CN113061576A - Immune cell NK frozen stock solution and activity research method thereof - Google Patents
Immune cell NK frozen stock solution and activity research method thereof Download PDFInfo
- Publication number
- CN113061576A CN113061576A CN202110206293.9A CN202110206293A CN113061576A CN 113061576 A CN113061576 A CN 113061576A CN 202110206293 A CN202110206293 A CN 202110206293A CN 113061576 A CN113061576 A CN 113061576A
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- immune
- solution
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 53
- 239000011550 stock solution Substances 0.000 title claims abstract description 26
- 230000000694 effects Effects 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims abstract description 114
- 239000000243 solution Substances 0.000 claims abstract description 46
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 238000002474 experimental method Methods 0.000 claims abstract description 8
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 230000012010 growth Effects 0.000 claims abstract description 7
- 238000000338 in vitro Methods 0.000 claims abstract description 4
- 210000000822 natural killer cell Anatomy 0.000 claims description 19
- 238000005119 centrifugation Methods 0.000 claims description 13
- 238000004113 cell culture Methods 0.000 claims description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 210000000601 blood cell Anatomy 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 210000005259 peripheral blood Anatomy 0.000 claims description 6
- 239000011886 peripheral blood Substances 0.000 claims description 6
- 230000003247 decreasing effect Effects 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 4
- 229920002307 Dextran Polymers 0.000 claims description 3
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 3
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 3
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 230000022131 cell cycle Effects 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000003792 electrolyte Substances 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229940093181 glucose injection Drugs 0.000 claims description 3
- 230000000415 inactivating effect Effects 0.000 claims description 3
- 229940090044 injection Drugs 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000008354 sodium chloride injection Substances 0.000 claims description 3
- 238000007447 staining method Methods 0.000 claims description 3
- 210000003954 umbilical cord Anatomy 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims 12
- 238000000684 flow cytometry Methods 0.000 abstract description 4
- 230000001640 apoptogenic effect Effects 0.000 abstract description 3
- 230000006907 apoptotic process Effects 0.000 abstract description 2
- 230000035755 proliferation Effects 0.000 abstract description 2
- 230000004987 nonapoptotic effect Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 2
- 206010061623 Adverse drug reaction Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Organic Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physiology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a frozen stock solution of immune cell NK and an activity research method thereof, which comprises the steps of separating the immune cell NK, carrying out serum-free in-vitro amplification on the cell NK, analyzing a growth curve of the cell NK, analyzing the surface antigen expression of the cell NK, analyzing the cycle of the cell NK, carrying out frozen stock and recovery on the immune cell NK and detecting various indexes of the immune cell. The separated immune cells NK have stronger proliferation capacity, the cell activity in four cell freezing solutions is the highest after the cells frozen by the cell freezing solution 1 are recovered, the cell activity is more than 90%, the detection result of a flow cytometry shows that the immunophenotype of the cells before the frozen storage and the cells after the frozen storage are recovered has no obvious difference and has the immunophenotype of the immune cells, and the detection experiment of apoptosis by flow cytometry shows that the number of late apoptotic cells is the minimum and the number of non-apoptotic cells is the maximum after the cells frozen by the cell freezing solution 1 are recovered.
Description
Technical Field
The invention relates to a frozen stock solution of immune cells NK and an activity research method thereof, in particular to a frozen stock solution of immune cells NK and an activity research method thereof, belonging to the technical field of cell research.
Background
Tumors are a common problem faced by human beings, no effective curing means exists at present, and the life quality of patients is greatly influenced. Many complications of blood tumor, long treatment time, large adverse drug reaction and high operation cost, and brings great family pressure and social burden.
NK cells (Natural killer cells) are the first line of defense of human body against tumors, have important function in the innate immune system, and can directly kill tumor cells without being sensitized in advance. Although NK cells have been used for cell therapy for tumor immunity and have a good effect in clinical treatment of tumors (such as leukemia, lymphoma and melanoma); however, the long-term efficient storage of NK cells is always a problem, both for third party cell banks and for clinical administration of delivery and application.
Disclosure of Invention
The present invention aims to solve the problems and provide a frozen stock solution of immune cells NK and a method for studying the activity of the frozen stock solution.
The invention realizes the purpose through the following technical scheme: a frozen stock solution of immune cells NK and an activity research method thereof comprise the following steps:
the first step, the separation of immune cell NK comprises the following processes:
(1) in a biological safety cabinet, dividing the peripheral blood into 50ml centrifuge tubes equally according to that each tube is not more than 35ml, setting the centrifugal parameters to rise and fall by 7 and fall by 9, and centrifuging for 8min at 2000 rmp;
(2) after centrifuging peripheral blood, firstly absorbing upper plasma into a 50ml centrifuge tube, taking four EP tubes, adding 1.0ml plasma into each tube, inactivating at 56 ℃ for 30min in a water bath, centrifuging at 3000rpm for 10min, and taking upper plasma into a clean 50ml centrifuge tube for later use;
(3) diluting the blood cells with NA such that the ratio of blood cells: NA is 1: 1.
Step two, the serum-free in vitro amplification of the cell NK comprises the following steps:
(1) initial culture of immune cells;
all cells were collected by centrifugation, resuspended in 30ml NK medium (1.5 ml of autologous serum, 5%) and 2ml of NK amplification reagent were added, centrifugation parameters were set, 9-7 was increased and decreased, 2000rpm was applied, centrifugation was carried out for 8min, and the remaining supernatant was discarded. Sucking 15ml of NK cell culture solution into a centrifuge tube by using an electric suction aid, suspending and precipitating, blowing and uniformly mixing, adding into a culture bottle, adding a 15ml centrifuge tube of the NK cell culture solution, transferring into the culture bottle, and horizontally placing in a 7.5% CO2 incubator at 37 ℃ for culture.
(2) Serum-free culture of immune cells;
centrifuging, changing liquid and changing bottles when the cells are cultured to the 3 rd day: cells were harvested and centrifuged at 1500 rpm for 5 minutes. Adding 30ml of NK cell culture solution containing 5% autologous plasma into the centrifuged cell sediment for resuspension, adding solution according to the actual growth condition on days 4-6, and transferring a culture bag: counting on the 7 th day, if the number of cells is more than 1 × 108, adding 4ml of amplification reagent, and if the number of cells is 3-8 × 107, adding the amplification reagent until the 8 th day. NK Cell culture medium containing 5% autologous plasma was added to give a total Cell concentration of 1.0 x 106 cells/m. The culture solution with volume more than 200ml or the number of cells more than 1 x 108 can be transferred to a culture bag for culture; observing every day on 8-11 days, and adding 1% autologous plasma-containing NK Cell culture solution to make the Cell concentration about 1 × 106 Cell/ml; on day 12-14, the cells were added to a concentration of about 2 × 106 cells/ml. When the cells grow to the required number, the cells can be collected for use or frozen for storage. In general, it is recommended to collect the cells for 13 or 14 days.
And step three, analyzing the growth curve of the cell NK.
And step four, analyzing the expression of the cell NK surface antigen.
And step five, analyzing the cell NK cycle.
And step six, freezing and recovering the immune cells NK, wherein the experimental group is provided with four groups of freezing experiments of the immune cells NK and a group of control group, and five groups of experiments are respectively frozen for 10 days and 30 days.
And seventhly, detecting various indexes of the immune cells.
As a still further scheme of the invention: in the first step, uniformly and slowly adding the mixture to the upper layer of the corresponding solution A to form a complete interface, setting a centrifugal parameter to rise and fall by 4 and 1600rpm, centrifuging for 20min, removing the supernatant of the first layer, carefully and gently sucking the white cell layer of the second layer into a clean 50ml centrifuge tube, adding NA into each tube, diluting and uniformly mixing, fixing the volume to 50 ml/tube, setting a centrifugal parameter to rise and fall by 7 and 2000rpm, centrifuging for 8min, removing the supernatant, adding 10ml NA/tube, and resuspending the cells.
As a still further scheme of the invention: and in the third step, detecting the activity of the cells by adopting a trypan blue staining method, and drawing a corresponding cell growth curve.
As a still further scheme of the invention: in the fourth step, the cell surface CD3, CD56 and CD8 antigens are detected by adopting a flow cytometer.
As a still further scheme of the invention: and in the fifth step, detecting the cell cycle by adopting a flow cytometer.
As a still further scheme of the invention: in the sixth step, the four experimental groups and the one control group are respectively:
(1) and cell frozen stock solution 1: solution A: 20% of 20% human serum albumin, 10% of 15% dextran/5% glucose injection; and B, liquid B: 31.25 percent of Bomaili A (compound electrolyte injection), 31.25 percent of 5 percent of glucose/0.45 percent of sodium chloride injection and 7.5 percent of DMSO, subpackaging the mixture into 15ml of centrifuge tubes after the preparation, and standing at-20 ℃ for later use;
(2) cell frozen stock solution 2: adopting the B solution frozen and stored by the umbilical cord tissue, subpackaging the obtained solution into 15ml centrifuge tubes after purchase, and standing at-20 ℃ for later use;
(3) friend's health frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(4) the panono frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(5) and a control group: fresh cells.
As a still further scheme of the invention: in the seventh step, various indexes of the NK immune cells can be detected through nuclear cell counting, living cell detection, colony culture, immune cell detection and the like.
The invention has the beneficial effects that: the frozen stock solution of the immune cell NK and the activity research method thereof are reasonable in design, a serum-free culture medium suspension culture system is applied, the immune cell NK obtained by separation has stronger proliferation capacity, after the cells frozen by the cell frozen stock solution 1 are recovered, the cell activity is the highest in the four cell frozen stock solutions and is more than 90%, the detection result of a flow cytometer shows that the immunophenotypes of the cells before freezing and the cells after freezing and recovering have no obvious difference and all have the immunophenotypes of the immune cells, and the experiment of detecting apoptosis by flow cytometry shows that after the cells frozen by the cell frozen stock solution 1 are recovered, the number of late-stage apoptotic cells is the least, and the number of the cells which are not apoptotic is the most.
Drawings
FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a frozen stock solution of immune cells NK and a method for studying the activity thereof, comprising the following steps:
the first step, the separation of immune cell NK comprises the following processes:
(4) in a biological safety cabinet, dividing the peripheral blood into 50ml centrifuge tubes equally according to that each tube is not more than 35ml, setting the centrifugal parameters to rise and fall by 7 and fall by 9, and centrifuging for 8min at 2000 rmp;
(5) after centrifuging peripheral blood, firstly absorbing upper plasma into a 50ml centrifuge tube, taking four EP tubes, adding 1.0ml plasma into each tube, inactivating at 56 ℃ for 30min in a water bath, centrifuging at 3000rpm for 10min, and taking upper plasma into a clean 50ml centrifuge tube for later use;
(6) diluting the blood cells with NA such that the ratio of blood cells: NA is 1: 1.
Step two, the serum-free in vitro amplification of the cell NK comprises the following steps:
(2) initial culture of immune cells;
all cells were collected by centrifugation, resuspended in 30ml NK medium (1.5 ml of autologous serum, 5%) and 2ml of NK amplification reagent were added, centrifugation parameters were set, 9-7 was increased and decreased, 2000rpm was applied, centrifugation was carried out for 8min, and the remaining supernatant was discarded. Sucking 15ml of NK cell culture solution into a centrifuge tube by using an electric suction aid, suspending and precipitating, blowing and uniformly mixing, adding into a culture bottle, adding a 15ml centrifuge tube of the NK cell culture solution, transferring into the culture bottle, and horizontally placing in a 7.5% CO2 incubator at 37 ℃ for culture.
(2) Serum-free culture of immune cells;
centrifuging, changing liquid and changing bottles when the cells are cultured to the 3 rd day: cells were harvested and centrifuged at 1500 rpm for 5 minutes. Adding 30ml of NK cell culture solution containing 5% autologous plasma into the centrifuged cell sediment for resuspension, adding solution according to the actual growth condition on days 4-6, and transferring a culture bag: counting on the 7 th day, if the number of cells is more than 1 × 108, adding 4ml of amplification reagent, and if the number of cells is 3-8 × 107, adding the amplification reagent until the 8 th day. NK Cell culture medium containing 5% autologous plasma was added to give a total Cell concentration of 1.0 x 106 cells/m. The culture solution with volume more than 200ml or the number of cells more than 1 x 108 can be transferred to a culture bag for culture; observing every day on 8-11 days, and adding 1% autologous plasma-containing NK Cell culture solution to make the Cell concentration about 1 × 106 Cell/ml; on day 12-14, the cells were added to a concentration of about 2 × 106 cells/ml. When the cells grow to the required number, the cells can be collected for use or frozen for storage. In general, it is recommended to collect the cells for 13 or 14 days.
And step three, analyzing the growth curve of the cell NK.
And step four, analyzing the expression of the cell NK surface antigen.
And step five, analyzing the cell NK cycle.
And step six, freezing and recovering the immune cells NK, wherein the experimental group is provided with four groups of freezing experiments of the immune cells NK and a group of control group, and five groups of experiments are respectively frozen for 10 days and 30 days.
And seventhly, detecting various indexes of the immune cells.
Further, in the embodiment of the present invention, in the first step, after mixing, the mixture is uniformly and slowly added to the upper layer of the corresponding solution a to form a complete interface, the centrifugation parameter is set to be increased and decreased by 4, 1600rpm, centrifugation is performed for 20min, the supernatant of the first layer is discarded, the white cell layer of the second layer is carefully and gently sucked into a clean 50ml centrifuge tube, NA is added into each tube to dilute and mix the mixture, the volume is fixed to 50 ml/tube, the centrifugation parameter is set to be increased and decreased by 9, 2000rpm, centrifugation is performed for 8min, the supernatant is discarded, 10ml NA/tube is added, and cells are resuspended.
Further, in the third step of the present invention, trypan blue staining method is used to detect the cell activity, and a corresponding cell growth curve is drawn.
Further, in the fourth step of the present invention, the cell surface CD3, CD56, CD8 antigens are detected by flow cytometry.
Further, in the embodiment of the present invention, in the fifth step, a flow cytometer is used to detect the cell cycle.
Further, in the present embodiment, in the sixth step, the four experimental groups and the one control group are respectively:
(1) and cell frozen stock solution 1: solution A: 20% of 20% human serum albumin, 10% of 15% dextran/5% glucose injection; and B, liquid B: 31.25 percent of Bomaili A (compound electrolyte injection), 31.25 percent of 5 percent of glucose/0.45 percent of sodium chloride injection and 7.5 percent of DMSO, subpackaging the mixture into 15ml of centrifuge tubes after the preparation, and standing at-20 ℃ for later use;
(2) cell frozen stock solution 2: adopting the B solution frozen and stored by the umbilical cord tissue, subpackaging the obtained solution into 15ml centrifuge tubes after purchase, and standing at-20 ℃ for later use;
(3) friend's health frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(4) the panono frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(5) and a control group: fresh cells.
Further, in the seventh embodiment of the present invention, in the seventh step, indexes of the NK immune cells may be detected by counting of nuclear cells, detection of viable cells, colony culture, detection of immune cells, and the like.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A frozen stock solution of immune cells NK and an activity research method thereof are characterized in that: the method comprises the following steps:
the first step, the separation of immune cell NK comprises the following processes:
(1) in a biological safety cabinet, dividing the peripheral blood into 50ml centrifuge tubes equally according to that each tube is not more than 35ml, setting the centrifugal parameters to rise and fall by 7 and fall by 9, and centrifuging for 8min at 2000 rmp;
(2) after centrifuging peripheral blood, firstly absorbing upper plasma into a 50ml centrifuge tube, taking four EP tubes, adding 1.0ml plasma into each tube, inactivating at 56 ℃ for 30min in a water bath, centrifuging at 3000rpm for 10min, and taking upper plasma into a clean 50ml centrifuge tube for later use;
(3) diluting the blood cells with NA such that the ratio of blood cells: NA is 1: 1.
Step two, the serum-free in vitro amplification of the cell NK comprises the following steps:
(1) initial culture of immune cells;
all cells were collected by centrifugation, resuspended in 30ml NK medium (1.5 ml of autologous serum, 5%) and 2ml of NK amplification reagent were added, centrifugation parameters were set, 9-7 was increased and decreased, 2000rpm was applied, centrifugation was carried out for 8min, and the remaining supernatant was discarded. Sucking 15ml of NK cell culture solution into a centrifuge tube by using an electric suction aid, suspending and precipitating, blowing and uniformly mixing, adding into a culture bottle, adding a 15ml centrifuge tube of the NK cell culture solution, transferring into the culture bottle, and horizontally placing in a 7.5% CO2 incubator at 37 ℃ for culture;
(2) serum-free culture of immune cells;
centrifuging, changing liquid and changing bottles when the cells are cultured to the 3 rd day: cells were harvested and centrifuged at 1500 rpm for 5 minutes. Adding 30ml of NK cell culture solution containing 5% autologous plasma into the centrifuged cell sediment for resuspension, adding solution according to the actual growth condition on days 4-6, and transferring a culture bag: counting on the 7 th day, if the number of cells is more than 1 × 108, adding 4ml of amplification reagent, and if the number of cells is 3-8 × 107, adding the amplification reagent until the 8 th day. NK Cell culture medium containing 5% autologous plasma was added to give a total Cell concentration of 1.0 x 106 cells/m. The culture solution with volume more than 200ml or the number of cells more than 1 x 108 can be transferred to a culture bag for culture; observing every day on 8-11 days, and adding 1% autologous plasma-containing NK Cell culture solution to make the Cell concentration about 1 × 106 Cell/ml; on day 12-14, the cells were added to a concentration of about 2 × 106 cells/ml. When the cells grow to the required number, the cells can be collected for use or frozen for storage. In general, it is recommended to collect cells for 13 or 14 days;
step three, analyzing a growth curve of the cell NK;
step four, analyzing the expression of cell NK surface antigen;
step five, analyzing the cell NK cycle;
freezing and recovering the immune cells NK, wherein an experimental group is provided with four groups of freezing experiments of the immune cells NK and a group of control group, and five groups of experiments are respectively frozen for 10 days and 30 days;
and seventhly, detecting various indexes of the immune cells.
2. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: in the step (3), uniformly and slowly adding the mixture to the upper layer of the corresponding solution A to form a complete interface, setting a centrifugal parameter to rise and fall by 4 and 1600rpm, centrifuging for 20min, removing the supernatant of the first layer, carefully and gently sucking the white cell layer of the second layer into a clean 50ml centrifuge tube, adding NA into each tube, diluting and uniformly mixing, fixing the volume to 50 ml/tube, setting a centrifugal parameter to rise and fall by 7 and 2000rpm, centrifuging for 8min, removing the supernatant, adding 10ml NA/tube, and re-suspending the cells.
3. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: and in the third step, detecting the activity of the cells by adopting a trypan blue staining method, and drawing a corresponding cell growth curve.
4. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: in the fourth step, the cell surface CD3, CD56 and CD8 antigens are detected by adopting a flow cytometer.
5. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: and in the fifth step, detecting the cell cycle by adopting a flow cytometer.
6. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: in the sixth step, the four experimental groups and the one control group are respectively:
(1) and cell frozen stock solution 1: solution A: 20% of 20% human serum albumin, 10% of 15% dextran/5% glucose injection; and B, liquid B: 31.25 percent of Bomaili A (compound electrolyte injection), 31.25 percent of 5 percent of glucose/0.45 percent of sodium chloride injection and 7.5 percent of DMSO, subpackaging the mixture into 15ml of centrifuge tubes after the preparation, and standing at-20 ℃ for later use;
(2) cell frozen stock solution 2: adopting the B solution frozen and stored by the umbilical cord tissue, subpackaging the obtained solution into 15ml centrifuge tubes after purchase, and standing at-20 ℃ for later use;
(3) friend's health frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(4) the panono frozen stock solution: after purchase, subpackaging into 15ml centrifuge tubes, and standing at-20 ℃ for later use;
(5) and a control group: fresh cells.
7. The immune cell NK cryopreservation solution and the activity research method thereof according to claim 1, wherein the immune cell NK cryopreservation solution comprises: in the seventh step, various indexes of the NK immune cells can be detected through nuclear cell counting, living cell detection, colony culture, immune cell detection and the like.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110206293.9A CN113061576A (en) | 2021-02-24 | 2021-02-24 | Immune cell NK frozen stock solution and activity research method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110206293.9A CN113061576A (en) | 2021-02-24 | 2021-02-24 | Immune cell NK frozen stock solution and activity research method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113061576A true CN113061576A (en) | 2021-07-02 |
Family
ID=76558956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110206293.9A Withdrawn CN113061576A (en) | 2021-02-24 | 2021-02-24 | Immune cell NK frozen stock solution and activity research method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113061576A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106064A (en) * | 2021-02-24 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Frozen stock solution of immune cell CIK and activity research method thereof |
| CN114041456A (en) * | 2021-12-09 | 2022-02-15 | 安徽中盛溯源生物科技有限公司 | Clinical-grade NK cell cryopreservation liquid and using method thereof |
-
2021
- 2021-02-24 CN CN202110206293.9A patent/CN113061576A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106064A (en) * | 2021-02-24 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Frozen stock solution of immune cell CIK and activity research method thereof |
| CN114041456A (en) * | 2021-12-09 | 2022-02-15 | 安徽中盛溯源生物科技有限公司 | Clinical-grade NK cell cryopreservation liquid and using method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Buckner et al. | Leukapheresis* by continuous flow centrifugation (CFC) in patients with chronic myelocytic leukemia (CML) | |
| CN106591233B (en) | A kind of external evoked amplification of immunocyte and the method frozen | |
| CN108251365B (en) | Immune cell culture medium system | |
| CN113061576A (en) | Immune cell NK frozen stock solution and activity research method thereof | |
| CN104498434B (en) | A kind of preparation method of a large amount of BMDCs, gained BMDC | |
| CN112391344B (en) | In-vitro amplification and culture method for non-coated NK cells | |
| CN113151168B (en) | Human NK cell culture system and preparation method thereof | |
| AU2005281481A1 (en) | Apheresis tubing set | |
| CN111454903B (en) | Immune cell in vitro culture, induction, activation and cryopreservation method and cell bank establishment thereof | |
| CN111394309A (en) | Method for in-vitro amplification culture of NK (natural killer) cells | |
| CN110564683A (en) | Method for co-culture induced amplification of gamma delta T cells and NK cells | |
| CN106754704B (en) | Method for inducing and expanding immune cells in vitro | |
| CN110438077B (en) | Method for simultaneously culturing NK and gamma delta T cells | |
| Nagel et al. | Cell-mediated immunity against malignant melanoma in monozygous twins | |
| US20150218518A1 (en) | Industrial preparation of natural killer cells (nks) and injection using human allo-geneic karyocytes | |
| CN101914497B (en) | Clinical N-CIK cell culture and quality control and identification kit and application | |
| CN114058584B (en) | Preparation method of clinical natural killer cells | |
| US20030190306A1 (en) | HLA matching donor-originating activated lymphocytes to be used in prevention/treatment of tumors, infectious diseases and autoimmune diseases, treatment method achieved by using the lymphocytes, formula having the lymphocytes as a main constituent thereof, method for manufacturing the formula and preparation kit to be used to prepare the formula | |
| CN111548994A (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN106566807A (en) | Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof | |
| CN114438028B (en) | Method for in-vitro amplification of peripheral blood NK | |
| CN114807031A (en) | Construction method of human peripheral blood immune cell bank and stem cell bank | |
| CN107090434A (en) | A kind of blood anticoagulant and store method for improving CIK cell culture effect | |
| CN110747167B (en) | Preparation method and application of hemizygous BAK cell | |
| CN114480279A (en) | Efficient separation culture technology for human blood immune cells CD4T |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20210702 |
|
| WW01 | Invention patent application withdrawn after publication |