A method for improving the efficiency and survival rate of dendritic cell electroporation

Abstract

The application belongs to the technical field of cell electroporation, and particularly relates to a method for improving the electroporation efficiency and survival rate of dendritic cells. The method comprises the following steps: S1, iDC pretreatment; S2, iDC electroporation; and S3, iDC induction of mDC. The method provided by the application significantly improves the electroporation efficiency and the survival rate of cells after electroporation. Moreover, the method is simple to operate and easy to popularize in the existing experimental system. The finally obtained DC cells have better state and function, and lay a solid foundation for downstream immunotherapy application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell electroporation, and particularly relates to a method for improving the electroporation efficiency and survival rate of dendritic cells. BACKGROUND

[0002] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells and are ideal carriers for cancer vaccines. They are crucial in the field of immunotherapy: in CAR-T and TCR-T therapies, DCs are key to screening and expanding specific T cells in vitro; in tumor vaccine strategies, DC vaccines loaded with tumor antigens (such as the marketed Provenge®) can directly activate and guide T cells to launch a precise attack on tumors. Therefore, efficiently genetically modifying DCs to express specific tumor antigens or immunomodulatory factors is one of the core links to enhance the effectiveness of these therapies.

[0003] Electroporation is currently the mainstream physical technology for introducing exogenous genes (such as mRNA or DNA) into DCs and other difficult-to-transfect cells due to its safety, high efficiency, and ease of clinical translation. However, the inherent limitations of this technology have severely hampered its application: traditional electroporation procedures are often accompanied by significant cell damage, leading to a sharp decline in cell survival rate; at the same time, the transfection efficiency is often unsatisfactory, making it difficult to achieve high levels of gene expression in living cells. These two major problems - high mortality and low efficiency - have become technical bottlenecks for obtaining high-quality engineered DCs.

[0004] Currently, researchers rely heavily on commercial electroporation systems (such as Lonza's Nucleofector™ series of kits), but these ready-made solutions still fail to adequately address the above shortcomings, with significant fluctuations in efficiency and cell survival rate and high costs. Although some studies have reported that optimizing the composition of the electroporation buffer can reduce cell damage to some extent, existing formulations still have significant shortcomings in improving the overall performance of DC electroporation (i.e., ensuring high survival rate and high efficiency), and there is a lack of a stable, efficient, and gentle standardized solution.

[0005] The technical problem to be solved by the present application is to overcome the core contradiction between transfection efficiency and cell survival rate in existing DC cell mRNA electroporation technology.

[0006] The prior art, including widely used commercialized electroporation kits, has obvious problems and defects in mediating mRNA transfection of DC cells: in order to pursue higher transfection efficiency, it is often necessary to apply strong electric shock parameters, but this will irreversibly damage the cell membrane structure and intracellular environment, leading to accelerated cell apoptosis and ultimately significantly reduced survival rate; on the contrary, if mild electric transfection conditions are used in order to maintain a high cell survival rate, it is difficult to ensure the effective introduction and expression of exogenous mRNA, resulting in low transfection efficiency. This "efficiency-survival rate" trade-off seriously limits the feasibility of obtaining a large number of high-quality, high-expression target antigen engineered DCs, and becomes a major technical bottleneck in the development and clinical transformation of DC immunotherapy.

[0007] In addition, the existing electric transfection buffer system has limited protection for DC cells, and its components are not optimized for DC, which is a fragile and sensitive immune cell, further exacerbating cell function damage and activity loss after electric shock.

[0008] Therefore, the present application aims to provide a completely new technical solution to fundamentally solve the above-mentioned contradictions. This solution can greatly improve the mRNA transfection efficiency while maximizing the survival rate and physiological function of DC cells through innovative electroporation systems and processes, thereby achieving the effect of "efficiency-survival rate" and providing better cell products for subsequent cell therapy. SUMMARY

[0009] The purpose of the present application is to provide a method for improving the efficiency and survival rate of dendritic cell electroporation in view of the existing problems.

[0010] The present application is realized by the following technical solutions:

[0011] A method for improving the efficiency and survival rate of dendritic cell electroporation, comprising the following steps:

[0012] S1, iDC pretreatment:

[0013] The collected iDCs are resuspended in fresh complete culture medium, the cell density is adjusted to 1x10 6 / mL, and placed in a cell incubator for 2h;

[0014] S2, iDC electroporation:

[0015] S201, the collected and pretreated iDCs are washed twice with Opti medium, and centrifuged at 400g for 5min at room temperature;

[0016] S202, according to the experimental grouping, the iDCs are resuspended with 100μL of pre-cooled DC-EB electroporation solution of different formulations, and the resulting cell suspension is mixed with mRNA;

[0017] The DC-EB electrotransformation liquid is prepared by mixing iodixanol and GlutaMAX TM are essential ingredients;

[0018] The mRNA is mRNA of a mouse MC38 colorectal cancer neoantigen polypeptide screened and verified in a preliminary stage before commercial synthesis; the mRNA encodes 4 tumor neoantigen polypeptides in series, the amino acid sequences of the 4 tumor neoantigen polypeptides are shown in SEQ ID NO. 1, 2, 3, and 4 in sequence, and the mRNA is dissolved in RNase-free water to prepare a stock solution with a concentration of 1 μg / μL, and then the stock solution is mixed thoroughly and stored in a dark place at -80°C after being divided into aliquots.

[0019] S203. Instant stable electroporation is performed using an electrotransformation system, and immediately after the instant stable electroporation, 400 μL of cell culture solution containing a Rock inhibitor preheated at 37°C is added to the electrotransformation cup, and the cup is placed in a 37°C cell incubator for 10-15 min;

[0020] The instant stable electroporation is controlled at a pulse voltage of 150-300 v, a pulse duration of 5-50 ms, and a pulse number of 1-3 times.

[0021] S204. Subsequently, the electrotransformed cells are seeded in a cell plate preheated in advance, and the cell culture density is 1×10 6 cells / mL, and the culture conditions are 37°C and 5% CO2 in a carbon dioxide incubator, to obtain iDC cells loaded with mRNA expressing an antigen;

[0022] S3. iDC induction of mDC:

[0023] After the iDC loaded with mRNA expressing an antigen is stably cultured in a cell incubator for 4-6 h, a maturation inducer IFNγ and LPS are added, and maturation is induced overnight to obtain mDC cells loaded with mRNA expressing an antigen.

[0024] Further, the preparation of iDC in step S1 includes the following steps:

[0025] (1) Obtain C57 / BL6 mouse bone marrow cells:

[0026] C57BL / 6 mice are sacrificed by cervical dislocation, and the obtained mouse bone marrow cells are sterilized in 70% ethanol for 5 min. Red blood cells are lysed by adding 5-8 mL of red blood cell lysis solution for 5 min, and then centrifuged at 300 g for 5 min. The supernatant is discarded, and then washed once with 5 mL of PBS, centrifuged at 300 g for 5 min, and the supernatant is discarded. The cells are resuspended in 1 mL of RPMI1640 complete medium, counted, and the cell concentration is adjusted to 1×10 6 / mL.

[0027] (2) DC induction:

[0028] Day 0: Add recombinant mouse GM-CSF 20 ng / mL and IL-4 10 ng / mL to the above adjusted cell density of mouse bone marrow cells, and place in a 37°C, 5% CO2 incubator, which is the 0th day of culture;

[0029] Day 3: Add medium and add cytokines GM-CSF 20 ng / mL and IL-4 10 ng / mL;

[0030] Day 5: Harvest the suspended cells and semi-adherent cells as iDC.

[0031] Further, the complete culture solution in step S1 is RPMI1640 complete culture medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4.

[0032] Further, the experimental grouping in step S202 includes blank control, experimental group: electric conversion buffer formula 1-formula 4.

[0033] The composition of formula 1 is: K2HPO410mM, HEPES 25mM, KCl 3.6mM, anhydrous MgCl25.4mM, CaCl2·2H2O 0.88mM, sodium succinate 10mM, mannitol 25mM, sucrose 125mM, EDTA 0.2mM, pH 7.2, osmotic pressure 290mOsm / L;

[0034] The composition of formula 2 is: iodixanol 19%, K2HPO410mM, HEPES 25mM, KCl 3.6mM, anhydrous MgCl25.4mM, CaCl2·2H2O 0.88mM, sodium succinate 10mM, mannitol 25mM, sucrose 95.8mM, EDTA 0.2mM, pH 7.2, osmotic pressure 294mOsm / L;

[0035] The composition of formula 3 is: iodixanol 19%, K2HPO410mM, HEPES 25mM, KCl 3.6mM, anhydrous MgCl25.4mM, CaCl2·2H2O 0.88mM, sodium succinate 10mM, mannitol 25mM, sucrose 95.8mM, GlutaMAX TM 2%, EDTA 0.2mM, pH 7.2, osmotic pressure 310mOsm / L;

[0036] Formulation 4: Iodixanol 19%, K2HPO4 10 mM, HEPES 25 mM, KCl 3.6 mM, anhydrous MgCl2 5.4 mM, CaCl2 2H2O 0.88 mM, sodium succinate 10 mM, mannitol 25 mM, sucrose 75 mM, GlutaMAX TM 3%, EDTA 0.2 mM, pH 7.2, osmotic pressure 308 mOsm / L.

[0037] Further, the number of cells in the cell suspension in step S202 is 5 x 10 5 -2 x 10 6 cells; mRNA mass is 0-10 μg.

[0038] Further, the cell culture solution containing Rock inhibitor in step S203 is: RPMI1640 complete medium additionally added with 10 μM Y-27632;

[0039] The RPMI1640 complete medium is: 10% serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 20 ng / mL GM-CSF and 10 ng / mL IL-4 are added to the RPMI1640 medium.

[0040] The volume of each well is 600 μL / well, which is prepared in advance and preheated in a 37°C cell incubator.

[0041] Further, all operations are carried out under sterile conditions.

[0042] Further, the IFNγ in step S3 is 500 U / mL and the LPS is 100 ng / mL.

[0043] Compared with the prior art, the present application has the following advantages:

[0044] The method of the present application significantly improves the efficiency of electroporation. The cell survival rate after electroporation is significantly improved. The operation is simple and easy to popularize in existing experimental systems. The final obtained DC cells are in a better state and have better functions, which lay a solid foundation for downstream immunotherapy applications. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Figure for the effect of different formulations of Example 2 on mouse DC cells after 24 h of mRNA electroporation;

[0046] Figure 2 Figure for the comparison results of cell survival rate and recovery rate of mouse DC cells after 24 h of mRNA electroporation in Example 3;

[0047] Figure 3 The ratio of EGPF positive cells of the mouse DC cells after 24 hours of mRNA electroporation in Example 3 is compared;

[0048] Figure 4 The microscopic representation of the EGPF fluorescence intensity of the mouse DC cells after 24 hours of mRNA electroporation in Example 3 is compared;

[0049] Figure 5 The statistical results of the EGPF fluorescence intensity of the mouse DC cells after 24 hours of mRNA electroporation in Example 3 are compared;

[0050] Figure 6 The Elispot plate hole spot experiment chart and statistical comparison results of Example 3 are compared;

[0051] Figure 7 The effect chart of the Rock inhibitor post-treatment on the cells after 24 hours of mRNA electroporation in Example 4 is compared;

[0052] Figure 8 The effect chart of the different density cells after 24 hours of mRNA electroporation in Example 5 is compared;

[0053] Figure 9 The MC38 tumor antigen expression plasmid constructed by the application. DETAILED DESCRIPTION

[0054] In order to further explain the application, the following specific examples are combined for illustration.

[0055] Example 1: Preparation of the electroporation buffer for dendritic cells

[0056] 1. The raw materials are prepared according to the formula in Table 1 below:

[0057] Table 1: Electroporation buffer (electrotransformation liquid) formula

[0058] Ingredients Formulation 1 Formulation 2 Formulation 3 Formulation 4 Iodixanol 0 19% 19% 19% K2HPO4 10 mM 10 mM 10 mM 10 mM HEPES 25 mM 25 mM 25 mM 25 mM KCl 3.6 mM 3.6 mM 3.6 mM 3.6 mM Anhydrous MgCl2 5.4 mM 5.4 mM 5.4 mM 5.4 mM CaCl2·2H2O 0.88 mM 0.88 mM 0.88 mM 0.88 mM Sodium succinate 10 mM 10 mM 10 mM 10 mM Mannitol 25 mM 25 mM 25 mM 25 mM Sucrose 125 mM 95.8 mM 95.8 mM 75 mM GlutaMAX TM ]] 0 0 2% 3% EDTA 0.2 mM 0.2 mM 0.2 mM 0.2 mM PH 7.2 7.2 7.2 7.2 Osmotic pressure / mOsm / L 290 294 310 308

[0059] and prepared according to the following method:

[0060] (1) K2HPO4 is weighed according to the formula, and the use amount of 1M HEPES mother liquor is stirred until completely transparent. The pH is adjusted to 7.2 with 1M NaOH / HCl;

[0061] (2) Other solutes are added according to the formula amount (each addition needs to be completely dissolved): sodium succinate, mannitol, KCl, EDTA, anhydrous MgCl2, and sucrose;

[0062] (3) The amount of 1M CaCl2·2H2O mother liquor, GlutaMAX TM and iodixanol solution (60% w / v) was calculated according to the formula, and then added to the beaker of step (2);

[0063] (4) Before the solution was filled to volume, the pH was adjusted to 7.2 again using 1M NaOH / HCl, and then filtered through a 0.22μm filter membrane. After aliquoting, it was stored at 4℃.

[0064] Example 2: Preparation of tumor neoantigen mRNA dendritic cells and experiment of the effect of different formula electrotransfection solutions on the viability of DCs

[0065] 1. mRNA synthesis

[0066] The mRNA of the mouse MC38 colorectal cancer neoantigen polypeptide (patent publication number: CN119798421A) screened and verified in the early stage of commercial synthesis (four tumor neoantigen polypeptides in series TP01-TP04, the sequences are YEGICNGVLM; VTVRNVTVI; YSLLNENYV; INYSSSLPL in turn) was dissolved in RNase-free water to prepare a mother liquor with a concentration of 1μg / μL. After thorough mixing, it was aliquoted, stored in the dark at -80℃.

[0067] 2. Preparation of C57 / BL6 mouse DCs

[0068] 1) Obtain C57 / BL6 mouse bone marrow cells: C57BL / 6 mice were sacrificed by cervical dislocation, immersed in 70% ethanol for 5min, and the obtained mouse bone marrow cells were added with 5-8mL red blood cell lysis solution to lyse red blood cells for 5min, centrifuged at 300g for 5min, the supernatant was discarded, washed once with 5mL PBS, centrifuged at 300g for 5min, the supernatant was discarded, resuspended with 1mL RPMI1640 complete medium, counted the cells, and adjusted the cell concentration to 1×10 6 / mL;

[0069] 2) DC induction

[0070] Day 0: The above mouse bone marrow cells with adjusted cell density were added with recombinant mouse GM-CSF (20ng / mL) and IL-4 (10ng / mL), and placed in a 37℃, 5% CO2 incubator for culture, which was the 0th day of culture;

[0071] Day 3: Add culture medium and cytokines GM-CSF (20ng / mL) and IL-4 (10ng / mL);

[0072] Day 5: Harvest the suspended cells and semi-adherent cells, which are iDCs.

[0073] 3. Electroporation

[0074] 1) Pretreatment of iDC: collected iDC were resuspended in fresh complete medium (RPMI1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4) and the cell density was adjusted to 1 x 10 6 / mL, and the cells were incubated in the cell incubator for 2 h;

[0075] 2) Electroporation of iDC

[0076] a. Rock inhibitor cell culture medium: RPMI1640 complete medium containing 20 ng / mL GM-CSF, 10 ng / mL IL-4 and 10 μM Y-27632 inhibitor; volume 600 μL / well, prepared in advance and preheated in a 37°C cell incubator;

[0077] b. The pretreated iDC were collected and washed twice with Opti medium, and centrifuged at 400 g for 5 min at room temperature;

[0078] c. According to the experimental grouping (including blank control, experimental group: electroporation buffer formula 1-formula 4), the iDC were resuspended with 100 μL of pre-cooled DC-EB electroporation buffer of different formulas to obtain a cell suspension (the number of cells was 5 x 10 5 -2 x 10 6 cells) mixed with mRNA (the amount of mRNA was 0-10 μg);

[0079] In this example, the number of cells in the DC cell suspension was about 1 x 10 6 cells, and the amount of mRNA added was 4 μg;

[0080] d. The U.S. Bio-Rad Gene Pulser Xcell TM transient stable electroporation system was used for transient stable electroporation (pulse voltage 150-300 v), pulse duration (5-50 ms), and pulse number 1-3 times;

[0081] In this example, the pulse voltage was 200 v, the pulse duration was 10 ms, the pulse number was 2 times with an interval of 1 s; after transient stable electroporation, 400 μL of cell culture medium containing Rock inhibitor preheated at 37°C was immediately added to the electroporation cup, and the mixture was placed in a 37°C cell incubator for 10-15 min;

[0082] e. Subsequently, the electroporated cells were seeded in a preheated cell plate, and the cell culture density was 1 x 10 6cells / mL, and the culture conditions were 37℃, 5% CO2 in a carbon dioxide incubator, to obtain iDC cells loaded with mRNA expressing antigens;

[0083] All operations were performed under sterile conditions.

[0084] 4. iDC induced mDC (mature DC cells)

[0085] After the iDCs were instantaneously stably transfected in the cell incubator for 4-6h, mature induction agents IFNγ (500U / mL) and LPS (100ng / mL) were then added to induce maturation overnight (16-24h), to obtain mDC cells loaded with mRNA expressing antigens.

[0086] 5. Transfection efficiency and viability detection of DCs after transfection

[0087] The mDCs in each group after transfection were collected, centrifuged (300xg, 5min), the supernatant was discarded, and the cells were washed twice with PBS (containing 2% FBS) and resuspended to 1x10 7 cells / 100μL.

[0088] Cell viability detection and recovery rate: 20μL of mDCs after transfection were taken in a 1.5mL centrifuge tube, 20μL of AOPI cell dye was added, mixed well, and then the cell viability was detected by a cell counter (Cellometer® K2). The cell recovery rate was calculated according to the formula: (total number of harvested cells / total number of cells input for electroporation) x 100%

[0089] Transfection efficiency detection: 5x10 5 DC cells were taken, 0.5μL of dead cell dye eBioscience™ Fixable Viability Dye eFluor™ 660 was added, and incubated at 4℃ for 30min. The percentage of live cells expressing GFP+ (transfection efficiency) and the average fluorescence intensity were detected by flow cytometry.

[0090] As shown in Figure 1 compared with the traditional electroporation solution formula 1, the addition of iodixanol and GlutaMAX TM in the electroporation solution formula 2 showed significantly higher mRNA transfection efficiency, cell survival rate and recovery rate.

[0091] Cell survival rate: the average survival rate of the formula 2 with the addition of iodixanol was 71.8, which was significantly higher than that of the traditional electroporation solution formula 1 (64%), and the cell survival rate of the formula 3 and 4 with the addition of iodixanol and GlutaMAX TM was the highest, about 80%, which was about 25% higher than that of the electroporation solution formula 1.

[0092] Cell recovery: average cell recovery of formulation 3 and 4 with Iodixanol and GlutaMAX added was about 90%, which was significantly higher than that of traditional electroporation buffer formulation 1 (67%), with a relative increase of about 34%. TM

[0093] Transfection efficiency: average transfection efficiency of formulation 2, 3 and 4 was 86.5%, 88.5% and 89.7% respectively, with no significant difference, but significantly higher than that of traditional electroporation buffer 1 (72.6%), with a relative increase of about 19%.

[0094] The above results show that Iodixanol and GlutaMAX TM are indispensable in the present formulation, and have a synergistic effect when used with other ingredients. Compared with traditional electroporation buffer, the present formulation significantly improves cell survival rate, cell recovery rate and the proportion of EGFP positive cells after electroporation.

[0095] Example 3: Comparison of transfection efficiency and cell viability of the present electroporation method and commercial kits

[0096] The following examples further illustrate the present application. In the following examples, the instrument used in the present application is the American Bio-Rad Gene Pulser Xcell TM electroporation system, and the electroporation conditions are the optimized electroporation parameters of the present application. The comparative example uses the Lonza commercial kit (Nucleofector™ 2b kit For Mouse Dendritic cells, batch number F-16041) and the German Lonza electroporation instrument, and the electroporation program code is DCimmature mouse: Y-001.

[0097] The same healthy mouse bone marrow-derived iDCs were induced and cultured under the same conditions, and then divided into two groups. The first group was electroporated according to the operation process of the Lonza commercial kit. The second group was electroporated using the special formulation 3 buffer of the present application and the optimized electroporation parameters. Both groups were transfected with mRNA (4 μg / 10 6 cells) encoding green fluorescent protein (GFP). After transfection, the cells were incubated at 37°C, 5% CO2 for 4 h, and then mature induction agent was added for 16-24 h to obtain mature DCs transfected with mRNA.

[0098] Fluorescence microscope: direct observation of GFP fluorescence expression in cells.

[0099] Cell counter and flow cytometry were used to detect cell viability, recovery rate and transfection efficiency.

[0100] ​Elispot assay to evaluate the antigen presentation function of mDC: synthetic antigen peptides and immunize C57 / BL6 healthy mice once every 5 days for a total of 3 times. Collect mouse spleen cells 7 days after immunization. According to the experimental design, group (including positive control, negative control and experimental group), the mDC after electroporation or untransfected mDC are co-cultured with mouse spleen cells in proportion (1:10) for 24h, 5x10 5 spleen cells are added to each well, and 5x10 4 mDC are added at the same time. ELISpot kit (Dako, batch number 250701) is used to detect the number of IFN-γ secreting spot forming cells.

[0101] As Figures 2-6 shown, compared with the existing Lonza commercial kit method, the electroporation method provided by the present application exhibits significant superior effects in many aspects, including: ① better cell viability: the cell survival rate under the method of the present application is close to 80%, which is much higher than the survival rate of about 65.8% of the commercial kit method, proving that the permeability change of the cell membrane under the method of the present application is more reversible, and the cell damage is smaller (p<0.0001) Figure 2 ). ② higher transfection efficiency: the transfection efficiency of the method of the present application can reach more than 85%, which is significantly higher than 64.4% of the commercial kit method, ensuring that most cells can successfully express target antigens (p<0.0001) Figure 3 ). ③ stronger protein expression level: the higher average fluorescence intensity indicates that the amount of target protein expressed by each cell under the method of the present application is higher, which is conducive to stronger immunogenicity presentation (p<0.0001) Figure 4 and Figure 5 ). ④ better antigen presentation function of DC cells: the average spot number of IFNγ under the method of the present application is more than that of the commercial kit method. Statistical analysis shows that the spot number produced by the method of the present application is increased by about 87.5% compared with the commercial kit method, and the difference has extremely significant statistical significance (p<0.0001) Figure 6 .

[0102] The above results show that, compared with the existing commercial kit method, the method of the present application for electroporation buffer transfection of tumor neoantigen mRNA-EGFP significantly improves the proportion of living cells, the proportion of EGFP positive cells, the average fluorescence intensity of EGFP, and enhances the antigen presentation function of DC cells after transfection and their ability to activate antigen-specific T cell immune response. This provides key functional data support for the application of the present application in the fields of tumor immunotherapy, vaccine development, etc.

[0103] Example 4: Effect of Y-27632 post-treatment on the viability and function of DC after electroporation

[0104] To verify the beneficial effects of the present application, the following experiments were performed:

[0105] The dendritic cells of C57 / BL mice were divided into two groups, and the same conditions (the same equipment, procedure, DC-EB buffer formula 3 and mRNA dose) were used for electroporation.

[0106] Experimental group (10 μM ROCK inhibitor group): After electroporation, the cells were inoculated in complete medium containing 10 μM Y-27632.

[0107] Control group (0 μM ROCK inhibitor group): After electroporation, the cells were inoculated in complete medium without Y-27632.

[0108] After transfection, the cells were recovered in complete medium for 4 h, and then induced with maturation inducers IFNγ (500 U / mL) and LPS (100 ng / mL) overnight (16-24 h) to obtain mRNA-loaded mDC cells expressing antigens. AOPI staining was used to detect cell viability, and a live cell counter was used to calculate cell recovery rate=(total number of harvested cells / total number of cells input at electroporation) x 100%; flow cytometry was used to detect EGFP+ expression rate and CD11c+CD86+ ratio to evaluate DC cell maturation function index.

[0109] As shown in Table 1, compared with the control group, the average survival rate of the experimental group inoculated in complete medium containing 10 μM Y-27632 was 79.7, which was significantly higher than that of the control group (73.6%), with a relative increase of about 8.2%. Figure 7

[0110] The above results show that, compared with conventional recovery culture, the use of the ROCK inhibitor Y-27632 for post-treatment after transfection significantly improves the survival rate of cells after electroporation; there is no significant difference in cell recovery rate, EGFP positive cell ratio and DC cell maturation, indicating that Y-27632 does not affect the maturation of DC cells.

[0111] Example 5: Effect of different electroporation cell densities on dendritic cell viability and recovery rate

[0112] The same batch of C57 / BL mouse iDC cells were collected, resuspended in electroporation solution, and adjusted to the following four densities (Table 2). The same conditions (the same equipment, procedure, DC-EB buffer formula 3 and mRNA dose) were used for electroporation.

[0113] Table 2 Electroporation cell densities corresponding to different proportions

[0114] Experimental groups Electroporation density (cells / 100 μL) Comparative Example 1 2.5 x 10 5 ]]> Comparative Example 2 5 x 10 5 ]]> Examples 2-4 1 x 10 6 ]] Comparative Example 3 2 x 10 6 ]]

[0115] ​Cells were cultured at 37°C in a 5% CO2 incubator. Cell viability, recovery rate, and transfection efficiency were assessed 24 hours after electrotransfection. Cell viability: 20 μL of cell suspension was mixed with an equal volume of PI staining solution, and the percentage of viable cells was calculated using an automated cell counter. Recovery rate = (total number of viable cells harvested / total number of viable cells introduced during electrotransfection) × 100%. Transfection efficiency: The percentage of cells expressing EGFP+ was detected using flow cytometry.

[0116] like Figure 8 As shown, the cell viability of Examples 2-4 was significantly higher than that of Comparative Examples 1 and 2. Electrotransfection of cells in Comparative Examples 1 and 2 (low density) caused severe damage, with cell viability <62% and recovery rate <60% after transfection. The average cell viability and average EGFP+ cell ratio of Comparative Example 3 (high density group) were not significantly different from those of Examples 2-4, but the recovery rate of Examples 2-4 (88.5%) was significantly higher than that of Comparative Example 3 (65%).

[0117] The above results indicate that the optimal cell electrotransfection density using the method of this invention is 1×10⁻⁶. 6 cells / 100μL.

[0118] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of improving the efficiency and survival of dendritic cells upon electroporation, comprising, Comprising the following steps: S1, iDC pretreatment: The collected iDCs were resuspended in fresh complete medium, cell density adjusted to 1 x 10 6 / mL, and incubated in a cell incubator for 2 h; S2, iDC electroporation: S201, after the iDCs collected and pretreated, they were washed twice with Opti medium, centrifuged at 400g for 5min at room temperature; S202, the iDCs were resuspended with 100μL pre-cooled DC-EB electroporation solution, and the resulting cell suspension was mixed with mRNA; The formula of the DC-EB electroporation solution is formula 2, formula 3 or formula 4. The composition of formula 2 is: iodixanol 19%, K2HPO4 10mM, HEPES 25mM, KCl 3.6mM, anhydrous MgCl2 5.4mM, CaCl2·2H2O 0.88mM, sodium succinate 10mM, mannitol 25mM, sucrose 95.8mM, EDTA 0.2mM, pH 7.2, osmotic pressure 294mOsm / L; Composition of Formula 3: Iodixanol 19%, K2HPO4 10 mM, HEPES 25 mM, KCl 3.6 mM, anhydrous MgCl2 5.4 mM, CaCl2 2H2O 0.88 mM, sodium succinate 10 mM, mannitol 25 mM, sucrose 95.8 mM, GlutaMAX TM 2%, EDTA 0.2 mM, pH 7.2, osmotic pressure 310 mOsm / L; Composition of Formula 4: Iodixanol 19%, K2HPO4 10 mM, HEPES 25 mM, KCl 3.6 mM, anhydrous MgCl2 5.4 mM, CaCl2 2H2O 0.88 mM, sodium succinate 10 mM, mannitol 25 mM, sucrose 75 mM, GlutaMAX TM 3%, EDTA 0.2 mM, pH 7.2, osmotic pressure 308 mOsm / L; The mRNA encodes four tumor neoantigen polypeptides in series, the amino acid sequences of the four tumor neoantigen polypeptides are shown in SEQ ID NO. 1, 2, 3, and 4, respectively, and are prepared into a stock solution in RNase-free water with a concentration of 1μg / μL. After thorough mixing, store in a dark place at -80℃. S203, use the instant stable electroporation system for instant stable electroporation. Immediately after instant stable electroporation, add 400μL of 37℃ preheated cell culture medium containing 10μM Y-27632 to the electroporation cup, and place it in a 37℃ cell incubator for 10-15min. The pulse voltage is controlled at 150-300v, the pulse duration is 5-50ms, and the pulse number is 1-3 times during instant stable electroporation. S204、Subsequently, the electrotransformed cells are seeded in a pre-warmed cell plate, with a cell culture density of 1 x 10 6 cells / mL, and cultured in a 37°C, 5% CO2 carbon dioxide incubator to obtain iDC cells loaded with mRNA expressing antigens; S3, iDC induced mDC: After the iDCs loaded with mRNA expressing antigens are stably cultured in the cell incubator for 4-6h, add the maturation inducer IFNγ and LPS, and induce maturation overnight to obtain mDC cells loaded with mRNA expressing antigens.

2. The method of claim 1, wherein the dendritic cells are derived from peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells (CBMCs). The preparation of iDCs in step S1 comprises the following steps: (1) Obtain C57 / BL6 mouse bone marrow cells: C57BL / 6 mice were sacrificed by cervical dislocation, and the mice were sterilized in 70% ethanol for 5 min. The obtained mouse bone marrow cells were lysed with 5-8 mL of red blood cell lysis solution for 5 min, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were washed once with 5 mL of PBS, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of RPMI1640 complete medium, counted, and adjusted to a cell concentration of 1 x 10 6 / mL. (2) DC induction: Day 0: Add recombinant mouse GM-CSF 20ng / mL and IL-4 10ng / mL to the above adjusted mouse bone marrow cells, and place them in a 37℃, 5% CO2 incubator for culture. This is the 0th day of culture. Day 3: Add medium and add cytokines GM-CSF 20ng / mL and IL-4 10ng / mL. Day 5: Harvest the suspended cells and semi-adherent cells as iDCs.

3. The method of claim 1, wherein the dendritic cells are transfected with a nucleic acid molecule encoding a tumor antigen. The complete culture solution in step S1 is RPMI1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4.

4. The method of claim 1, wherein the dendritic cells are transfected with a nucleic acid molecule encoding a tumor antigen. The number of cells in the cell suspension described in step S202 is 5 x 10 5 -2 x 10 6 cells; the mRNA mass is 4-10 μg.

5. The method of claim 1, wherein the method is for improving the electroporation efficiency and survival rate of dendritic cells. The cell culture medium in step S203 is RPMI1640 complete medium. The RPMI1640 complete medium is: 10% serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 20 ng / mL GM-CSF and 10 ng / mL IL-4 are added to the basis of RPMI1640 medium; The volume of each hole is 600 μL / hole, which is prepared in advance and preheated in a 37°C cell incubator.

6. The method of claim 1, wherein the dendritic cells are transfected with a nucleic acid molecule encoding a tumor antigen. All operations are carried out under sterile conditions.

7. The method of claim 1, wherein the method further comprises the step of: The IFNγ in step S3 is 500 U / mL and the LPS is 100 ng / mL. ​

Images