CN117617225A - NK cell cryopreservation liquid, preparation method, cryopreservation method and kit - Google Patents
NK cell cryopreservation liquid, preparation method, cryopreservation method and kit Download PDFInfo
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- 238000005138 cryopreservation Methods 0.000 title claims abstract description 88
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an NK cell cryopreservation liquid, a preparation method, a cryopreservation method and a kit, wherein the NK cell cryopreservation liquid comprises dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, resveratrol, betulinic acid, apigenin and HEPES solution. On the premise of not affecting the freezing and preserving effect, the invention combines the glycerol and the dimethyl sulfoxide to reduce the dosage of the dimethyl sulfoxide, greatly weakens the toxicity of the dimethyl sulfoxide to cells or human bodies, combines the permeable freezing protective agent and the impermeable freezing protective agent to protect the cells, reduces the mechanical damage of ice crystals in the freezing and preserving process, can also protect the cell swelling and cracking damage caused by the rapid entry of water into the cells in the cell recovery process, and improves the survival rate of NK cell recovery.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to NK cell cryopreservation liquid, a preparation method, a cryopreservation method and a kit.
Background
NK cells are the first line of defense against tumors in the human body, play an important role in the innate immune system, can directly kill tumor cells without advanced sensitization, are already used for cell therapy of tumor immunity, have a good effect in clinical treatment of tumors, are treated by fresh cells in more than 97% of cell therapy clinical tests at present, are greatly limited by regions, and enable certain patients to not receive effective treatment in the first time, so that low-temperature storage is important for realizing trans-regional cell transportation and clinical application of cell immunotherapy.
The low temperature storage technology is to freeze and keep alive cells at ultra-low temperature, reduce the metabolism speed of the cells, and inhibit the activity of various enzymes for regulating and controlling the growth and metabolism of the cells, so that the inherent biochemical reaction of the cells is very slow, even the growth is stopped, thereby keeping the activity of the cells and prolonging the storage life of the cells. The existing NK cell cryopreservation liquid containing dimethyl sulfoxide is high in dosage, high in toxicity and unreasonable in component design, the NK cells are easily damaged in the cryopreservation process, the survival rate of the NK cells is affected when the NK cells are resuscitated, phenotype detection is carried out on the NK cells, the integrity inside and outside the cell phenotype is also greatly damaged, and then the killing activity of the NK cells after resuscitating is affected.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
The invention provides NK cell cryopreservation liquid, a preparation method, a cryopreservation method and a kit, and aims to solve the technical problems in the background art part.
The invention comprises the following steps:
the first aspect of the invention provides an NK cell cryopreservation solution comprising a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, and monomeric components of traditional Chinese medicine, and a PH buffer; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent and antioxidant comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the PH buffer comprises HEPES solution.
In an alternative embodiment of the first aspect of the present invention, in the NK cell cryopreservation solution, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10. Mu.M, betulinic acid is 0.1-10. Mu.M, apigenin is 0.1-10. Mu. M, HEPES solution is 10-25 mM.
In an alternative embodiment of the first aspect of the present invention, in the NK cell freezing solution, dimethyl sulfoxide is 5mL/100mL, glycerol is 5mL/100mL, hydroxyethyl starch is 10g/100mL, trehalose is 5g/100mL, raffinose is 5g/100mL, D-glucose is 3g/100mL, glutamine is 0.5g/100mL, sodium pyruvate is 0.5g/100mL, glycine is 0.005g/100mL, vitamin C is 1g/100mL, vitamin B12 is 0.003g/100mL, resveratrol is 0.6. Mu.M, betulinic acid is 0.3. Mu.M, apigenin is 0.5. Mu. M, HEPES solution is 20mM.
The second aspect of the invention provides a method for preparing NK cell cryopreservation liquid, which comprises the following steps:
providing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, antioxidants and herbal monomeric components, a PH buffer, a PH adjuster, and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer component comprises resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell frozen stock solution semi-finished product;
and determining the pH value of the NK cell cryopreservation liquid semi-finished product, and adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain the NK cell cryopreservation liquid finished product.
In an alternative embodiment of the second aspect of the present invention, the target volume comprises 100mL, the target value is 7 to 7.5, and the concentration of the NaOH solution is 1M.
In an alternative embodiment of the second aspect of the present invention, in the NK cell frozen stock solution finished product, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10 μm, betulinic acid is 0.1-10 μm, apigenin is 0.1-10 μm, and solution is 10-25 mM.
In a third aspect, the invention provides a method for cryopreserving NK cells comprising:
preparing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, a herbal monomer component, a pH buffer, a pH regulator and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell frozen stock solution semi-finished product;
determining the pH value of the NK cell cryopreservation liquid semi-finished product, adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain an NK cell cryopreservation liquid finished product;
resuspension of NK cells by using the NK cell frozen stock solution finished product, adjusting the density of the NK cells to a preset density, and then loading the NK cells into a frozen stock tube;
and directly placing the freezing tube in a refrigerator with negative temperature for temporary storage, and transferring the tube into liquid nitrogen for long-term storage after overnight.
In an alternative embodiment of the third aspect of the present invention, the NK cells are cells obtained by a pre-culture treatment, wherein the NK cell pre-culture treatment is performed as follows: and culturing NK cells according to the designed culture conditions, centrifuging the cultured NK cell suspension, discarding supernatant, and then washing with physiological saline or PBS solution to obtain the required NK cells.
In an alternative embodiment of the third aspect of the present invention, the target volume includes 100mL, the target value is 7 to 7.5, and the preset density is 5×10 6 ～5×10 7 The negative temperature is minus 60 ℃ to minus 100 ℃ per mL.
The fourth aspect of the invention provides an NK cell cryopreservation kit comprising an NK cell cryopreservation solution according to any one of the above.
The beneficial effects are that: the invention provides an NK cell cryopreservation liquid, a preparation method, a cryopreservation method and a kit, wherein the NK cell cryopreservation liquid comprises dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, resveratrol, betulinic acid, apigenin and HEPES solution. According to the invention, on the premise of not affecting the freezing effect, glycerol and dimethyl sulfoxide are used in a combined way, so that the dosage of dimethyl sulfoxide is reduced, the toxicity of dimethyl sulfoxide to cells or human bodies is greatly weakened, a permeable freezing protective agent and an impermeable freezing protective agent are used in a combined way, the cells are protected by both inner and outer consideration, the mechanical damage of ice crystals in the freezing process is reduced, the cell swelling and cracking damage caused by the rapid entry of water into cells in the cell recovery process can be also protected, the survival rate of NK cell recovery is improved, and the cell phenotype and killing activity of NK cells can be well maintained.
Drawings
FIG. 1 is a comparative bar graph of NK cell recovery survival rates of each example and comparative example before and after various times of cryopreservation according to the present invention.
FIG. 2 is a comparative bar graph showing the killing rate of K562 cells by NK cells of each of the examples and comparative examples before and after the different times of the freezing in accordance with the present invention.
FIG. 3 is a graph comparing the results of the effects on NK cell phenotype after 6 months of cryopreservation of the respective examples of the present invention and the comparative examples.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and effects of the present invention more clear and distinct. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The first aspect of the invention provides an NK cell cryopreservation solution comprising a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, and monomeric components of traditional Chinese medicine, and a PH buffer; wherein the permeable cryoprotectant comprises dimethyl sulfoxide (DMSO) and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer component comprises resveratrol, betulinic acid and apigenin; the PH buffer comprises HEPES solution.
Summarizing, the NK cell cryopreservation solution has the following beneficial effects:
1. the NK cell frozen stock solution disclosed by the invention has definite components, does not contain animal-derived components, and does not have the risk of introducing animal-derived antigens;
2. the NK cell cryopreservation liquid has low dimethyl sulfoxide (DMSO) content, greatly reduces toxicity to human bodies or cryopreserved cells, is safer, and is expected to be prepared into clinical NK cell cryopreservation liquid;
3. the NK cell cryopreservation liquid does not need gradient cooling operation, and cells to be frozen can be directly placed into a refrigerator at-80 ℃ for overnight and then transferred to liquid nitrogen, so that the operation is convenient, and the purchase cost of a cryopreservation box or a program cooling instrument required by gradient freezing is saved;
4. the NK cell freezing solution can better maintain the activity, phenotype and killing capacity of freezing NK cells, and reduce the damage to cells in the freezing process;
5. the NK cell cryopreservation liquid combines the permeable cryoprotectant and the impermeable cryoprotectant, can protect cells from both inside and outside, reduce mechanical damage of ice crystals in the cryopreservation process, and also can protect cell swelling, cracking and damage caused by water entering cells rapidly in the cell resuscitation process;
6. the NK cell frozen stock solution is added with the resveratrol, betulinic acid and apigenin which are traditional Chinese medicine monomer components, so that the killing activity of NK cells can be maintained.
In an alternative embodiment of the first aspect of the present invention, in the NK cell cryopreservation solution, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10. Mu.M, betulinic acid is 0.1-10. Mu.M, apigenin is 0.1-10. Mu. M, HEPES solution is 10-25 mM.
In an alternative embodiment of the first aspect of the present invention, in the NK cell freezing solution, dimethyl sulfoxide is 5mL/100mL, glycerol is 5mL/100mL, hydroxyethyl starch is 10g/100mL, trehalose is 5g/100mL, raffinose is 5g/100mL, D-glucose is 3g/100mL, glutamine is 0.5g/100mL, sodium pyruvate is 0.5g/100mL, glycine is 0.005g/100mL, vitamin C is 1g/100mL, vitamin B12 is 0.003g/100mL, resveratrol is 0.6. Mu.M, betulinic acid is 0.3. Mu.M, apigenin is 0.5. Mu. M, HEPES solution is 20mM.
In an alternative embodiment of the first aspect of the present invention, the NK cell lysate further comprises a PH adjuster for adjusting the final PH of the NK cell lysate to 7-7.5, e.g. 7.4, and sterile water.
The second aspect of the invention provides a method for preparing NK cell cryopreservation liquid, which comprises the following steps:
providing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, a herbal monomer component, a PH buffer, a PH adjuster, and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell frozen stock solution semi-finished product;
and determining the pH value of the NK cell cryopreservation liquid semi-finished product, and adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain the NK cell cryopreservation liquid finished product.
In an alternative embodiment of the second aspect of the present invention, the target volume comprises 100mL, the target value is 7 to 7.5, and the concentration of the NaOH solution is 1M.
In an alternative embodiment of the second aspect of the present invention, in the NK cell frozen stock solution finished product, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10 μm, betulinic acid is 0.1-10 μm, apigenin is 0.1-10 μm, and solution is 10-25 mM.
In a third aspect, the invention provides a method for cryopreserving NK cells comprising:
preparing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, antioxidants and herbal monomer components, a PH buffer, a PH adjuster and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell frozen stock solution semi-finished product;
determining the pH value of the NK cell cryopreservation liquid semi-finished product, adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain an NK cell cryopreservation liquid finished product;
resuspension of NK cells by using the NK cell frozen stock solution finished product, adjusting the density of the NK cells to a preset density, and then loading the NK cells into a frozen stock tube;
and directly placing the freezing tube in a refrigerator with negative temperature for temporary storage, and transferring the tube into liquid nitrogen for long-term storage after overnight.
In an alternative embodiment of the third aspect of the present invention, the NK cells are cells obtained by a pre-culture treatment, wherein the NK cell pre-culture treatment is performed as follows: and culturing NK cells according to the designed culture conditions, centrifuging the cultured NK cell suspension, discarding supernatant, and then washing with physiological saline or PBS solution to obtain the required NK cells.
In an alternative embodiment of the third aspect of the present invention, the target volume includes 100mL, the target value is 7 to 7.5, and the preset density is 5×10 6 ～5×10 7 Exemplary, e.g., 3X 10, of 7 The negative temperature is-60 ℃ to-100 ℃, for example-80 ℃, per mL.
The fourth aspect of the invention provides an NK cell cryopreservation kit comprising an NK cell cryopreservation solution according to any one of the above. The NK cell cryopreservation liquid comprises a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, a traditional Chinese medicine monomer component and a PH buffer; wherein the permeable cryoprotectant comprises dimethyl sulfoxide (DMSO) and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer component comprises resveratrol, betulinic acid and apigenin; the PH buffer comprises HEPES solution.
Example 1: an NK cell cryopreservation solution comprises the following components: dimethyl sulfoxide (DMSO) 5mL/100mL, glycerol 5mL/100mL, hydroxyethyl starch 10g/100mL, trehalose 5g/100mL, raffinose 5g/100mL, D-glucose 3g/100mL, glutamine 0.5g/100mL, sodium pyruvate 0.5g/100mL, glycine 0.005g/100mL, vitamin C1g/100mL, vitamin B120.003g/100mL, resveratrol 0.6. Mu.M, betulinic acid 0.3. Mu.M, apigenin 0.5. Mu.M, HEPES solution 20mM.
The NK cell cryopreservation liquid is prepared and cryopreserved by the following method: fully and uniformly mixing dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, HEPES solution, resveratrol, betulinic acid, apigenin and a proper amount of sterile water, adding 1M NaOH solution to adjust the pH value to 7.4, and fixing the volume of the residual water to obtain NK cell frozen stock solution; resuspending NK cells with the obtained NK cell frozen stock solution to adjust cellsDensity of 1X 10 7 Filling the mixture into a 1mL freezing tube; the obtained frozen tube is directly placed in a refrigerator at the temperature of-80 ℃ (no programmed cooling is needed), and is transferred into liquid nitrogen for long-term storage after overnight.
Example 2: an NK cell cryopreservation solution comprises the following components: dimethyl sulfoxide (DMSO) 5mL/100mL, glycerol 5mL/100mL, hydroxyethyl starch 10g/100mL, trehalose 5g/100mL, raffinose 5g/100mL, D-glucose 3g/100mL, glutamine 0.5g/100mL, sodium pyruvate 0.5g/100mL, glycine 0.005g/100mL, vitamin C1g/100mL, vitamin B120.003g/100mL, resveratrol 0.6. Mu.M, betulinic acid 0.3. Mu.M, apigenin 0.5. Mu. M, HEPES solution 20mM.
The NK cell cryopreservation liquid is prepared and cryopreserved by the following method: fully and uniformly mixing dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, HEPES solution, resveratrol, betulinic acid, apigenin and a proper amount of sterile water, adding 1M NaOH solution to adjust the pH value to 7.2, and fixing the volume of the residual water to obtain NK cell frozen stock solution; resuspending NK cells with the NK cell frozen stock solution obtained in the above step, and adjusting the cell density to 1×10 7 Filling 1mL of frozen tube into each mL of frozen tube; and (3) directly placing the frozen tube obtained in the previous step in a refrigerator at the temperature of-80 ℃ (no programmed cooling is needed), and transferring the frozen tube into liquid nitrogen for long-term storage after overnight.
Example 3: the serum-free NK cell cryopreservation solution comprises the following components: dimethyl sulfoxide (DMSO) 5mL/100mL, glycerol 5mL/100mL, hydroxyethyl starch 10g/100mL, trehalose 5g/100mL, raffinose 5g/100mL, D-glucose 3g/100mL, glutamine 0.5g/100mL, sodium pyruvate 0.5g/100mL, glycine 0.005g/100mL, vitamin C1g/100mL, vitamin B120.003g/100mL, resveratrol 0.6. Mu.M, betulinic acid 0.3. Mu.M, apigenin 0.5. Mu. M, HEPES solution 20mM.
The NK cell cryopreservation liquid is prepared and cryopreserved by the following method: dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, HEPES solution, resveratrol, betulinic acid, apigenin, and appropriate amount of non-essential componentsThe bacteria water is fully and uniformly mixed, 1M NaOH solution is added to adjust the pH value to 7.4, and the NK cell frozen stock solution is obtained after the volume of the residual water is fixed; resuspending NK cells with the obtained de NK cell frozen stock solution to adjust the cell density to 3×10 7 Filling 1mL of frozen tube into each mL of frozen tube; the obtained cell cryopreservation tube is directly placed in a refrigerator at-80 ℃ without programmed cooling, and is transferred into liquid nitrogen for long-term storage after overnight.
Comparative example 1: an NK cell cryopreservation solution comprises the following components: dimethyl sulfoxide (DMSO) 5mL/100mL, glycerol 5mL/100mL, hydroxyethyl starch 10g/100mL, trehalose 5g/100mL, raffinose 5g/100mL, D-glucose 3g/100mL, glutamine 0.5g/100mL, sodium pyruvate 0.5g/100mL, glycine 0.005g/100mL, vitamin C1g/100mL, vitamin B120.003g/100mL, HEPES solution 20mM.
The NK cell cryopreservation liquid is prepared and cryopreserved by the following method: fully and uniformly mixing dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, HEPES solution and a proper amount of sterile water, adding a 1M NaOH solution to adjust the pH value to 7.4, and fixing the volume of the residual water to obtain frozen stock solution; resuspending NK cells with the obtained NK cell frozen stock solution to adjust the cell density to 1×10 7 Filling 1mL of frozen tube into each mL of frozen tube; the obtained cell cryopreservation tube is directly placed in a refrigerator at-80 ℃ without programmed cooling, and is transferred into liquid nitrogen for long-term storage after overnight.
Comparative example 2: a commercially available NK cell cryopreservation solution comprises dimethyl sulfoxide, FBS and SCGM. Prepared and frozen by the following method: dimethyl sulfoxide, FBS and SCGM basal medium were mixed at 1:2:7, mixing the components in proportion, and uniformly mixing the components to obtain NK cell frozen stock solution; resuspension of NK cells with the obtained NK cell frozen stock solution to adjust cell density to 1×10 7 Filling 1mL of frozen tube into each mL of frozen tube; the obtained frozen tube is directly placed in a refrigerator at the temperature of minus 80 ℃ and is transferred into liquid nitrogen for long-term storage after being overnight.
Survival rate test: the method for detecting the survival rate of NK cells before and after freezing is as follows: NK cells before and after cryopreservation and resuscitation were subjected to AO/PI staining, and cell counts were used for cell counting, and cell viability was calculated, and the results are shown in Table 1 and FIG. 1 below.
TABLE 1 comparison of NK cell recovery survival rates of comparative examples of examples before and after different times of cryopreservation
| Group of | 0 month (%) | 3 months (%) | 6 months (%) |
| Example 1 | 96.56±0.43 | 94.98±0.71 | 93.04±0.72 |
| Example 2 | 96.56±0.43 | 93.55±1.10 | 89.09±1.67 |
| Example 3 | 96.56±0.43 | 93.11±0.30 | 88.85±1.46 |
| Comparative example 1 | 96.56±0.43 | 92.31±0.92 | 88.03±1.43 |
| ComparisonExample 2 | 96.56±0.43 | 74.04±1.16 | 63.79±1.34 |
Killing ability test: the method for detecting the killing capacity of NK cells before and after freezing is as follows: and detecting NK killing activity before freezing and after resuscitating by adopting an LDH release experiment.
Taking K562 cells as target cells and NK cells as effector cells, preparing single cell suspension by centrifugally dispersing K562 cells growing in logarithmic phase, performing AO/PI staining and counting, and regulating the cell density to 2×10 5 individual/mL; the K562 cell suspension was added to 96-well plates at 50uL per well, effector cells (NK) at 10:1 effect/target ratio, again 50uL per well: simultaneously setting up natural release holes of effector cells and target cells, maximum release holes of target cells and natural release holes of culture medium, correcting volume of control holes, wherein the volume of each hole is 100uL, and setting up 3 compound holes; placing at 37deg.C and 5% CO 2 Incubating in an incubator for 12 hours, and adding 10uL of lysate into each of the maximum release holes of the target cells 45min before the reaction is finished; after the reaction is finished, 50uL of LDH enzyme reaction solution and 50uL of supernatant are sucked from each hole, the mixture is added into another new 96-well plate, the mixture is subjected to light-proof reaction for 30min at room temperature, 50uL of reaction stopping solution is added, and an enzyme-labeled instrument is used for detecting the OD value of the mixture; the killing activity was calculated according to the following formula: killing activity (%) = (assay tube OD value-target cell natural release tube OD value-effector cell natural release tube OD value)/(target cell maximum release tube OD value-target cell natural release tube OD value) ×100%. The results of the killing activity of the resuscitated NK cells against K562 cells of the different examples and comparative examples are shown in Table 2 and FIG. 2. In addition, taking 6 months of freezing as an example, a comparative graph of the results of the effects on NK cell phenotype after 6 months of freezing of the respective examples of the present invention and the comparative examples is shown in FIG. 3.
TABLE 2 comparison of NK cell killing rate against K562 cells for each example before and after different time of cryopreservation
| Group of | 0 month (%) | 3 months (%) | 6 months (%) |
| Example 1 | 91.26±2.26 | 88.77±0.85 | 84.87±0.82 |
| Example 2 | 91.26±2.26 | 86.79±0.94 | 81.28±1.05 |
| Example 3 | 91.26±2.26 | 86.23±0.53 | 80.07±1.01 |
| Comparative example 1 | 91.26±2.26 | 76.61±1.74 | 70.06±2.04 |
| Comparative example 2 | 91.26±2.26 | 66.61±1.40 | 54.31±1.90 |
In summary, the invention provides an NK cell cryopreservation solution, a preparation method, a cryopreservation method and a kit, wherein the NK cell cryopreservation solution comprises dimethyl sulfoxide, glycerol, hydroxyethyl starch, trehalose, raffinose, D-glucose, glutamine, sodium pyruvate, glycine, vitamin C, vitamin B12, resveratrol, betulinic acid, apigenin and HEPES solution. On the premise of not affecting the freezing and preserving effect, the invention combines the glycerol and the dimethyl sulfoxide to reduce the dosage of the dimethyl sulfoxide, greatly weakens the toxicity of the dimethyl sulfoxide to cells or human bodies, combines the permeable freezing protective agent and the impermeable freezing protective agent to protect the cells, reduces the mechanical damage of ice crystals in the freezing and preserving process, can also protect the cell swelling and cracking damage caused by the rapid entry of water into the cells in the cell recovery process, and improves the survival rate of NK cell recovery.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An NK cell cryopreservation solution is characterized by comprising a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, traditional Chinese medicine monomer components and a PH buffer; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the PH buffer comprises HEPES solution.
2. The NK cell cryopreservation solution according to claim 1, wherein in the NK cell cryopreservation solution, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10 μΜ, betulinic acid is 0.1-10 μΜ, apigenin is 0.1-10 μ M, HEPES solution is 10-25 mM.
3. The NK cell cryopreservation solution according to claim 2, wherein in the NK cell cryopreservation solution, dimethyl sulfoxide is 5mL/100mL, glycerol is 5mL/100mL, hydroxyethyl starch is 10g/100mL, trehalose is 5g/100mL, raffinose is 5g/100mL, D-glucose is 3g/100mL, glutamine is 0.5g/100mL, sodium pyruvate is 0.5g/100mL, glycine is 0.005g/100mL, vitamin C is 1g/100mL, vitamin B12 is 0.003g/100mL, resveratrol is 0.6 μΜ, betulinic acid is 0.3 μΜ, apigenin is 0.5 μ M, HEPES solution is 20mM.
4. A method for preparing an NK cell cryopreservation solution, comprising:
providing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, a herbal monomer component, a PH buffer, a PH adjuster, and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell cryopreservation liquid semi-finished product;
and determining the pH value of the NK cell cryopreservation liquid semi-finished product, and adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain the NK cell cryopreservation liquid finished product.
5. The method according to claim 4, wherein the target volume is 100mL, the target value is 7 to 7.5, and the concentration of the NaOH solution is 1M.
6. The method according to claim 5, wherein in the NK cell frozen stock solution, dimethyl sulfoxide is (1-10) mL/100mL, glycerol is (1-10) mL/100mL, hydroxyethyl starch is (5-30) g/100mL, trehalose is (1-10) g/100mL, raffinose is (1-10) g/100mL, D-glucose is (1-5) g/100mL, glutamine is (0.1-1) g/100mL, sodium pyruvate is (0.1-1) g/100mL, glycine is (0.001-0.01) g/100mL, vitamin C is (0.1-5) g/100mL, vitamin B12 is (0.001-0.01) g/100mL, resveratrol is 0.1-10. Mu.M, betulinic acid is 0.1-10. Mu.M, apigenin is 0.1-10. Mu. M, HEPES and the solution is 10-5225 mM.
7. A method of cryopreserving NK cells comprising:
preparing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, a herbal monomer component, a pH buffer, a pH regulator and sterile water; wherein the permeable cryoprotectant comprises dimethyl sulfoxide and glycerol; the impermeable protective agent comprises hydroxyethyl starch, trehalose and raffinose; the nutritional agent comprises D-glucose, glutamine, sodium pyruvate, glycine, vitamin C and vitamin B12; the traditional Chinese medicine monomer components comprise resveratrol, betulinic acid and apigenin; the pH buffer comprises HEPES solution, and the pH regulator comprises NaOH solution;
uniformly mixing a permeable cryoprotectant, a non-permeable protectant, a nutritional agent, an antioxidant, traditional Chinese medicine monomer components, a PH buffer and a certain amount of sterile water to obtain an NK cell cryopreservation liquid semi-finished product;
determining the pH value of the NK cell cryopreservation liquid semi-finished product, adjusting the pH value of the NK cell cryopreservation liquid semi-finished product to a target value based on the difference value from the current volume to the target volume of the NK cell cryopreservation liquid semi-finished product by using the pH regulator and the rest sterile water so as to obtain an NK cell cryopreservation liquid finished product;
resuspension of NK cells by using the NK cell frozen stock solution finished product, adjusting the density of the NK cells to a preset density, and then loading the NK cells into a frozen stock tube;
and directly placing the freezing tube in a refrigerator with negative temperature for temporary storage, and transferring the tube into liquid nitrogen for long-term storage after overnight.
8. The method for cryopreserving NK cells according to claim 7, wherein the NK cells are cells obtained by a pre-culture treatment, and the NK cell pre-culture treatment is performed as follows: and culturing NK cells according to the designed culture conditions, centrifuging the cultured NK cell suspension, discarding supernatant, and then washing with physiological saline or PBS solution to obtain the required NK cells.
9. The method of claim 7, wherein the target volume comprises 100mL, the target value is 7-7.5, and the predetermined density is 5 x 10 6 ～5×10 7 The negative temperature is minus 60 ℃ to minus 100 ℃ per mL.
10. An NK cell cryopreservation kit comprising the NK cell cryopreservation solution of any one of claims 1-3.
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| WO2016063208A1 (en) * | 2014-10-20 | 2016-04-28 | Stempeutics Research Pvt. Ltd. | A cryopreservation composition and methods thereof |
| CN107787959A (en) * | 2016-08-30 | 2018-03-13 | 广州市金航生物科技有限公司 | A kind of immunocyte frozen stock solution, its preparation method, cryopreservation methods and application |
| CN111925986A (en) * | 2020-09-24 | 2020-11-13 | 深圳市一五零生命科技有限公司 | Umbilical cord mesenchymal stem cell serum-free culture medium and preparation method thereof |
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| CN116473051A (en) * | 2023-04-25 | 2023-07-25 | 太仓超云生物信息咨询服务有限公司 | Serum-free non-program cell frozen stock solution and preparation method and application thereof |
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