Cryopreservation protection liquid, cryopreservation method and recovery method of CIK (Cytokine Induced Killer) cells

Abstract

The invention provides a cryopreservation protective solution for CIK (cytokine-induced killer) cells. The cryopreservation protective solution for a CIK cell injection comprises the following components in percentage by volume: 5-10% of dimethyl sulfoxide, 20-40% of human serum albumin and the balance of compound electrolyte. The cryopreservation protection liquid for the CIK cells is simple in component and low in raw material cost, the safety risk caused by high-concentration DMSO is reduced, meanwhile, the cell viability after resuscitation of the cryopreserved cells is improved, the CIK cells are cryopreserved through the cryopreservation protection liquid, the CIK cells can be directly used after being unfrozen and centrifugally cleaned, and activation culture does not need to be conducted again.

Description

Technical Field

[0001] The present invention relates to the technical field of cell cryopreservation and recovery, and specifically, to a cryopreservation protective solution, a cryopreservation method, and a recovery method for CIK cells. Background Art

[0002] In recent years, CIK cells (Cytokine-induced killer cells) have been considered as the first cells for the new generation of adoptive cell immunotherapy for anti-tumor. The killing of tumor cells by CIK cells is non-MHC restricted, that is, it is not restricted by the cancer tissue type, and can automatically identify and kill tumor cells in a wide range of parts of the human body. Some clinical studies have shown that CIK cells have safety and promising efficacy in the treatment of autologous and allogeneic solid and hematological malignancies.

[0003] According to the usage standard of cytokine-induced killer cells, at least 1×10 9 CIK cells are required to obtain a clinically meaningful therapeutic effect. However, current CIK cell culture protocols all require 2 - 3 weeks of culture, with a long time cycle and unable to meet some temporary and urgent cell requirements. Therefore, a cryopreservation protocol is needed to cryopreserve the amplified CIK cell injection so that when there is a temporary cell requirement, the CIK cell cryopreservation injection can be directly recovered for use. The cell viability and quality of the cells recovered after cryopreservation directly affect the quality of subsequent treatment and healthcare.

[0004] The cell cryopreservation solution is an important factor directly affecting the cell viability and quality after cryopreservation. However, after cryopreserving CIK cells with the existing CIK cell cryopreservation solution, it is necessary to culture for several days after recovery to meet the usage requirements. Summary of the Invention

[0005] Aiming at the defects in the prior art, the purpose of the present invention is to provide a cryopreservation protective solution, a cryopreservation method, and a recovery method for CIK cells. The cryopreservation protective solution for CIK cells provided by the present invention has simple components and low raw material costs, reduces the safety risks brought by high-concentration DMSO, and at the same time, improves the cell survival rate of the cryopreserved cells after recovery.

[0006] The first aspect of the present invention provides a cryopreservation protective solution for CIK cells. The cryopreservation solution of the CIK cell injection is composed of the following components by volume percentage: 5% - 10% dimethyl sulfoxide, 20% - 40% human albumin, and the balance is compound electrolyte.

[0007] In an embodiment of the present invention, the dimethyl sulfoxide is clinical-grade dimethyl sulfoxide, the content of dimethyl sulfoxide in the clinical-grade dimethyl sulfoxide is ≥ 99.5%, the water content in the clinical-grade dimethyl sulfoxide is ≤ 0.2%, the dimethyl sulfone in the clinical-grade dimethyl sulfoxide is ≤ 0.1%, and the non-volatile residue in the clinical-grade dimethyl sulfoxide should be ≤ 0.1%.

[0008] In an embodiment of the present invention, the human albumin used is human albumin for injection. The protein content in the human albumin for injection is 190 - 210 g / L, the purity should be not less than 96.0% of the total protein amount, the pH value is 6.4 - 7.4, and the osmotic molality is 210 - 400 mOsmol / kg.

[0009] In an embodiment of the present invention, the compound electrolyte comprises raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L;

[0010] The osmotic molality ratio of the compound electrolyte is 0.9 - 1.1.

[0011] In an embodiment of the present invention, the volume percentage of the dimethyl sulfoxide is 5% or 10%; the volume percentage of the human albumin is 20% or 40%.

[0012] Preferably, the cryopreservation protective solution for CIK cells comprises, by volume percentage: 75% compound electrolyte, 20% human albumin, 5% dimethyl sulfoxide;

[0013] Preferably, the cryopreservation protective solution for CIK cells comprises, by volume percentage: 55% compound electrolyte, 40% human albumin, 5% dimethyl sulfoxide;

[0014] Preferably, the cryopreservation protective solution for CIK cells comprises, by volume percentage: 70% compound electrolyte, 20% human albumin, 10% dimethyl sulfoxide;

[0015] Preferably, the cryopreservation protective solution for CIK cells comprises, by volume percentage: 50% compound electrolyte, 40% human albumin, 10% dimethyl sulfoxide.

[0016] The second aspect of the present invention provides a preparation method of the above-mentioned cryopreservation protective solution for CIK cells, comprising the following steps:

[0017] Measure the formula amounts of the compound electrolyte, human albumin, and dimethyl sulfoxide;

[0018] Add the dimethyl sulfoxide to the compound electrolyte and mix evenly to obtain a mixed solution;

[0019] After the temperature of the mixed solution drops to 18-30 °C, human albumin is added and mixed evenly to obtain a cryopreservation protective solution for CIK cells.

[0020] The third aspect of the present invention provides a cryopreservation method for CIK cells, comprising the following steps:

[0021] The above cryopreservation protective solution or the cryopreservation protective solution prepared by the above preparation method is pre-cooled at 2-8 °C for more than 30 minutes;

[0022] The CIK cells are resuspended with the pre-cooled cryopreservation protective solution to obtain a cell suspension, which is then aliquoted into cryopreservation tubes or cryopreservation bags. The cryopreservation tubes are placed in a programmed cooling box and transferred to an environment of -80 °C, and after 24 hours, they are placed in a liquid nitrogen atmosphere for storage; or the cryopreservation bags are placed in a programmed cooler, and after cooling to -80 °C, they are placed in a liquid nitrogen atmosphere for storage.

[0023] In an embodiment of the present invention, in the cell suspension, the cell concentration is 0.5×10 8 ~1×10 8 cells / ml.

[0024] In an embodiment of the present invention, the cooling rate of the programmed cooling box or the programmed cooler is 1 °C / min.

[0025] The fourth aspect of the present invention provides a method for resuscitating CIK cells, which comprises:

[0026] The cryopreservation tube or cryopreservation bag contains cryopreserved CIK cells, and the cryopreserved CIK cells are cryopreserved by the above CIK cell cryopreservation method;

[0027] The cryopreservation tube or cryopreservation bag of cells is placed in a water bath at 35-42 °C, thawed within 1-3 minutes, transferred to a centrifuge tube, centrifuged to discard the supernatant, resuspended and washed with an electrolyte solution, centrifuged again, and then resuspended with an electrolyte solution to obtain resuscitated CIK cells.

[0028] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0029] 1. The cryopreservation protective solution for CIK cells provided by the embodiments of the present invention has simple components and low raw material costs, reduces the safety risks brought by high-concentration DMSO, and at the same time, improves the cell viability after resuscitation of cryopreserved cells.

[0030] 2. When using the cryopreservation protective solution provided by the embodiments of the present invention to cryopreserve CIK cells, after thawing, it can be directly used after centrifuging to discard the supernatant, resuspending and washing with an electrolyte solution, centrifuging again, and resuspending with an electrolyte solution, without the need for re-activation and culture; among them, the electrolyte solution can be physiological saline containing 1% HSA or a compound electrolyte solution. Brief Description of the Drawings

[0031] Other features, objectives, and advantages of the present invention will become more apparent by reading the following detailed description of non - restrictive embodiments with reference to the accompanying drawings:

[0032] Figure 1 It is an analysis chart of the resuscitation count and flow cytometry detection results after cryopreservation for 3 months in Examples 1 - 6 and Comparative Example 1. Detailed Description of the Embodiments

[0033] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can be made. These all fall within the protection scope of the present invention.

[0034] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise defined, all professional terms, symbols, and other technical and scientific terms or proprietary vocabulary used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with conventional understood meanings are defined herein for the purpose of clarification and / or facilitating citation. The inclusion of such definitions herein should not be construed as indicating a significant difference from the conventional understanding in the art.

[0036] After cryopreserving CIK cells using the existing CIK cell cryopreservation solution, it is necessary to culture for several days after resuscitation to meet the usage requirements. Aiming at the above - mentioned disadvantages and deficiencies of the prior art, the present invention proposes a cryopreservation protective solution, a cryopreservation method, and a resuscitation method for CIK cells. After cryopreserving CIK cells using the cryopreservation protective solution proposed by the present invention, after thawing, the supernatant is discarded by centrifugation, the cells are resuspended and washed by centrifugation using an electrolyte solution, and then resuspended in the electrolyte solution after discarding the supernatant, and can be directly used without the need to culture for several days, saving time cost and raw material cost, and being able to meet the purpose of the urgent need for CIK cells.

[0037] The human albumin used in Examples 1 - 6 and Comparative Example 1 of the present invention is all human albumin for injection. The protein content in the selected human albumin for injection is 190 - 210 g / L, the purity is not less than 96.0% of the total protein, the pH value is 6.4 - 7.4, and the osmotic molality is 210 - 400 mOsmol / kg; for example, the human albumin for injection used in Examples 1 - 6 and Comparative Example 1 of the present invention can be selected from the same brand and the same batch of human albumin for injection produced by the same company.

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0039] Example 1

[0040] This example provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolytes, human albumin, and dimethyl sulfoxide. The volume ratio of compound electrolytes, human albumin, and dimethyl sulfoxide is 75:20:5.

[0041] The compound electrolytes in this example include raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolytes is 0.9 - 1.1.

[0042] The dimethyl sulfoxide in this example is clinical-grade dimethyl sulfoxide. The content of dimethyl sulfoxide in clinical-grade dimethyl sulfoxide is ≥99.5%, the water content is ≤0.2%, the dimethyl sulfone is ≤0.1%, and the non-volatile residue should be ≤0.1%.

[0043] The human albumin in this example is human albumin for injection.

[0044] The cryopreservation protective solution for CIK cells in this example is prepared by the following steps: slowly add 0.4 ml of DMSO (dimethyl sulfoxide) to 6 ml of compound electrolytes, mix well, and after the mixture has dissipated heat, add 1.6 ml of HSA (human albumin), mix well, and store in a 4°C refrigerator for pre-cooling and standby.

[0045] Example 2

[0046] This example provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolytes, human albumin, and dimethyl sulfoxide. The volume ratio of compound electrolytes, human albumin, and dimethyl sulfoxide is 55:40:5.

[0047] The compound electrolytes in this example include raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolytes is 0.9 - 1.1.

[0048] The dimethyl sulfoxide in this embodiment is clinical-grade dimethyl sulfoxide. The content of dimethyl sulfoxide in clinical-grade dimethyl sulfoxide is ≥99.5%, the water content is ≤0.2%, the dimethyl sulfone content is ≤0.1%, and the non-volatile residue should be ≤0.1%.

[0049] The human albumin in this embodiment is human albumin for injection.

[0050] The cryopreservation protective solution for CIK cells in this embodiment is prepared by the following steps: Slowly add 0.4 ml of DMSO to 4.4 ml of compound electrolyte, mix well. After the mixture cools down, add 3.2 ml of HSA, mix well, and store in a 4°C refrigerator for pre-cooling and standby.

[0051] Example 3

[0052] This embodiment provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolyte, human albumin, and dimethyl sulfoxide. The volume ratio of compound electrolyte, human albumin, and dimethyl sulfoxide is 70:20:10.

[0053] The compound electrolyte in this embodiment includes raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolyte is 0.9 - 1.1.

[0054] The dimethyl sulfoxide in this embodiment is clinical-grade dimethyl sulfoxide. The content of dimethyl sulfoxide in clinical-grade dimethyl sulfoxide is ≥99.5%, the water content is ≤0.2%, the dimethyl sulfone content is ≤0.1%, and the non-volatile residue should be ≤0.1%.

[0055] The human albumin in this embodiment is human albumin for injection.

[0056] The cryopreservation protective solution for CIK cells in this embodiment is prepared by the following steps: Slowly add 0.8 ml of DMSO to 5.6 ml of compound electrolyte, mix well. After the mixture cools down, add 1.6 ml of HSA, mix well, and store in a 4°C refrigerator for pre-cooling and standby.

[0057] Example 4

[0058] This embodiment provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolyte, human albumin, and dimethyl sulfoxide. The volume ratio of compound electrolyte, human albumin, and dimethyl sulfoxide is 50:40:10.

[0059] The compound electrolyte in this example includes raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolyte is 0.9 - 1.1.

[0060] The dimethyl sulfoxide in this example is clinical - grade dimethyl sulfoxide. In the clinical - grade dimethyl sulfoxide, the content of dimethyl sulfoxide ≥ 99.5%, the water content ≤ 0.2%, the dimethyl sulfone ≤ 0.1%, and the non - volatile residue should ≤ 0.1%.

[0061] The human albumin in this example is human albumin for injection.

[0062] The cryopreservation protective solution for CIK cells in this example is prepared through the following steps: slowly add 0.8 ml of DMSO to 4 ml of the compound electrolyte, mix well. After the mixture cools down, add 3.2 ml of HSA, mix well, and store it in a 4°C refrigerator for pre - cooling and standby.

[0063] Example 5

[0064] This example provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolyte, human albumin and dimethyl sulfoxide. The volume ratio of the compound electrolyte, human albumin and dimethyl sulfoxide is 52:40:8.

[0065] The compound electrolyte in this example includes raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolyte is 0.9 - 1.1.

[0066] The dimethyl sulfoxide in this example is clinical - grade dimethyl sulfoxide. In the clinical - grade dimethyl sulfoxide, the content of dimethyl sulfoxide ≥ 99.5%, the water content ≤ 0.2%, the dimethyl sulfone ≤ 0.1%, and the non - volatile residue should ≤ 0.1%.

[0067] The human albumin in this example is human albumin for injection.

[0068] The cryopreservation protective solution for CIK cells in this example is prepared through the following steps: slowly add 0.64 ml of DMSO to 4.16 ml of the compound electrolyte, mix well. After the mixture cools down, add 3.2 ml of HSA, mix well, and store it in a 4°C refrigerator for pre - cooling and standby.

[0069] Example 6

[0070] This example provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolytes, human albumin, and dimethyl sulfoxide. The volume ratio of compound electrolytes, human albumin, and dimethyl sulfoxide is 60:30:10.

[0071] The compound electrolytes in this example include raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolytes is 0.9 - 1.1.

[0072] The dimethyl sulfoxide in this example is clinical-grade dimethyl sulfoxide. The content of dimethyl sulfoxide in clinical-grade dimethyl sulfoxide is ≥99.5%, the water content is ≤0.2%, the dimethyl sulfone is ≤0.1%, and the non-volatile residue should be ≤0.1%.

[0073] The human albumin in this example is human albumin for injection.

[0074] The cryopreservation protective solution for CIK cells in this example is prepared by the following steps: slowly add 0.8 ml of DMSO to 4.8 ml of compound electrolytes, mix well, and after the mixture has dissipated heat, add 2.4 ml of HSA, mix well, and place it in a 4°C refrigerator for pre-cooling and standby.

[0075] Comparative Example 1

[0076] This example provides a cryopreservation protective solution for CIK cells. The cryopreservation protective solution for CIK cells includes compound electrolytes, human albumin, dimethyl sulfoxide, and glycine. The volume ratio of compound electrolytes, human albumin, and dimethyl sulfoxide is 50:40:10, and the final concentration of glycine is 10 μg / ml.

[0077] The compound electrolytes in this example include raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolytes is 0.9 - 1.1.

[0078] The dimethyl sulfoxide in this example is clinical-grade dimethyl sulfoxide. The content of dimethyl sulfoxide in clinical-grade dimethyl sulfoxide is ≥99.5%, the water content is ≤0.2%, the dimethyl sulfone is ≤0.1%, and the non-volatile residue should be ≤0.1%.

[0079] The human albumin in this example is human albumin for injection.

[0080] The cryopreservation protective solution for CIK cells in this example is prepared through the following steps: slowly add 0.8 ml of DMSO to 3.9 ml of compound electrolyte, mix well, and after the mixture has dissipated heat, add 3.2 ml of HSA, mix well, and finally add 0.1 ml of sterile glycine solution prepared and filtered with compound electrolyte, mix well and place in a 4°C refrigerator for pre-cooling and standby.

[0081] The preparation process of the glycine solution in this example is to weigh 0.008 g of glycine powder, add it to 10 ml of compound electrolyte for dissolution and then filter it with a 0.22 μm needle filter (800 μg / ml).

[0082] Test Example

[0083] The method for detecting cell viability in this test example is as follows: Take a small amount of cell suspension into an EP tube for cell counting. Mix the cell suspension in the EP tube, pipette 20 μl of the cell suspension into a new EP tube, then add 20 μl of 0.2% trypan blue and stain for 1 - 3 min (the volume ratio of the cell suspension to the trypan blue solution is 1:1), mix well, pipette 20 μl of the stained cell suspension into a cell counting chamber, and use a Rigel fully automatic cell fluorescence analyzer for counting. According to the counting results, dilute the cell suspension with physiological saline to make the viable cell concentration 1 - 2×10 6 cells / ml. Pipette 20 μl of the diluted cell solution into 3 new EP tubes respectively, then add 20 μl of 0.2% trypan blue to each tube and stain for 1 - 3 min, mix well, pipette 20 μl of the stained cell suspension into a cell counting chamber respectively, and use a Rigel fully automatic cell fluorescence analyzer for counting, and record the counting data (viable cell concentration, cell viability, total number of viable cells).

[0084] The method for flow cytometry detection of cell phenotypes in this experimental example is as follows: Take a cell suspension containing 5 million cells into a 15-ml centrifuge tube, add 5 ml of PBS solution containing 2% FBS to resuspend, centrifuge at 1000 rpm for 5 min. Discard the supernatant, then add 1 ml of PBS solution containing 2% FBS to resuspend, centrifuge at 1000 rpm for 5 min. Add 500 μl of PBS solution containing 2% FBS to resuspend, mix well and dispense into 5 EP tubes, 100 μl / tube. The 5 EP tubes are labeled respectively: batch number + blank tube, batch number + Mouse Anti-Human CD3 tube, batch number + Mouse Anti-Human CD8 tube, batch number + Mouse Anti-Human CD56 tube, batch number + Mouse Anti-Human CD3 / CD8 / CD56 tube. Then add the corresponding antibodies to the labeled contents, mix well and incubate at 2-8 °C in the dark for 30 min. After incubation, add 900 μl of PBS solution containing 2% FBS to each EP tube to resuspend and wash, then centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 1 ml of PBS solution to each tube to resuspend, centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 150 μl of PBS solution to each tube to resuspend, and then use the CytoFLEX flow cytometer for on-machine analysis.

[0085] In this experimental example, 80 ml of peripheral blood was drawn from the veins of screened and qualified volunteers. After separating PBMC, CIK cells were induced and cultured. After 14 days of induction and amplification, CIK cells were collected, sampled for cell counting and flow cytometry detection of cell phenotypes (CD3+, CD3+CD8+, CD3+CD56+), and cryopreservation operations were performed on 7 kinds of cryopreservation solutions according to the above screening groups in sequence. After 3 months of cryopreservation, the cells were taken out from the liquid nitrogen tank, thawed, and cell counting and flow cytometry detection of cell phenotypes (CD3+, CD3+CD8+, CD3+CD56+) were performed.

[0086] In this experimental example, 80 ml of peripheral blood was drawn from the veins of screened and qualified volunteers. After separating PBMC, CIK cells were induced and cultured. After 14 days of induction and amplification, CIK cells were collected, sampled for cell counting, and according to the counting results, the cell suspension was dispensed into 14 centrifuge tubes;

[0087] The 14 centrifuge tubes were divided into 7 groups, with two in each group. The first group was resuspended with the pre-cooled cryopreservation solution prepared in Example 1 at cryopreservation densities of 5×10 7 cells / ml and 1×10 8 cells / ml, then dispensed into sterile cryopreservation tubes, placed in a programmable cooling box and transferred to an -80 °C refrigerator, and transferred to liquid nitrogen storage after 24 h; The second group was resuspended at 5×10 7cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Example 2, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage; The third group is at 5×10 7 cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Example 3, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage; The fourth group is at 5×10 7 cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Example 4, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage; The fifth group is at 5×10 7 cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Example 5, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage; The sixth group is at 5×10 7 cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Example 6, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage; The seventh group is at 5×10 7 cells / ml and 1×10 8 cells / ml, resuspend the cells with the pre-cooled cryopreservation solution prepared in Comparative Example 1, then aliquot them into sterile cryotubes, place them in a programmable freezing container and transfer them to an -80°C refrigerator. After 24 hours, transfer them to liquid nitrogen for storage.

[0088] Before resuspending the cells with the cryopreservation solution for each group of cells, flow cytometry detection of cell phenotypes (CD3+, CD3+CD8+, CD3+CD56+) was performed.

[0089] After 3 months of cryopreservation, the cells were taken out from the liquid nitrogen tank and thawed in a 37°C water bath. The thawing process should be completed within 2 minutes. The thawed cells were transferred to a PBS solution containing 2% FBS (the volume ratio of the cell suspension to the PBS solution containing 2% FBS was 1:5), and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended with 5 ml of PBS solution containing 2% FBS, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with 5 ml of PBS solution containing 2% FBS. Sampling was performed for cell counting and flow cytometry detection of cell phenotypes (CD3+, CD3+CD8+, CD3+CD56+). The detection results are shown in Table 1.

[0090] Table 1 Experimental result data and differences of Examples 1-6 and Comparative Example

[0091]

[0092]

[0093]

[0094] The bar chart of the difference analysis between the cell counting and flow cytometry detection results after thawing and recovery of the cryopreserved cells for 3 months and the results before cryopreservation is as Figure 1 shown:

[0095] By comparing the experimental data of Comparative Example 1 and Example 4, it was found that there was no significant difference between the two groups of data, indicating that on the basis of the embodiments of the present invention, adding glycine, which is commonly used in cell cryopreservation solutions to improve cell cryopreservation effects, to the cryopreservation solution formula has no obvious enhancing effect on the protection effect of the cryopreservation solution.

[0096] From the above test results, it can be seen that after cryopreserving CIK cells for 3 months using the CIK cell cryopreservation protective solution provided by the present invention, the cells can be directly used after thawing and recovery without further culture and activation; among them, the cryopreservation solution formula is compound electrolyte: human albumin: dimethyl sulfoxide = 55:40:5, and the cryopreservation density is 1×10 8 cells / ml, and the cryopreservation effect is the best. Based on the above data, when the cryopreservation solution of the CIK cell injection is within the range of 5% - 10% dimethyl sulfoxide, 20% - 40% human albumin, and the balance is compound electrolyte by volume percentage, the cryopreservation effect is better. After 3 months of cryopreservation, the decline range of the survival rate after recovery is within 10%, and the decline range of cell phenotypes is also about 10%.

[0097] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A cryopreservation protective solution for CIK cells, characterized in that, the cryopreservation solution of the CIK cell injection is composed of the following components by volume percentage: 5% - 10% dimethyl sulfoxide, 20% - 40% human albumin, and the balance is compound electrolyte.

2. The cryopreservation protective solution for CIK cells according to claim 1, characterized in that, the dimethyl sulfoxide is clinical-grade dimethyl sulfoxide, the content of dimethyl sulfoxide in the clinical-grade dimethyl sulfoxide is ≥ 99.5%, the water content in the clinical-grade dimethyl sulfoxide is ≤ 0.2%, the dimethyl sulfone in the clinical-grade dimethyl sulfoxide is ≤ 0.1%, and the non-volatile residue in the clinical-grade dimethyl sulfoxide should be ≤ 0.1%.

3. The cryopreservation protective solution for CIK cells according to claim 1, characterized in that, the human albumin used is human albumin for injection, the protein content in the human albumin for injection is 190 - 210 g / L, the purity should be not less than 96.0% of the total protein, the pH value is 6.4 - 7.4, and the osmotic molality is 210 - 400 mOsmol / kg.

4. The cryopreservation protective solution for CIK cells according to claim 1, characterized in that, the compound electrolyte includes raw materials with the following mass concentrations: sodium chloride 5.26 g / L, sodium gluconate 5.02 g / L, sodium acetate 3.68 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L; the osmotic molality ratio of the compound electrolyte is 0.9 - 1.

1.

5. The cryopreservation protective solution for CIK cells according to claim 1, characterized in that, the volume percentage of dimethyl sulfoxide is 5% or 10%; the volume percentage of human albumin is 20% or 40%.

6. The cryopreservation protective solution for CIK cells according to any one of claims 1 - 5, characterized in that, the cryopreservation protective solution for CIK cells includes by volume percentage: 75% compound electrolyte, 20% human albumin, 5% dimethyl sulfoxide; or, the cryopreservation protective solution for CIK cells includes by volume percentage: 55% compound electrolyte, 40% human albumin, 5% dimethyl sulfoxide; or, the cryopreservation protective solution for CIK cells includes by volume percentage: 70% compound electrolyte, 20% human albumin, 10% dimethyl sulfoxide; or, the cryopreservation protective solution for CIK cells includes by volume percentage: 50% compound electrolyte, 40% human albumin, 10% dimethyl sulfoxide.

7. A preparation method of a cryopreservation protective solution for CIK cells, characterized in that, comprises the following steps: Measure the formulated amounts of compound electrolyte, human albumin, and dimethyl sulfoxide; Add dimethyl sulfoxide to the compound electrolyte and mix evenly to obtain a mixed solution; After the temperature of the mixed solution drops to 18 - 30 °C, add human albumin and mix evenly to obtain the cryopreservation protective solution for CIK cells.

8. A cryopreservation method for CIK cells, characterized in that, comprises the following steps: The cryopreservation solution described in any one of claims 1-6 or the cryopreservation solution prepared as described in claim 7 is pre-cooled at 2-8 °C for more than 30 minutes; The CIK cells are resuspended with the pre-cooled cryopreservation solution to obtain a cell suspension, which is then aliquoted into cryotubes or cryobags; The cryotubes are placed in a programmable freezing container and transferred to an environment at -80 °C, and then stored in a liquid nitrogen atmosphere after 24 hours; or the cryobags are placed in a programmable freezer and stored in a liquid nitrogen atmosphere after cooling to -80 °C.

9. The method for cryopreserving CIK cells according to claim 8, wherein, In the cell suspension, the cell concentration is 0.5×10 8 ~1×10 8 cells / ml.

10. The method for cryopreserving CIK cells according to claim 8, wherein, the cooling rate of the programmable freezing container or the programmable freezer is 1 °C / min.

11. A method for thawing CIK cells, which comprises: a cryotube or cryobag containing cryopreserved CIK cells, and the cryopreserved CIK cells are cryopreserved by the method for cryopreserving CIK cells described in any one of claims 8-10; The cryotube or cryobag is placed in a water bath at 35-42 °C, thawed within 1-3 minutes, transferred to a centrifuge tube, centrifuged to discard the supernatant, resuspended and washed with an electrolyte solution, centrifuged again, and the supernatant is discarded, and then the CIK cells are resuspended with the electrolyte solution to obtain thawed CIK cells.

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