A method for freezing mature dendritic cells

Abstract

The present invention discloses a method for freezing mature dendritic cells, wherein the method for freezing mature dendritic cells comprises a dendritic cell freezing solution, wherein the dendritic cell freezing solution is mainly made of the following raw materials in percentage by volume: human albumin injection 15-16 v / v, antifreeze protein solution 1-3 v / v, compound dextran 40 injection 17-20 v / v, compound electrolyte injection 17-20 v / v, CryoStor® CS10 41-50 v / v%, and Setanaxib, wherein the concentration of Setanaxib in the freezing solution is 20 μmol / L. The freezing solution is used for the freezing treatment of mature dendritic cells, and the synergistic enhancement between different components in the freezing solution makes the survival rate of mature dendritic cells after freezing ≥80%, and the antigen presentation ability thereof can be preserved to a large extent.

Description

Technical Field

[0001] The present application relates to the technical field of cryopreservation protective fluids, and in particular to a method for cryopreserving mature dendritic cells. Background Art

[0002] Dendritic cells (DC) are an important type of immune cells with unique morphological and functional characteristics, playing a key role in initiating and regulating immune responses. Dendritic cells were discovered by Ralph M. Steinman in 1973 and are named for their stellate polymorphism or dendritic processes on their surface. DCs are the most powerful professional antigen presenting cells (APCs), which can efficiently take up, process and present antigens to activate naive T cells. At present, scholars at home and abroad hope to stimulate immune responses in vivo with the help of DC's strong antigen loading and information presentation capabilities, such as inducing monocytes to transform into immature DCs through cytokines, loading and inducing them to become mature DCs with tumor-like composite antigens, and using them as a tumor therapeutic vaccine. After subcutaneous injection, cytotoxic T lymphocytes can be activated in the human body, thereby achieving effective recognition and killing of tumor cells.

[0003] During the process, the DCs induced to mature need to be frozen within a specific period of time to ensure that the DCs have enough time for quality inspection and release and to effectively present antigen information.

[0004] The effect of cryopreservation on dendritic cells is an important research area. Because DC plays a key role in immune response, it is often used in clinical applications. The following are some research results on the effect of cryopreservation on DC: ① "Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells", M. Martikainen et al., this article compared the overall immune reactivity of fresh and frozen peripheral blood mononuclear cells (PBMCs), and specifically studied the effect of cryopreservation on circulating antigen-presenting cells (APCs) in PBMCs. The study found that frozen PBMCs produced fewer cytokines than fresh cells, but the response was comparable. Cryopreservation had an effect on the relative proportion of APCs, and the stimulation-induced response was reproducible, but weaker than that observed in fresh cells. This suggests that cryopreservation may have a certain effect on the function of DC, but the specific extent may depend on the cryopreservation technology and conditions. ② "Preparation and preliminary stability test of autologous dendritic cell injection", Liu Junyun et al., this article studied the effect of cryopreservation temperature on DC survival and explored the effect of different cryopreservation temperatures on DC survival rate. Studies have shown that choosing a suitable freezing method is crucial to maintaining the survival rate and activity of frozen DCs. ③ "Effects of homemade cell freezing solution on the survival rate and activity of dendritic cells", Zhao Mancang et al. This article studied the cell survival rate of DCs after freezing DCs using homemade improved cell freezing solution (containing DMSO, hydroxyethyl starch and cell stabilizer) and the killing effect of in vitro induced CIK on tumor cells. The results showed that the survival rate and activity of DCs frozen using homemade freezing solution decreased slightly with the extension of freezing time, but there was no significant difference compared with two commonly used freezing solutions (1. RPMI1640 culture solution containing 10v / v% dimethyl sulfoxide (DMSO) and 20v / v% bovine serum; 2. Cellbanker cell freezing solution of Japan ZENOAQ). This shows that homemade improved cell freezing solution can be used as a substitute for traditional freezing solution and imported freezing solution, and has a good application prospect.

[0005] In summary, cryopreservation technology has a certain impact on the immune reactivity and survival rate of DCs, but these effects can be reduced by improving the cryopreservation method and maintaining the function and activity of DCs. Therefore, choosing appropriate cryopreservation fluid and cryopreservation conditions is critical for the clinical application of DCs. Summary of the invention

[0006] The present invention provides a method for freezing mature dendritic cells, which achieves effective freezing of DCs through the synergistic enhancement of CryoStor®CS10, human albumin injection, compound dextran 40 injection, compound electrolyte injection, antifreeze protein solution, and Setanaxib components contained in the freezing solution, as well as a specific programmed freezing temperature during the freezing process, that is, the survival rate after resuscitation is ≥80%, and the antigen presentation ability of DCs is preserved to a large extent.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a dendritic cell freezing solution, which is prepared from CryoStor®CS10, human albumin injection, compound dextran 40 injection, compound electrolyte injection, antifreeze protein solution, and Setanaxib; wherein the volume percentage of CryoStor®CS10 is 41-50%, the volume percentage of human albumin injection is 15-16%, the volume percentage of compound dextran 40 injection is 17-20%, the volume percentage of compound electrolyte injection is 17-20%, the volume percentage of antifreeze protein solution is 1-3%, and the concentration of Setanaxib is 20μmoL / L.

[0009] Preferably, the volume percentage of the CryoStor® CS10 is 50%.

[0010] Preferably, the volume percentage of the human albumin injection is 15%.

[0011] Preferably, the volume percentage of the compound dextran 40 injection is 17%.

[0012] Preferably, the volume percentage of the compound electrolyte injection is 17%.

[0013] Preferably, the volume percentage of the antifreeze protein solution is 1%.

[0014] In a second aspect, the present invention provides a method for freezing dendritic cells, and the specific operating steps are as follows:

[0015] Step 1: Purchase human peripheral blood mononuclear cells (PBMCs) from a third-party blood product supplier (i.e., Xiamen Sany Hematopoietic Technology Co., Ltd.), obtain target cells (i.e., dendritic cells) by CD14 magnetic bead positive selection, place the target cells in culture medium and culture for 120 hours, during which the medium is changed once, 72 hours after the start of culture, and the replaced medium is the original culture medium. Add the target antigen on the 6th day, add the maturation inducer 4-6 hours later and continue culturing, and collect the mature dendritic cells within 20-50 hours after the addition of the target antigen;

[0016] Step 2: Count the collected DCs, then centrifuge and wash 1-3 times, and remove the supernatant;

[0017] Step 3: Add the dendritic cell freezing solution to DC to obtain a cell suspension, the concentration of which is 2.0×10 6 -2.5×10 7 cells / mL, and then the cell suspension was dispensed into cryopreservation bags, 26 mL / bag;

[0018] Step 4: programmed cooling, that is, placing the packaged cell suspension into a programmed cooling device, and gradually lowering the temperature according to a preset cooling rate, wherein the preset cooling rate is:

[0019] T1: When the temperature in the programmed cooling chamber reaches 6±0.5℃, put the packaged cells in, select a cell sample, and connect the temperature probe;

[0020] T2: The temperature in the chamber is maintained at 6±0.5℃, waiting for the sample temperature to drop to 8±0.5℃;

[0021] T3: The cooling rate of the programmed temperature cooling instrument is 1.0-1.5℃ / min, and the sample temperature is cooled from 8±0.5℃ to -4±0.5℃;

[0022] T4: The cooling rate of the programmed cooling instrument is 20-21℃ / min, and the sample temperature is cooled to -60±0.5℃;

[0023] T5: The heating rate of the programmed cooling instrument is 7-8℃ / min, and the sample temperature is heated to -25±0.5℃;

[0024] T6: The cooling rate of the programmed cooling instrument is 1.0±0.5℃ / min, and the sample temperature is cooled to -60±0.5℃;

[0025] T7: The cooling rate of the programmed cooling instrument is 10-11℃ / min, and the sample temperature is cooled to -120±0.5℃;

[0026] T8: The sample temperature is -120±0.5℃ for 10min;

[0027] T9: End.

[0028] Step 5: After the programmed cooling is completed, transfer the cell freezing bag to vapor phase liquid nitrogen for long-term storage.

[0029] The immature induction complete medium described in step 1 is composed of 10v / v% FBS, 500IU / mL recombinant human IL-4 and 500IU / mL GM-CSF added to x-vivo medium; the maturation inducer is the maturation inducer produced by Near Shore Protein Company, product number: GMP-1806; the addition amount is: 1.0v / v%; the target antigen is purchased from MABTECH, product number 3616-1, and the addition amount is: 2µg / mL.

[0030] In the present invention:

[0031] (1) CryoStor CS10 is a uniquely formulated, serum-free, animal-free cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) that protects cells at low temperatures (-80°C or -196°C), providing a safe and protective environment for cells and tissues during freezing, storage, and thawing.

[0032] (2) Antifreeze proteins (AFPs) are a class of protein compounds that can improve the antifreeze ability of organisms. They were originally discovered in the serum of marine fish in the Antarctic and Arctic regions. They are a specific protein that can bind to ice crystals. They can prevent the formation and growth of ice nuclei in body fluids and maintain the non-frozen state of body fluids. The role of antifreeze proteins (AFPs) in cell cryopreservation is mainly reflected in the following aspects: 1. Ice recrystallization inhibition (IRI) activity: AFPs have IRI activity, which can inhibit the growth and recrystallization of ice crystals, thereby reducing the damage to cells during the freezing process; 2. Improving the survival rate of cells after freezing: By using AFPs as cryoprotectants, the survival rate and vitality of cells after freezing and thawing can be improved; 3. Reducing the use of toxic cryoprotectants. Traditional cell freezing methods often use some toxic organic solvents as cryoprotectants, while AFPs provide a safer and more effective alternative.

[0033] (3) The main components of compound electrolyte injection, each 500ml contains: sodium chloride 2.63g, sodium gluconate 2.51g, sodium acetate 1.84g, potassium chloride 0.185g, magnesium chloride 0.15g. This product is compatible with blood and blood components, and can be infused before or after blood transfusion (i.e. as a priming solution) using the same administration device, can be added to the blood components being infused, or used as a diluent for blood cells.

[0034] (4) The role of compound dextran 40 injection in cell cryopreservation is mainly related to its characteristics as a plasma substitute. Specifically for cell cryopreservation, compound dextran 40 injection may be used in the following aspects: 1. As a component of the cryopreservation medium, as part of the cell cryopreservation solution, it helps protect cells from freezing damage; 2. Prevent cell aggregation, by reducing cell-cell adhesion, reducing the possibility of cells forming large aggregates during the freezing process; 3. Maintain osmotic pressure balance, during the freezing and thawing process, it helps maintain the osmotic pressure balance inside and outside the cell, and reduces cell rupture caused by osmotic pressure changes.

[0035] (5) The role of human serum albumin (HSA) in cell cryopreservation is mainly reflected in the following aspects: 1. Maintaining osmotic pressure balance. Human serum albumin is the most abundant protein in plasma. It can maintain the osmotic pressure of extracellular fluid and prevent cells from excessive dehydration or swelling due to osmotic pressure changes during cryopreservation. 2. As a cryoprotectant, human serum albumin can be used as a non-permeable cryoprotectant. It reduces the content of extracellular free water and the formation of ice crystals by binding to water molecules, thereby protecting cells from mechanical damage caused by ice crystals. 3. Antioxidant effect. Human serum albumin has antioxidant properties and can bind and neutralize free radicals, reducing the damage of oxidative stress to cells. This has a protective effect on oxidative damage that may occur during cryopreservation. 4. Stabilize cell membranes. Human albumin can interact with the phospholipids on the cell membrane, stabilize the cell membrane structure, and reduce damage to the cell membrane during the freezing process. 5. Transport and storage functions. Human albumin can bind to and transport a variety of molecules, including nutrients and metabolites, which helps maintain the stability of the intracellular environment during the freezing process. 6. Promote cell recovery. During the cell recovery process, human albumin helps maintain the stability of the extracellular environment, reduce cell damage during the thawing process, and improve cell survival rate and functional recovery after recovery.

[0036] (6) Setanaxib, also known as GKT137831, is a dual NADPH oxidase 1 and 4 (NOX1 / 4) inhibitor. Its application in cell cryopreservation has gradually attracted attention in recent years. Role and mechanism in cell cryopreservation: 1. Antioxidant protection. Setanaxib reduces the production of intracellular reactive oxygen species (ROS) by inhibiting NOX1 and NOX4. During cell cryopreservation, the generation of ROS may cause cell damage and death, and the antioxidant properties of Setanaxib can help protect the activity of cells during cryopreservation and recovery. 2. Improve cell survival rate. During cell cryopreservation, ice crystal formation and cell membrane damage are the main challenges. Setanaxib reduces oxidative stress and improves the integrity of cell membranes, thereby improving the survival rate of cells after cryopreservation. 3. Promote cell recovery. The use of Setanaxib helps cells recover quickly after thawing. By reducing oxidative damage in cells, Setanaxib may improve cell function and vitality and promote cell recovery. 4. Potential combined application. Setanaxib can be used in combination with other cryoprotectants (such as DMSO) to enhance the effect of cell cryopreservation. By comprehensively utilizing the protective effects of different mechanisms, the success rate of cell cryopreservation may be improved.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The use of the freezing solution and freezing method defined in the present invention can effectively improve the survival rate of mature DCs after freezing.

[0039] 2. The cryopreservation time point defined in the present invention can effectively preserve the antigen presentation ability of mature DC after cryopreservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : DCs were cryopreserved in cryopreservation solution 1 12, 20, 36, 50 and 72 hours after being loaded with target antigen, and co-cultured with autologous T cells for 24 hours after recovery. The negative control was cryopreserved in cryopreservation solution 1 for 12 hours without antigen loading, and co-cultured with autologous T cells after recovery. h represents hours (the number in the upper left corner is the number of spots).

[0041] Figure 2 : Changes in immune spots after co-culture of frozen DCs and non-frozen DCs with autologous T cells (the number in the upper left corner is the number of spots).

[0042] Figure 3 : As Figure 2 Statistical description: The statistical method was t-test, and ns means there is no significant statistical difference.

[0043] Figure 4: Example diagram of the programmed cooling box for conventional freezing methods.

[0044] Figure 5 : The viability changes of DCs from three batches of donors after being cryopreserved by the programmed cooling method defined by the present invention and the conventional cryopreservation method were detected for 3 consecutive days. ns indicates no significant statistical difference, * indicates p values ​​of the two groups are <0.05, and ** indicates p values ​​of the two groups are <0.01.

[0045] Figure 6 :DCs revived by two cryopreservation methods were co-cultured with autologous T cells, and the Elispot experiment was performed to detect the statistical changes in the number of spots. The statistical method was t-test. ** indicates that the p value of the two groups is <0.01.

[0046] Figure 7 : To detect the changes in the viability of DCs from three batches of donors after being revived in 6 kinds of cryopreservation solutions for 3 consecutive days.

[0047] Figure 8 :for Figure 7 Comparison of activity changes between cryopreservation solution 1 and cryopreservation solution 5.

[0048] Fig. 9 :for Figure 7 Comparison of activity changes between cryopreservation solution 1 and cryopreservation solution 4.

[0049] Fig.10 :for Figure 7 Comparison of activity changes between cryopreservation solution 1 and cryopreservation solution 6. DETAILED DESCRIPTION

[0050] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0051] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0052] Some raw materials and their sources are as follows:

[0053] Human peripheral blood mononuclear cells: purchased from Xiamen Sany Hematopoietic Technology Co., Ltd.

[0054] HSA (20% human albumin): purchased from CSL Behring LLC, catalog number: 0077900300;

[0055] Antifreeze protein solution: purchased from Nanjing Anfei Biotechnology Co., Ltd.;

[0056] Compound dextran 40 injection: purchased from Jiangsu Zhengda Fenghai Pharmaceutical Co., Ltd., national medicine standard number: H32024886;

[0057] Compound electrolyte injection: purchased from Shanghai Better Medical Products Co., Ltd., national medicine approval number: H20000475;

[0058] CS10: purchased from CryoStor CS10, Stemcell, catalog number: 100-1016;

[0059] Setanaxib: purchased from MCE, medchemexpress, CAS: 1218942-37-0;

[0060] Freezing bag: purchased from Miltenyi Biotec, Freezing Bag 50;

[0061] Program cooling instrument: purchased from Thermos, model: 7455;

[0062] Elispot kit: purchased from Dakoway, catalog number: 1110002;

[0063] X-vivo culture medium: purchased from Lonza, catalog number: 04-418Q;

[0064] PBS: purchased from Solebol, catalog number: P1020;

[0065] IL-4: purchased from ABMOLE, product number M9363;

[0066] GM-CSF: purchased from ABMOLE, product number M9344;

[0067] Maturation inducer: purchased from Nearshore Protein, catalog number: GMP-1806;

[0068] Target antigen: purchased from MABTECH, catalog number: 3616-1;

[0069] The steps for testing the viability and antigen presentation ability of mature dendritic cells after preservation and resuscitation are as follows:

[0070] a. Collect 20 ml of peripheral blood, separate human peripheral blood mononuclear cells (PBMCs), select CD14+ monocytes by magnetic beads, and select the remaining CD14- cells (i.e. the remaining cells after CD14+ monocytes are removed) by CD4 and CD8 magnetic beads to obtain T cells;

[0071] bT cells were added with 2 ml of freezing solution (90 v / v% FBS (fetal bovine serum) + 10 v / v% DMSO) and placed in a programmed cooling box, and directly transferred to a -80°C refrigerator for storage;

[0072] c. Prepare DC immature induction complete medium: the immature induction complete medium is composed of adding 10v / v% FBS, 500IU / mL recombinant human IL-4 and 500IU / mL GM-CSF to x-vivo medium; CD14+ monocytes are cultured at 2.0×10 5 Cells / mL density culture;

[0073] d. After culturing CD14+ monocytes with DC immature induction complete medium for 72 h, centrifuge and replace with the same volume of DC immature induction complete medium;

[0074] e. After 48 hours of continuous culture, the cells were divided into 6 parts, one of which was used as a negative control without antigen loading, and the other 5 parts were loaded with antigen and the time was recorded. The antigen addition amount was 2 ug / mL, and 1.0 / v% maturation inducer was added after 7 hours;

[0075] f. Collect and wash the cells by centrifugation at 12, 20, 36, 50 and 72 hours after antigen loading, and freeze mature DC in batches. The negative control group and the 12-hour group are frozen together. The freezing solution refers to the freezing solution in Table 1, and the cooling program refers to the T1-T9 cooling program. Finally, transfer to gas phase liquid nitrogen for storage;

[0076] g. After all groups have completed cryopreservation, thaw the T cells stored in step b, perform counting and viability tests, centrifuge and wash off the cryopreservation solution, and transfer to an incubator to recover for 2 hours;

[0077] h. After T cell recovery was complete, mature DCs from the six groups were revived uniformly for viability and antigen presentation ability tests. Specifically, 2.0×10 4 mature DC, 2.0×10 5 autologous T cells and an Elispot plate required for the test, the Elispot plate being: an Elispot plate coated with IFN-γ antibody;

[0078] i. After 20 hours, perform development according to the instructions of the Elispot kit.

[0079] The specific formula components of the freezing solution are shown in Table 1.

[0080] Table 1 Specific ingredients and ratios of freezing solution

[0081] Cryopreservation medium Cryopreservation Solution 1 Cryopreservation medium 2 Cryopreservation Solution 3 Cryopreservation medium 4 Cryopreservation Solution 5 Cryopreservation Solution 6 CryoStor® CS10 v / v% 50 41 45 50 50 50 Human albumin injection v / v% 15 16 16 15 15 15 Compound Dextran 40 Injection v / v% 17 20 19 17 17 17 Compound electrolyte injection v / v% 17 20 18 17 17 17 Antifreeze protein solution v / v% 1 3 2 0 1 0 Setanaxib µmol / L 20 20 20 20 0 0

[0082] Note: The missing raw materials of Cryopreservation Solution 4, Cryopreservation Solution 5 and Cryopreservation Solution 6 can be supplemented with deionized sterile water.

[0083] The cooling procedure is:

[0084] T1: When the temperature in the programmed cooling chamber reaches 6±0.5℃, put the packaged cells in, select a cell sample, and connect the temperature probe;

[0085] T2: The temperature in the chamber is maintained at 6±0.5℃, waiting for the sample temperature to drop to 8±0.5℃;

[0086] T3: The cooling rate of the programmed temperature cooling instrument is 1.0-1.5℃ / min, and the sample temperature is cooled from 8±0.5℃ to -4±0.5℃;

[0087] T4: The cooling rate of the programmed cooling instrument is 20-21℃ / min, and the sample temperature is cooled to -60±0.5℃;

[0088] T5: The heating rate of the programmed cooling instrument is 7-8℃ / min, and the sample temperature is heated to -25±0.5℃;

[0089] T6: The cooling rate of the programmed cooling instrument is 1.0±0.5℃ / min, and the sample temperature is cooled to -60±0.5℃;

[0090] T7: The cooling rate of the programmed cooling instrument is 10-11℃ / min, and the sample temperature is cooled to -120±0.5℃;

[0091] T8: The sample temperature is -120±0.5℃ for 10min;

[0092] T9: End.

[0093] Experiment 1: Experiment to determine the freezing time

[0094] In order to verify the feasibility of the cryopreservation time point selection of the present invention, this experiment selected DCs to be cryopreserved 12, 20, 36, 50 and 72 hours after antigen loading. The mature DCs after recovery were co-cultured with autologous T cells for 24 hours. 2.0×10 4 mature DC, 2.0×10 5 The autologous T cells and the Elispot plate required for the experiment are: Elispot plates coated with IFN-γ antibodies. The changes in immune spots after co-culture are detected to evaluate the effect of the selection of freezing time points on signal presentation (refer to the above experimental steps ai for details. The freezing solution used in this experiment is freezing solution 1, and the freezing program is T1-T9). For details, please refer to Figure 1 .

[0095] The experimental results show that the number of spots at 12 hours and 72 hours is significantly less than the average values ​​of the three middle time points. This result proves that the time interval between 12 hours and 72 hours exceeds the range of signal presentation, so the time range from DC loading antigen to freezing is tightened to 20-50 hours.

[0096] Experiment 2: Antigen presentation ability preservation test of DC after cryopreservation

[0097] To verify whether cryopreservation of mature DCs affects their antigen presentation, fresh mature DCs and thawed frozen DCs from three donors were co-cultured with autologous T cells for 16 h. 2.0×10 4 mature DCs and 2.0×10 5 The autologous T cells were loaded with antigens in the experimental group and unloaded in the control group, respectively. The effects of cryopreservation were evaluated by incubating Elispot plates coated with IFN-γ antibodies and detecting changes in immune spots (refer to the experimental steps ai above for details. The cryopreservation solution used in this experiment was cryopreservation solution 1, and the cryopreservation procedure was T1-T9).

[0098] The test results are as follows Figure 2 and Figure 3 As shown, there is no significant statistical difference in the number of spots produced by co-culture with T cells before and after DC freezing, which proves that the freezing process defined in the present invention can ensure that the ability of DC to present antigen signals will not be significantly changed.

[0099] Experiment 3: Effect of different cryopreservation methods on DC viability

[0100] Conventional freezing method: 30 minutes before DC preparation is completed, place the programmed cooling box ( Figure 4 ) Pre-cool in a 2-8℃ refrigerator. After DC preparation is completed, fill it into a 2ml cell cryopreservation tube and transfer it to a pre-cooled program cooling box. Then, place the box in a -80℃ refrigerator for 24-48 hours, and finally transfer it to gas phase liquid nitrogen for long-term stable storage;

[0101] Programmed freezing method: refer to freezing program T1-T9;

[0102] Cryopreservation medium: Cryopreservation medium 1;

[0103] The experimental method is based on ai.

[0104] like Figure 5 As shown in the figure, the cryopreservation procedure of the present invention can obtain DCs with higher viability than conventional cryopreservation methods. The conclusion of Experiment 1 shows that the time range from DC loading antigen to cryopreservation is 20-50 hours. If the DCs after resuscitation do not recover to the normal viability state in time during this period, the ability of DCs to present signals may be affected. This hypothesis is Figure 6 The change in activity rate shows that the cryopreservation procedure of the present invention is superior to the conventional cryopreservation method.

[0105] as follows Figure 6As shown, DCs from three batches of donors were cryopreserved by two cryopreservation methods, and were co-cultured with autologous T cells immediately after thawing for 20 hours. Elispot experiments were performed, and the changes in the number of spots showed that the cryopreservation procedure of the present invention was more conducive to ensuring the signal presentation of DCs, which further demonstrated the superiority of this method.

[0106] Experiment 4: Effect of different cryopreservation solutions on DC viability

[0107] For cryopreservation solutions 1-6, six cryopreservation solutions with different component ratios, mature DCs were divided into 6 parts, and DCs were cultured and cryopreserved according to the established process (step ai, cooling program T1-T9). The effects of the six cryopreservation solutions were evaluated by comparing the changes in viability just after recovery (Day0), the first day of recovery (Day1), and the second day of recovery (Day2). The specific data are shown in Table 2.

[0108] Table 2 Effects of different cryopreservation solutions on DC viability

[0109] Time Cryopreservation Solution 1 Cryopreservation medium 2 Cryopreservation Solution 3 Cryopreservation medium 4 Cryopreservation Solution 5 Cryopreservation Solution 6 Day 0 87.34 86.49 85.23 78.36 73.52 70.39 Day 1 86.91 84.36 85.91 72.29 70.36 68.42 Day 2 86.74 84.29 85.76 71.61 68.41 67.38

[0110] like Figure 7-Figure 10 As shown, cryopreservation solution 6 is equivalent to the traditional cryopreservation solution, but has the worst freezing effect on DC; the comparison of cryopreservation solutions 1, 5 and 1, 4 respectively demonstrates that antifreeze protein and Setanaxib are indispensable in this formulation; and by comparing cryopreservation solution 1 with cryopreservation solution 4, cryopreservation solution 5, and cryopreservation solution 6, it is further demonstrated that antifreeze protein and Setanaxib have a synergistic effect when used together with other ingredients in this formulation, confirming the applicability and necessity of the cryopreservation solution provided by the present invention.

[0111] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. A dendritic cell freezing solution, characterized in that: The freezing solution is made of CryoStor®CS10, human albumin injection, compound dextran 40 injection, compound electrolyte injection, antifreeze protein solution and Setanaxib; wherein the volume percentage of CryoStor®CS10 is 41-50%, the volume percentage of human albumin injection is 15-16%, the volume percentage of compound dextran 40 injection is 17-20%, the volume percentage of compound electrolyte injection is 17-20%, the volume percentage of antifreeze protein solution is 1-3%, and the concentration of Setanaxib is 20 μmol / L; The compound dextran 40 injection is a compound dextran 40 injection with the national medicine standard number: H32024886; The compound electrolyte injection is a compound electrolyte injection with a national medicine standard number: H20000475; The antifreeze protein solution was purchased from Nanjing Anfei Biotechnology Co., Ltd. The human albumin injection is a 20wt% human albumin injection.

2. A method for freezing dendritic cells, characterized in that: The mature dendritic cells are cryopreserved using the dendritic cell cryopreservation solution described in claim 1.

3. The cryopreservation method according to claim 2, characterized in that: The following steps are involved: Step 1: Use CD14 magnetic bead positive selection method to select human peripheral blood mononuclear cells to obtain CD14+ monocytes, and place CD14+ monocytes in immature induction complete medium for culture for 120 hours, during which the medium is changed once, and the medium change time is 72 hours after the start of culture. The replaced medium is immature induction complete medium; on the 6th day, add the target antigen, add the maturation inducer 4-6 hours after the addition of the target antigen, and continue to culture. Collect mature dendritic cells within 20-50 hours after the addition of the target antigen; Step 2: Count the collected mature dendritic cells, then centrifuge and wash them 1-3 times, and remove the supernatant; Step 3: Add the dendritic cell freezing solution to the mature dendritic cells to obtain a cell suspension, wherein the density of the cell suspension is 2.0×10 6 -2.5×10 7 cells / mL, and then the cell suspension was dispensed into freezing bags; Step 4: programmed cooling; Step 5: Transfer the cryopreserved cells that have completed programmed cooling to vapor phase liquid nitrogen for storage.

4. The cryopreservation method according to claim 3, characterized in that: The specific procedures of the programmed cooling are as follows: T1: When the temperature in the programmed cooling chamber reaches 6±0.5℃, put the packaged cells in, select a cell sample, and connect the temperature probe; T2: The temperature in the chamber is maintained at 6±0.5℃, waiting for the sample temperature to drop to 8±0.5℃; T3: The cooling rate of the programmed temperature cooling instrument is 1.0-1.5℃ / min, and the sample temperature is cooled from 8±0.5℃ to -4±0.5℃; T4: The cooling rate of the programmed cooling instrument is 20-21℃ / min, and the sample temperature is cooled to -60±0.5℃; T5: The heating rate of the programmed cooling instrument is 7-8℃ / min, and the sample temperature is heated to -25±0.5℃; T6: The cooling rate of the programmed cooling instrument is 1.0±0.5℃ / min, and the sample temperature is cooled to -60±0.5℃; T7: The cooling rate of the programmed cooling instrument is 10-11℃ / min, and the sample temperature is cooled to -120±0.5℃; T8: The sample temperature is -120±0.5℃ for 10min; T9: End.

5. The cryopreservation method according to claim 3, characterized in that: The immature induction complete medium is composed of 10 v / v% FBS, 500 IU / mL recombinant human IL-4 and 500 IU / mL GM-CSF added to the x-vivo medium; the maturation inducer is the maturation inducer produced by Near Shore Protein Company, product number: GMP-1806; the addition amount is: 1.0 v / v%.

6. The cryopreservation method according to claim 3, characterized in that: The target antigen is produced by MABTECH, with a product number of 3616-1 and an added amount of 2 µg / ml.

7. Use of the dendritic cell freezing solution according to claim 1 in cryopreserving mature dendritic cells.

8. Use of the dendritic cell cryopreservation method according to any one of claims 3 to 6 in cryopreserving mature dendritic cells.

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