000 to 200000U/mL and a solvent of 0.3 to 0.7 vol% human serum albumin;
the fourth infection-promoting agent is: the final concentration of Phytohaemagglutinin (PHA) is 1-20 mg/mL of aqueous solution.
The kit for assisting lentivirus with CAR to transfect primary NK cells contains four infection aids, which respectively contain polybrene, IL-2, IL-12 and PHA, and the synergistic effect of the four agents can improve the transfection efficiency of lentivirus with CAR on primary NK cells and obtain more CAR-NK cells.
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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/mL and a solvent of 0.5 vol% 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 lentivirus with CAR into a primary NK cell according to the MOI of 2.5-20, then adding a first infection adjuvant, culturing for 5-7 h, and then adding the lentivirus with CAR into a transfection system according to the MOI of 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: a polybrene final concentration of 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: a solution of human serum albumin with a final concentration of interleukin-12 of 20000 to 200000U/ml and a solvent of 0.3 to 0.7 vol%;
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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/mL and a solvent of 0.5 vol% 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: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-20, then adding a first infection adjuvant, centrifuging, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 0-10.
According to the scheme, after the lentivirus with the CAR is added into the primary NK cells, the first infection assisting reagent is added, and centrifugation is performed before culture, so that the first infection assisting reagent can be fully mixed with the lentivirus with the CAR and the primary NK cells, transfection of the lentivirus with the CAR on the primary NK cells is facilitated, and the infection positive rate of the NK cells is effectively improved.
Preferably, the conditions of centrifugation are: 800-1200 g at 30-37 ℃ for 0.5-5 h.
Preferably, in the above method for preparing CAR-NK cells, step (1) is: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-10, then adding a first infection adjuvant, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 2.5-10.
Preferably, in the above method for preparing CAR-NK cells, step (1) is: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-10, then adding a first infection adjuvant, centrifuging, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 2.5-10.
According to the scheme, the CAR-NK cells are prepared by adding the lentiviruses with the CAR step by step, after the lentiviruses with the CAR and the first auxiliary infectious agent are added into the primary NK cells according to the MOI of 2.5-10, centrifugal treatment is carried out, then culture is carried out for 5-7 h, and then the lentiviruses with the CAR are added into a transfection system according to the MOI of 2.5-10, so that the transfection efficiency of the NK cells is higher, and more CAR-NK cells are obtained.
Preferably, the conditions of centrifugation are: 30-37 ℃, 800-1200 g, 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 invention has the beneficial effects that: 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 adjuvant and simultaneously performing centrifugation after the infection adjuvant 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within 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 the MOI of 20, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 10 mu g/mL), and putting the 12-well plate into an incubator for culturing for 7 h;
(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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final 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 the MOI of 10, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 10 mu g/mL), and putting the 12-well plate into an incubator for culturing for 7 h;
(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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% 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 the MOI of 5, adding a first auxiliary infection agent (the final concentration of the first auxiliary infection agent in a transfection system is 8 mu g/mL), and putting the 12-well plate into an incubator for culturing for 6 h;
(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: the polybrene final concentration is 8mg/mL water solution;
the second infection-promoting agent is: a solution of interleukin-2 with a final concentration of 300000U/mL and a solvent of 0.7 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 100000U/ml and a solvent of 0.7 vol% 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 the MOI of 2.5, adding a first auxiliary infectious agent (the final concentration of the first auxiliary infectious agent in a transfection system is 6 mu g/mL), and putting the 12-well plate into an incubator for culturing for 5 h;
(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 48h 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-promoting agent is: interleukin-2 with a final concentration of 500000U/mL and a solvent of 0.3 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 20000U/ml and a solvent of 0.3 vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final 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 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), adding a first auxiliary infectious agent (the final concentration of the first auxiliary infectious agent in a transfection system is 10 mu g/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 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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution having a final phytohemagglutinin 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 for 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 ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) To a 12-well plate was added a lentivirus with the corresponding CAR at an MOI of 20, followed by the addition of a first co-infectious agent (the final concentration of the first co-infectious 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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final 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 a 12-well plate was added a lentivirus with the corresponding CAR at an MOI of 20, followed by the addition of a first co-infectious agent (the final concentration of the first co-infectious 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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% human serum albumin;
the fourth infection-promoting agent is: an aqueous solution of phytohemagglutinin at a final 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 a 12-well plate was added a lentivirus with the corresponding CAR at MOI-10, followed by the addition of a first co-infectious agent (the final concentration of the first co-infectious 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; putting the 12-hole plate into an incubator for culturing for 7h, and then adding the lentivirus with the CAR into a transfection system according to the MOI of 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 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 0.5 vol% human serum albumin;
the third infection-promoting agent is: interleukin-12 with a final concentration of 200000U/ml and a solvent of 0.5 vol% human serum albumin;
the fourth infection-assisting 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) Lentivirus with the corresponding CAR was added to 12-well plates at MOI of 20, and the 12-well plates were cultured in an incubator for 36 hours 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 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 the MOI of 10, putting the 12-well plate into an incubator for culturing for 7h, then adding the lentivirus with the CAR into a transfection system according to the MOI of 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 lentivirus with corresponding CAR to 12-well plate according to MOI of 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-well 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 ratio of 10-100 ten thousand per well (not more than 1 mL).
(2) Adding lentiviruses with corresponding CAR to 12-well plates at MOI of 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; and (3) putting the 12-well plate into an incubator for culturing for 7h, then adding the lentivirus with the CAR into a transfection system according to the MOI of 10, and putting the 12-well plate into the incubator for culturing for 36h to obtain the 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 adjuvant.
Test example
1. Experimental construction mode
The CAR-NK cells prepared in examples 1 to 10 and comparative examples 1 to 5 (transfection system containing lentivirus and infection-assisting agent) were subjected to virus removal and sampling, and detected by flow cytometry, and since lentivirus was labeled with GFP (green fluorescent protein), GFP-positive cells were successfully transfected during flow detection, and the cells that were successfully transfected (CAR-NK cells) and negative control (untreated) were counted, and the infection-positive rate, i.e., the lentivirus transfection efficiency of NK cells, was obtained by calculating the ratio of CAR-NK cells to untreated cells. The calculation formula is as follows: the lentiviral transfection efficiency of NK cells is 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 1-10 and comparative examples 1-5 are shown in Table 1. As can be seen from Table 1, compared with comparative examples 1-5, the transfection efficiency of the primary NK cells transfected by the lentiviruses with CAR assisted by four infection promoters in examples 1-10 is remarkably improved, and is more than 73%. 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, and it was demonstrated 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 (10)
1. A kit for assisting lentivirus with CAR in transfecting primary NK cells, comprising: 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 assistant is: a polybrene final concentration of 8-100 mg/mL;
the second infection-assisting agent is: a solution of human serum albumin with a final concentration of 500000-5000000U/mL interleukin-2 and a solvent of 0.3-0.7 vol%;
the third infection-assisting agent is: a solution of human serum albumin with a final concentration of interleukin-12 of 20000 to 200000U/mL and a solvent of 0.3 to 0.7 vol%;
the fourth infection-assisting agent is: the final concentration of the phytohemagglutinin is 1-20 mg/mL.
2. A kit for the transfection of primary NK cells with a CAR-bearing lentivirus according to claim 1, characterized in that: 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 assistant is: an aqueous solution with a final polybrene concentration of 80 mg/mL;
the second infection-assisting agent is: interleukin-2 with a final concentration of 5000000U/mL and a solvent of 0.5 vol% human serum albumin;
the third infection-assisting agent is: interleukin-12 with a final concentration of 200000U/mL and a solvent of 0.5 vol% human serum albumin;
the fourth infection-assisting agent is: an aqueous solution of phytohemagglutinin at a final concentration of 10 mg/mL.
3. Use of a kit according to claim 1 or 2 for the assisted transfection of primary NK cells with a lentivirus carrying a CAR in the preparation of CAR-NK cells.
4. A method for producing CAR-NK cells, comprising the steps of:
(1) adding lentivirus with CAR into primary NK cells according to the MOI of 2.5-20, then adding a first infection adjuvant, culturing for 5-7 h, and then adding the lentivirus with CAR into a transfection system according to the MOI of 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 the CAR-NK cells;
the first infection assistant is: a polybrene final concentration of 8-100 mg/mL;
the second infection-assisting agent is: a solution of human serum albumin with a final concentration of 500000-5000000U/mL interleukin-2 and a solvent of 0.3-0.7 vol%;
the third infection-assisting agent is: a solution of human serum albumin with a final concentration of interleukin-12 of 20000 to 200000U/mL and a solvent of 0.3 to 0.7 vol%;
the fourth infection-assisting agent is: the final concentration of the phytohemagglutinin is 1-20 mg/mL.
5. The method for producing CAR-NK cells according to claim 4, wherein step (1) is: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-20, then adding a first infection adjuvant, centrifuging, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 0-10.
6. The method of claim 5 for producing CAR-NK cells, wherein: the centrifugation conditions were: 800-1200 g at 30-37 ℃ for 0.5-5 h.
7. The method for producing CAR-NK cells according to claim 4, wherein step (1) is: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-10, then adding a first infection assisting agent, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 2.5-10.
8. The method for producing CAR-NK cells according to claim 7, wherein step (1) is: adding the CAR-carrying lentivirus into primary NK cells according to the MOI of 2.5-10, then adding a first infection adjuvant, centrifuging, culturing for 5-7 h, and then adding the CAR-carrying lentivirus into a transfection system according to the MOI of 2.5-10.
9. The method of claim 8 for producing CAR-NK cells, wherein: the centrifugation conditions were: 800-1200 g at 30-37 ℃ for 0.5-5 h.
10. The method of claim 4 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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| CN114921416A (en) * | 2022-05-12 | 2022-08-19 | 广东普罗凯融生物医药科技有限公司 | NK cell and preparation method thereof |
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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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| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| 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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| CN114807237A (en) * | 2022-05-12 | 2022-07-29 | 广东普罗凯融生物医药科技有限公司 | Preparation method and application of NK (natural killer) cells for over-expressing CD16a |
| CN114921416A (en) * | 2022-05-12 | 2022-08-19 | 广东普罗凯融生物医药科技有限公司 | NK cell and preparation method thereof |
| WO2023217183A1 (en) * | 2022-05-12 | 2023-11-16 | 广东普罗凯融生物医药科技有限公司 | Nk cell and preparation method therefor |
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