CN114231488A - Culture solution for in vitro culture of TH1 cells and application thereof, and in vitro culture method of TH1 cells - Google Patents
Culture solution for in vitro culture of TH1 cells and application thereof, and in vitro culture method of TH1 cells Download PDFInfo
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Abstract
The invention relates to the field of immune cell therapy, in particular to a culture solution for culturing TH1 cells in vitro and application thereof, and an in vitro culture method for TH1 cells. The invention adds monoclonal antibody CD3, monoclonal antibody CD28 and CpG-A ODN, vitamin A, heparin, gamma-secretase inhibitor DAPT, allicin, IL-18, IL-12 and IL-2 at the same time when starting to culture cells, and can induce TH1 cells to activate and proliferate; the serum-free culture medium containing heparin, IL-18, IL-12 and IL-2 is continuously added in the culture process, so that the growth and proliferation of TH1 cells can be further promoted, the number of TH1 cells is increased, the expanded TH cells maintain good cell activity, and the clinical value of TH1 cells is improved. The culture solution and the corresponding culture method provided by the invention can effectively improve the quantity and quality of TH1 cells cultured in vitro.
Description
Technical Field
The invention relates to the field of immune cell therapy, in particular to a culture solution for culturing TH1 cells in vitro and application thereof, and an in vitro culture method for TH1 cells.
Background
Helper T cell (Th) 1 is used as the earliest discovered CD4+ helper T cell subgroup, has the functions of promoting cell immunity and enhancing anti-tumor, and its proliferation and differentiation are regulated by tumor microenvironment and various immune cells to participate in various tumor immunity.
Th1 cells mainly secrete IL-2, IL-12, IFN-gamma and TNF-and the like, mediate immune responses related to cytotoxicity and local inflammation, participate in cellular immunity and formation of delayed type hypersensitivity inflammation, are also called inflammatory T cells and can be regarded as TDTH cells. Th1 cells play an important role in combating intracellular pathogen (viral, bacterial and parasitic) infections. Upon intracellular bacterial infection, Th1 cells preferentially develop and elicit phagocyte-mediated host defense responses. Th1 cells have strong persistent response, and may be associated with organ-specific autoimmune disease, contact dermatitis, chronic inflammatory diseases with unknown cause, delayed hypersensitivity, etc.
It has been shown that the differentiation of TH1 cells is influenced by a variety of factors, such as transcription factors including activator of transcription (STAT)4, T-beta, Interferon Regulatory Factor (IRF)1, such as IL-12, Interferon (IFN) -gamma, IL-27, and co-stimulatory molecules, which are important regulators of Th1 cell differentiation.
Currently, the prior art generally obtains TH1 cells in two different ways: one is to sort out some of the T cells (e.g., Naive CD4+ T cells) by magnetic bead separation or flow cytometry (FACS), and then to activate these cells in vitro and differentiate them into specific Th1 subpopulations, which, while yielding higher purity Th1 cells, is costly and presents a risk for subsequent use in clinical treatment due to the use of magnetic beads or flow sorting; secondly, PBMC is directly separated from peripheral blood, anti-CD 3 and anti-CD 28 antibodies are added, and IL-12 induces TH1 cells, the method is simple and convenient to operate, but the obtained TH1 cells have low purity and are difficult to meet the requirement of clinical treatment.
Disclosure of Invention
In order to solve the problems, the invention provides a culture solution for culturing TH1 cells in vitro and an application thereof, and an in vitro culture method of TH1 cells. The culture solution and the matched culture method provided by the invention can effectively improve the purity, quantity and safety of TH1 cells obtained by in vitro culture.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a culture solution for culturing TH1 cells in vitro, which comprises a culture solution A and a culture solution B;
the A culture solution comprises the following components in concentration: 3500-1000 ng/mL of monoclonal antibody CD, 28500-1000 ng/mL of monoclonal antibody CD, 0.05-3 nmol/mL of CpG-A ODN, 0.5-2 mu mol/mL of vitamin A, 20-100 nmol/mL of gamma-secretase inhibitor DAPT, 50-150 ng/mL of allicin, 10-100 IU/mL of heparin, 1810-50 IU/mL of IL-1250-200 IU/mL of IL-2100-1000 IU/mL of IL-A;
the culture solution B comprises a serum-free culture medium and effective substances, wherein the effective substances comprise the following components in concentration: 10-100 IU/mL of heparin, 1810-50 IU/mL of IL-1250-200 IU/mL of IL-2100-1000 IU/mL of IL-.
Preferably, the serum-free medium comprises ALyS 505N-0.
The invention also provides application of the culture solution in culturing TH1 cells in vitro.
The invention provides an in vitro culture method of TH1 cells, which comprises the following steps:
preparing peripheral blood mononuclear cells into cell suspension by using a serum-free culture medium, mixing the cell suspension with the culture solution A to obtain a mixture a, and beginning to culture, wherein the mixture a is marked as Day 0;
mixing the mixture a with the culture solution B as described in claim 1 or 2 when Day3 to obtain a mixture B;
mixing the mixture B with the culture solution B according to claim 1 or 2 when Day5 to obtain a mixture c;
mixing the mixture c with the culture solution B according to claim 1 or 2 when Day7 to obtain a mixture d;
TH1 cells were harvested from mixture d at Day 9.
Preferably, the cell density of the peripheral blood mononuclear cells in the cell suspension is (1.0-1.5) multiplied by 106one/mL.
Preferably, in the mixture b, the mixture c and the mixture d, the density of the cells is more than or equal to 0.5X 106one/mL.
Preferably, the temperature of the culture is 37 ℃ and the concentration of carbon dioxide is 5%.
Preferably, the cell density of peripheral blood mononuclear cells in the cell suspension is (1.0-1.5) multiplied by 106one/mL.
The invention also provides application of the in vitro culture method of the TH1 cell in rapidly culturing the TH1 cell.
Preferably, the TH1 cells harvested by the culture method at Day 9 have the proliferation multiple of 228-235 for TH1 cells and the percentage of cells which are double positive for CD4+ IFN-gamma + is 71.5% -73.4%.
The invention adds monoclonal antibody CD3, monoclonal antibody CD28 and CpG-A ODN, vitamin A, heparin, gamma-secretase inhibitor DAPT, allicin, IL-18, IL-12 and IL-2 at the same time when starting to culture cells, and can induce TH1 cells to activate and proliferate; the serum-free culture medium (namely B culture solution) containing heparin, IL-18, IL-12 and IL-2 is continuously added in the culture process, so that the growth and proliferation of TH1 cells can be further promoted, the number of TH1 cells is increased, the expanded TH cells keep better cell activity, and the clinical value of TH1 cells is improved. The embodiment proves that the culture solution and the corresponding culture method provided by the invention can effectively improve the quantity and quality of TH1 cells cultured in vitro.
Drawings
FIG. 1 is a flow chart of the detection of the CD4+ IFN-. gamma. + phenotype by day 9 of the in vitro culture of TH1 cells in example 1;
FIG. 2 is a flow chart of the detection of the CD4+ IFN-. gamma. + phenotype by day 9 of the in vitro culture of TH1 cells in example 2;
FIG. 3 is a flow chart of the detection of the CD4+ IFN-. gamma. + phenotype by day 9 of the in vitro culture of TH1 cells in example 3;
FIG. 4 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by day 9 of in vitro culture of TH1 cells in comparative example 1;
FIG. 5 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by day 9 of in vitro culture of TH1 cells in comparative example 2;
FIG. 6 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by day 9 of in vitro culture of TH1 cells in comparative example 3;
FIG. 7 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by day 9 of in vitro culture of TH1 cells in comparative example 4;
FIG. 8 is a flow chart of the detection of the CD4+ IFN-. gamma. + phenotype by day 9 of the in vitro culture of TH1 cells in comparative example 5;
FIG. 9 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by day 9 of in vitro culture of TH1 cells in comparative example 6;
FIG. 10 is a flow chart of the detection of CD4+ IFN-. gamma. + phenotype by TH1 cells cultured in vitro on day 9 in comparative example 7.
Detailed Description
The present invention will be further described with reference to the following embodiments. It should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
The experimental environment, experimental materials and instrument equipment which need to be prompted and explained in the invention are as follows:
1. the experimental environment is as follows: operating in an ultra clean bench in a laboratory in a GMP environment.
2. Reagent: phosphate buffer DPBS (Biotech company, Kyoho), ALyS505N-0 (CSTI, Kyoho institute of cell science), human lymphocyte separation, heparin sodium blood collection tube, CD3 monoclonal antibody, CD28 monoclonal antibody, CpG-A ODN, vitamin A, gamma-secretase inhibitor DAPT, allicin, heparin, IL-18, IL-12, IL-2.
3. Instruments and equipment: centrifuge (Thermo, USA), T75 suspension culture bottle, T175 suspension culture bottle, NIPRO cell culture bag (NIPRO corporation, Japan), and CO2Incubator (Sanyo, China) and super clean bench (Zhijing, China).
Example 1
A method of culturing TH1 cells in vitro, comprising the steps of:
(1) obtaining PBMC by density gradient centrifugation, and culturing in serum-free cell culture mediumAlyS505N-0 was prepared as a mononuclear cell suspension at a cell concentration of 1.0X 106one/mL.
(2) Adding 20mL of the cell suspension prepared in the step (1) into a T75 culture bottle, adding monoclonal antibody CD 3500 ng/mL, monoclonal antibody CD 28500 ng/mL, CpG-A ODN 0.05nmol/mL, vitamin A1 μmol/mL, gamma-secretase inhibitor DAPT 30nmol/mL, allicin 50ng/mL, heparin 50IU/mL, IL-1820 IU/mL, IL-12100 IU/mL and IL-2500 IU/mL into the culture bottle, and adding 5% CO at 37 ℃ to obtain a mixture2The incubator of (2) for cultivation.
(3) After inoculating cells on DAY0, DAY3 was supplemented with AlyS505N-0 medium containing heparin 50IU/mL, IL-1820 IU/mL, IL-12100 IU/mL, IL-2500 IU/mL;
DAY5 adding AlyS505N-0 culture medium containing heparin 50IU/mL, IL-1820 IU/mL, IL-12100 IU/mL and IL-2500 IU/mL;
DAY7 was supplemented with AlyS505N-0 medium at 50IU/mL, IL-1820 IU/mL, IL-12100 IU/mL, and IL-2500 IU/mL heparin.
The cell density was kept at 1.0X 10 after each fluid infusion6one/mL. At 37 ℃ 5% CO2Culturing in an incubator.
(4) Mature TH1 cells are harvested when the cells are cultured to the 9 TH day, the number of the cells is sampled and detected, the proliferation multiple is calculated, the purity of TH1 cells is detected by flow detection, and the detection result is shown in Table 1.
TABLE 1 fold increase of TH1 cells in example 1 and flow assay results
Example 2
A method of culturing TH1 cells in vitro, comprising the steps of:
(1) obtaining PBMC by density gradient centrifugation, preparing mononuclear cell suspension with serum-free cell culture medium AlyS505N-0, and making into cell suspension with cell concentration of 1.3 × 106one/mL.
(2) Adding 20mL of the cell suspension prepared in the step (1) into a T75 culture bottle, adding monoclonal antibody CD 3800 ng/mL,Monoclonal antibody CD 28800 ng/mL, CpG-A ODN 1nmol/mL, vitamin A1.5 μmol/mL, gamma-secretase inhibitor DAPT 50nmol/mL, allicin 100ng/mL, heparin 60IU/mL, IL-1810 IU/mL, IL-12150 IU/mL, and IL-2700 IU/mL, at 37 deg.C, 5% CO2Culturing in an incubator.
(3) After inoculating cells on DAY0, DAY3 was supplemented with AlyS505N-0 medium containing heparin 60IU/mL, IL-1810 IU/mL, IL-12150 IU/mL, IL-2700 IU/mL;
DAY5 adding AlyS505N-0 culture medium containing heparin 60IU/mL, IL-1810 IU/mL, IL-12150 IU/mL and IL-2700 IU/mL;
DAY7 was supplemented with AlyS505N-0 medium containing heparin 60IU/mL, IL-1810 IU/mL, IL-12150 IU/mL, or IL-2700 IU/mL, and the cell density was maintained at 1.0X 10 after each rehydration6one/mL. At 37 ℃ 5% CO2Culturing in an incubator.
(4) Mature TH1 cells were harvested by culturing until day 9, and the number of cells was sampled and examined to calculate the proliferation fold and the purity of TH1 cells was examined by flow-assay, the results are shown in Table 2.
TABLE 2 fold increase of TH1 cells in example 2 and flow assay results
Example 3
A method of culturing TH1 cells in vitro, comprising the steps of:
(1) obtaining PBMC by density gradient centrifugation, preparing mononuclear cell suspension with serum-free cell culture medium AlyS505N-0, and making into cell suspension with cell concentration of 1.5 × 106one/mL.
(2) Adding 20mL of the cell suspension prepared in the step (1) into a T75 culture bottle, adding monoclonal antibody CD 31000 ng/mL, monoclonal antibody CD 281000 ng/mL, CpG-A ODN 3nmol/mL, vitamin A0.5 μmol/mL, gamma-secretase inhibitor DAPT 100nmol/mL, allicin 80ng/mL, heparin 100IU/mL, IL-1840 IU/mL, IL-12120 IU/mL, IL-21000 IU/mL, and adding 5% CO at 37 DEG C2Culturing in an incubator.
(3) After inoculating cells on DAY0 (i.e., culturing in step 2), DAY3 was supplemented with AlyS505N-0 medium containing heparin 100IU/mL, IL-1840 IU/mL, IL-12120 IU/mL, IL-21000 IU/mL;
DAY5 adding AlyS505N-0 culture medium containing heparin 100IU/mL, IL-1840 IU/mL, IL-12120 IU/mL, IL-21000 IU/mL;
DAY7 was supplemented with AlyS505N-0 medium containing heparin 100IU/mL, IL-1840 IU/mL, IL-12120 IU/mL, and IL-21000 IU/mL, and the cell density was maintained at 1.0X 10 after each fluid replacement6one/mL. At 37 ℃ 5% CO2Culturing in an incubator.
(4) Mature TH1 cells were harvested by culturing until day 9, and the number of cells was sampled and examined to calculate the proliferation fold and the purity of TH1 cells was examined by flow-assay, the results are shown in Table 3.
TABLE 3 fold increase of TH1 cells in example 3 and flow assay results
Comparative example 1
The differences between comparative example 1 and example 1 are as follows:
DAY0 adding monoclonal antibody CD 3500 ng/mL, monoclonal antibody CD 28500 ng/mL and IL-2500 IU/mL;
DAY3 was supplemented with AlyS505N-0 medium containing IL-2500 IU/mL;
DAY5 was supplemented with AlyS505N-0 medium containing IL-2500 IU/mL;
the same procedure as in example 1 was repeated except that the medium was supplemented with DAY7 in AlyS505N-0 medium containing IL-2500 IU/mL.
Comparative example 2
The differences between comparative example 2 and example 1 are as follows:
the CpG-A ODN added in step (2) was omitted, and the procedure was as in example 1.
Comparative example 3
The differences between comparative example 3 and example 1 are as follows:
the vitamin A added in step (2) was omitted, and the rest was the same as in example 1.
Comparative example 4
The differences between comparative example 4 and example 1 are as follows:
the gamma-secretase inhibitor DAPT added in step (2) was omitted and the procedure was otherwise the same as in example 1.
Comparative example 5
The differences between comparative example 5 and example 1 are as follows:
allicin added in the step (2) was omitted, and the procedure was the same as in example 1.
Comparative example 6
The differences between comparative example 6 and example 1 are as follows:
the steps (2) and (3) were omitted and the procedure was as in example 1.
Comparative example 7
The differences between comparative example 7 and example 1 are as follows:
IL-18 added in steps (2) and (3) was omitted, and the procedure was the same as in example 1.
Application example 1
(1) The number of cells in example 1 and comparative examples 1 to 7 on the 0 th and 9 th days of culture was counted by trypan blue staining, and the proliferation fold of each group was calculated.
(2) Flow-type phenotypic detection: the cells cultured up to day 9 in example 1, comparative examples 1 to 7 were stained with flow antibody, and the percentage of CD4+ IFN-. gamma. + double positive cells of TH1 cells was determined. The results are shown in Table 4. The flow charts of the phenotypic assay of CD4+ IFN-. gamma. + for examples 1 to 3 and comparative examples 1 to 7 are shown in FIGS. 1 to 10.
TABLE 4 multiplication factor of TH1 cells and flow assay results in example 1 and comparative examples 1 to 7
As is clear from Table 4, the culture method of example 1 showed the highest cell growth rate and the highest number of cells harvested on day 9. In comparative examples 1 to 7, TH1 cells were prepared in vitro by a conventional method, or TH1 cells were obtained without some of the components of the present invention, and cell numbers and cell purities were reduced. It can be seen from each of the control groups 1-7 that, after some components in the method for preparing the TH1 cells in vitro are omitted, the TH1 cell culture effect is not as good as that of the embodiment of the present invention, and the synergistic effect of the components is further explained to improve the quantity and purity of the immunocytes.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A culture solution for culturing TH1 cells in vitro, which comprises a culture solution A and a culture solution B;
the A culture solution comprises the following components in concentration: 3500-1000 ng/mL of monoclonal antibody CD, 28500-1000 ng/mL of monoclonal antibody CD, 0.05-3 nmol/mL of CpG-A ODN, 0.5-2 mu mol/mL of vitamin A, 20-100 nmol/mL of gamma-secretase inhibitor DAPT, 50-150 ng/mL of allicin, 10-100 IU/mL of heparin, 1810-50 IU/mL of IL-1250-200 IU/mL of IL-2100-1000 IU/mL of IL-A;
the culture solution B comprises a serum-free culture medium and effective substances, wherein the effective substances comprise the following components in concentration: 10-100 IU/mL of heparin, 1810-50 IU/mL of IL-1250-200 IU/mL of IL-2100-1000 IU/mL of IL-.
2. The culture solution according to claim 1, wherein the serum-free medium comprises ALyS 505N-0.
3. Use of the culture solution of claim 1 or 2 for culturing TH1 cells in vitro.
4. A method for in vitro culture of TH1 cells, comprising the steps of:
preparing peripheral blood mononuclear cells into a cell suspension by using a serum-free culture medium, mixing the cell suspension with the culture solution A as defined in claim 1 or 2 to obtain a mixture a, and beginning to culture the mixture a, which is marked as Day 0;
mixing the mixture a with the culture solution B as described in claim 1 or 2 when Day3 to obtain a mixture B;
mixing the mixture B with the culture solution B according to claim 1 or 2 when Day5 to obtain a mixture c;
mixing the mixture c with the culture solution B according to claim 1 or 2 when Day7 to obtain a mixture d;
TH1 cells were harvested from mixture d at Day 9.
5. The in vitro culture method according to claim 4, wherein the cell density of peripheral blood mononuclear cells in the cell suspension is (1.0 to 1.5). times.106one/mL.
6. The in vitro culture method according to claim 4, wherein the density of cells in the mixture b, the mixture c and the mixture d is not less than 0.5X 106one/mL.
7. The in vitro culture method according to claim 4, wherein the temperature of said culture is 37 ℃ and the concentration of carbon dioxide is 5%.
8. The in vitro culture method according to claim 4, wherein the cell density of peripheral blood mononuclear cells in the cell suspension is (1.0 to 1.5). times.106one/mL.
9. Use of the TH1 cell culture method of claims 4-8 in the rapid culture of TH1 cells.
10. The use of claim 9, wherein the culture method comprises harvesting TH1 cells at Day 9, wherein the proliferation fold of TH1 cells is 228-235, and the percentage of cells that are double positive for CD4+ IFN- γ + is 71.5-73.4%.
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