A NK cell transport storage solution and its preparation method and application

Abstract

The present invention relates to the field of immune cells, and in particular to a NK cell transport storage solution and a preparation method and application thereof. The NK cell transport storage solution provided by the present invention uses ultrapure water as a solvent, and the storage solution also includes the following ingredients added to the ultrapure water: mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin. The NK cell transport storage solution of the present invention reduces costs, has a simple preparation method, and has high NK cell activity after transportation and storage, stable quality, will not change the NK cell phenotype, and can maintain good tumor killing activity, which plays a good protective role on NK cells and is conducive to ensuring the quality of NK cells during transportation and storage.

Description

Technical Field

[0001] The present invention relates to the field of immune cells, and in particular to a NK cell transport storage fluid and a preparation method and application thereof. Background Art

[0002] Immune cell therapy is considered to be one of the effective methods for treating tumors. It mainly improves immune cells to make them have higher tumor killing activity, so as to better achieve the purpose of killing tumor cells. At present, it has been applied in the treatment of some malignant tumors, among which natural killer cells (NK cells) are the ideal choice in immune cell therapy. NK cells are natural immune effector cells in the human body. They can achieve specific immunity without pre-sensitization, and can quickly remove and eliminate aging and malignant cells in the body. They play an important role in anti-tumor treatment.

[0003] NK cell therapy requires the collection of cells, induction culture, in vitro expansion, and transfusion into the patient, among which the transportation method of NK cells is one of the important links to ensure cell quality. At present, the main method of cell transportation is ultra-low temperature preservation of cells, including dry ice transportation and liquid nitrogen transportation. Both methods mainly stop cell growth by freezing, but the transportation cost is high and the transportation process is complicated. In addition, cell metabolism can be reduced by transporting and storing at room temperature, thereby realizing cell storage and transportation. However, the existing NK cell transportation and storage has the problems of high cost and poor cell activity.

[0004] Therefore, it is necessary to develop a transport storage fluid that has a good protective effect on NK cells, reduce transportation costs, and ensure the survival rate and killing activity of NK cells after in vitro storage to meet the application of NK cells in cellular immunotherapy. Summary of the invention

[0005] In order to solve the above-mentioned technical problems existing in the prior art, the first aspect of the present invention provides a NK cell transport storage solution, which has a safe formula and can effectively ensure the original activity of NK cells.

[0006] The second aspect of the present invention provides a method for preparing a NK cell transport storage solution, which has a simple process and is easy to operate.

[0007] The third aspect of the present invention provides the use of NK cell transport and storage fluid in protecting NK cells. The present invention uses the above-mentioned NK cell transport and storage fluid to store and transport cells, which reduces the transportation cost and ensures the quality of NK cells during transportation and storage.

[0008] In order to achieve the above object, the technical solution adopted in the first aspect of the present invention is:

[0009] A NK cell transport storage solution, wherein the storage solution uses ultrapure water as a solvent and further comprises the following components added to the ultrapure water: mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin.

[0010] Preferably, the final concentrations of the components added to the ultrapure water are: mannitol 1.5-5 mg / mL, shikimic acid 4.5-9 μg / mL, sodium chloride 1.2-5.8 mg / mL, sodium pyrophosphate 20-28 ng / mL, calcium chloride 0.2-3.5 μg / mL, L-glutamine 10-14 μg / mL, L-lysine 15-25 ng / mL, and icariin 5-15 μg / mL.

[0011] Preferably, the final concentrations of the components added to the ultrapure water are: mannitol 3 mg / mL, shikimic acid 6.5 μg / mL, sodium chloride 3 mg / mL, sodium pyrophosphate 25 ng / mL, calcium chloride 1.2 μg / mL, L-glutamine 12 μg / mL, L-lysine 20 ng / mL, and icariin 10 μg / mL.

[0012] The technical solution adopted in the second aspect of the present invention is:

[0013] The preparation method of the above-mentioned NK cell transport storage solution comprises the following steps: under sterile conditions, mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin are dissolved in ultrapure water according to the formula and mixed evenly.

[0014] The technical solution adopted in the third aspect of the present invention is:

[0015] The application of the above NK cell transport storage solution in protecting NK cells is as follows:

[0016] Resuspend the NK cells with NK cell transport storage solution to obtain a mixed cell suspension, which is then dispensed into storage containers for storage and transportation.

[0017] Preferably, the concentration of the cell suspension is 2-6×10 7 / mL.

[0018] Preferably, the storage temperature is 2-6°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention provides a NK cell transport storage solution, which reduces costs. The shikimic acid in the storage solution can improve the stability of NK cells in the transport storage solution, maintain cell survival rate, and prolong storage time; icariin can help cells quickly restore their killing activity.

[0021] 2. The present invention also provides a method for preparing the above-mentioned NK cell transport storage solution. The preparation method of the present invention is simple, easy to operate, and conducive to large-scale production.

[0022] 3. The present invention also provides the use of NK cell transport storage solution in protecting NK cells. The NK cells preserved by the NK cell transport storage solution of the present invention have a high survival rate and stable quality. In addition, a high effector cell ratio and tumor killing activity can be maintained, fully ensuring the quality and safety of NK cells during transportation and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow cytometry test result diagram of NK cells after transportation and storage in Example 1 of the present invention;

[0024] Figure 2 This is a flow cytometry test result diagram of NK cells after transportation and storage in Comparative Example 1 of the present invention;

[0025] Figure 3 This is a flow cytometry test result diagram of NK cells after transportation and storage in Comparative Example 2 of the present invention;

[0026] Figure 4 This is a flow cytometry test result diagram of NK cells after transportation and storage in Comparative Example 3 of the present invention;

[0027] Figure 5 This is a flow cytometry test result diagram of NK cells after transportation and storage in Comparative Example 4 of the present invention;

[0028] Figure 6 This is a diagram of the NK cell tumor killing activity after transportation and storage of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0030] Example 1

[0031] A NK cell transport storage solution uses ultrapure water as a solvent, and the storage solution also includes the following components added to the ultrapure water: 3 mg / mL mannitol, 6.5 μg / mL shikimic acid, 3 mg / mL sodium chloride, 25 ng / mL sodium pyrophosphate, 1.2 μg / mL calcium chloride, 12 μg / mL L-glutamine, 20 ng / mL L-lysine, and 10 μg / mL icariin.

[0032] A method for preparing NK cell transport storage solution is as follows: under room temperature conditions, in an ultra-clean workbench, mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin are dissolved in ultrapure water according to the above formula, and mixed evenly to obtain the NK cell transport storage solution, which is prepared and used immediately.

[0033] An application of a NK cell transport storage solution in protecting NK cells specifically comprises the following steps:

[0034] (1) Isolation of mononuclear cells from peripheral blood: Collect 50 mL of healthy peripheral blood, mix it with normal saline, slowly add lymphocyte separation solution (the volume ratio of peripheral blood to lymphocyte separation solution is 2:1), centrifuge at 3000 rpm for 10 min, then carefully discard the upper plasma, absorb the mononuclear cell layer (i.e., buffy coat layer), then wash twice with PBS buffer, centrifuge at 3000 rpm for 10 min, and wash twice with PBS buffer to collect peripheral blood mononuclear cells (PBMC);

[0035] (2) The PBMCs collected in step (1) were added to AIM-V serum-free medium containing 10% inactivated autologous plasma, 60 ng / mL IL-21, and 1000 IU / mL IL-2. The culture conditions were 37°C, 5% CO2, and the medium was replenished every 2 days to maintain the density at 3×10 6 / mL, cultured for proliferation until 14 days, and NK cells were collected;

[0036] (3) Resuspend the NK cells collected in step (2) with the above NK cell transport storage solution to obtain a mixed cell suspension with a cell concentration of 2×10 7 / mL, dispense into cell freezing tubes, and store and transport at 4℃.

[0037] Example 2

[0038] A NK cell transport storage solution uses ultrapure water as a solvent, and the storage solution also includes the following components added to the ultrapure water: 1.5 mg / mL mannitol, 4.5 μg / mL shikimic acid, 1.2 mg / mL sodium chloride, 20 ng / mL sodium pyrophosphate, 0.2 μg / mL calcium chloride, 10 μg / mL L-glutamine, 15 ng / mL L-lysine, and 5 μg / mL icariin.

[0039] A method for preparing NK cell transport storage solution is as follows: under room temperature conditions, in an ultra-clean workbench, mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin are dissolved in ultrapure water according to the above formula, and mixed evenly to obtain the NK cell transport storage solution, which is prepared and used immediately.

[0040] An application of a NK cell transport storage solution in protecting NK cells specifically comprises the following steps:

[0041] Steps (1) and (2) are the same as those in Example 1. Step (3) is to resuspend the NK cells collected in step (2) with the NK cell transport storage solution to obtain a mixed cell suspension with a cell concentration of 4×10 7 / mL, dispense into cell freezing tubes, and store and transport at 6℃.

[0042] Example 3

[0043] A NK cell transport storage solution uses ultrapure water as a solvent, and the storage solution also includes the following components added to the ultrapure water: 5 mg / mL mannitol, 9 μg / mL shikimic acid, 5.8 mg / mL sodium chloride, 28 ng / mL sodium pyrophosphate, 3.5 μg / mL calcium chloride, 14 μg / mL L-glutamine, 25 ng / mL L-lysine, and 15 μg / mL icariin.

[0044] A method for preparing NK cell transport storage solution is as follows: under room temperature conditions, in an ultra-clean workbench, mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin are dissolved in ultrapure water according to the above formula, and mixed evenly to obtain the NK cell transport storage solution, which is prepared and used immediately.

[0045] An application of a NK cell transport storage solution in protecting NK cells specifically comprises the following steps:

[0046] Steps (1) and (2) are the same as those in Example 1. Step (3) is to resuspend the NK cells collected in step (2) with the NK cell transport storage solution to obtain a mixed cell suspension so that the cell concentration is 6×10 7 / mL, dispense into cell freezing tubes, and store and transport at 2℃.

[0047] Comparative Example 1

[0048] The only difference between this comparative example and Example 1 is that the concentration of shikimic acid in the NK cell transport storage solution is adjusted to 1 μg / mL.

[0049] Comparative Example 2

[0050] The only difference between this comparative example and Example 1 is that the concentration of shikimic acid in the NK cell transport storage solution is adjusted to 15 μg / mL.

[0051] Comparative Example 3

[0052] The only difference between this comparative example and Example 1 is that shikimic acid is not added to the NK cell transport storage solution.

[0053] Comparative Example 4

[0054] The only difference between this comparative example and Example 1 is that icariin is not added to the NK cell transport storage solution.

[0055] Test Example 1 Trypan blue staining method to detect cell activity during NK cell transport and storage

[0056] The cell cryopreservation tubes containing NK cells were taken out on the 1st, 2nd and 3rd day of transportation and storage, respectively, and placed in a 37°C water bath for resuscitation. After being blown and mixed, the cell suspensions of Example 1 and Comparative Examples 1 to 4 after transportation and storage for 1, 2 and 3 days were stained with 0.4% trypan blue solution (volume ratio was 9:1). After 2 minutes, the number of cells was counted with a cell counting plate. Three groups of cells were taken from each of Example 1 and Comparative Examples 1 to 4 for cell counting, and the cell survival rate was calculated according to the following formula, and the average value was calculated. The average survival rate of NK cells before storage and transportation was 99.38%.

[0057]

[0058] The results of cell viability are shown in Table 1:

[0059] Table 1

[0060]

[0061] The results are shown in Table 1. After NK cells were transported and stored for 1d, 2d, and 3d, the mean cell survival rates of Example 1 were 97.98%, 96.05%, and 94.40%, respectively. The cell survival rate was higher than that of Comparative Examples 1 to 4. Comparative Examples 1 and 2 adjusted the dosage range of shikimic acid. It can be seen that the concentration of shikimic acid has a certain effect on the viability of cells. Comparative Example 3 omitted shikimic acid, and the cell survival rate dropped significantly to 68.41%. Comparative Example 4 omitted icariin, and the cell survival rate was 85.56% after 3d of transportation and storage. Therefore, the above results show that shikimic acid can improve the stability of NK cells in the transport storage solution, maintain cell survival rate, and prolong the storage time.

[0062] Test Example 2 Phenotypic detection of NK cells after 3 days of transportation and storage

[0063] The NK cells of Example 1 and Comparative Examples 1-4 were transported and stored for 3 days and then revived in a 37°C water bath, washed with PBS buffer, centrifuged at 2000 rpm for 5 min, the washing supernatant was discarded, and then resuspended with PBS buffer to maintain the cell density at 1×10 6The cells were added to the flow cytometer tubes, 100 μL of cell suspension and 10 μL of CD56 monoclonal antibody (CD56-APC) labeled with allophycocyanin (APC) and CD3 monoclonal antibody (CD3-FITC) labeled with fluorescein isothiocyanate (FITC) were added to the tubes, and IgG labeled with fluorescein isothiocyanate (FITC) was used as a negative control. The cells were cultured for 20 minutes in a dark environment, and then washed twice with PBS buffer. After resuspending, the proportion of effector cells was detected by flow cytometry. The results are shown in the figure. Figures 1 to 5 shown.

[0064] NK cells are highly efficient effector cells, and their phenotype is CD3-CD56+. Figures 1 to 5 The flow cytometry test results showed that the effector cells of the NK cells transported and stored for 3 days in Example 1 reached 90.25% after proliferation culture, while Comparative Examples 1 and 2 changed the concentration range of shikimic acid, Comparative Example 3 did not add shikimic acid, and Comparative Example 4 did not add icariin, which all had a significant effect on the proportion of effector cells. Therefore, the above results show that shikimic acid and icariin play an irreplaceable role in the cell transport storage fluid.

[0065] Test Example 3 Cytotoxicity of NK cells against human chronic myeloid leukemia cells (K562)

[0066] The NK cells of Example 1 and Comparative Examples 1-4 after transportation and storage for 1 day, 2 days, and 3 days were revived in a 37°C water bath, centrifuged at 2000 rpm for 5 minutes, and then resuspended in PBS buffer to prepare 2×10 6 K562 cells in the logarithmic growth phase were prepared at a cell concentration of 2×10 5 The cell concentration of 100 μg / mL was used as the target cells. The effector cells and target cells were inoculated into 96-well plates at an effector-target ratio of 20:1 and mixed evenly as the experimental group. A separate K562 cell blank group and a separate NK cell blank group were set up at the same time. The total volume of each group was 200 μL. Five replicate wells were set up and cultured at 37°C for 15 h. Then 20 μL of CCK-8 reagent was added to each well. After incubation at 37°C in the dark for 3 h, the absorbance values ​​of the above wells at a wavelength of 490 nm were detected by an automatic microplate reader. The average value of each group was taken to calculate the tumor killing activity of NK cells.

[0067]

[0068] The results of NK cell killing activity test on K562 cells are as follows Figure 6As shown in the figure, the results show that the NK cells can still maintain a high killing activity after the NK cell transport storage solution prepared in Example 1 is stored for 1d, 2d and 3d. After 3 days of storage, the killing activities of Comparative Examples 1 to 3 are 76.5%, 74.4% and 73.1%, respectively. In Comparative Example 4, no icariin is added, and its killing rate is 67.1% after 3 days of storage, which is the lowest killing activity. Therefore, the above results show that icariin can help cells quickly restore killing activity.

[0069] In summary, the NK cell transport storage solution provided by the present invention has a simple formula and reduces costs, wherein the role of shikimic acid in the transport storage solution is to improve the stability of cells in the storage solution and prolong the storage time. Icariin helps cells to quickly restore killing activity, and can effectively ensure the quality of NK cells during transportation and storage. Therefore, the NK cell transport storage solution provided by the present invention has a protective effect on NK cells, can not only prolong the storage time of NK cells, but also maintain the killing activity of NK cells against tumor cells, and will not change the phenotype of NK cells, and can be used as a protective agent for NK cells.

[0070] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A NK cell transport storage solution, characterized in that: The storage solution uses ultrapure water as a solvent, and the storage solution also includes the following components added to the ultrapure water: mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine, and icariin. The final concentrations of the components added to the ultrapure water are: mannitol 3 mg / mL, shikimic acid 6.5 μg / mL, sodium chloride 3 mg / mL, sodium pyrophosphate 25 ng / mL, calcium chloride 1.2 μg / mL, L-glutamine 12 μg / mL, L-lysine 20 ng / mL, and icariin 10 μg / mL.

2. The method for preparing the NK cell transport storage solution according to claim 1, characterized in that: The method comprises the following steps: under sterile conditions, mannitol, shikimic acid, sodium chloride, sodium pyrophosphate, calcium chloride, L-glutamine, L-lysine and icariin are dissolved in ultrapure water according to a formula and mixed evenly to obtain the preparation method.

3. The use of the NK cell transport storage solution in protecting NK cells according to claim 1, characterized in that: The application is as follows: Resuspend the NK cells with NK cell transport storage solution to obtain a mixed cell suspension, which is then dispensed into storage containers for storage and transportation.

4. The use of the NK cell transport storage solution in protecting NK cells as claimed in claim 3, characterized in that: The cell concentration in the cell suspension is 2-6×10 7 Pieces / mL.

5. The use of the NK cell transport storage solution in protecting NK cells as claimed in claim 3, characterized in that: The storage temperature is 2-6°C.

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