Immune cell freezing medium and application thereof

Abstract

The invention discloses an immune cell cryopreservation solution and application thereof. The cryopreservation solution comprises a basic solution and a cryoprotectant, wherein the basic solution comprises a DMEM (Dulbecco Modified Eagle Medium), and nicotinamide mononucleotide with the final concentration of 300-600 mu M and betaine with the final concentration of 20-40 mM are contained in the DMEM; the cryoprotectant comprises a polyhydric alcohol aqueous solution with the concentration of 414 to 669g / L, an erucamide propyl dimethyl betaine solution with the concentration of 0.5 to 2.5 g / L and a sugar composition solution with the concentration of 30 to 80g / L; the sugar composition comprises chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of (1-2.5): (2-5): 2. In the cryoprotectant of the cell cryopreservation liquid, chondroitin sulfate, tamarind seed polysaccharide and trehalose have certain ice recrystallization inhibitor characteristics, damage of ice crystals to cells is reduced, the survival rate and growth amplification rate of immune cells after recovery are greatly increased, the cryopreservation liquid does not contain DMSO components, and toxic and side effects of the cryopreservation liquid to the cells are reduced.

Description

Technical Field

[0001] The invention relates to a cell freezing technology, in particular to a preparation component of an immune cell freezing solution and an application thereof in immune cell freezing. Background Art

[0002] In recent years, cell cryopreservation has become an important supporting technology for various cell-based applications, such as stem cell therapy, tissue engineering, assisted human reproduction and transfusion medicine. At present, cell cryopreservation is mainly carried out under ultra-low temperature conditions of -196℃ liquid nitrogen. Under ultra-low temperature conditions, the chemical, biological and physical processes that usually occur at the cellular level may remain suspended for a long time, so that the cells can be preserved for a long time; when the cells are needed, the frozen cells are taken out and quickly thawed in a 37℃ water bath to restore the activity and function of the cells.

[0003] However, in the existing cell freezing methods, cells are inevitably subjected to freezing damage, including solution damage and ice damage, during the freeze-thaw cycle. Excessive dehydration caused by freezing concentrations can damage cells and lead to solution damage. In particular, among immune cells, such as dendritic cells, NK cells, monocytes, T cells, CIK cells, etc., due to the complex surface synaptic structure of various cells, the existing cryoprotectants (CPAs) containing dimethyl sulfoxide (DMSO) will cause varying degrees of damage to the surface synaptic structure of immune cells during contact with immune cells, which greatly reduces the proliferation and growth efficiency of immune cells after recovery, leading to problems such as easy aging of cells, poor activity of frozen cells after recovery, and low survival rate. Summary of the invention

[0004] Based on the above problems, the problem to be solved by the present invention is to provide a cryopreservation solution and a cryopreservation method that are conducive to efficient proliferation of immune cells with low damage.

[0005] The technical solution of the present invention is as follows:

[0006] An immune cell freezing solution, comprising a base solution and a cryoprotectant; wherein:

[0007] The basal solution includes a DMEM culture medium, which contains nicotinamide mononucleotide at a final concentration of 300 to 600 μM and betaine at a final concentration of 20 to 40 mM;

[0008] The cryoprotectant includes, calculated by mass concentration, 41.4-66.9% of a polyol aqueous solution, 0.05-0.25% of an erucic acid amide propyl dimethyl betaine solution and 3.0-8.0% of a sugar composition solution; wherein the sugar composition solution includes chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of 1-2.5:2-5:2.

[0009] Preferably, in the cryoprotectant of the immune cell freezing solution, the polyol is ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol or 2,3-butanediol; preferably, the polyol is ethylene glycol.

[0010] Preferably, in the cryoprotectant of the immune cell freezing solution, the molecular weight of the tamarind seed polysaccharide is 90-200 kD; and the molecular weight of the chondroitin sulfate is 10-20 kD.

[0011] The preparation process steps of the above-mentioned cryoprotectant are as follows:

[0012] Adding polyol into deionized water and mixing them to obtain a polyol aqueous solution;

[0013] Add erucamidopropyl dimethyl betaine and the sugar composition to the polyol aqueous solution, add the balance of deionized water, stir and mix at 60-70° C. for 1-2 hours, cool to room temperature, and set aside;

[0014] When the sugar composition is added to the polyol aqueous solution, the chondroitin sulfate is first added to the polyol aqueous solution and mixed and dissolved; then tamarind seed polysaccharide is added and mixed and dissolved; finally trehalose is added and mixed and dissolved; a cryoprotectant containing 41.4-66.9% of the polyol aqueous solution, 0.05-0.25% of erucic acid amide propyl dimethyl betaine solution and 3.0-8.0% of the sugar composition solution is prepared; wherein the sugar composition solution comprises chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of 1-2.5:2-5:2.

[0015] The present invention also provides an immune cell cryopreservation method, comprising the following steps:

[0016] At room temperature, resuspend the immune cells using the above-mentioned basal solution to obtain a cell suspension;

[0017] Adding the above-mentioned cryoprotectant precooled to 2-8° C. to the cell suspension, with the volume ratio of the base liquid to the cryoprotectant being 1:2-3, to obtain a cell mixture;

[0018] The cell mixture was dispensed into cryopreservation tubes, programmed to cool to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0019] Preferably, in the immune cell freezing method, the density of the immune cells in the cell suspension after the immune cells are resuspended in the basal solution is maintained at 0.5 to 3×10 7 Pieces / mL.

[0020] Preferably, in the immune cell freezing method, after the cell mixture is dispensed into cryopreservation tubes, the cooling process is performed at a cooling rate of 4°C / min when the temperature drops to -80°C.

[0021] Preferably, in the present invention, the immune cells include any one of dendritic cells (DC cells), NK cells, monocytes, T cells, and CIK cells.

[0022] Chondroitin sulfate, tamarind seed polysaccharide and trehalose in the cryoprotectant of the present invention each have certain ice recrystallization inhibitor properties. The sugar composition of the three reacts with polyols and erucamidopropyl dimethyl betaine to form a highly efficient ice recrystallization inhibitor, which has a micellar network structure at low temperatures, can reduce the formation of ice crystals during cell freezing, reduce the damage of ice crystals to cells, and greatly improve the survival rate and growth expansion rate of immune cells after recovery; the cryopreservation solution of the present invention does not contain DMSO components, thereby reducing the toxic and side effects of the cryopreservation solution on cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The cell viability bar graphs of immune cells before cryopreservation, 30-day cryopreservation recovery, and 24-hour culture after recovery in Examples 1 to 4;

[0024] Figure 2 The growth curves of NK and CIK cells in Examples 2, 3, 5 and 6 after being frozen for 30 days and then revived and cultured for 16 days;

[0025] Figure 3 This is a logistic histogram of the markers detected after the cryopreserved DC cells were revived and cultured for 24 hours in Example 1 and Example 4;

[0026] Figure 4 This is a bar graph showing the EGFP expression rate of the frozen DC cells detected after 24 hours of culture after recovery in Example 1 and Example 4. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0028] The base liquid of the present invention contains nicotinamide mononucleotide and betaine; the cryoprotectant includes polyol, erucamidopropyl dimethyl betaine, a sugar composition and deionized water, and the sugar composition includes chondroitin sulfate, tamarind seed polysaccharide and trehalose; the polyol is ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol or 2,3-butylene glycol, etc.

[0029] In the base solution, nicotinamide mononucleotide (NMN) is a direct derivative of nicotinamide adenine dinucleotide (NAD+). NMN can effectively improve the disorder of mitochondrial homeostasis, the generation of reactive oxygen species, DNA repair and cell survival in cells caused by NAD+ deficiency; betaine is a natural zwitterion with good biocompatibility and blood compatibility. It can be used as a significant osmotic protectant to prevent cells from osmotic damage.

[0030] Among cryoprotectants, the modified erucic acid amide propyl dimethyl betaine reacts with polyols and sugar molecules in a polyol aqueous solution to form a micellar network structure, which has strong low-temperature elasticity, can reduce the surface tension of the liquid, and make the overall solution freezing point far below 0°C, which can effectively reduce the damage of ice crystals to cells.

[0031] Chondroitin sulfate, tamarind seed polysaccharides and trehalose are all antifreeze agents. The combination of the three sugar compositions can synergistically and efficiently inhibit ice recrystallization, reduce the formation of ice crystals during cell freezing, reduce the damage of ice crystals to cells, and greatly improve the survival rate and growth expansion rate of immune cells after recovery.

[0032] 1. Preparation of Cryopreservation Solution

[0033] The immune cell freezing solution provided by the present invention comprises a base solution and a cryoprotectant; wherein:

[0034] The basal solution includes a DMEM culture medium, which contains nicotinamide mononucleotide at a final concentration of 300 to 600 μM and betaine at a final concentration of 20 to 40 mM;

[0035] Preparation of cryoprotectant: First, according to the mass concentration, for example, 41.4-66.9% polyol aqueous solution, 0.05-0.25% erucic acid amide propyl dimethyl betaine solution and 3.0-8.0% sugar composition; 41.4-66.9g polyol is added to deionized water, and mixed to obtain a polyol aqueous solution; then, 0.05-0.25g erucic acid amide propyl dimethyl betaine and 3.0-8.0g sugar are added to the polyol aqueous solution. The composition is supplemented with excess deionized water; stirred and mixed at 60-70°C for 1-2h, cooled to room temperature, and a cryoprotectant is prepared for standby use; wherein the sugar composition comprises chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of 1-2.5:2-5:2; when the sugar composition is added to the polyol aqueous solution, the chondroitin sulfate is first added to the polyol aqueous solution, mixed and dissolved; then the tamarind seed polysaccharide is added, mixed and dissolved; and finally the trehalose is added, mixed and dissolved.

[0036] Preferably, in the immune cell freezing solution, the basal solution includes DMEM culture medium, which contains nicotinamide mononucleotide with a final concentration of 200-500 μM and betaine with a final concentration of 25-35 mM; the cryoprotectant includes 45-60% polyol aqueous solution, 0.1-0.2% erucamidopropyl dimethyl betaine solution and 4.0-7.0% sugar composition solution; the sugar composition solution includes chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of 1-2.5:2-5:2.

[0037] Preferably, in the immune cell freezing solution, the basal solution includes DMEM culture medium, which contains nicotinamide mononucleotide with a final concentration of 300-400 μM and betaine with a final concentration of 25-30 mM; the cryoprotectant includes 50-55% polyol aqueous solution, 0.1-0.15% erucamidopropyl dimethyl betaine solution and 5.0-6.0% sugar composition solution; the sugar composition solution includes chondroitin sulfate, tamarind seed polysaccharide and trehalose in a mass ratio of 1-2.5:2-5:2.

[0038] Preferably, in the above-mentioned cryoprotectant, the polyol may be ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol or 2,3-butylene glycol; in the present invention, the preferred polyol is ethylene glycol.

[0039] Preferably, in the cryoprotectant, the molecular weight of tamarind seed polysaccharide is 90-200 kD; the molecular weight of chondroitin sulfate is 10-20 kD; or, the molecular weight of tamarind seed polysaccharide is 100-180 kD; the molecular weight of chondroitin sulfate is 12-18 kD; or, the molecular weight of tamarind seed polysaccharide is 120-150 kD; the molecular weight of chondroitin sulfate is 14-16 kD, etc.

[0040] In the cryoprotectant of the present invention, polyol is dissolved in deionized water to form an alcohol-water mixed solution, and this mixed solution is used as a mixed solvent for dissolving erucamidopropyl dimethyl betaine and the sugar composition, so that the entire reaction system forms a multi-component mixed solution, that is, a cryoprotectant.

[0041] 2. Cryopreservation of immune cells

[0042] The present invention also provides a cryopreservation method for cryopreserving immune cells using the cryopreservation solution, comprising the following steps:

[0043] At room temperature, resuspend the immune cells using the basic solution prepared in the first item of the present invention to obtain a cell suspension;

[0044] Adding the cryoprotectant prepared in the first item of the present invention, which has been precooled to 2-8° C., to the cell suspension, with the volume ratio of the base liquid to the cryoprotectant being 1:2-3, to obtain a cell mixture;

[0045] The cell mixture was dispensed into cryopreservation tubes, programmed to cool to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0046] Preferably, in the immune cell freezing method, the density of the immune cells in the cell suspension after the immune cells are resuspended in the basal solution is maintained at 0.5 to 3×10 7 Pieces / mL.

[0047] Preferably, in the immune cell freezing method, after the cell mixture is dispensed into cryopreservation tubes, the temperature is programmed to cool to -80°C at a cooling rate of 4°C / min.

[0048] Preferably, in the present invention, the immune cells include any one of dendritic cells (DC cells), NK cells, monocytes, T cells, and CIK cells.

[0049] 3. Cryopreservation and recovery of immune cells

[0050] Take out the frozen immune cells from liquid nitrogen and thaw them in a 40-42℃ dry cell resuscitation instrument for 1.5-2 minutes until no ice particles are visible to the naked eye;

[0051] Aspirate cells from the cryopreservation tube and slowly drip into a 15 ml centrifuge tube containing 5 ml of 4°C precooled serum-free cell culture medium, and make up to 15 ml with 4°C precooled serum-free cell culture medium;

[0052] Take the fixed volume of cells, centrifuge at 500g for 8 minutes, remove the supernatant and resuspend the cell pellet in room temperature serum-free cell culture medium, then centrifuge at 500g for 8 minutes to obtain the cell pellet for cell culture after recovery.

[0053] The technical solution of the present invention is further described in detail below through specific embodiments.

[0054] (I) Cell cryopreservation

[0055] Example 1

[0056] 1.1. Preparation of freezing solution

[0057] Prepare 100 mL of basal solution: Based on DMEM culture medium, the culture medium contains nicotinamide mononucleotide with a final concentration of 450 μM and betaine with a final concentration of 35 mM;

[0058] Prepare 100 mL of cryoprotectant: first, take 50 g of ethylene glycol and dissolve it in 40 mL of deionized water to obtain an ethylene glycol aqueous solution; then, add 0.1 g of erucic acid amide propyl dimethyl betaine to the ethylene glycol aqueous solution and mix it to dissolve; secondly, add 1.43 g of chondroitin sulfate to mix the solvent; then add 2.14 g of tamarind seed polysaccharide to mix the solvent, and finally add 1.43 g of trehalose to mix and dissolve, and add 4.9 mL of deionized water; the entire mixing process is stirred and mixed at 65°C for 1.5 hours, cooled to room temperature, and a cryoprotectant is obtained; wherein, the mass ratio of chondroitin sulfate (molecular weight 15kD), tamarind seed polysaccharide (molecular weight 150kD) and trehalose is 2:3:2.

[0059] 1.2. DC cell cryopreservation

[0060] DC cells loaded with antigen for 24 hours (electroporated with mRNA antigen containing EGFP reporter gene), washed with PBS 1-2 times, centrifuged at 500g for 6 minutes; the supernatant was removed to obtain the cell precipitate, i.e., DC cells loaded with antigen;

[0061] Take 10 mL of the basic solution prepared in step 1.1 of this example and resuspend 3×10 8 antigen-loaded DC cells to obtain a DC cell suspension;

[0062] Take 20 mL of the cryoprotectant prepared in step 1.1 of this example, precool it at 4°C for 4 min, add it to the DC cell suspension, mix well, and control the cell density to 1×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0063] Example 2

[0064] 2.1. Preparation of cryopreservation solution

[0065] Prepare 100 mL of basal solution: Based on DMEM culture medium, the culture medium contains nicotinamide mononucleotide with a final concentration of 600 μM and betaine with a final concentration of 20 mM;

[0066] Preparation of 100 mL of cryoprotectant: First, take 41.4 g of 1,3-propylene glycol and dissolve it in 40 mL of deionized water to obtain a 1,3-propylene glycol aqueous solution; then, add 0.25 g of erucic acid amide propyl dimethyl betaine to the 1,3-propylene glycol aqueous solution and mix it to dissolve; secondly, add 1 g of chondroitin sulfate to mix the solvent; then add 5 g of tamarind seed polysaccharide to mix the solvent, and finally add 2 g of trehalose to mix and dissolve, and add 10.35 mL of deionized water; the entire mixing process is stirred and mixed at 70°C for 1 hour, cooled to room temperature, and a cryoprotectant is obtained; wherein, the mass ratio of chondroitin sulfate (molecular weight 10 kD), tamarind seed polysaccharide (molecular weight 200 kD) and trehalose is 1:5:2.

[0067] 2.2. NK cell cryopreservation

[0068] Take 10 mL of the basic solution prepared in step 2.1 of this example and resuspend 2×10 8 NK cells to obtain NK cell suspension;

[0069] Take 30 mL of the cryoprotectant prepared in step 2.1 of this example, precool it at 2°C for 6 min, add it to the NK cell suspension, mix well, and control the cell density to 0.5×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0070] Example 3

[0071] 3.1. Preparation of cryopreservation solution

[0072] Prepare 100 mL of basal solution: Based on DMEM culture medium, the culture medium contains nicotinamide mononucleotide with a final concentration of 300 μM and betaine with a final concentration of 40 mM;

[0073] Prepare 100 mL of cryoprotectant: first, take 66.9 g of 2,3-butanediol and dissolve it in 25 mL of deionized water to obtain a 2,3-butanediol aqueous solution; then, add 0.05 g of erucic acid amide propyl dimethyl betaine to the 2,3-butanediol aqueous solution and mix it to dissolve; secondly, add 1.16 g of chondroitin sulfate to mix the solvent; then add 0.92 g of tamarind seed polysaccharide to mix the solvent, and finally add 0.92 g of trehalose to mix and dissolve, and add 5.05 mL of deionized water; the entire mixing process is stirred and mixed at 60°C for 2 hours, cooled to room temperature, and a cryoprotectant is obtained; wherein, the mass ratio of chondroitin sulfate (molecular weight 20 kD), tamarind seed polysaccharide (molecular weight 90 kD) and trehalose is 2.5:2:2.

[0074] 3.2. Cryopreservation of CIK cells

[0075] Take 10 mL of the basic solution prepared in step 3.1 of this example and resuspend 7.5×10 8 CIK cells were obtained to obtain CI K cell suspension;

[0076] Take 25 mL of the cryoprotectant prepared in step 3.1 of this example, precool it at 8°C for 2 min, add it to the CIK cell suspension, mix well, and control the cell density to 3×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0077] Example 4 (Comparative Example 1)

[0078] 4.1. Preparation of cryopreservation solution

[0079] Prepare 100 mL of basal solution: Use DMEM medium as the basal solution;

[0080] Prepare 100 mL of cryoprotectant: First, dissolve 44.6 g of ethylene glycol in 30 mL of deionized water and mix well to obtain an ethylene glycol aqueous solution; then, add 5 g of trehalose to the ethylene glycol aqueous solution and mix well to dissolve; finally, add 10 mL of DSMO with a concentration of and mix well to dissolve, and then add 10.4 mL of deionized water; the entire mixing process is carried out at 65°C, stirring and mixing for 1.5 hours, and then cooling to room temperature to obtain a cryoprotectant.

[0081] 4.2 DC cell cryopreservation

[0082] Take the DC cells that have been cultured for 24 hours and loaded with antigen (electroporated with mRNA antigen containing EGFP reporter gene), wash them with PBS 1-2 times, centrifuge them at 500g for 6 minutes, remove the supernatant, and obtain the DC cells loaded with antigen;

[0083] Take 10 mL of the basic solution prepared in step 4.1 of this example and resuspend 3×10 8 antigen-loaded DC cells to obtain a DC cell suspension;

[0084] Take 20 mL of the cryoprotectant prepared in step 4.1 of this example, precool it at 4°C for 4 min, add it to the DC cell suspension, mix well, and control the cell density to 1×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0085] Example 5 (Comparative Example 2)

[0086] 5.1. Preparation of freezing solution

[0087] Prepare 100 mL of basal solution: Based on DMEM culture medium, the culture medium contains nicotinamide mononucleotide with a final concentration of 600 μM and betaine with a final concentration of 20 mM;

[0088] Prepare 100 mL of cryoprotectant: First, dissolve 41.4 g of 1,3-propylene glycol in 45 mL of deionized water and mix well to obtain a 1,3-propylene glycol aqueous solution; then, add 0.25 g of erucamidopropyl dimethyl betaine to the 1,3-propylene glycol aqueous solution and mix well to dissolve; finally, add 8 g of trehalose and mix well to dissolve, and add 5.35 mL of deionized water; the entire mixing process is stirred and mixed at 65°C for 1.5 hours, cooled to room temperature, and the cryoprotectant is obtained.

[0089] 5.2. NK cell cryopreservation

[0090] Take 10 mL of the basic solution prepared in step 5.1 of this example and resuspend 2×10 8 NK cells to obtain NK cell suspension;

[0091] Take 30 mL of the cryoprotectant prepared in step 5.1 of this example, precool it at 2°C for 6 min, add it to the NK cell suspension, mix well, and control the cell density to 0.5×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0092] Example 6 (Comparative Example 3)

[0093] 6.1. Preparation of cryopreservation solution

[0094] Prepare 100 mL of basal solution: Based on DMEM culture medium, the culture medium contains nicotinamide mononucleotide with a final concentration of 300 μM and betaine with a final concentration of 40 mM;

[0095] Prepare 100 mL of cryoprotectant: First, dissolve 66.9 g of 2,3-butanediol in 25 mL of deionized water and mix well to obtain a 2,3-butanediol aqueous solution; then, add 0.05 g of erucamidopropyl dimethyl betaine to the 2,3-butanediol aqueous solution and mix well to dissolve; finally, add 3 g of trehalose and mix well to dissolve, and add 5.05 mL of deionized water; the entire mixing process is stirred and mixed at 65°C for 1.5 hours, cooled to room temperature, and the cryoprotectant is obtained.

[0096] 6.2. Cryopreservation of CIK cells

[0097] Take 10 mL of the basic solution prepared in step 6.1 of this example and resuspend 7.5×10 8 CIK cells were obtained to obtain CI K cell suspension;

[0098] Take 25 mL of the cryoprotectant prepared in step 6.1 of this example, precool it at 8°C for 2 min, add it to the CIK cell suspension, mix well, and control the cell density to 3×10 7 / mL, dispense into 2 ml cryotubes, cool to -80℃ at a cooling rate of 4℃ / min, and then cryopreserve in liquid nitrogen.

[0099] (II) Cell recovery and functional test

[0100] The cells used for the cell function test in this part are taken from the frozen cells in Examples 1 to 4, respectively, and obtained according to the third item "cryopreservation and recovery of immune cells" of the present invention. The culture time after recovery is determined according to the test requirements.

[0101] Example 7

[0102] 7.1. Cell viability detection

[0103] Samples of cells recovered and cultured for 24 hours before freezing (day 0) and after freezing for 30 days in Examples 1 to 4 were taken respectively. The viability was measured using 7-AAD stain and flow cytometry. The statistical results are shown in Tables 1 and Figure 1 As shown; Figure 1 The data are drawn based on Table 1.

[0104] Table 1 Cell cryopreservation and resuscitation viability test results

[0105]

[0106] The data in Table 1 showed significant differences after statistical analysis, p < 0.05. Figure 1 It can be seen that the cell viability in Examples 1 to 3 is better than that in Example 4 (Comparative Example 1), and the cell viability in Comparative Example 1 decreases significantly after the cells are cryopreserved and revived. This shows that the sugar composition composed of chondroitin sulfate, tamarind seed polysaccharide and trehalose in the cryoprotectant of the present invention reacts with polyols and erucic acid amide propyl dimethyl betaine to form a highly efficient ice recrystallization inhibitor, which has a micellar network structure at low temperatures, can reduce the formation of ice crystals during cell cryopreservation, reduce the damage of ice crystals to cells, and greatly improve the survival rate and growth expansion rate of immune cells after recovery. The freezing solution does not contain DMSO components, which also reduces the toxic side effects of the freezing solution on cells, further improving the survival rate of cells.

[0107] 7.2. Cryopreserved Cell Recovery and Proliferation Growth Test

[0108] The cells in Examples 2, 3, 5 and 6 were cryopreserved for 30 days and then revived and cultured for 16 days. The number of cells was counted using a cell counter on day 0, day 4, day 7, day 10, day 13 and day 16, respectively. The results are shown in Tables 2 and Figure 2 As shown; among them, Figure 2 The figures are drawn based on the data in Table 2.

[0109] Table 2 Count of cell cryopreservation and recovery growth

[0110]

[0111] From Table 2 and Figure 2 It can be seen that in Examples 2 and 3, the cells were cultured for 16 days after recovery, and the expansion multiples of the cells were 211.1 and 179.2 times, which were much higher than the expansion multiples of the cells in Examples 5 and 6 (i.e., Comparative Examples 2 and 3), which were 116.1 and 104.5 times, respectively. This shows that the sugar composition composed of chondroitin sulfate, tamarind seed polysaccharide and trehalose in the cryoprotectant of the present invention, after reacting with polyols and erucic acid amide propyl dimethyl betaine, forms a highly efficient ice recrystallization inhibitor, which has a micellar network structure at low temperatures, can reduce the formation of ice crystals during cell freezing, reduce the damage of ice crystals to cells, and greatly improve the survival rate of immune cells after recovery. The freezing solution does not contain DMSO components, which also reduces the toxic side effects of the freezing solution on cells, thereby improving the survival rate of cells.

[0112] 7.3. Detection of surface markers after DC cell cryopreservation and thawing

[0113] The frozen DC cells in Example 1 and Example 4 (Comparative Example 1) were revived and cultured for 24 h, and PBS solution was added to adjust the cell concentration to 1×10 6 / mL. Flow cytometry was used to detect cell surface markers CD80, CD11c, CD86 and HLA-DR, as shown in Table 3 and Figure 3 As shown; Figure 3 The data are drawn based on Table 3.

[0114] Table 3 DC cell marker ratio statistics

[0115]

[0116] From Table 3 and Figure 3 It can be seen that the ratio of markers CD80, CD11 c and CD86 represents the maturity and biological function activity (antigen presenting ability) of DC, and the ratio of marker HLA-DR represents the activation state of DC cells. In Examples 1 and 4, the markers CD80, CD11 c, CD86 and HLA-DR are all lower than before cryopreservation, indicating that cryopreservation has weakened the biological function activity of DC, but the ratio of markers CD80, CD11 c, CD86 and HLA-DR in Example 4 decreased more significantly, and the decrease in HLA-DR was nearly 30% ((91.61-65.18) / 91.61*100%=28.55%). This is because: the chondroitin sulfate, tamarind seed polysaccharide and trehalose in the cryoprotectant of the present invention can synergistically inhibit the formation of ice crystals as antifreeze agents, and the micellar network structure formed by the reaction of the three-combination sugar composition with polyols and erucic acid amide propyl dimethyl betaine can further inhibit the recrystallization of ice, reduce the damage of ice crystals to cells during the cell freezing process, greatly improve the survival rate and amplification growth of immune cells after recovery, and the freezing solution does not contain DMSO components, which also reduces the toxic side effects of the freezing solution on cells, thereby further improving the survival rate of cells.

[0117] 7.4. Detection of EGFP expression rate of antigen-loaded DC cells

[0118] The frozen DC cells in Example 1 and Example 4 (Comparative Example 1) were revived and incubated for 24 h, and then sampled, washed once with PBS, centrifuged at 400 g for 4 min, and directly loaded for flow cytometry. The fluorescence channel corresponding to EGFP was selected, and the DC without antigen loading was used as the control to draw the gate to detect the EGFP expression rate. The test results are shown in Table 4 and Figure 3 As shown, Figure 4 The data are drawn based on Table 4.

[0119] Table 4 EGFP expression rate of DC cells loaded with antigen

[0120]

[0121] From Table 4 and Figure 4 It can be seen that the EGFP expression rate of DC cells after cryopreservation and resuscitation for 24 hours in Example 1 is much higher than the EGFP expression rate of DC cells after cryopreservation and resuscitation for 24 hours in Example 4. This shows that the mRNA antigen containing the EGFP reporter gene transferred into DC cells will express EGFP protein, and a positive test means a higher mRNA transfer rate. From the results, the EGFP expression rate of Example 1 is higher, indicating that the physiological activity of the revived DC is better restored and has a higher expression maintenance rate (86.08 / 94.26*100%=91.32%), while the expression maintenance rate of Example 4 (Comparative Example 1) is less than 60% of the original level (55.7 / 94.26*100%=59.09%), which further illustrates that the sugar composition composed of chondroitin sulfate, tamarind seed polysaccharide and trehalose in the cryoprotectant of the present invention, after reacting with polyols and erucic acid amide propyl dimethyl betaine, forms a highly efficient ice recrystallization inhibitor, which has a micellar network structure at low temperature, can reduce the formation of ice crystals during cell freezing, reduce the damage of ice crystals to cells, greatly improve the survival rate and amplification growth of immune cells after recovery, and the freezing solution does not contain DMSO components, which also reduces the toxic side effects of the freezing solution on cells, thereby improving the survival rate of cells.

[0122] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. An immune cell freezing solution, characterized in that: The freezing solution comprises a base solution and a cryoprotectant; wherein: The basal solution includes a DMEM culture medium, which contains nicotinamide mononucleotide with a final concentration of 300 to 600 μM and betaine with a final concentration of 20 to 40 mM; The cryoprotectant, calculated by mass concentration, includes 41.4-66.9% of a polyol aqueous solution, 0.05-0.25% of an erucic acid amide propyl dimethyl betaine solution, and 3.0-8.0% of a sugar composition solution; the sugar composition solution includes chondroitin sulfate, tamarind seed polysaccharide, and trehalose in a mass ratio of 1-2.5:2-5:

2.

2. The immune cell freezing solution according to claim 1, characterized in that: The polyol is ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol or 2,3-butylene glycol.

3. The immune cell freezing solution according to claim 1, characterized in that: The polyol is ethylene glycol.

4. The immune cell freezing solution according to claim 1, characterized in that: The molecular weight of the tamarind seed polysaccharide is 90-200 kD; the molecular weight of the chondroitin sulfate is 10-20 kD.

5. A method for preparing the cryoprotectant as claimed in claim 1, characterized in that: The steps include: Adding polyol into deionized water and mixing them to obtain a polyol aqueous solution; Add erucamidopropyl dimethyl betaine and the sugar composition to the polyol aqueous solution, add the balance of deionized water, stir and mix at 60-70° C. for 1-2 hours, cool to room temperature, and prepare a cryoprotectant for later use; When the sugar composition is added to the polyol aqueous solution, the chondroitin sulfate is first added to the polyol aqueous solution and mixed and dissolved; then the tamarind seed polysaccharide is added and mixed and dissolved; finally, the trehalose is added and mixed and dissolved.

6. A method for freezing immune cells, characterized in that: The steps include: At room temperature, resuspend the immune cells using the basic solution as described in claim 1 to obtain a cell suspension; Adding the cryoprotectant as claimed in claim 1 which has been precooled at 2 to 8° C. for 2 to 6 minutes to the cell suspension, wherein the volume ratio of the base liquid to the cryoprotectant is 1:2 to 3, to obtain a cell mixture; The cell mixture was dispensed into cryopreservation tubes, programmed to cool to -80°C, and then transferred to liquid nitrogen for long-term storage.

7. The immune cell cryopreservation method according to claim 6, characterized in that: In the cell suspension after the immune cells are resuspended in the basal solution, the density of immune cells is maintained at 0.5 to 3×10 7 Pieces / mL.

8. The immune cell cryopreservation method according to claim 6, characterized in that: When the temperature is lowered to -80°C, the temperature is lowered at a rate of 4°C / min.

9. The immune cell cryopreservation method according to claim 6, characterized in that: The immune cells include any one of dendritic cells, NK cells, monocytes, T cells, and CIK cells.

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