CN108893493A - A method of promoting NK cell transfecting efficiency - Google Patents
A method of promoting NK cell transfecting efficiency Download PDFInfo
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- CN108893493A CN108893493A CN201810830835.8A CN201810830835A CN108893493A CN 108893493 A CN108893493 A CN 108893493A CN 201810830835 A CN201810830835 A CN 201810830835A CN 108893493 A CN108893493 A CN 108893493A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/86—Viral vectors
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Abstract
The present invention relates to NK cell expansion ex vivo technical fields, in particular to a kind of method for promoting NK cell transfecting efficiency.The method of this kind promotion NK cell transfecting efficiency, when NK cell carries out slow-virus infection, it is added while the slow virus carrier comprising CAR is added in first subinfection and helps infectious agent 1, be added while the slow virus carrier comprising CAR is added in the second subinfection and help infectious agent 2, help infectious agent 3 and help infectious agent 4；It is described to help infectious agent 1 to contain polybrene solution；It is described to help infectious agent 2 for the solution containing IL2；It is described to help infectious agent 3 for the solution containing IL12；It is described to help infectious agent 4 for the solution containing PHA.The method of Car-NK provided by the invention transfection, by be added during transfection it is different help infectious agent, improve the transfection efficiency of NK cell, can be by the slow-virus transfection efficiency of NK cell by being increased to 50%~60% less than 10%.
Description
Technical field
The present invention relates to NK cell expansion ex vivo technical fields, in particular to a kind of promotion NK cell transfecting efficiency
Method.
Background technique
Car-NK is mainly the cellular immunotherapy for being applied to tumour, is mainly for the cellular immunotherapy of tumour at present
Car-T technology, for Car-NK using less, main cause is the transfection of NK cell difficulty.For experiment in vitro, NK cell is compared with T cell
It is higher to the killing activity of tumour, and T cell is due to immunogenicity height, it is impossible to be used in allosome is fed back, and NK cell immunogenicity is low,
It can be used for allosome feedback and the application of Car-NK limited due to the characteristic of NK cell difficulty transfection, therefore, improve turning for NK cell
It is most important to contaminate efficiency.
The method for mainly using slow virus carrier to infect CAR-NK gene modification at present, it is unprocessed under normal circumstances
Slow virus carrier virus titer it is low, moreover, NK cell is difficult to infect again, therefore, the CAR-NK of general method preparation is effective
Cells ratio is low, and total effective cell number is low.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for promoting NK cell transfecting efficiency, improve the transfection effect of NK cell
Rate, can be by the slow virus carrier transfection efficiency of NK cell by being increased to 50%~60% less than 10%.
A method of NK cell transfecting efficiency being promoted, when NK cell carries out slow virus carrier infection, the first subinfection adds
It is added while entering the slow virus carrier comprising CAR and helps infectious agent 1, the slow virus carrier comprising CAR is added in the second subinfection
It is added simultaneously and helps infectious agent 2, helps infectious agent 3 and help infectious agent 4；
It is described to help infectious agent 1 to contain polybrene solution；
It is described to help infectious agent 2 for the solution containing IL2；
It is described to help infectious agent 3 for the solution containing IL12；
It is described to help infectious agent 4 for the solution containing PHA.
The method of Car-NK provided by the invention transfection, by be added during transfection it is different help infectious agent, have
Effect improves the transfection efficiency of NK cell.
Wherein, polybrene is the english abbreviation of polybrene, also known as is hexadimethrine bromide.IL2 is interleukin 2
English abbreviation.IL12 is the english abbreviation of interleukin 12.PHA is the english abbreviation of polyhydroxyalkanoate.
Further, the NK cell by newly separate or the PBMC cell of cryopreservation resuscitation screening culture obtain.
Slow virus carrier changes the infectious effect of NK cell with NK cell cultivation process, therefore, preferably, NK is thin
At born of the same parents' amplification in vitro culture the 5-7 days, by 3 × 105-8×105The first subinfection of the hole /ml progress slow virus.
If pressing 1 × 106The hole /ml, then will appear infection ratio reduces situation.
Further, the time interval of the first subinfection and the second subinfection is 5-7h.
Further, the Car-NK slow virus carrier is prepared by the following method：
PsPAX2, pMD2.0G and Plenti-CAR-NK pack slow virus, and the slow virus of collection suspends through ultracentrifugation
It arrives.
Further, psPAX2, pMD2.0G and Plenti-CAR-NK, by 3-3.5:1.5-2.0:4.5-5.5 weight ratio
Example configuration, such as can be in 3.2 μ g, 1.8 μ g, the preparation of 5 μ g ratios.
First day transfection 293T cell, for 24 hours, 48h, 72h collect supernatant, ultracentrifugation concentration obtains slow virus.
Further, the packaging slow virus using 10cm plate pack, for 24 hours, 48h, 72h collect 3 times totally 30 ± 2ml it is slow
Virus, after centrifugation, being suspended with 100 μ l DPBS is a slow virus carrier.
Further, described be centrifuged is：50,000g 0-4 DEG C of centrifugation 100-150min, sufficiently to obtain collection virus.
Further, the slow virus carrier obtained is saved in -80 DEG C.
Further, described to help the infectious agent 1 to be：The aqueous solution of the final concentration of 8mg/ml of polybrene；
It is described to help the infectious agent 2 to be：The final concentration of 500,000U/ml of IL2, the solution that solvent is 0.5%BSA；
It is described to help the infectious agent 3 to be：The final concentration of 20,000U/ml of IL12, the solution that solvent is 0.5%BSA；
It is described to help the infectious agent 4 to be：The aqueous solution of the final concentration of 1mg/ml of PHA.
Further, 0.2 μm of membrane filtration after helping the dissolution of infectious agent 1.
Further, described that infectious agent 1-3 is helped to save in -80 DEG C, it is described that infectious agent 4 is helped to save in -20 DEG C.
Further, when the first subinfection, final concentration of 7-9 μ g/ml of the polybrene of addition in infection system；Second
When subinfection, the final concentration of 18- of the IL2 final concentration of 480-520U/ml in infection system, IL12 in infection system
The final concentration of 0.8-1.2 μ g/ml of 22U/ml, PHA in infection system.
Such as, the cell liquid volume of NK cell is 0.8-1.5ml, when the first subinfection, one include CAR slow virus carrier
1 μ l is added helps infectious agent 1；When the second subinfection, a slow virus carrier comprising CAR is added 1 μ l and helps infectious agent 2,1 μ l
Help the μ of infectious agent 3 and 1 l's to help infectious agent 4.Infectious agent ingredient is helped by the way that addition is appropriate, transfection is effectively promoted, is promoted and turned
Contaminate efficiency.
In the present invention, the culture of NK cell expansion ex vivo is conventionally carried out, and can such as be prepared using following steps：
Take the peripheral blood mononuclear cells 20,000,000 or 30,000,000 newly separated；Or recovery freezes peripheral blood mononuclear cells 2-
3；Or recovery peripheral blood mononuclear cells 3-4 branch, inoculating cell density is adjusted to by >=2*10^ according to above-mentioned cell quantity
Required NK cell-stimulating culture solution (10-15ml) is added in T25 or T75 bottles in 6cells/ml.It is placed in saturated humidity, 37
DEG C, 5.0%CO2It is cultivated in incubator, culture bottle is put vertically.Start to cultivate cell at this time, be denoted as the 0th day (D0).
Liquid, centrifugal condition 300g, 10min (raising speed are changed to cell centrifugation after 72h：150s, reduction of speed：150s).Again will
Density is adjusted to >=1~0.8*10^6cells/ml, is incubated at a new T75 culture bottle after being resuspended with NK cell amplification cultivation liquid
In (must keep sample after operation in case Quality Control detect).
5th day, cell growth state is observed, cell is dispelled, counted, fluid infusion when necessary, adjustment cell density is not more than
1.2*10^6cells/ml (must be kept sample after operation in case Quality Control detects) not less than 0.8*10^6cells/ml.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention in different ways carry out slow virus carrier infection, by control be added slow virus carrier when
Between put and help addition time point and the dosage of infectious agent, effectively increase the transfection efficiency of NK cell.
(2) the slow virus carrier efficiency of infection of NK cell is increased to 50%~60% less than 10% by existing.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
State when Fig. 1 is NK cell culture 5 days in embodiment；
Fig. 2 is state after NK cell transfecting culture in embodiment 1；
Fig. 3 is 1 transfection efficiency quantitative fluorescence analysis figure of embodiment；
Fig. 4 is state after NK cell transfecting culture in embodiment 2；
Fig. 5 is state after NK cell transfecting culture in embodiment 3；
Fig. 6 is state after NK cell transfecting culture in embodiment 4；
Fig. 7 is state after NK cell transfecting culture in embodiment 5；
Fig. 8 is state after NK cell transfecting culture in comparative example 1；
Fig. 9 is 1 transfection efficiency quantitative fluorescence analysis schematic diagram of comparative example；
Figure 10 is state after NK cell transfecting culture in comparative example 2.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK cell expansion ex vivo the 5th day such as Fig. 1, by 5 × 105(culture solution of 12 orifice plates, every hole is in the hole /ml
Slow virus carrier infection 0.8ml) is carried out, the first subinfection is added 1 and includes the slow virus carrier of CAR, while helping for 1 μ l is added
Infectious agent 1；
1 slow virus carrier comprising CAR is added in second subinfection after 5h, 1 μ l is added, infectious agent 2,1 μ l is helped to help infection
Agent 3,1 μ l help infectious agent 4, and gently piping and druming is uniformly mixed, and are normally cultivated；
Preparing for each raw material being related to is as follows：
1. slow virus carrier is concentrated：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：
PsPAX2, pMD2.0G and Plenti-CAR-NK are prepared, first day transfection 293T in 3.2 μ g, 1.8 μ g, 5 μ g ratios, for 24 hours,
48h, 72h collect the supernatant that 3 total 30ml contain slow virus, and using ultracentrifuge, 50,000g 4 DEG C of centrifugation 2h are used
100 μ l DPBS suspend as a slow virus carrier comprising CAR, -80 DEG C of preservations；
2. helping infectious agent 1：It weighs polybrene to dissolve using sterile water, final concentration of 8mg/ml, 0.2 μm of filter membrane mistake after dissolution
Filter；
3. helping infectious agent 2：It is that 0.5%BSA dissolves that IL2, which is used concentration, final concentration of 500,000U/ml, -80 after packing
DEG C save；
4. helping infectious agent 3：It is that 0.5%BSA dissolves that IL12, which is used concentration, final concentration of 20,000U/ml, -80 after packing
DEG C save；
5. helping infectious agent 4：PHA (polyhydroxyalkanoate) is dissolved using sterile water, final concentration of 1mg/ml, -20 DEG C
It saves.
State is as shown in Figure 2 after NK cell transfecting culture.
Detection, as a result as shown in Figure 3.In Fig. 3, R1 indicates the ratio of NK cells on total cells, and R1 indicates that CAR-NK accounts for NK
The ratio of cell, obtains, and the slow-virus transfection efficiency of NK cell is 56.12%.
Embodiment 2
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 6th day, by 5 × 105The hole /ml (12 orifice plates, the culture solution in every hole are 1ml) carries out slow
Viral vector infection, 1 slow virus carrier comprising CAR of the first subinfection addition, while 1 μ l's of addition helps infectious agent 1；
1 slow virus carrier comprising CAR is added in second subinfection after 6h, 1 μ l is added, infectious agent 2,1 μ l is helped to help infection
Agent 3,1 μ l help infectious agent 4, and gently piping and druming is uniformly mixed, and are normally cultivated；
Preparing for each raw material being related to is as follows：
1. slow virus carrier is concentrated：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：
PsPAX2, pMD2.0G and Plenti-CAR-NK are prepared, first day transfection 293T in 3.2 μ g, 1.8 μ g, 5 μ g ratios, for 24 hours,
48h, 72h collect the supernatant that 3 total 30ml contain slow virus, using ultracentrifuge, 50,000g 4 DEG C of centrifugation 100min,
It is suspended using 100 μ l DPBS as a slow virus carrier comprising CAR, -80 DEG C of preservations；
2. helping infectious agent 1：It weighs polybrene to dissolve using sterile water, final concentration of 8mg/ml, 0.2 μm of filter membrane mistake after dissolution
Filter；
3. helping infectious agent 2：It is that 0.5%BSA dissolves that IL2, which is used concentration, final concentration of 500,000U/ml, -80 after packing
DEG C save；
4. helping infectious agent 3：It is that 0.5%BSA dissolves that IL12, which is used concentration, final concentration of 20,000U/ml, -80 after packing
DEG C save；
5. helping infectious agent 4：PHA (polyhydroxyalkanoate) is dissolved using sterile water, final concentration of 1mg/ml, -20 DEG C
It saves.
State is as shown in Figure 4 after NK cell transfecting culture.
Detection, the slow-virus transfection efficiency of NK cell are 60.01%.
Embodiment 3
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 7th day, by 8 × 105The hole /ml (12 orifice plates, the culture solution in every hole are 1.5ml) carries out
Slow virus carrier infection, 1 slow virus carrier comprising CAR of the first subinfection addition, while 1 μ l's of addition helps infectious agent 1；
1 slow virus carrier comprising CAR is added in second subinfection after 7h, 1 μ l is added, infectious agent 2,1 μ l is helped to help infection
Agent 3,1 μ l help infectious agent 4, and gently piping and druming is uniformly mixed, and are normally cultivated；
Preparing for each raw material being related to is as follows：
1. slow virus is concentrated：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：
PsPAX2, pMD2.0G and Plenti-CAR-NK are prepared, first day transfection 293T in 3.5 μ g, 2.0 μ g, 5.5 μ g ratios, the
For 24 hours, 48h, 72h collect the supernatant that 3 total 30ml contain slow virus, and using ultracentrifuge, 50,000g 4 DEG C of centrifugations two are small
When, it is suspended using 100 μ l DPBS as a slow virus carrier comprising CAR, -80 DEG C of preservations；
2. helping infectious agent 1：It weighs polybrene to dissolve using sterile water, final concentration of 8mg/ml, 0.2 μm of filter membrane mistake after dissolution
Filter；
3. helping infectious agent 2：It is that 0.5%BSA dissolves that IL2, which is used concentration, final concentration of 500,000U/ml, -80 after packing
DEG C save；
4. helping infectious agent 3：It is that 0.5%BSA dissolves that IL12, which is used concentration, final concentration of 20,000U/ml, -80 after packing
DEG C save；
5. helping infectious agent 4：PHA (polyhydroxyalkanoate) is dissolved using sterile water, final concentration of 1mg/ml, -20 DEG C
It saves.
State is as shown in Figure 5 after NK cell transfecting culture.
Detection, the slow-virus transfection efficiency of NK cell are 55.32%.
Embodiment 4
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 5th day, by 6 × 105The hole /ml (12 orifice plates, the culture solution in every hole are 1ml) carries out slow
Viral vector infection, 1 slow virus carrier comprising CAR of the first subinfection addition, while 1 μ l's of addition helps infectious agent 1；
1 slow virus carrier comprising CAR is added in second subinfection after about 6h, 1 μ l is added, infectious agent 2,1 μ l is helped to help sense
Stain 3,1 μ l help infectious agent 4, and gently piping and druming is uniformly mixed, and are normally cultivated；
Preparing for each raw material being related to is as follows：
1. slow virus is concentrated：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：
PsPAX2, pMD2.0G and Plenti-CAR-NK are prepared, first day transfection 293T in 3 μ g, 1.5 μ g, 4.5 μ g ratios, for 24 hours,
48h, 72h collect the supernatant that 3 total 30ml contain slow virus, and using ultracentrifuge, 50,000g 4 DEG C are centrifuged two hours,
It is suspended using 100 μ l DPBS as a slow virus carrier comprising CAR, -80 DEG C of preservations；
2. helping infectious agent 1：It weighs polybrene to dissolve using sterile water, final concentration of 8mg/ml, 0.2 μm of filter membrane mistake after dissolution
Filter；
3. helping infectious agent 2：It is that 0.5%BSA dissolves that IL2, which is used concentration, final concentration of 500,000U/ml, -80 after packing
DEG C save；
4. helping infectious agent 3：It is that 0.5%BSA dissolves that IL12, which is used concentration, final concentration of 20,000U/ml, -80 after packing
DEG C save；
5. helping infectious agent 4：PHA (polyhydroxyalkanoate) is dissolved using sterile water, final concentration of 1mg/ml, -20 DEG C
It saves.
State is as shown in Figure 6 after NK cell transfecting culture.
Detection, the slow-virus transfection efficiency of NK cell are 57.41%.
Embodiment 5
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 6th day, by 1 × 106The hole /ml (12 orifice plates, the culture solution in every hole are 1ml) carries out slow
Viral vector infection, 1 slow virus carrier comprising CAR of the first subinfection addition, while 1 μ l's of addition helps infectious agent 1；
1 slow virus carrier comprising CAR is added in second subinfection after about 6h, 1 μ l is added, infectious agent 2,1 μ l is helped to help sense
Stain 3,1 μ l help infectious agent 4, and gently piping and druming is uniformly mixed, and are normally cultivated；
Preparing for each raw material being related to is as follows：
1. slow virus is concentrated：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：
PsPAX2, pMD2.0G and Plenti-CAR-NK are prepared, first day transfection 293T in 3.2 μ g, 1.8 μ g, 5 μ g ratios, for 24 hours,
48h, 72h collect the supernatant that 3 total 30ml contain slow virus, and using ultracentrifuge, 50,000g 4 DEG C are centrifuged two hours,
It is suspended using 100 μ l DPBS as a slow virus carrier comprising CAR, -80 DEG C of preservations；
2. helping infectious agent 1：It weighs polybrene to dissolve using sterile water, final concentration of 8mg/ml, 0.2 μm of filter membrane mistake after dissolution
Filter；
3. helping infectious agent 2：It is that 0.5%BSA dissolves that IL2, which is used concentration, final concentration of 500,000U/ml, -80 after packing
DEG C save；
4. helping infectious agent 3：It is that 0.5%BSA dissolves that IL12, which is used concentration, final concentration of 20,000U/ml, -80 after packing
DEG C save；
5. helping infectious agent 4：PHA (polyhydroxyalkanoate) is dissolved using sterile water, final concentration of 1mg/ml, -20 DEG C
It saves.
State is as shown in Figure 7 after NK cell transfecting culture.
Detection, the slow-virus transfection efficiency of NK cell are 50.55%.
Comparative example 1
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 5th day, by 5 × 105The hole /ml (12 orifice plates, the culture solution in every hole are 1ml) carries out slow
1 slow virus carrier comprising CAR is added in virus infection, the first subinfection；
1 slow virus carrier comprising CAR is added in second subinfection after about 6h, and gently piping and druming is uniformly mixed, and carries out normal
Culture；
Slow virus concentration：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：psPAX2,
PMD2.0G and Plenti-CAR-NK is prepared, first day transfection 293T in 3.2 μ g, 1.8 μ g, 5 μ g ratios, for 24 hours, 48h, 72h
The supernatant that 3 total 30ml contain slow virus is collected, using ultracentrifuge, 50,000g 4 DEG C are centrifuged two hours, use 100 μ
It is a Car-NK slow virus, -80 DEG C of preservations that l DPBS, which suspends,.
State is as shown in Figure 8 after NK cell transfecting culture.
Detection, as a result as shown in Figure 9.In Fig. 9, R1 indicates the ratio of NK cells on total cells, and R1 indicates that CAR-NK accounts for NK
The ratio of cell, obtains, and the slow-virus transfection efficiency of NK cell is 6.45%.
Comparative example 2
A method of NK cell transfecting efficiency being promoted, steps are as follows：
At the culture of NK amplification in vitro the 5th day, by 1 × 106The hole /ml (12 orifice plates, the culture solution in every hole are 1ml) carries out slow
1 Car-NK slow virus is added in virus infection, the first subinfection；
1 Car-NK slow virus is added in second subinfection after about 6h, and gently piping and druming is uniformly mixed, and is normally cultivated；
Slow virus concentration：Slow virus is packed using 10cm plate, slow virus packaging includes three plasmids, composition：psPAX2,
PMD2.0G and Plenti-CAR-NK is prepared, first day transfection 293T in 3.2 μ g, 1.8 μ g, 5 μ g ratios, for 24 hours, 48h, 72h
The supernatant that 3 total 30ml contain slow virus is collected, using ultracentrifuge, 50,000g 4 DEG C are centrifuged two hours, use 100 μ
It is a slow virus, -80 DEG C of preservations that l DPBS, which suspends,.
State is as shown in Figure 10 after NK cell transfecting culture.
Detection, the slow-virus transfection efficiency of NK cell are 5.02%.
From the above, it is seen that a kind of method for promoting NK cell transfecting efficiency provided by the invention, improves NK
The transfection efficiency of cell, can be by the slow-virus transfection efficiency of NK cell by being increased to 50%~60% less than 10%.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of method for promoting NK cell transfecting efficiency, which is characterized in that when NK cell carries out slow-virus infection, feel for the first time
It is added while the slow virus carrier comprising CAR is added in dye and helps infectious agent 1, the second subinfection is added the slow virus comprising CAR and carries
It is added while body and helps infectious agent 2, helps infectious agent 3 and help infectious agent 4；
It is described to help infectious agent 1 to contain polybrene solution；
It is described to help infectious agent 2 for the solution containing IL2；
It is described to help infectious agent 3 for the solution containing IL12；
It is described to help infectious agent 4 for the solution containing PHA.
2. the method according to claim 1, wherein the NK cell is by newly separating or the PBMC of cryopreservation resuscitation
Cell screening culture obtains.
3. the method according to claim 1, wherein the time of the first subinfection is the culture of NK cell expansion ex vivo
At the 5-7 days.
4. the method according to claim 1, wherein pressing 3 × 105-8×105The hole /ml carries out slow virus carrier
The first subinfection.
5. according to the method described in claim 2, it is characterized in that, the time interval of the first subinfection and the second subinfection is 5-
7h。
6. the method according to claim 1, wherein the slow virus carrier comprising CAR is by the following method
Preparation：
PsPAX2, pMD2.0G and Plenti-CAR-NK pack slow virus, by 3-3.5:1.5-2.0:4.5-5.5 weight ratio is matched
It sets, through being centrifuged, suspension obtains the slow virus of collection.
7. according to the method described in claim 4, it is characterized in that, the packaging slow virus using 10cm plate pack, for 24 hours,
48h, 72h collect 3 totally 30 ± 2ml slow virus, and after centrifugation, being suspended with 100 μ l DPBS is a slow virus carrier.
8. the method according to the description of claim 7 is characterized in that the centrifugation is：50,000g0-4 DEG C of centrifugation 100-
150min。
9. helping the infectious agent 1 to be the method according to claim 1, wherein described：The final concentration of 8mg/ml of polybrene
Aqueous solution；
It is described to help the infectious agent 2 to be：The final concentration of 500,000U/ml of IL2, the solution that solvent is 0.5%BSA；
It is described to help the infectious agent 3 to be：The final concentration of 20,000U/ml of IL12, the solution that solvent is 0.5%BSA；
It is described to help the infectious agent 4 to be：The aqueous solution of the final concentration of 1mg/ml of PHA；
Further, 0.2 μm of membrane filtration after helping the dissolution of infectious agent 1.
10. -9 described in any item methods according to claim 1, which is characterized in that when the first subinfection, the polybrene of addition exists
Final concentration of 7-9 μ g/ml in infection system；When the second subinfection, final concentration of 480-520U/ of the IL2 in infection system
The final concentration of 0.8-1.2 μ g/ml of ml, the IL12 final concentration of 18-22U/ml in infection system, PHA in infection system.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122579A (en) * | 2021-04-14 | 2021-07-16 | 河北森朗生物科技有限公司 | Method for transfecting immune cells by lentivirus |
| CN114438129A (en) * | 2022-01-12 | 2022-05-06 | 广东普罗凯融生物医药科技有限公司 | Kit for assisting lentivirus with CAR to transfect primary NK cells and application of kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122579A (en) * | 2021-04-14 | 2021-07-16 | 河北森朗生物科技有限公司 | Method for transfecting immune cells by lentivirus |
| CN113122579B (en) * | 2021-04-14 | 2022-03-29 | 河北森朗生物科技有限公司 | Method for transfecting immune cells by lentivirus |
| CN114438129A (en) * | 2022-01-12 | 2022-05-06 | 广东普罗凯融生物医药科技有限公司 | Kit for assisting lentivirus with CAR to transfect primary NK cells and application of kit |
| CN114438129B (en) * | 2022-01-12 | 2023-01-06 | 广东普罗凯融生物医药科技有限公司 | Kit for assisting lentivirus with CAR to transfect primary NK cells and application of kit |
| CN115505600A (en) * | 2022-09-30 | 2022-12-23 | 北京奇迈永华生物科技有限公司 | Method for efficiently infecting human NK cells by lentiviruses |
| CN115505600B (en) * | 2022-09-30 | 2024-01-30 | 北京奇迈永华生物科技有限公司 | Method for efficiently infecting human NK cells by slow viruses |
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