CN114075545A - Preparation method of antiviral specific T cells - Google Patents
Preparation method of antiviral specific T cells Download PDFInfo
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Abstract
The invention provides a culture medium for culturing antiviral specific T cells, which comprises potassium chloride with the concentration of more than 1g/L and less than or equal to 4g/L, preferably 2g/L of X-VIVO culture medium, GT-T551 culture medium and ALYS505N-0 culture medium. Based on the culture medium, the invention also provides a culture method of the antiviral specific T cells, and in the culture process of the specific T cells, potassium chloride with the concentration of more than 1g/L and less than or equal to 4g/L is added into the culture medium. The cell product cultured by the culture medium and the culture method provided by the invention has higher ratio of anti-virus specific cells, low cost, simple and convenient operation and suitability for popularization.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a culture method of antiviral specific T cells.
Background
A virus is a noncellular organism that is small in size, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic in living cells and proliferated in a replicative manner. Viral infection causes various health problems, and general symptoms of viral infection mainly comprise systemic poisoning symptoms such as fever, headache, cough and the like and local symptoms (diarrhea, rash, liver function damage and the like) caused by inflammatory injury caused by virus hosts and invading tissues and organs. Some viruses also cause myocardial damage and viral myocarditis, depending on the symptoms of the virus.
Antiviral immunity is the immunity of the body against viruses. The immune system comprises body immune modes such as cellular immunity, humoral immunity and the like. Can effectively resist, restrain and eliminate the infection and damage of the virus to the organism. Is an important guarantee for the body to adapt to the natural environment. The antiviral specific T cells complete the elimination of the virus through cellular immunity.
The antiviral specific T lymphocyte is a subgroup of T cells, and is proliferated and differentiated under the assistance of antigen stimulation and cytokines to be activated into the T cells with specific killing activity. In the existing antiviral treatment, the treatment purpose can be achieved by directly eliminating viruses or exciting the immune response of an organism to eliminate the viruses by transferring immune cells with antiviral activity to a virus infected patient. The precondition for realizing the therapy is that a large amount of antiviral specific T cells are obtained by in vitro culture.
The effect of the specific T cell medium composition on the culture effect is still under extensive study.
Chinese patent 201810955224.6 discloses an immune cell culture medium and its application, which explores the influence of various inorganic salts such as anhydrous calcium chloride, various amino acids such as L-leucine, interleukin 2, interleukin 4 and other components on immune cell culture. However, the culture medium provided by the invention has complicated and variable component types and large component concentration range, and the specific component concentration of the culture medium with the optimal culture effect cannot be determined, thereby bringing great difficulty in culture selection.
The general concentration of potassium chloride in a general specific T cell or immune cell culture medium is lower (Chinese patent 201810955224.6: 310.5 mg/L-311.3 mg/L; Chinese patent 201711319524.7: 10-1000 mg/L; Chinese patent 201711321217.2: 10-1000 mg/L; Chinese patent 201710797665.3: 250-500mg/L), the highest concentration of potassium chloride conventionally used in the field is 1g/L, and the proportion of the anti-virus specific T cells in a cell culture final product is improved by optimizing the concentration of potassium chloride in the culture medium.
Disclosure of Invention
The invention provides a culture medium and a culture method for culturing antiviral specific T cells. The content of potassium chloride in the conventional culture medium of the antiviral specific T cells is up to 1g/L, and the invention unexpectedly discovers that the ratio of the antiviral specific cells in cell products cultured by the culture medium containing high-concentration potassium chloride is higher.
In one aspect, the invention provides a culture medium for antiviral specific T cell culture.
The concentration of potassium chloride in the antiviral specific T cell culture medium is more than 1g/L and less than or equal to 4 g/L.
Preferably, the antiviral specific T cell culture medium comprises potassium chloride with the concentration of 2 g/L.
The types of antiviral specific T cell culture media include, but are not limited to, serum-free media; the culture medium includes, but is not limited to KBM581 medium, GT-T551 medium, X-VIVO medium, GT-T505 medium, StemXVivo medium, TexMACS GMP medium, EX-CELL medium, ImmunoCult medium or ALYS505N-0 medium.
The components of the culture medium include but are not limited to: anhydrous calcium chloride, lipoic acid, sodium pyruvate, magnesium chloride, vitamins, ethanolamine, sodium chloride, nicotinamide, amino acids and antibiotics.
In some embodiments, the medium is X-VIVO medium supplemented with 2g/L potassium chloride.
In some embodiments, the medium is GT-T551 medium supplemented with 2g/L potassium chloride.
In some embodiments, the medium is ALYS505N-0 supplemented with 2g/L potassium chloride.
The culture medium can culture, but is not limited to, cytomegalovirus resistant specific T cells, hepatitis virus resistant specific T cells, leprosy virus resistant specific T cells, EB virus resistant specific T cells, polyoma virus resistant specific T cells, human papilloma virus resistant specific T cells and herpes virus resistant specific T cells.
Preferably, the culture medium also comprises human IL-2; the final concentration of the human IL-2 is 0.1 × 106-7×106U/L, preferably 1X 106U/L。
In another aspect, the invention provides a method for culturing antiviral specific T cells.
The culture method of the antiviral specific T cells comprises the following steps:
(1) blood sampling: taking peripheral blood of a patient with positive virus detection;
(2) separation: obtaining mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method;
(3) resuspending the culture solution: adding 1-4g/L potassium chloride serum-free culture solution to resuspend the mononuclear cells obtained in the step (2);
(4) culturing: and (3) inoculating the resuspended mononuclear cells into a pre-coated culture flask treated by fibronectin and anti-human CD3 antibody together for amplification to obtain a product of the antiviral specific T cells.
The patient with positive virus detection in the step (1) can be a patient with positive cytomegalovirus, hepatitis virus, leprosy virus and EB virus detection.
The step (2) further comprises a cell sorting step, wherein the cell sorting step comprises the following steps:
the obtained mononuclear cells are incubated with anti-human PD-1 antibody of IgG4 subtype, then incubated with biotinylated anti-human IgG4 antibody, added with magnetic beads coated with anti-biotin antibody for incubation, and magnetically separated to obtain PD-1 positive mononuclear cells.
Preferably, the cell sorting step is as follows:
the obtained mononuclear cells are incubated with anti-human PD-1 antibody (IgG4 subtype) for 10min at 4 ℃, then incubated with biotinylated anti-human IgG4 antibody for 15min at 25 ℃, then added with magnetic beads coated with anti-biotin antibody for incubation for 10min at 4 ℃, and then subjected to magnetic separation to obtain PD-1 positive mononuclear cells.
The concentration of the potassium chloride in the serum-free culture medium in the step (3) is preferably 2 g/L.
Preferably, the blood-free medium of step (3) is added with human IL-2; the final concentration of human IL-2 is 0.1 × 106-7×106U/L, preferably 1X 106U/L。
Preferably, in the step (4), the fibronectin content in the pre-coated culture flask is 0.5-10 μ g/mL, and the anti-human CD3 antibody content is 0.1-4 μ g/mL.
Further preferably, in the step (4), the fibronectin content in the pre-coated culture flask is 6 μ g/mL, and the anti-human CD3 antibody content is 1.5 μ g/mL.
Preferably, half of the culture solution is changed every other day during the culture in the step (4), and a new culture bottle is changed after 4 days.
Preferably, the culture time of step (4) is 10 days.
In some embodiments, the culture method further comprises a detection step to determine the proportion of anti-virus specific T cells in the final culture.
In yet another aspect, the invention also provides a product comprising antiviral specific T cells.
The product contains the antiviral specificity T cell final product obtained by the culture method.
The product types include, but are not limited to, pharmaceuticals and vaccines.
Compared with the prior culture method of specific T cells, the culture method provided by the invention is simple to operate and low in cost, and the final cell product has a higher proportion of antiviral specific cells, so that the culture method has a good industrial application prospect.
Drawings
FIG. 1 shows the flow-type assay result of the anti-cytomegalovirus specific T cell content in the final culture under the culture conditions of the culture medium containing potassium chloride of different concentrations. From this figure, the specific T cell content (percentage) of each experimental group can be judged.
FIG. 2 is a flow-type detection result of the proportion of anti-cytomegalovirus specific T cells in PD-1 positive T cell subsets under culture conditions of culture media containing different concentrations of potassium chloride. From this figure, the specific T cell content (percentage) of each experimental group can be judged.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 Effect of high concentration of Potassium chloride on the proportion of anti-Cytomegalovirus-specific T cells
(1) The screening result of HLA-A02: 01 gene is positive and peripheral blood of the patient with the cytomegalovirus positive is taken, 1 patient is related in the embodiment, the patient is the patient with the cytomegalovirus positive after the hematopoietic stem cell transplantation, and is selected from tumor hospitals in Henan province, and the experiment has obtained the patient permission.
(2) And (3) obtaining the mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method.
(3) Dividing the mononuclear cells separated in the step (2) into five groups, and respectively resuspending the five groups of cells by using the following culture solutions:
firstly, human IL-2 (final concentration of 10) was added without adding potassium chloride and serum-free medium (0g/L)6U/L)；
② adding 1g/L potassium chloride serum-free culture solution, adding human IL-2 (final concentration is 10)6U/L)；
③ 2g/L potassium chloride serum-free culture solution, and human IL-2 (final concentration is 10)6U/L)；
Fourthly, adding 3g/L potassium chloride serum-free culture solution and adding human IL-2 (the final concentration is 10)6U/L)；
Adding 4g/L potassium chloride serum-free culture solution, and adding human IL-2 (final concentration of 10)6U/L)。
Serum-free medium was purchased from Lonza corporation.
And (3) respectively inoculating the resuspended mononuclear cells into pre-coated culture flasks treated by 6 mu g/mL fibronectin and 1.5 mu g/mL anti-human CD3 antibodies for amplification, changing half of the cells every other day, changing the culture flasks after 4 days, and obtaining a product containing the anti-cytomegalovirus specific T cells after 10 days.
(4) The cell benefit obtained in the step (3)The proportion of anti-cytomegalovirus specific T cells contained therein was determined by flow cytometry. The specific method comprises the following steps: 10 of each group of cells were individually removed6Cells were washed 2 times with phosphate buffer and incubated for 30 minutes at room temperature with the addition of 10. mu.L of fluorescent tetramer (HLA-A02: 01pp65(NLVPMVATV) from MBL). Thereafter 5 μ L of a mixture of anti-human CD8 fluorescent antibody and anti-human CD3 fluorescent antibody was added and incubation continued for 20 minutes. After incubation, the cells were washed with phosphate buffer for 1 time, resuspended in phosphate buffer containing 0.5% paraformaldehyde, incubated at 4 ℃ for 1 hour and then examined by flow cytometry.
As shown in FIG. 1, the results showed that the cells cultured in the medium containing potassium chloride at a high concentration (1-3g/L) contained T cells specific to human cytomegalovirus antigen pp65(NLVPMVATV) at a higher level than the cells cultured in the medium containing potassium chloride at a lower concentration. However, the addition of higher concentrations of potassium chloride (4g/L) resulted in a decrease in the specific T cell fraction described above.
Example 2 Effect of high concentration of Potassium chloride on the proportion of anti-Virus specific T cells in a subset of PD-1 Positive T cells
(1) The peripheral blood of the patient with positive HLA-A02: 01 gene screening result and cytomegalovirus positive is taken, 1 patient is related in the embodiment, the patient is the cytomegalovirus positive patient after hematopoietic stem cell transplantation and is selected from tumor hospitals in Henan province, and the experiment has obtained patient permission.
(2) And (3) obtaining the mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method.
(3) And (3) incubating the mononuclear cells obtained in the step (2) with an anti-human PD-1 antibody (IgG4 subtype) at 4 ℃ for 10min, then incubating with a biotinylated anti-human IgG4 antibody at 25 ℃ for 15min, then adding magnetic beads coated with an anti-biotin antibody, incubating at 4 ℃ for 10min, and performing magnetic separation to obtain PD-1 positive mononuclear cells.
(4) Dividing the PD-1 positive mononuclear cells obtained in the step (2) into two groups, and respectively resuspending the two groups by using the following culture solutions:
firstly, human IL-2 (final concentration of 10) was added without adding potassium chloride and serum-free medium (0g/L)6U/L)；
② adding 2g/L potassium chlorideSerum-free culture medium, human IL-2 (final concentration of 10)6U/L) serum-free medium from Lonza.
And (3) respectively inoculating the resuspended mononuclear cells into pre-coated culture flasks treated by 6 mu g/mL fibronectin and 1.5 mu g/mL anti-human CD3 antibodies for amplification, changing half of the cells every other day, changing the culture flasks after 4 days, and obtaining a product containing the anti-cytomegalovirus specific T cells after 10 days.
(5) The proportion of anti-cytomegalovirus-specific T cells was determined as in step (4) of example 1.
The results of the relevant experiments are shown in FIG. 2, which shows that the content of specific T cells against human cytomegalovirus antigen pp65(NLVPMVATV) is higher in cells cultured in the medium with high concentration (2g/L) of potassium chloride than in the culture group without high concentration of potassium chloride.
Example 3 Effect of high concentration of Potassium chloride on the proportion of T cells specific for anti-EB Virus
(1) Peripheral blood of patients with positive HLA-A02: 01 gene screening results and positive EB virus is taken, 5 patients are involved in the experiment and selected from tumor hospitals in Henan province, and the experiment is approved by the patients.
(2) And (3) obtaining the mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method.
(3) Dividing the mononuclear cells separated in the step (2) into five groups, and respectively resuspending the five groups of cells by using the following culture solutions:
firstly, human IL-2 (final concentration of 10) was added without adding potassium chloride and serum-free medium (0g/L)6U/L)；
② adding 1g/L potassium chloride serum-free culture solution, adding human IL-2 (final concentration is 10)6U/L)；
③ 2g/L potassium chloride serum-free culture solution, and human IL-2 (final concentration is 10)6U/L)；
Fourthly, adding 3g/L potassium chloride serum-free culture solution and adding human IL-2 (the final concentration is 10)6U/L)；
Adding 4g/L potassium chloride serum-free culture solution, and adding human IL-2 (final concentration of 10)6U/L)。
Serum-free medium was purchased from Lonza corporation.
And (3) respectively inoculating the resuspended mononuclear cells into pre-coated culture flasks treated by 6 mu g/mL fibronectin and 1.5 mu g/mL anti-human CD3 antibodies for amplification, half-replacing all cells every other day, replacing the culture flasks with new ones after 4 days, and obtaining a product containing the anti-EB virus specific T cells after 10 days.
(4) And (4) detecting the proportion of the anti-EB specific T cells contained in the cells obtained in the step (3) by using a flow cytometer.
The results showed that the cells cultured in the medium containing potassium chloride at a high concentration (1 to 3g/L) had a higher content of T cells specific to the EB virus antigen than the cells cultured in the medium containing potassium chloride at a lower concentration.
Claims (10)
1. An antiviral specific T cell culture medium, wherein the concentration of potassium chloride in the culture medium is greater than 1g/L and less than or equal to 4 g/L.
2. The antiviral specific T cell culture medium of claim 1, wherein the culture medium comprises potassium chloride at a concentration of 2 g/L.
3. The antiviral specific T cell culture medium of claim 1, wherein the culture medium is a serum-free medium; the culture medium is KBM581 culture medium, GT-T551 culture medium, X-VIVO culture medium, GT-T505 culture medium, StemXVivo culture medium, TexMACS GMP culture medium, EX-CELL culture medium, ImmunoCult culture medium or ALYS505N-0 culture medium.
4. The antiviral specific T cell culture medium of claim 1, wherein the antiviral specific T cell is an anti-cytomegalovirus specific T cell, an anti-hepatitis virus specific T cell, an anti-leprosy virus specific T cell, an anti-epstein barr virus specific T cell, an anti-polyoma virus specific T cell, an anti-human papilloma virus specific T cell, or an anti-herpes virus specific T cell.
5. A method for culturing antiviral specific T cells, comprising the steps of:
(1) blood sampling: taking peripheral blood of a patient with positive virus DNA/RNA detection or serological detection result;
(2) separation: obtaining mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method;
(3) resuspending the culture solution: resuspending the mononuclear cells obtained in the step (2) in a serum-free culture solution, wherein the serum-free culture solution contains potassium chloride with the concentration of more than 1g/L and less than or equal to 4 g/L;
(4) culturing: and (3) inoculating the resuspended mononuclear cells into a pre-coated culture flask treated by fibronectin and anti-human CD3 antibody together for amplification to obtain a product of the antiviral specific T cells.
6. The method for culturing T cells with antiviral specificity as claimed in claim 5, wherein the patient with positive virus detection in step (1) is a patient with positive detection of cytomegalovirus, hepatitis virus, leprosy virus, Epstein-Barr virus, polyoma virus, human papillomavirus or herpes virus.
7. The method for culturing T cells with antiviral specificity as claimed in claim 5, wherein the blood-free medium of step (3) is supplemented with human IL-2; the final concentration of human IL-2 is 0.1 × 106-7×106U/L。
8. The method of claim 5, wherein the amount of fibronectin and anti-human CD3 antibody in the pre-coated flasks in step (4) is 0.5-10 μ g/mL and 0.1-4 μ g/mL, respectively.
9. The method of claim 5, wherein the step (2) further comprises a cell sorting step, said cell sorting step comprising:
the obtained mononuclear cells are incubated with anti-human PD-1 antibody of IgG4 subtype, then incubated with biotinylated anti-human IgG4 antibody, added with magnetic beads coated with anti-biotin antibody for incubation, and magnetically separated to obtain PD-1 positive mononuclear cells.
10. An antiviral specific T cell-containing product comprising the antiviral specific T cell produced by the culture method according to any one of claims 5 to 9.
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| CN106148278A (en) * | 2016-08-08 | 2016-11-23 | 安徽惠恩生物科技股份有限公司 | A kind of mescenchymal stem cell culture medium |
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