CN108064840B - NKT cell cryopreservation solution and preparation method thereof - Google Patents
NKT cell cryopreservation solution and preparation method thereof Download PDFInfo
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Abstract
The invention discloses NKT cell cryopreservation liquid and a preparation method thereof, wherein the cryopreservation liquid comprises the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, plantain herb extract, tartary buckwheat brass, tremella polysaccharide, laminarin and hydroxyethyl starch. The frozen stock solution does not contain animal-derived serum, can avoid the risk of introducing pollution and allergen, and has higher clinical safety compared with the conventional cell frozen stock solution. The cryopreservation liquid has a simple formula, all components are matched with each other, the cell state after cryopreservation recovery can be kept to be basically close to the state before cryopreservation under the synergistic effect, the activity is higher, and the cryopreservation effect of NKT cells can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of cell cryopreservation solution, and particularly relates to NKT cell cryopreservation solution and a preparation method thereof.
Background
NKT (natural killer T) cells are a distinct subset of T cells with both T cell receptors TCR and NK cell receptors on the cell surface. In recent years, research on the phenotypic characteristics, distribution and development of NKT cells, the immunological effects and relationship with diseases, tumor treatment, autoimmune treatment and the like has been greatly developed. Because the research of NKT cells in the aspects of tumor treatment, immune disease treatment and the like also determines the importance of cryopreservation of the NKT cells after culture, the low cell activity caused by delayed culture can be avoided in the aspect of treatment, the cost increase caused by delayed culture is avoided in the aspect of economy, and in the aspect of research, the NKT cells in different periods can be cryopreserved to research the activity of the NKT cells in different cryopreservation times.
However, at present, NKT cells are usually cryopreserved by using a cryopreservation solution prepared by adding fetal bovine serum and dimethyl sulfoxide in different proportions, but the addition of dimethyl sulfoxide is only to prevent the formation of ice crystals in cells from damaging the cells during cryopreservation, and the addition of fetal bovine serum is to protect the cells from being damaged by dimethyl sulfoxide and provide nutrition for the cells. However, the fetal calf serum belongs to a heterogeneous substance, has complex components, has risks of introducing pollution and allergen, is not suitable for clinical application, and particularly in cell therapy, the existence of the heterogeneous protein can cause unknown adverse reactions and seriously affect the treatment result.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the NKT cell cryopreservation solution and the preparation method thereof, and the cryopreservation solution has high clinical safety and can well maintain the activity of the cryopreserved cells.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
an NKT cell cryopreservation solution, which comprises the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin and hydroxyethyl starch; wherein, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 15-25: 8-15: 6-10: 5-10: 5-10; the concentration of herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin, and hydroxyethyl starch is 5-15mg/ml, 1-4mg/ml, 1-5mg/ml, 2-4mg/ml, and 15-20mg/ml respectively.
Further, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 20: 12: 8: 8: 8.
further, the mass volume percentage concentration of albumin in the human serum albumin injection is 20%.
Further, the concentration of the plantain herb extract, the buckwheat flavone, the tremella polysaccharide, the laminarin and the hydroxyethyl starch is respectively 8mg/ml, 2mg/ml, 4mg/ml, 3mg/ml and 18 mg/ml.
Further, the plantain herb extract is prepared by the following method: cleaning herba plantaginis, air drying, pulverizing to 80-100 mesh, mixing with anhydrous ethanol at weight ratio of 1-3:1, and performing CO2Performing supercritical extraction, centrifuging to remove impurities to obtain fat-soluble extract, adding 5-6 times of distilled water into the extraction residue, heating at 90-95 deg.C for 6-8 hr, filtering, concentrating, vacuum freeze drying to obtain water-soluble extract, and mixing the two extracts to obtain herba plantaginis extractive solution.
The method for preparing the NKT cell cryopreservation solution comprises the following steps:
(1) mixing dimethyl sulfoxide, polyethylene glycol and hydroxyethyl starch to obtain a solution A;
(2) mixing the glucose injection and the normal saline injection, then adding the plantain extract, the tartary buckwheat flavone, the tremella polysaccharide and the laminarin, and uniformly mixing to obtain a solution B;
(3) mixing the solution A and the solution B, adding the mixture into human serum albumin injection, and uniformly mixing to obtain the injection.
The NKT cell cryopreservation solution and the preparation method thereof provided by the invention have the following beneficial effects:
(1) the frozen stock solution does not contain animal-derived serum, can avoid the risk of introducing pollution and allergen, and has higher clinical safety compared with the conventional cell frozen stock solution.
(2) The cryopreservation liquid disclosed by the invention is simple in formula, the components are matched with each other, the cell state after cryopreservation recovery can be kept to be basically close to the state before cryopreservation under the synergistic effect, the activity is higher, and the cryopreservation effect of NKT cells can be obviously improved.
Detailed Description
Example 1
An NKT cell cryopreservation solution, which comprises the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin and hydroxyethyl starch; wherein, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 15: 8: 6: 5: 5; the mass volume percentage concentration of albumin in the human serum albumin injection is 20 percent; the concentrations of herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin, and hydroxyethyl starch are 5mg/ml, 1mg/ml, 2mg/ml and 15mg/ml respectively.
Wherein, the plantain herb extract is prepared by the following method: cleaning herba plantaginis, air drying, pulverizing to 100 mesh, mixing with anhydrous ethanol at weight ratio of 1:1, and performing CO extraction2Performing supercritical extraction, centrifuging to remove impurities to obtain fat soluble extract, adding 5 times of distilled water to the extraction residue, heating at 90 deg.C for 8 hr, filtering, concentrating, vacuum freeze drying to obtain water soluble extract,mixing the two extracts to obtain herba plantaginis extractive solution.
The method for preparing the NKT cell cryopreservation solution comprises the following steps:
(1) mixing dimethyl sulfoxide, polyethylene glycol and hydroxyethyl starch to obtain a solution A;
(2) mixing the glucose injection and the normal saline injection, then adding the plantain extract, the tartary buckwheat flavone, the tremella polysaccharide and the laminarin, and uniformly mixing to obtain a solution B;
(3) mixing the solution A and the solution B, adding the mixture into human serum albumin injection, and uniformly mixing to obtain the injection.
Example 2
An NKT cell cryopreservation solution, which comprises the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin and hydroxyethyl starch; wherein, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 25: 15: 10: 10: 10; the mass volume percentage concentration of albumin in the human serum albumin injection is 20 percent; the concentrations of herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin, and hydroxyethyl starch are 15mg/ml, 4mg/ml, 5mg/ml, 4mg/ml and 20mg/ml respectively.
Wherein, the plantain herb extract is prepared by the following method: cleaning herba plantaginis, air drying, pulverizing to 100 mesh, mixing with anhydrous ethanol at weight ratio of 3:1, and performing CO extraction2Performing supercritical extraction, centrifuging to remove impurities to obtain fat-soluble extract, adding 6 times of distilled water to the extraction residue, heating at 95 deg.C for 6 hr, filtering, concentrating, vacuum freeze drying to obtain water-soluble extract, and mixing the two extracts to obtain herba plantaginis extractive solution.
The method for preparing the NKT cell cryopreservation solution comprises the following steps:
(1) mixing dimethyl sulfoxide, polyethylene glycol and hydroxyethyl starch to obtain a solution A;
(2) mixing the glucose injection and the normal saline injection, then adding the plantain extract, the tartary buckwheat flavone, the tremella polysaccharide and the laminarin, and uniformly mixing to obtain a solution B;
(3) mixing the solution A and the solution B, adding the mixture into human serum albumin injection, and uniformly mixing to obtain the injection.
Example 3
An NKT cell cryopreservation solution, which comprises the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin and hydroxyethyl starch; wherein, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 20: 12: 8: 8: 8; the mass volume percentage concentration of albumin in the human serum albumin injection is 20 percent; the concentrations of herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin, and hydroxyethyl starch are 8mg/ml, 2mg/ml, 4mg/ml, 3mg/ml and 18mg/ml respectively.
Wherein, the plantain herb extract is prepared by the following method: cleaning herba plantaginis, air drying, pulverizing to 100 mesh, mixing with anhydrous ethanol at weight ratio of 2:1, and performing CO extraction2Performing supercritical extraction, centrifuging to remove impurities to obtain fat-soluble extract, adding 7 times of distilled water to the extraction residue, heating at 95 deg.C for 6 hr, filtering, concentrating, vacuum freeze drying to obtain water-soluble extract, and mixing the two extracts to obtain herba plantaginis extractive solution.
The method for preparing the NKT cell cryopreservation solution comprises the following steps:
(1) mixing dimethyl sulfoxide, polyethylene glycol and hydroxyethyl starch to obtain a solution A;
(2) mixing the glucose injection and the normal saline injection, then adding the plantain extract, the tartary buckwheat flavone, the tremella polysaccharide and the laminarin, and uniformly mixing to obtain a solution B;
(3) mixing the solution A and the solution B, adding the mixture into human serum albumin injection, and uniformly mixing to obtain the injection.
Comparative example 1
Comparative example 1 is a conventional cell cryopreservation solution, DMSO containing 10% FBS.
Comparative example 2
Comparative example 2 the same as example 3 except that the extract solution of plantain was absent as compared with example 3.
Comparative example 3
Comparative example 3 compared to example 3, tremella polysaccharide and laminarin were absent and the rest was the same as example 3.
Comparative example 4
Comparative example 4 compared to example 3, tartary buckwheat flavone is absent, and the rest is the same as example 3.
Test examples
1. Isolation of peripheral blood mononuclear cells
(1) Peripheral blood is extracted, and the physiological saline and the peripheral blood are mixed according to the volume ratio of 1:1, mixing to obtain peripheral blood diluent;
(2) according to the volume ratio of the lymphocyte separation liquid to the peripheral blood diluent of 1: 2, slowly adding peripheral blood diluent above the lymphocyte separation solution along the tube wall, and centrifuging for 30min at 800 g;
(3) after centrifugation is finished, the mixture is divided into 4 layers from bottom to top, namely a granulocyte layer and red blood cell layer, a lymphocyte separation liquid layer, a PBMC layer and a plasma layer, the PBMC layer is sucked into another centrifuge tube by a suction tube, and is washed by adding normal saline and centrifuged for 5min at 400 g;
(4) and after the centrifugation is finished, discarding the supernatant, taking the residual cell precipitate as PBMC, re-suspending the cell precipitate by using an RPMI1640 culture medium, and counting the cells.
2. Culture of NKT cells
(1) Resuspend the collected PBMC in RPMI1640 medium at 1X 106Inoculating into culture flask at density of one/mL, adding growth factors IL-2500U/mL and IL-1530ng/mL, placing at 37 deg.C and 5% CO2Culturing in an incubator;
(2) after 5 days of induction culture, fluid infusion is carried out, IL-2500U/mL and IL-1530ng/mL are supplemented in total amount, and the cell density before fluid infusion is not more than 3 multiplied by 106Cell density of 0.5-1.0 × 10 after fluid infusion6cells/mL, were replenished and supplemented with cell growth factors every 3 days, thus cultured until after 14 days, and the cells were harvested.
3. Freezing experiment
The cultured NKT cells were resuspended in the frozen stocks prepared in examples 1 to 3 and comparative examples 1 to 4 to a cell density of 1.0X 106And each freezing tube is 1mL, then the freezing tube is firstly placed into a freezing box, then the freezing box is placed into a program cooling instrument for freezing, and then the freezing tube is quickly transferred into liquid nitrogen for freezing.
4. Resuscitation
(1) Heating a constant-temperature water bath to 37 ℃, quickly taking out the groups of cell cryopreservation tubes which are cryopreserved in liquid nitrogen for 1 year, transferring the groups of cell cryopreservation tubes into the water bath, continuously oscillating the groups of cell cryopreservation tubes until the groups of cell cryopreservation tubes are completely dissolved, and transferring the dissolved cell suspension into a centrifugal tube added with a complete culture medium for washing and centrifugation;
(2) the centrifuged cells were suspended and cultured for 24 hours in a complete medium at 1.0X 106Each/mL cell was inoculated into a culture flask, and IL-2500U/mL and IL-1530ng/mL cell growth factors were added in total, and the mixture was placed at 37 ℃ in 5% CO2Culturing in an incubator.
5. Cell morphology detection
The cell morphology of each group of NKT cells before and after cryopreservation is observed under a microscope, and the result shows that the recovered cell morphology has no obvious difference from the cell morphology before cryopreservation, the cells are full, bright and glossy, and more cells are agglomerated.
6. Cell viability assay
The NKT cells before and after the cryopreservation were stained and counted to calculate the cell viability, and the cell viability of examples 1 to 3 and comparative examples 1 to 4 were 96.7%, 97.4%, 98.9%, 81.5%, 90.1%, 85.2%, and 88.7%, respectively. It can be seen that the cell viability recovered after cryopreservation of the cryopreservation solution prepared by the invention is close to that before cryopreservation, especially the cell viability in example 3 is the best, compared with the comparative example, the cell viability in example is obviously higher, and when the components of the cryopreservation solution are not complete, the cell viability is obviously reduced but is higher than that of the cryopreservation solution prepared by the conventional method.
7. Cell killing viability assay
Adopting a lactic acid dehydrogenase method, taking K562 cells as target cells, wherein the effective target ratio is respectively 40: 1. 20: 1. 10: 1 and 5: and 1, detecting the killing activity of NKT cells before and after cryopreservation. The detection result shows that the killing activity of each group of NKT cells to K562 cells is enhanced along with the increase of the effective target ratio, the killing activity of the cells after recovery of the cells in the examples 1-3 is not obviously different from that of the cells before freezing, the killing power of the cells after recovery of the cells in the comparative examples 1-4 is lower than that of the cells before freezing in different effective target ratios, and the killing power of the cells in the comparative example 1 is the lowest.
8. Immunophenotypic testing before and after cryopreservation
Resuspending NKT cells collected before cryopreservation and after recovery with PBS at a density of 1.0X 106And adding FITC labeled CD3 and PE labeled CD56 antibody into the mixture per mL, incubating the mixture for 30min at room temperature in a dark place, and detecting the mixture by using a flow cytometer. According to the detection results, the immunophenotyping detection results of examples 1-3 are higher than those of comparative examples 1-4, and the result of comparative example 1 is the lowest, which shows that the cryopreservation liquid provided by the invention can obviously improve the cryopreservation effect of NKT cells.
Claims (6)
1. An NKT cell cryopreservation solution, which is characterized by comprising the following components: human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection, normal saline injection, herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin and hydroxyethyl starch; wherein, the volume ratio of the human serum albumin injection to the dimethyl sulfoxide to the polyethylene glycol to the glucose injection to the normal saline injection is 15-25: 8-15: 6-10: 5-10: 5-10; the concentration of herba plantaginis extract, radix Et rhizoma Fagopyri Tatarici flavone, Tremella polysaccharide, laminarin, and hydroxyethyl starch is 5-15mg/ml, 1-4mg/ml, 1-5mg/ml, 2-4mg/ml, and 15-20mg/ml respectively.
2. The NKT cell cryopreservation solution of claim 1, wherein the volume ratio of human serum albumin injection, dimethyl sulfoxide, polyethylene glycol, glucose injection and physiological saline injection is 20: 12: 8: 8: 8.
3. the NKT cell lysate according to claim 1 or 2, wherein the albumin concentration in the human albumin injection is 20% by weight/volume.
4. The NKT cell cryopreservation solution of claim 1, wherein the concentration of the plantain extract, the buckwheat flavone, the tremella polysaccharide, the laminarin and the hydroxyethyl starch is 8mg/ml, 2mg/ml, 4mg/ml, 3mg/ml and 18mg/ml respectively.
5. The NKT cell cryopreservation solution of claim 1 or 4, wherein an extract of Plantago asiatica is prepared by the following method: cleaning herba plantaginis, air drying, pulverizing to 80-100 mesh, mixing with anhydrous ethanol at weight ratio of 1-3:1, and performing CO2Performing supercritical extraction, centrifuging to remove impurities to obtain fat-soluble extract, adding 5-6 times of distilled water into the extraction residue, heating at 90-95 deg.C for 6-8 hr, filtering, concentrating, vacuum freeze drying to obtain water-soluble extract, and mixing the two extracts to obtain herba plantaginis extractive solution.
6. The method for producing a NKT cell lysate according to any one of claims 1 to 5, which comprises:
(1) mixing dimethyl sulfoxide, polyethylene glycol and hydroxyethyl starch to obtain a solution A;
(2) mixing the glucose injection and the normal saline injection, then adding the plantain extract, the tartary buckwheat flavone, the tremella polysaccharide and the laminarin, and uniformly mixing to obtain a solution B;
(3) mixing the solution A and the solution B, adding the mixture into human serum albumin injection, and uniformly mixing to obtain the injection.
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| AU2022274645A1 (en) * | 2021-05-14 | 2024-01-04 | Gi Cell, Inc. | Composition for nk cell cryopreservation, and cryopreservation formulation comprising same |
| CN115843784B (en) * | 2022-12-30 | 2024-04-30 | 广东康盾高新技术产业集团股份公司 | CAR-NK cell preservation solution, preparation method and preservation method thereof |
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