CN111948195A - Method for evaluating activity of immune cells after cryopreservation - Google Patents
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- CN111948195A CN111948195A CN202010714672.4A CN202010714672A CN111948195A CN 111948195 A CN111948195 A CN 111948195A CN 202010714672 A CN202010714672 A CN 202010714672A CN 111948195 A CN111948195 A CN 111948195A
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- 210000002865 immune cell Anatomy 0.000 title claims abstract description 20
- 230000000694 effects Effects 0.000 title claims abstract description 19
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 79
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 239000003085 diluting agent Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 16
- 239000002504 physiological saline solution Substances 0.000 claims description 12
- 238000002965 ELISA Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 abstract 1
- 239000012530 fluid Substances 0.000 description 18
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 8
- 230000002503 metabolic effect Effects 0.000 description 4
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000012054 celltiter-glo Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229960002378 oftasceine Drugs 0.000 description 3
- XIAYFENBYCWHGY-UHFFFAOYSA-N 2-[2,7-bis[[bis(carboxymethyl)amino]methyl]-3-hydroxy-6-oxoxanthen-9-yl]benzoic acid Chemical compound C=12C=C(CN(CC(O)=O)CC(O)=O)C(=O)C=C2OC=2C=C(O)C(CN(CC(O)=O)CC(=O)O)=CC=2C=1C1=CC=CC=C1C(O)=O XIAYFENBYCWHGY-UHFFFAOYSA-N 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- -1 acetyl methyl Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method for evaluating the activity of immune cells after cryopreservation, which is characterized by comprising a step of preparing a test reagent reaction solution (reagent) and a step of diluting and detecting cell sap. The invention can rapidly and visually evaluate the cell activity of the immune cells after cryopreservation by detecting the recovered ATP value of the immune cells through fluorescence. The assessment is more intuitive and quick.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for evaluating activity of immune cells after cryopreservation.
Background
In recent years, many immunotherapies for treating various diseases have been established, and high-quality immune cells are required to grow. The demand of immune cells for patients with various diseases is increasing day by day, and the immune cells must be preserved at a deep temperature so as to supply the demand of the patients for the immune cells at any time after recovery.
However, the activity of immune cells after cryopreservation may be affected, and the result is unreliable if immune cells with unknown cellular activity are used for treatment or clinical trials. Therefore, it is essential to evaluate the cellular activity of immune cells after cryopreservation.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for evaluating the activity of immune cells after cryopreservation.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
a method of assessing the activity of an immune cell following cryopreservation comprising:
preparation of a test reagent reaction solution (reagent):
preparing a base solution and a buffer solution of a test reagent to obtain a test reagent reaction solution (reagent) for later use, wherein the test reagent reaction solution is an ATP reaction solution such as CellTiter-Glo;
cell sap dilution detection:
cell sap was diluted to 4 x 105Obtaining cell detection solution after the cell concentration of/ml;
uniformly mixing the cell detection solution, adding 1ml of the cell detection solution into a first test tube with 500ul of physiological saline, and uniformly mixing to obtain a first test tube cell diluent;
taking 500ul of the first test tube cell diluent, adding the first test tube cell diluent into a second test tube with 500ul of physiological saline, and uniformly mixing to obtain a second test tube cell diluent;
adding 500ul of the second test tube cell diluent into a third test tube with 500ul of physiological saline, and uniformly mixing to obtain a third test tube cell diluent;
taking 500ul of the third test tube cell diluent, adding the third test tube cell diluent into a fourth test tube containing 500ul of physiological saline, and uniformly mixing to obtain a fourth test tube cell diluent;
taking 500ul of the fourth test tube cell diluent, adding the fourth test tube cell diluent into a fifth test tube containing 500ul of physiological saline, and uniformly mixing to obtain fifth test tube cell diluent;
and (3) placing 50ul of each of the first to fifth test tube cell dilutions in a 96-well plate, continuously adding 50ul of test reagent reaction solution (reagent), uniformly mixing for 9-11 minutes, and reading chemiluminescence values on an enzyme-linked immunosorbent assay (ELISA) instrument.
In a preferred embodiment of the present invention, the mixing time after adding 50ul of the test reagent reaction solution (reagent) is 10 minutes.
The invention has the beneficial effects that:
the invention can rapidly and visually evaluate the cell activity of the immune cells after cryopreservation by detecting the recovered ATP value of the immune cells through fluorescence. The assessment is more intuitive and quick.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the present invention.
Fig. 2 is a schematic diagram illustrating a detection effect of the present invention.
Fig. 3 is a schematic diagram of the detection effect of the present invention.
Detailed Description
The invention evaluates the cell activity of immune cells after cryopreservation quickly and visually by detecting the recovered ATP value of the immune cells.
Specifically, as shown in FIG. 1, the fluorescence value of ATP after cell lysis is measured by the GTC method:
luciferase catalyses luminescence after the monooxygenation of luciferin in the presence of Mg2+, ATP and molecular oxygen.
Example 1: detection of NK cells prior to cryopreservation
A detection step:
1. 5 EP tubes (1.8 ml) were used and numbered 1, 2, 3, 4 and 5, respectively.
2. Mixing the cells to be detected, taking 1ml of cell liquid in a No. 1 EP tube, mixing uniformly and counting.
3. EP tubes No. 2, No. 3, No. 4 and No. 5 are taken, and 500ul of physiological saline is added into each EP tube.
4. 500ul of cell fluid was taken from the No. 1 EP tube and mixed with the cell fluid in the No. 2 EP tube.
5. 500ul of cell fluid was taken out of the EP tube No. 2 and mixed with the cell fluid in the EP tube No. 3.
6. 500ul of cell fluid was taken from the No. 3 EP tube and mixed with the cell fluid in the No. 4 EP tube.
7. 500ul of cell fluid was taken out of the No. 4 EP tube and mixed with the cell fluid in the No. 5 EP tube.
In EP tubes 8.1, 2, 3, 4, 5, 50ul of each cell fluid was placed in a 96-well opaque white plate, with 3 replicate wells per set.
8. 50ul of the well containing 50ul of the sample was placed in a 96 well plate and 50ul of the well containing CellTiter-Glo reagent was added.
9. After mixing for 10 minutes, chemiluminescence values were read on a microplate reader as shown in fig. 2, and the cell metabolic constant a was determined to be 272.36.
Example 2: and (5) carrying out fluorescence monitoring on the recovered NK cells.
A detection step:
1. 5 EP tubes (1.8 ml) were used and numbered 1, 2, 3, 4 and 5, respectively.
2. Mixing the cells to be detected, taking 1ml of cell liquid in a No. 1 EP tube, mixing uniformly and counting.
3. EP tubes No. 2, No. 3, No. 4 and No. 5 are taken, and 500ul of physiological saline is added into each EP tube.
4. 500ul of cell fluid was taken from the No. 1 EP tube and mixed with the cell fluid in the No. 2 EP tube.
5. 500ul of cell fluid was taken out of the EP tube No. 2 and mixed with the cell fluid in the EP tube No. 3.
6. 500ul of cell fluid was taken from the No. 3 EP tube and mixed with the cell fluid in the No. 4 EP tube.
7. 500ul of cell fluid was taken out of the No. 4 EP tube and mixed with the cell fluid in the No. 5 EP tube.
In EP tubes 8.1, 2, 3, 4, 5, 50ul of each cell fluid was placed in a 96-well opaque white plate, with 3 replicate wells per set.
8. 50ul of the well containing 50ul of the sample was placed in a 96 well plate and 50ul of the well containing CellTiter-Glo reagent was added.
9. After mixing for 10 minutes, the chemiluminescence values were read on a microplate reader, as shown in fig. 3, and the cell metabolic constant a was determined to be 225.38.
Comparing the metabolic constants of the cells before and after cryopreservation, the activity of the recovered cells is only about 80% of the activity of the recovered cells. (the higher the value of the metabolic constant a, the better the cell activity)
Whereas in the prior art staining methods are commonly used to assess cell viability, such as Calcein-AM (acetyl methyl Calcein). Calcein-AM is a cell staining reagent that can fluorescently label living cells, and Calcein-AM can easily penetrate living cell membranes because of enhanced hydrophobicity based on Calcein (Calcein). When it enters the cytoplasm, esterase will hydrolyze it to Calcein (Calcein) which remains inside the cell, emitting strong green fluorescence.
However, the cell membrane after cryopreservation is damaged by DMSO (dimethyl sulfoxide) in the frozen stock solution, so that the dye is easier to enter the cell membrane, and the detection result is inaccurate. The ATP Glo method is used for determining the cell activity by measuring the ATP content in each unit cell through chemiluminescence, is not influenced by a frozen solution, and has more accurate detection results.
In addition, the method of fluorescence labeling the living cells can only measure the survival rate of the cells, but cannot really quantify the vitality and the state of the cells, and the ATP Glo method can be used for achieving the purpose, so that the vitality and the state of the cells can be better judged and evaluated.
Claims (2)
1. A method of assessing the activity of an immune cell following cryopreservation comprising:
preparation of a test reagent reaction solution (reagent):
preparing a base solution and a buffer solution of a test reagent to obtain a test reagent reaction solution (reagent) for later use, wherein the test reagent reaction solution is an ATP reaction solution;
cell sap dilution detection:
cell sap was diluted to 4 x 105Obtaining cell detection solution after the cell concentration of/ml;
uniformly mixing the cell detection solution, adding 1ml of the cell detection solution into a first test tube with 500ul of physiological saline, and uniformly mixing to obtain a first test tube cell diluent;
taking 500ul of the first test tube cell diluent, adding the first test tube cell diluent into a second test tube with 500ul of physiological saline, and uniformly mixing to obtain a second test tube cell diluent;
adding 500ul of the second test tube cell diluent into a third test tube with 500ul of physiological saline, and uniformly mixing to obtain a third test tube cell diluent;
taking 500ul of the third test tube cell diluent, adding the third test tube cell diluent into a fourth test tube containing 500ul of physiological saline, and uniformly mixing to obtain a fourth test tube cell diluent;
taking 500ul of the fourth test tube cell diluent, adding the fourth test tube cell diluent into a fifth test tube containing 500ul of physiological saline, and uniformly mixing to obtain fifth test tube cell diluent;
and (3) placing 50ul of each of the first to fifth test tube cell dilutions in a 96-well plate, continuously adding 50ul of test reagent reaction solution (reagent), uniformly mixing for 9-11 minutes, and reading chemiluminescence values on an enzyme-linked immunosorbent assay (ELISA) instrument.
2. The method of claim 1, wherein the mixing time after adding 50ul of the test reagent reaction solution (reagent) is 10 minutes.
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| CN202010714672.4A CN111948195A (en) | 2020-07-23 | 2020-07-23 | Method for evaluating activity of immune cells after cryopreservation |
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| CN202010714672.4A CN111948195A (en) | 2020-07-23 | 2020-07-23 | Method for evaluating activity of immune cells after cryopreservation |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031292A1 (en) * | 1998-11-25 | 2000-06-02 | Kikkoman Corporation | Method for counting living cells |
| CN101889206A (en) * | 2007-11-09 | 2010-11-17 | 健赞公司 | Under the situation of not using control cells, measure the method for cell viability |
| CN103045712A (en) * | 2013-01-27 | 2013-04-17 | 罗奇志 | NK (Natural Killer) cell activity detection method suitable for teaching and scientific researching |
| CN204989188U (en) * | 2015-08-10 | 2016-01-20 | 广州市雷德生物科技有限公司 | Evaluation immune ability kit |
| CN105823882A (en) * | 2015-01-08 | 2016-08-03 | 广州市雷德生物科技有限公司 | Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof |
-
2020
- 2020-07-23 CN CN202010714672.4A patent/CN111948195A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031292A1 (en) * | 1998-11-25 | 2000-06-02 | Kikkoman Corporation | Method for counting living cells |
| CN101889206A (en) * | 2007-11-09 | 2010-11-17 | 健赞公司 | Under the situation of not using control cells, measure the method for cell viability |
| CN103045712A (en) * | 2013-01-27 | 2013-04-17 | 罗奇志 | NK (Natural Killer) cell activity detection method suitable for teaching and scientific researching |
| CN105823882A (en) * | 2015-01-08 | 2016-08-03 | 广州市雷德生物科技有限公司 | Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof |
| CN204989188U (en) * | 2015-08-10 | 2016-01-20 | 广州市雷德生物科技有限公司 | Evaluation immune ability kit |
Non-Patent Citations (2)
| Title |
|---|
| 李磊等: "细胞活性检测方法之比较" * |
| 杨慧芳;侯粉霞;鱼涛;: "自然杀伤细胞活性测定方法的可行性研究" * |
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