CN109439632A - A method of improving CAR-T cell transfecting efficiency - Google Patents
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Abstract
The invention belongs to oncotherapy technical fields, disclose a kind of method for improving CAR-T cell transfecting efficiency.CAR plasmid is constructed, guarantees plasmid concentration in 1500ng/ul or more；After the completion of CAR plasmid construction, the building of CAR liposome is carried out；It is packed using CAR plasmid pair slow virus, virus is received after 48 hours；Blood is diluted using PBS, ingredient mixes, and extracts to PBMC cell；It is infected during t cell activation, PBMC is added in coated 24 orifice plate of CD3 antibody and carries out t cell activation overnight after extraction, then carries out electricity turn, liposome transfection and slow-virus infection, and it is normal incubation medium that liquid is changed after 6h.The present invention has higher efficiency compared to electricity turn, liposome transfection, slow-virus infection for T cell；Carried out during t cell activation infection than activation before/activation after infected, the T cell of CAR positive infection increases.
Description
Technical field
The invention belongs to oncotherapy technical field more particularly to a kind of methods for improving CAR-T cell transfecting efficiency.
Background technique
Currently, the prior art commonly used in the trade is such that
Chimeric antigen receptor modifies T cell immunotherapy (Chimeric Antigen Receptor T-Cell
Immunotherapy, CAR-T) it is the popular domain that current oncotherapy is studied, it is the Typical Representative of adoptive immunotherapy.
By utilizing gene transfer technology, so that T cell surface is stablized expression has specific recognition tumor associated antigen ability
Single chain variable fragment (single-chain variable fragment, scFv), and with CD3 ζ and other T cell costimulations
It is connected between molecule such as CD28/CD137, it is otherwise by the restricted knowledge of non-MHC, high using antigen-antibody affinity, special
Anisotropic strong feature specifically, efficiently activates T cell, and then generates killing to the tumour cell for expressing the tumor associated antigen
Effect is the therapeutic modality that more mature, targeting is strong, specificity is high at present, to kill tumor range wide, effect is lasting.It is treating
Extremely strong effect has been shown in the non-physical knurls such as urgency/chronic lymphocytic leukemia, non-Hodgkin lymphoma, has been greatly improved
The life cycle of patient slows down recurrence, increases patient's remission rate 90%, reachable using the complete incidence graph life span of rear patient
2 years.CAR is still in initial stage in the treatment of solid tumor, accordingly obtains certain effect.However however external long time-histories training
It supports and will lead to the reduction of T cell activity, since culture medium nutritional deficiency also can largely reduce T cell during transfection (infection)
Activation.Larger, generally more than 10000bp or so additionally, due to the gene order of Chimeric antigen receptor (CAR) is transfected (infection)
Difficulty is big, inefficiency, is more not easy to be transferred to for the difficult T cell of transfection.
In conclusion problem of the existing technology is:
In the prior art since the gene order of Chimeric antigen receptor (CAR) is larger, generally more than 10000bp or so turns
It is big to contaminate (infection) difficulty, inefficiency is more not easy to be transferred to for the difficult T cell of transfection.
Solve the difficulty and meaning of above-mentioned technical problem:
Clinicing aspect: in CAR-T cell therapy, the T cell of the only CAR positive can be functioned.Due to preparation
The T cell of CAR-T cell is mainly derived from human peripheral, is limited to acquiring way and collects quantity, can obtain for infecting
T cell number it is few, under this restrictive condition, look for improve infection CAR positive T cell number method, for clinic
It treats significant.
Technical aspect: T cell mainly passes through the methods of electricity turn, liposome transfection and slow-virus infection and is transfected at present,
And the efficiency for transfecting (infection) is heavily dependent on plasmid size and cell state.CAR plasmid size usually larger one
As in 8000~10000bp, therefore previous experiments show that common electricity turns and liposome Cell Transfection Conditions can not be effective
So big plasmid is transferred in T cell, even if ideal efficiency of infection can not be reached by carrying out slow-virus transfection T cell.
Therefore we need to find more effective way, while reducing transfection (infection) influences cell state, and guarantee transfection
The positive rate of (infection) cell afterwards.And then enough CAR positive T cells can be prepared in certain amount peripheral blood for facing
Bed treatment.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of methods for improving CAR-T cell transfecting efficiency.
The invention is realized in this way a method of CAR-T cell transfecting efficiency is improved, specifically includes the following steps:
Step 1: building CAR plasmid guarantees plasmid concentration in 1500ng/ul or more；
Step 2: after the completion of CAR plasmid construction, the building of CAR liposome is carried out；
Step 3: being packed using CAR plasmid pair slow virus, and virus is received after 48；
Step 4: being diluted blood using PBS, and ingredient mixes, and extracts to PBMC cell；
Step 5: being infected during t cell activation, and PBMC is added in coated 24 orifice plate of CD3 antibody after extraction
T cell activation is carried out overnight, then carries out electricity turn, liposome transfection and slow-virus infection, and it is normal incubation medium that liquid is changed after 6h.
Further, in step 2, the building of CAR liposome, specifically:
In EP pipe 1: being added in CAR plasmid and 100ulOpti-MEM, 5ulP3000 is added and mixes well；
In EP pipe 2: 100ulOpti-MEM+5ul Lipo3000, pipe 1 and 2 stand 15min after mixing at room temperature.
Further, in step 3, slow virus is packed using CAR plasmid, specifically:
(1) in EP pipe 1: 5ug CAR plasmid+3.75ugPSPAX2+1.25ugPMD2.G is added in 250ulOpti-MEM,
12ulP3000 is added to mix well, in EP pipe 2: 250ulOpti-MEM+12ul Lipo3000, after the mixing of pipe 1 and 2 at room temperature,
Stand 15min；
(2) replacement 293T culture medium is serum-free Opti-MEM without double antibody or serum-free DMEM culture medium 5ml without double antibody；
(3) mixed liquor is added dropwise in 293T after 15min；7mlDMEM culture medium is changed to after 6h；
Virus is received after (4) 48.
Further, in step 4, PBMC cell is extracted, specifically includes the following steps:
(1) PBS mixes well one times of hemodilution；
(2) 5mlFicoll is added in 15mL centrifuge tube, 10ml blood is added, according to blood: the ratio of Ficoll=2:1,
Haemocyte is added slowly to the upper layer Ficoll；
(3) 500g is centrifuged 20min, 20 degrees Celsius, ACC1, DCC0, draws tunica albuginea confluent monolayer cells (PBMC)
(4) PBS is added, is settled to 50mL；
(5) 1700rpm is centrifuged 5min, carefully siphons away supernatant, and PBS continues washed once, PBMC cell count, carries out next
Step stimulation；
(6) it extracts PBMC cell and is divided into tri- groups of ABC, while being coated with 24 orifice plates using CD3 antibody 20ug/ml.
In conclusion advantages of the present invention and good effect are as follows:
(1) present invention has been prompted compared to electricity turn, liposome transfection, and slow-virus infection has higher sense for T cell
Contaminate efficiency.
(2) present invention improves T cells to transfect (infection) low efficiency, and CAR positive T cell is not easy the problem obtained, thin in T
By slow-virus infection in born of the same parents' activation, by the transfection ratio of CAR positive T cell by being promoted less than 5% to 38.7%.
(see the table below)
Detailed description of the invention
Fig. 1 is the method flow diagram provided in an embodiment of the present invention for improving CAR-T cell transfecting efficiency.
Fig. 2 is the CAR slow virus plasmid schematic diagram of building EGFP green fluorescence provided in an embodiment of the present invention.
Fig. 3 is electricity turn, liposome transfection and the slow-virus infection situation schematic diagram of A group provided in an embodiment of the present invention.
Fig. 4 is electricity turn, liposome transfection and the slow-virus infection situation schematic diagram of B group provided in an embodiment of the present invention.
Fig. 5 is electricity turn, liposome transfection and the slow-virus infection situation schematic diagram of C group provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is described in detail with reference to the accompanying drawing.
As shown in Figure 1, the method provided in an embodiment of the present invention for improving CAR-T cell transfecting efficiency, specifically includes following
Step:
S101: building CAR plasmid guarantees plasmid concentration in 1500ng/ul or more；
After the completion of S102:CAR plasmid construction, the building of CAR liposome is carried out；
S103: being packed using CAR plasmid pair slow virus, and virus is received after 48 hours；
S104: being diluted blood using PBS, and ingredient mixes, and extracts to PBMC cell；
S105: being infected during t cell activation, and mistake in coated 24 orifice plate of CD3 antibody is added in PBMC after extraction
Night carries out t cell activation, then carries out electricity turn, liposome transfection and slow-virus infection, and it is normal incubation medium that liquid is changed after 6h.
In step S102, CAR liposome building provided in an embodiment of the present invention, specifically:
In EP pipe 1: being added in CAR plasmid and 100ulOpti-MEM, 5ulP3000 is added and mixes well；
In EP pipe 2: 100ulOpti-MEM+5ul Lipo3000, pipe 1 and 2 stand 15min after mixing at room temperature.
It is provided in an embodiment of the present invention to pack slow virus using CAR plasmid in step S103, specifically:
(1) in EP pipe 1: 5ug CAR plasmid+3.75ugPSPAX2+1.25ugPMD2.G is added in 250ulOpti-MEM,
12ulP3000 is added to mix well, in EP pipe 2: 250ulOpti-MEM+12ul Lipo3000, after the mixing of pipe 1 and 2 at room temperature,
Stand 15min；
(2) replacement 293T culture medium is serum-free Opti-MEM without double antibody or serum-free DMEM culture medium 5ml without double antibody；
(3) mixed liquor is added dropwise in 293T after 15min；7mlDMEM culture medium is changed to after 6h；
Virus is received after (4) 48.
In step S104, extraction PBMC cell provided in an embodiment of the present invention, specifically includes the following steps:
(1) PBS mixes well one times of hemodilution；
(2) 5mlFicoll is added in 15mL centrifuge tube, 10ml blood is added, according to blood: the ratio of Ficoll=2:1,
Haemocyte is added slowly to the upper layer Ficoll；
(3) 500g is centrifuged 20min, 20 degrees Celsius, ACC1, DCC0, draws tunica albuginea confluent monolayer cells (PBMC)
(4) PBS is added, is settled to 50mL；
(5) 1700rpm is centrifuged 5min, carefully siphons away supernatant, and PBS continues washed once, PBMC cell count, carries out next
Step stimulation；
(6) it extracts PBMC cell and is divided into tri- groups of ABC, while being coated with 24 orifice plates using CD3 antibody 20ug/ml.
Application principle of the invention is further elaborated combined with specific embodiments below；
Embodiment 1；
Since the gene order of Chimeric antigen receptor (CAR) is larger, generally more than 10000bp or so, transfection (infection) hardly possible
Degree is big, inefficiency, is more not easy to be transferred to for the difficult T cell of transfection.
Therefore, the present invention provides the methods that raising CAR is transferred to T cell efficiency；
1, material:
T cell culture medium: Sodium Pyruvate, L-Glutamine, HEPES, beta -mercaptoethanol, Gbico are added in 1640
Thermo serum, IL-2.
anti-huaman CD3:biolegend,clone:OKT3,cat:317304
Opti-MEM:thermo, REF:51985
PEI:polyscience,23966-2
3000 Transfection Reagent:invitrogen, L3000015
Nunceasyflask 25cm2:cat:156367
Nunceasyflask 75cm2:cat:156499
Nunc 24 well plate:cat 142475
2, method and steps:
As shown in Fig. 2, building CAR plasmid, construct EGFP green fluorescence CAR slow virus plasmid (plasmid size=
9825bp), guarantee plasmid concentration in 1500ng/ul or more.
3, CAR liposome constructs
In EP pipe 1: it is added in CAR plasmid and 100ulOpti-MEM, 5ulP3000 is added and mixes well, in EP pipe 2:
100ulOpti-MEM+5ul Lipo3000, pipe 1 and 2 stand 15min after mixing at room temperature.
4, slow virus is packed using CAR plasmid
Packaging virus: in EP pipe 1: 250ulOpti- is added in 5ug CAR plasmid+3.75ugPSPAX2+1.25ugPMD2.G
In MEM, 12ulP3000 is added and mixes well, in EP pipe 2: 250ulOpti-MEM+12ul Lipo3000, after pipe 1 and 2 mixes
15min is stood at room temperature；Replacing 293T culture medium is serum-free Opti-MEM without double antibody or serum-free DMEM culture medium without double antibody
5ml.Mixed liquor is added dropwise in 293T after 15min；7mlDMEM culture medium is changed to after 6h.Virus is received after 48 hours.
5, PBMC cell is extracted
1) PBS mixes well one times of hemodilution；
2) 5ml Ficoll is added in 15mL centrifuge tube, 10ml blood is added, according to blood: the ratio of Ficoll=2:1,
Haemocyte is added slowly to the upper layer Ficoll；
3) 500g is centrifuged 20min, 20 degrees Celsius, ACC 1, DCC 0, draws tunica albuginea confluent monolayer cells (PBMC)
4) PBS is added, is settled to 50mL；
5) 1700rpm is centrifuged 5min, carefully siphons away supernatant, and PBS continues washed once, PBMC cell count, carries out next
Step stimulation.
6) it extracts PBMC cell and is divided into tri- groups of ABC, while being coated with 24 orifice plates using CD3 antibody 20ug/ml.
6, different transfection (infection) modes compare:
A group: transfecting (infection) group before t cell activation, be added after carrying out electricity turn, liposome transfection and slow-virus infection 6h and change
Liquid continues to cultivate 12h, and coated 24 orifice plate of CD3 antibody then is added in cell and carries out t cell activation.
B group: transfecting (infection) group during t cell activation, mistake in coated 24 orifice plate of CD3 antibody is added in PBMC after extraction
Night carries out t cell activation, then carries out electricity turn, liposome transfection and slow-virus infection, and it is normal incubation medium that liquid is changed after 6h.
C group: transfecting (infection) group after t cell activation, PBMC is added in coated 24 orifice plate of CD3 antibody after extraction, culture 4
After it, electricity turn, liposome transfection and slow-virus infection are carried out, it is that normal incubation medium transfection (infection) is received for 72 hours afterwards that liquid is changed after 6h
Cell is taken, activate T cell with CD3 magnetic bead sorting and passes through flow cytometer detection CAR positive population.
As shown in figure 3, the electricity of A group turn, liposome transfection and slow-virus infection situation schematic diagram.
As shown in figure 4, the electricity of B group turn, liposome transfection and slow-virus infection situation schematic diagram.
As shown in figure 5, the electricity of C group turn, liposome transfection and slow-virus infection situation schematic diagram.
As a result:
(1) turn compared to electricity, liposome transfection, slow-virus infection has higher efficiency for T cell；
(2) carried out during t cell activation infection than activation before/activation after infected, the T of CAR positive infection is thin
Born of the same parents increase.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of method for improving CAR-T cell transfecting efficiency, which is characterized in that the raising CAR-T cell transfecting efficiency
Method, specifically includes the following steps:
Step 1: building CAR plasmid guarantees plasmid concentration in 1500ng/ul or more；
Step 2: after the completion of CAR plasmid construction, the building of CAR liposome is carried out；
Step 3: being packed using CAR plasmid pair slow virus, and virus is received after 48 hours；
Step 4: being diluted blood using PBS, and ingredient mixes, and extracts to PBMC cell；
Step 5: being infected during t cell activation, and PBMC is added in coated 24 orifice plate of CD3 antibody overnight after extraction
T cell activation is carried out, electricity turn, liposome transfection and slow-virus infection are then carried out, it is normal incubation medium that liquid is changed after 6h.
2. improving the method for CAR-T cell transfecting efficiency as described in claim 1, which is characterized in that in the step 2,
The building of CAR liposome, specifically:
In EP pipe 1: being added in CAR plasmid and 100ulOpti-MEM, 5ulP3000 is added and mixes well；
In EP pipe 2: 100ulOpti-MEM+5ul Lipo3000, pipe 1 and 2 stand 15min after mixing at room temperature.
3. improving the method for CAR-T cell transfecting efficiency as described in claim 1, which is characterized in that in the step 3, benefit
Slow virus is packed with CAR plasmid, specifically:
(1) in EP pipe 1: 5ug CAR plasmid+3.75ugPSPAX2+1.25ugPMD2.G is added in 250ulOpti-MEM, is added
12ulP3000 is mixed well, in EP pipe 2: 250ulOpti-MEM+12ul Lipo3000, after the mixing of pipe 1 and 2 at room temperature, is stood
15min；
(2) replacement 293T culture medium is serum-free Opti-MEM without double antibody or serum-free DMEM culture medium 5ml without double antibody；
(3) mixed liquor is added dropwise in 293T after 15min；7mlDMEM culture medium is changed to after 6h；
Virus is received after (4) 48.
4. improving the method for CAR-T cell transfecting efficiency as described in claim 1, which is characterized in that in the step 4, mention
PBMC cell is taken, specifically includes the following steps:
(1) PBS mixes well one times of hemodilution；
(2) 5ml Ficoll is added in 15mL centrifuge tube, 10ml blood is added, according to blood: the ratio of Ficoll=2:1, it will
Haemocyte is added slowly to the upper layer Ficoll；
(3) 500g is centrifuged 20min, 20 degrees Celsius, ACC 1, DCC 0, draws tunica albuginea confluent monolayer cells (PBMC)
(4) PBS is added, is settled to 50mL；
(5) 1700rpm is centrifuged 5min, carefully siphons away supernatant, and PBS continues washed once, PBMC cell count, carries out next step thorn
Swash；
(6) it extracts PBMC cell and is divided into tri- groups of ABC, while being coated with 24 orifice plates using CD3 antibody 20ug/ml.
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| CN109868260A (en) * | 2019-04-16 | 2019-06-11 | 上海汉尼生物细胞技术有限公司 | A kind of preparation method of CAR-T cell |
| CN110669871A (en) * | 2019-10-17 | 2020-01-10 | 河北森朗生物科技有限公司 | Method for measuring transduction titer of lentivirus |
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Application publication date: 20190308 |