CN113957046A - Medium and application for lentiviral infection of T cells - Google Patents
Medium and application for lentiviral infection of T cellsDownload PDFInfo
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Abstract
Translated fromChineseDescription
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium for T cell lentivirus infection and application thereof, and more particularly relates to a culture medium for T cell lentivirus infection and a method for infecting T cells by lentivirus.
Background
At present, the difficulty of T cell infection is very high, the efficiency of lentivirus infection of T cells is limited by virus titer and culture medium conditions, and the difficulty of realizing high-efficiency lentivirus infection is very high. After the T cells are activated by anti-CD3/CD28 antibody magnetic beads, the lentivirus infection efficiency is extremely low (less than 5%) when the T cells are cultured in a common T cell culture medium (only IL2 or IL-7, IL-15 and IL-21 are added).
Therefore, methods that can effectively improve the efficiency of viral infection are still in need of further development.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, aiming at the problem of low infection efficiency in the prior art, the inventor of the application adds infection promoting components (human serum and serum substitutes) into a T cell serum-free culture medium, searches the concentration of additives and an infection method, effectively improves the T cell infection efficiency of lentivirus, and forms a novel method for improving the T cell infection efficiency.
In a first aspect of the invention, the invention provides a culture medium for lentiviral infection of T cells. According to an embodiment of the invention, the medium comprises a T cell basal medium and human serum. The inventor finds that the effect of adding human serum to the culture medium is more remarkable in the aspect of improving the efficiency of infecting T cells by lentivirus compared with adding fetal bovine serum. The culture medium provided by the embodiment of the invention is used for lentivirus infected T cells, and greatly improves the virus infection efficiency on the premise of not increasing the virus titer (virus amount).
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to an embodiment of the present invention, the human serum is prepared by the following method: human whole blood was centrifuged at 1000g at 4 ℃ for 10 minutes to obtain a supernatant, which was human serum.
According to an embodiment of the invention, the medium further comprises serum replacement; preferably, the serum replacement is CTSTMImmune Cell serum replacement. The inventors found that the infection efficiency of the virus was further improved by further containing a serum replacement in the medium.
According to an embodiment of the invention, the volume fraction of the human serum in the culture medium is between 1.3% and 2.6%.
According to an embodiment of the invention, the serum replacement is present in the culture medium in a volume fraction of 1% to 2%.
The culture medium according to the embodiment of the present invention, in which the content of human serum or serum replacement is within the above range, can further improve the efficiency of T cell infection by lentivirus.
According to an embodiment of the invention, the volume ratio of the human serum to the serum substitute is (1-1.5): 1, preferably in a volume ratio of 1.3: 1.
the term "T cell basal medium" as used herein refers to a medium that can be used for T cell culture, and can be purchased commercially or prepared by itself. According to a specific embodiment of the present invention, the T cell basal Medium includes IMDM (Iscove's Modified Dulbecco's Medium) Medium, Human Serum Albumin (HSA), transferrin, insulin, unsaturated fatty acids, and cholesterol.
According to a specific embodiment of the invention, the content of the human serum albumin in the T cell basic culture medium is 1.3-1.8 g/L, the content of the transferrin in the T cell basic culture medium is 38-42 mg/L, the content of the insulin in the T cell basic culture medium is 0.1-10 mg/L, the content of the unsaturated fatty acid in the T cell basic culture medium is 5-50 μmol/L, and the content of the cholesterol in the T cell basic culture medium is 55-70 g/L.
According to a specific embodiment of the invention, the IMDM basal medium, HSA, transferrin, insulin, unsaturated fatty acids, cholesterol is provided as the tertiary medium CART-505 product.
According to an embodiment of the invention, the culture medium further comprises hIL7, hIL15 and hIL21, optionally, the concentrations of hIL7, hIL15 and hIL21 in the culture medium are respectively and independently 38-42U/mL, preferably 40U/mL.
In a second aspect of the invention, the invention features a method for lentivirus infection of T cells. According to an embodiment of the invention, the method comprises: activating and culturing the T cells in the culture medium; transfecting the activated T cells with lentiviruses in the presence of a transfection mixture based on the medium described above; lentiviral-transfected T cells were incubated and expanded. According to the method provided by the embodiment of the invention, the virus preparation cost is greatly saved, the virus infection efficiency is high, the operation is simple, and the cell survival rate after infection is higher and is more than 90%.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to an embodiment of the invention, the activation culture is performed by: t cells were included at 10 per ml of medium6The density of each cell is inoculated in a culture well plate; the inoculated T cells were subjected to primary culture in the presence of Dynabeads Human T-Activator CD3/CD 28.
Specifically, the first culture is at 37 ℃, 5% CO2And performing the reaction for 24 to 28 hours under the condition.
Specifically, the dosage ratio of the T cells to Dynabeads Human T-Activator CD3/CD28 is 106And (2) cell: 25 μ L.
Specifically, the T cells after the first culture are subjected to a second culture for 24 to 28 hours under a condition of a double amount of medium. Here, "two-fold amount of medium" means that the amount of medium used in the second culture is 2 times the amount of medium used in the first culture.
According to an embodiment of the present invention, the transfection mixture contains 0.32. mu.L polybrene per 300. mu.L matrix.
According to an embodiment of the invention, the lentivirus is provided in the form of a lentivirus liquid, 100 μ L of which is coupled to 10 of the activated lentivirus liquid per 300 μ L of matrix6Individual T cells were transfected with lentiviral fluid at an MOI of 1-2.
According to an embodiment of the present invention, after the transfection and before the incubation, the method further comprises centrifuging the lentivirus-transfected T cells for 40min at a rotation speed of 1000 g. The inventors have found that centrifugation of lentivirus-transfected T cells under the above conditions further increases the efficiency of lentivirus infection of T cells.
According to an embodiment of the invention, the incubation and expansion culture of the lentivirus-transfected T cells is performed in an expansion medium comprising a T cell basal medium and hIL7, hIL15, hIL 21.
Specifically, the T cell basal medium includes IMDM medium, human serum albumin, transferrin, insulin, unsaturated fatty acids, and cholesterol.
Specifically, the content of the human serum albumin in the T cell basic culture medium is 1.3-1.8 g/L, the content of the transferrin in the T cell basic culture medium is 38-42 mg/L, the content of the insulin in the T cell basic culture medium is 0.1-10 mg/L, the content of the unsaturated fatty acid in the T cell basic culture medium is 5-50 mu mol/L, and the content of the cholesterol in the T cell basic culture medium is 55-70 g/L. The concentrations of the hIL7, hIL15 and hIL21 in the amplification medium are respectively and independently 38-42U/mL, preferably 40U/mL.
According to a particular embodiment of the invention, the expansion medium is T cell Medium-2.
According to an embodiment of the invention, the incubation treatment time is 15-24 h.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a graph of the results of flow assay CAR + ratios under different lentivirus-infected T cell conditions according to an embodiment of the present invention; and
FIG. 2 shows the CAR + ratio results of flow assay after lentivirus infection of T cells in a medium containing 5% FBS or 1% SR according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
According to an embodiment of the present invention, the method for infecting T cells with lentiviruses proposed by the present invention comprises the following steps:
1. t cell activation culture
(1) Counting freshly isolated T cells or resuscitated T cells (hemacytometer or AO/PI fluorescence count);
(2) using T cell culture medium-1 (the concentration of serum substitute and human serum added in T cell basic culture medium is 1% -2%, 1.3% -2.6%, the concentration of hIL7, hIL15 and hIL21 are 40U/ml) to 106cells/mL were plated in culture well plates at a density of 25. mu.L/10, with addition of Dynabeads Human T-Activator CD3/CD28, which promotes T cell expansion and activation6cells，37℃，5％CO2Culturing in an incubator.
The T cell basic culture medium comprises an IMDM culture medium, Human Serum Albumin (HSA), transferrin, insulin, unsaturated fatty acid and cholesterol, wherein the content of the human serum albumin in the T cell basic culture medium is 1.3-1.8 g/L, the content of the transferrin in the T cell basic culture medium is 38-42 mg/L, the content of the insulin in the T cell basic culture medium is 0.1-10 mg/L, the content of the unsaturated fatty acid in the T cell basic culture medium is 5-50 mu mol/L, and the content of the cholesterol in the T cell basic culture medium is 55-70 g/L.
(3) The next day of activation, one time of T cell medium-1 was added (serum replacement and human serum were added to the T cell basal medium at concentrations of 1% -2%, 1.3% -2.6%, respectively, and all of hIL7, hIL15, and hIL21 were 40U/ml).
2. Infection of T cells
(1) On the third day of cell activation, cells were collected and placed on a DynaMag-15 magnetic frame for 2min, and the magnetic beads were removed.
(2) Collecting the cell suspension without the magnetic beads, 300g, normal temperature (RT), 5min, centrifuging and collecting the cells.
(3) The following virus infection systems were formulated in 24-well plates: 1 × 10^6 living cells +0.32 μ L polybrene (mother liquor concentration 10 μ g/μ L) +300 μ L T cell culture medium-1 (T cell basal medium with serum replacement and human serum added as well as hIL7, hIL15 and hIL21, serum replacement and human serum concentrations of 1% -2%, 1.3% -2.6%, hIL7, hIL15 and hIL21 concentrations were 40U/ml) + virus solution (volume 100 μ L, MOI 1-2).
The culture plate is centrifuged for 40min at normal temperature by using a horizontal rotary head centrifuge of 1000g, and then is placed in an incubator to be cultured overnight.
After 15-24h incubation of cells with virus, cells were collected, 300g, RT, 5min, counted by centrifugation and counted at 10%6Inoculation was carried out at a density of/ml, the medium being T cell medium-2 (T cell basal medium and hIL7, hIL15 and hIL21, hIL7, hIL15 and hIL21 all at a concentration of 40U/ml).
3. Detection of T cell lentivirus infection efficiency
(1) On the third day of virus infection, cells were collected, 300g, RT, 10min, and counted by centrifugation.
(2) A portion of the cells were collected and the expression of the lentiviral target protein was detected by flow assay.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1
First, experimental material
Cell: human PBMC isolated T cells were depleted of beads 3 days after stimulation with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation.
Reagent: cart medium (tertiary nip)Ocean CART-505) (used as T cell basal Medium in this example), Dynabeads Human T-Activator CD3/CD28, PBS, DAPI, Human IL-21, Human IL-7, Human IL-15, polybrene, GlutaMAX-1(100X), serum replacement CTSTMImmune Cell SR, homemade human serum (freshly collected human whole blood was centrifuged at 4 ℃ for 10 minutes at 1000g to collect the supernatant, i.e. the obtained homemade human serum);
equipment: cell culture box (Thermo 150i), clean bench (Suzhou Antai), Beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), 4 ℃ refrigerator (Hill HYC390), -20 ℃ refrigerator (Hill), negative pressure aspirator (fish jump), pipette (Eppendorf), BD flow cytometer, ice maker
The T cell culture medium-1 used in the following experiments comprises a T cell basic culture medium, serum, hIL7, hIL15 and hIL21, wherein the concentrations of hIL7, hIL15 and hIL21 are 40U/ml, and the serum is human serum with the concentration of 1.3%; or human serum at a concentration of 2.6%; or serum replacement and human serum at concentrations of 1%, 1.3%, respectively; or serum replacement and human serum at concentrations of 2%, 2.6%, respectively.
The T cell culture medium-2 comprises a T cell basic culture medium and hIL7, hIL15 and hIL21, wherein the concentrations of hIL7, hIL15 and hIL21 are all 40U/ml.
Second, Experimental methods
Experimental groups: t cells are cultured in CART culture media added with infection promoting components with different concentrations, and then are infected by lentivirus.
Control group: culturing T cells in CART culture medium added with infection promoting component, and infecting with lentivirus
1. T cell activation culture
(1) Counting freshly isolated T cells or resuscitated T cells (hemacytometer or AO/PI fluorescence count);
(2) experimental groups used 5ml of T cell medium-1 at 5 x 106cell T density in culture well plate, at the same time add 125. mu.L Dynabeads Human T-Activator CD3/CD28, 37 ℃, 5% CO2Culturing in an incubator. The control group was cultured with the same cell amount using volume of T cell medium-2, and other conditions were consistent;
(3) the following day of activation, the experimental groups were supplemented with 5ml of T cell Medium-1. The control group was supplemented with the same volume of T cell medium-2.
2. Infection of T cells
(1) On the third day of cell activation, cells were collected and placed on a DynaMag-15 magnetic frame for 2min, and the magnetic beads were removed.
(2) And (5) centrifuging the cell suspension after removing the magnetic beads for 5min at 300 g.
(3) Experimental groups the following virus infection systems were formulated in 24-well plates: 1*106Viable cells +0.32 μ L polybrene (stock solution concentration 10 μ g/μ L) +300 μ L T cell culture medium-1 + virus solution (MOI ═ 2) (Multiplicity of infection ═ number of virus/number of cells to be infected). T cell culture medium-2 is used when the control group T cells are infected, and other conditions are consistent;
(4) plates were incubated overnight at 1000g, RT, centrifuged for 40min and placed in an incubator.
(5) After the cells and the viruses are incubated for 15h, a culture medium is blown and beaten to form a cell suspension, the cell suspension is centrifuged, the cells are collected, the cell suspension is treated with 300g and RT for 5min, the cell suspension is centrifuged and counted, the cell suspension is inoculated at the density of 10^6/ml, and the culture medium of an experimental group is a T cell culture medium-2.
(6) Onday 2 of infection, cells were collected, 300g, RT, 5min, counted by centrifugation, and seeded at a density of 10^6/ml, culture medium being T cell culture medium-2.
3. T cell lentivirus infection efficiency assay (CAR membrane protein molecule assay)
The third day after the cells are infected with the virus, the cells are collected, and 1 x 10^6cells are respectively taken from the experimental group and the control group, and are centrifugally washed 2 times by PBS 1 ml/time and 300g 5 min.
0.5 x 10^6cells were stained in each group, 1. mu.L protein L (1mg/ml) was added to 100. mu.L of the stain buffer again, and after 1 hour of staining at 4 ℃, the cells were collected, washed 2 times with 1ml of the stain buffer and 300g for 5min, and resuspended in 200. mu.L of the stain buffer for flow analysis. The non-staining group followed the staining group except that Protein L was not added. Meanwhile, T cells were stained as a staining control. Flow detection was performed in a BD flow cytometer.
Third, conclusion of experiment
FIG. 1 is the CAR + ratio of cells after different approaches to T cell infection. The higher the CAR + ratio, the better the infection was shown. The infection efficiency is shown to be as follows from big to small: experimental group-2 (20.05%) > experimental group-1 (18.16%) > experimental group-4 (14.63%) > experimental group-3 (14.52%), from which the following conclusions were drawn: in the aspect of infection efficiency, the efficiency of adding human serum and a serum substitute is higher than the efficiency of adding human serum, the efficiency of adding human serum is higher than the efficiency of adding no human serum and serum substitute, the concentration of the added human serum and the serum substitute is not higher and better, the concentration of the added human serum and the serum substitute is respectively in the range of 1.3% -2.6% and 1% -2%, and the concentration ratio of the added human serum and the serum substitute is 1.3: 1, the virus infection efficiency is higher.
The novel infection method effectively improves the lentivirus infection efficiency of T cells.
Example 2
First, experimental material
Cell: human PBMC isolated T cells were depleted of beads 3 days after stimulation with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation.
Reagent: CART medium (tertiary CART-505) (as T cell basal medium in this example), Dynabeads Human T-Activator CD3/CD28, PBS, DAPI, Human IL-21, Human IL-7, Human IL-15, polybrene, GlutaMAX-1(100X), fetal bovine serum, serum replacement CTSTMImmune Cell SR, self-made human serum;
equipment: cell culture box (Thermo 150i), clean bench (Suzhou Antai), Beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), 4 ℃ refrigerator (Hill HYC390), -20 ℃ refrigerator (Hill), negative pressure aspirator (fish jump), pipette (Eppendorf), BD flow cytometer, ice maker
The T cell culture medium-1 used in the following experiments comprises a T cell basic culture medium, serum, hIL7, hIL15 and hIL21, wherein the concentrations of hIL7, hIL15 and hIL21 are all 40U/ml, and the serum is fetal bovine serum with the concentration of 5%; or serum replacement at a concentration of 1%; or serum replacement and human serum at concentrations of 1%, 1.3%, respectively.
The T cell culture medium-2 comprises a T cell basic culture medium and hIL7, hIL15 and hIL21, wherein the concentrations of hIL7, hIL15 and hIL21 are all 40U/ml.
Second, Experimental methods
Experimental groups: t cells are cultured in CART culture media added with infection promoting components with different concentrations, and then are infected by lentivirus.
Control group: culturing T cells in CART culture medium added with infection promoting component, and infecting with lentivirus
1. T cell activation culture
(1) Counting freshly isolated T cells or resuscitated T cells (hemacytometer or AO/PI fluorescence count);
(2) the experimental group used 5ml of T cell culture medium-1 at a density of 5 × 10^6cells T in culture well plates, and 125 μ L of Dynabeads Human T-Activator CD3/CD28, 37 ℃, 5% CO2Culturing in an incubator. The control group was cultured with the same cell amount using volume of T cell medium-2, and other conditions were consistent;
(3) the following day of activation, the experimental groups were supplemented with 5ml of T cell Medium-1. The control group was supplemented with the same volume of T cell medium-2.
2. Infection of T cells
(1) On the third day of cell activation, cells were collected and placed on a DynaMag-15 magnetic frame for 2min, and the magnetic beads were removed.
(2) And (5) centrifuging the cell suspension after removing the magnetic beads for 5min at 300 g.
(3) Experimental groups the following virus infection systems were formulated in 24-well plates: 1 × 10^6 viable cells +0.32 μ L polybrene (mother liquor concentration 10 μ g/μ L) +100 μ L T cell culture medium-1 + virus fluid (MOI ═ 2). T cell culture medium-2 is used when the control group T cells are infected, and other conditions are consistent;
(4) plates were incubated overnight at 1000g, RT, centrifuged for 40min and placed in an incubator.
(5) After the cells and the virus are incubated for 15h, the cells are collected, 300g is performed at RT for 5min, the cells are centrifugally counted and inoculated at the density of 10^6/ml, and the experimental group culture medium is a T cell culture medium-2.
(6) Onday 2 of infection, cells were collected, 300g, RT, 5min, counted by centrifugation, and seeded at a density of 10^6/ml, culture medium being T cell culture medium-2.
3. T cell lentivirus infection efficiency assay (CAR membrane protein molecule assay)
The third day after the cells are infected with the virus, the cells are collected, and 1 x 10^6cells are respectively taken from the experimental group and the control group, and are centrifugally washed 2 times by PBS 1 ml/time and 300g 5 min.
0.5 x 10 staining of each group6After cells were stained with 1. mu.L of protein L (1mg/ml) in 100. mu.L of the stain buffer at 4 ℃ for 1 hour, the cells were collected, centrifuged and washed 2 times at 1ml of the stain buffer at 300g for 5min, and resuspended in 200. mu.L of the stain buffer for flow analysis. The non-staining group followed the staining group except that Protein L was not added. Meanwhile, T cells were stained as a staining control. Flow detection was performed in a BD flow cytometer.
Third, conclusion of experiment
FIG. 2 is the CAR + ratio of cells after different approaches to T cell infection. The higher the CAR + ratio, the better the infection was shown. The result shows that the cell infection efficiency is not obviously improved by adding 5 percent fetal calf serum into the culture medium; 1% SR increased the lentivirus infection efficiency of T cells, but did not reach higher levels.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A culture medium for T cell lentivirus infection is characterized by comprising a T cell basic culture medium and human serum.
2. The culture medium according to claim 1, wherein the human serum is prepared by the following method: centrifuging human whole blood at 1000g for 10 minutes at 4 ℃ to obtain a supernatant, wherein the supernatant is human serum;
optionally, the volume fraction of the human serum in the culture medium is 1.3% -2.6%.
3. The culture medium of claim 1, further comprising a serum replacement; preferably, the serum replacement is CTSTMImmune Cell serum replacement;
optionally, the serum replacement is present in the culture medium at a volume fraction of 1% to 2%.
4. The culture medium according to claim 3, wherein the volume ratio of the human serum to the serum replacement is (1-1.5): 1, preferably in a volume ratio of 1.3: 1.
5. the culture medium of claim 1, wherein the T cell basal medium comprises IMDM medium, human serum albumin, transferrin, insulin, unsaturated fatty acids, and cholesterol;
optionally, the content of the human serum albumin in the T cell basic culture medium is 1.3-1.8 g/L, the content of the transferrin in the T cell basic culture medium is 38-42 mg/L, the content of the insulin in the T cell basic culture medium is 0.1-10 mg/L, the content of the unsaturated fatty acid in the T cell basic culture medium is 5-50 mu mol/L, and the content of the cholesterol in the T cell basic culture medium is 55-70 g/L;
optionally, the IMDM basal medium, transferrin, insulin, unsaturated fatty acids, cholesterol are provided as the tertiary media CART-505 product.
6. The culture medium according to any one of claims 1 to 5, wherein the culture medium further comprises hIL7, hIL15 and hIL21, and optionally the concentration of each of the hIL7, hIL15 and hIL21 in the culture medium is independently 38 to 42U/mL, preferably 40U/mL.
7. A method of lentivirus infection of T cells comprising:
performing activation culture on T cells in a culture medium of any one of claims 1-6;
transfecting lentivirus into activated T cells in the presence of a transfection mixture based on a medium according to any one of claims 1 to 6;
lentiviral-transfected T cells were incubated and expanded.
8. The method of claim 7, wherein the activation culture is performed by:
t cells were included at 10 per ml of medium6The density of each cell is inoculated in a culture well plate;
subjecting the inoculated T cells to a first culture in the presence of Dynabeads Human T-Activator CD3/CD28, optionally at 37 ℃ and 5% CO2The reaction is carried out for 24-28 hours under the condition, and the optional dosage ratio of the T cells to Dynabeads Human T-Activator CD3/CD28 is 106And (2) cell: 25 mu L of the solution;
optionally, the T cells after the first culture are subjected to a second culture for 24-28 hours under the condition of twice the amount of the culture medium.
9. The method of claim 7, wherein the transfection mixture comprises 0.32 μ L polybrene per 300 μ L matrix;
optionally, the lentivirus is provided in the form of a lentivirus fluid, 100 μ L of which is administered per 300 μ L of matrix to 10 of the activated lentivirus fluid6Performing transfection treatment on the T cells, wherein the MOI of the lentivirus solution is 1-2;
preferably, the method further comprises centrifuging the lentivirus-transfected T cells for 40min at a rotation speed of 1000g after the transfection treatment and before the incubation treatment.
10. The method of claim 9, wherein the incubating and expanding the lentivirus-transfected T cells is performed in an expanding medium comprising a T cell basal medium and hIL7, hIL15, hIL 21;
optionally, the T cell basal medium comprises IMDM medium, human serum albumin, transferrin, insulin, unsaturated fatty acids, and cholesterol;
optionally, the content of the human serum albumin in the T cell basal medium is 1.3-1.8 g/L, the content of the transferrin in the T cell basal medium is 38-42 mg/L, the content of the insulin in the T cell basal medium is 0.1-10 mg/L, the content of the unsaturated fatty acid in the T cell basal medium is 5-50 μmol/L, the content of the cholesterol in the T cell basal medium is 55-70 g/L, and the concentrations of the hIL7, the hIL15 and the hIL21 in the amplification medium are respectively and independently 38-42U/mL, preferably 40U/mL;
preferably, the incubation treatment time is 15-24 h.
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