CN115011559A - Method for reducing cell loss in cell collection process - Google Patents
Method for reducing cell loss in cell collection process Download PDFInfo
- Publication number
- CN115011559A CN115011559A CN202210654949.8A CN202210654949A CN115011559A CN 115011559 A CN115011559 A CN 115011559A CN 202210654949 A CN202210654949 A CN 202210654949A CN 115011559 A CN115011559 A CN 115011559A
- Authority
- CN
- China
- Prior art keywords
- hours
- centrifugal container
- sealing liquid
- cell
- centrifugal
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 53
- 230000006727 cell loss Effects 0.000 title claims abstract description 16
- 238000007789 sealing Methods 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000001035 drying Methods 0.000 claims abstract description 10
- 238000007710 freezing Methods 0.000 claims abstract description 9
- 230000008014 freezing Effects 0.000 claims abstract description 9
- 238000002791 soaking Methods 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims abstract description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- 239000002518 antifoaming agent Substances 0.000 claims description 12
- -1 polydimethylsiloxane Polymers 0.000 claims description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 5
- 239000006143 cell culture medium Substances 0.000 claims description 5
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 4
- 239000012091 fetal bovine serum Substances 0.000 claims description 4
- 239000012894 fetal calf serum Substances 0.000 claims description 4
- RBNPOMFGQQGHHO-UHFFFAOYSA-N glyceric acid Chemical compound OCC(O)C(O)=O RBNPOMFGQQGHHO-UHFFFAOYSA-N 0.000 claims description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 4
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 2
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 2
- 239000000194 fatty acid Substances 0.000 claims description 2
- 229930195729 fatty acid Natural products 0.000 claims description 2
- 229920001451 polypropylene glycol Polymers 0.000 claims description 2
- 125000004122 cyclic group Chemical group 0.000 claims 1
- 239000000839 emulsion Substances 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 229920001296 polysiloxane Polymers 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 18
- 238000004108 freeze drying Methods 0.000 abstract description 16
- 210000004027 cell Anatomy 0.000 description 66
- 230000000052 comparative effect Effects 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 239000002244 precipitate Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 239000000126 substance Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- XMSXQFUHVRWGNA-UHFFFAOYSA-N Decamethylcyclopentasiloxane Chemical compound C[Si]1(C)O[Si](C)(C)O[Si](C)(C)O[Si](C)(C)O[Si](C)(C)O1 XMSXQFUHVRWGNA-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229940086555 cyclomethicone Drugs 0.000 description 1
- 229940008099 dimethicone Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000006432 protein unfolding Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229920002545 silicone oil Polymers 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/05—Means for pre-treatment of biological substances by centrifugation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
-
- 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
- C12N2500/05—Inorganic 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic 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
-
- 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
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Hematology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for reducing cell loss in a cell collection process, which comprises the following steps: 1) filling a sealing liquid in a centrifugal container, soaking for 30-60min, and removing the sealing liquid; 2) cooling the centrifugal container treated in the step 1) from 4-8 ℃ to-80-60 ℃ within 30-60 minutes, and freezing for 5-6 hours; 3) applying vacuum 0.15-0.2bar to the centrifugal container treated in the step 2), drying the centrifugal container for 8-10 hours at-80 to-60 ℃, raising the temperature to-30 to-20 ℃ within 1-2 hours, incubating for 3-5 hours, and then incubating for 3-4 hours at-30 to-20 ℃ to 20-30 ℃ within 0.5-1 hour 。 The centrifugal container treated by the method of the invention is subjected to sealing pretreatment and freeze-drying in advance, so that the experimental steps of experimental operators can be reduced, and the experiment can be savedAnd the time is consumed, and meanwhile, the centrifugal container subjected to freeze-drying treatment can be stored at room temperature, and the cell loss caused by the generation of bubbles in the experimental process is further reduced by utilizing transportation and storage.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for reducing cell loss in a collection process.
Background
At present, methods such as centrifugation are often adopted during cell collection, a large number of cells can be poured out along with supernatant when waste liquid is poured, and a large number of target cells are lost to influence the collection effect.
The method for reducing the loss of the cell process commonly used at present is to carry out sealing treatment on an instrument in the experimental process, and commonly used sealing liquid is protein substances such as BSA, serum and the like. However, the existing method has the following problems: 1. the closed process requires the experiment operator to configure the relevant solution and operate the solution on site, which not only increases the complexity and time consumption of the experiment process, but also is easy to generate large errors on the experiment results because the experiment operator has no closed step due to negligence. 2. The protein substances used in the blocking process can cause a large amount of bubbles to be generated in the solution in the subsequent experimental process, and the generation of the bubbles can easily cause the loss of cells. This technical scheme directly seals preliminary treatment and freeze-drying to the required centrifugation container of experiment, and reducible experiment operator's experimental step saves experiment consuming time, and the centrifugation container through freeze-drying treatment can be preserved at room temperature simultaneously, utilizes transportation and save, and creative has added the defoaming agent in this confining liquid in addition, has further reduced the cell loss that leads to because of the bubble produces in the experimentation.
Disclosure of Invention
The invention provides a method for reducing cell loss in a cell collection process, which is characterized in that a centrifugal container is subjected to sealing liquid treatment and then is subjected to freeze-drying, so that the method can be used for a rare target cell recovery experiment, the loss of devices such as the centrifugal container and the like and reagent foaming in the experiment process to cell recovery can be obviously reduced, and the devices can be transported and stored at room temperature after being subjected to freeze-drying.
The invention provides a method for reducing cell loss in a cell collection process, which comprises the following steps:
1) filling a sealing liquid in a centrifugal container, soaking for 30-60min, and removing the sealing liquid;
2) cooling the centrifugal container treated in the step 1) from 4-8 ℃ to-80-60 ℃ within 30-60 minutes, and freezing for 5-6 hours;
3) applying vacuum (0.15-0.2bar) to the centrifugal container treated in the step 2), drying the centrifugal container for 8-10 hours at-80 to-60 ℃, raising the temperature to-30 to-20 ℃ within 1-2 hours, keeping the temperature for 3-5 hours, then incubating for 3-4 hours from-30 to-20 ℃ to 20-30 ℃ within 0.5-1 hour for later use.
The sealing liquid comprises fetal calf serum, calcium chloride, magnesium chloride and a defoaming agent.
The defoaming agent is selected from one or more of emulsified silicone oil, cyclomethicone, higher alcohol fatty acid ester compound, polyoxyethylene polyoxypropylene pentaerythritol ether, polyoxyethylene polyoxypropylene amine ether, polyoxypropylene glycerol ether, polyoxypropylene polyoxyethylene glycerol ether and dimethicone.
The concentration of the defoaming agent is 0.1-50 ppm.
The pH value of the sealing liquid is 7.0-7.4.
The preparation method of the sealing liquid comprises the following steps:
and respectively adding fetal calf serum, calcium chloride, magnesium chloride and a defoaming agent into the serum-free cell culture medium to prepare the confining liquid, and adjusting the pH of the confining liquid to 7.0-7.4.
The volume concentration of the fetal calf serum is 0.1-10%.
The concentration of the calcium chloride is 0.5-10 mM; the concentration of the magnesium chloride is 1-10 mM.
The centrifugal container prepared by the preparation method also belongs to the protection scope of the application.
The application of the centrifugal container in cell collection also belongs to the protection scope of the application.
The centrifugal container can be a centrifugal container with different specifications such as 1.5mL, 2.0mL, 5mL, 10mL, 15mL, 25mL, 50mL and the like which is common in laboratories, or other devices with other shapes or specifications which can be used for cell cleaning, staining and the like.
The serum-free cell culture Medium is preferably RPMI 1640 Medium.
The key point of the invention is that the sealing liquid is firstly contained in a centrifugal container, the centrifugal container is soaked for 30-60min, then the sealing liquid is removed, and then the device is frozen and dried. The method can reduce the experiment steps of experiment operators, save the experiment time consumption, meanwhile, the centrifugal container subjected to freeze-drying treatment can be stored at room temperature, and is transported and stored.
The present inventors have attempted to seal only the centrifuge vessel and found that the process loss rate of the cells is around 10% due to the generation of more bubbles.
The present inventors have attempted to treat only the bubbles generated during the experiment with a defoaming agent, and have found that the loss of the cell process can be reduced by 5% to 10%.
The preferred embodiments of the present invention are obtained after numerous alternatives are eliminated.
Compared with the prior art, the invention has the following beneficial effects:
1. the centrifugal container is freeze-dried after being treated by the sealing liquid and is used for the recovery experiment of rare target cells, so that the cell loss is avoided; meanwhile, the centrifugal container can be stored at room temperature, and is transported and stored;
2. the invention carries out freeze-drying treatment on a sealing liquid treatment centrifugal container, and firstly, the sealing liquid is contained in the centrifugal container, and the centrifugal container is removed after being soaked for 30-60 min; then the centrifugal container is cooled from 4 ℃ to 8 ℃ to 80 ℃ below zero to 60 ℃ below zero within 30-60 minutes and is frozen for 5-6 hours; then vacuum (0.15-0.2bar) is applied, the centrifugal container is dried for 8-10 hours at-80 to-60 ℃, the temperature is increased to-30 to-20 ℃ within 1-2 hours and incubated for 3-5 hours, and then the temperature is increased from-30 to-20 ℃ to 20-30 ℃ within 0.5-1 hour and incubated for 3-4 hours; as a solvent, the liquid crystalline state of water, the hydrogen bonding ability, lubricates or "elasticizes" the movement of proteins. Water controls protein structure through the sequential cleavage and reestablishment of hydrogen bonds in the hydrated shell and through the guidance of secondary and quaternary structures such as alpha-helices and beta-sheets by inter-peptide and side chain interactions. Although thermal energy at room temperature is sufficient to denature proteins, the hydrated shell fragments at Dh < 0.2, resulting in insufficient water molecules to perform the rearrangement of hydrogen bonds associated with unfolding of protein domains. Therefore, the invention adopts a freezing method. However, freeze-drying can generate stress which can also denature proteins to different degrees, so that the gradual freezing for realizing the room-temperature storage of the proteins becomes the first choice of the invention; applying vacuum to the frozen sample, and sublimating water at subzero temperature or evaporating ice phase (primary drying), and removing residual water by gradually raising temperature (secondary drying), wherein crystalline substance or powder is generated in the obtained freeze-dried product;
3. the sealing liquid is innovatively added with the defoaming agent, so that the cell loss caused by bubble generation in the experimental process is further reduced;
4. through the treatment of the centrifugal container, for the detection of peripheral blood Circulating Tumor Cells (CTC), the loss of the CTC which is originally very rare in the experimental process can be reduced, the detection sensitivity of the CTC is improved, and the false negative of clinical results is reduced.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that numerous modifications and adaptations can be made by those skilled in the art without departing from the inventive concepts herein. All falling within the scope of the present invention.
Examples 1 to 4
Examples 1-4 provide a method of reducing cell loss during cell collection comprising the steps of:
step 1, preparation of centrifugal tube
In examples 1 to 4, a 15mL centrifuge tube was used.
The centrifuge tube may be a common centrifuge tube with different specifications such as 1.5mL, 2.0mL, 5mL, 10mL, 25mL, 50mL in a laboratory, or other device with other shape or specification that can be used for cell washing, staining, etc.
Step 2, preparation of sealing liquid
The following substances were added to serum-free cell culture media according to the concentration and pH requirements in Table 1, respectively, to prepare blocking solutions.
TABLE 1
In table 1, product information of each substance is as follows:
calcium chloride (alatin, cat # C110766);
magnesium chloride (alatin, cat # M140788);
polydimethylsiloxane (Aladdin, cat # C194626);
fetal bovine serum (thermo, Gibco, cat # 10100147);
serum-free cell culture Medium (RPMI 1640 Medium) was obtained from (thermo, Gibco, cat # 11875093).
Step 3, freeze drying
Examples 1-2 the following freeze drying procedure was used:
the method comprises the steps of filling a sealing liquid in a centrifugal tube, soaking for 30min at 4 ℃, removing the sealing liquid, cooling the temperature of the centrifugal tube from 4 ℃ to-60 ℃ within 30min, freezing for 5h, applying vacuum (0.15bar), drying a sample at-60 ℃ for 8 h, raising the temperature to-30 ℃ within 2 h, incubating for 3 h, and incubating for 3 h from-30 ℃ to room temperature within 1h for later use.
Examples 3-4 the following freeze-drying procedure was used: the method comprises the steps of filling a centrifugal tube with a sealing solution, soaking at 8 ℃ for 60min, removing the sealing solution, cooling the centrifugal tube from 8 ℃ to-80 ℃ within 60min, freezing for 65 hours, applying vacuum (0.2bar), drying a sample at-80 ℃ for 10 hours, raising the temperature to-20 ℃ within 1 hour, incubating for 5 hours, and incubating for 4 hours from-20 ℃ to room temperature within 0.5 hour for later use.
Comparative example 1
The comparative example differs from example 1 in that no antifoam was added, the remainder being unchanged, the cell process loss rate being: 5 to 20 percent.
Comparative example 2
The comparative example differs from example 1 in that no fetal bovine serum was added, the remainder was unchanged, and the cell process loss rate was: 10 to 30 percent.
Comparative example 3
The present comparative example differs from example 1 in that no vacuum is applied during the freeze-drying process, specifically:
the method comprises the steps of filling a sealing liquid in a centrifugal tube, removing the sealing liquid after soaking for 30min, cooling the centrifugal tube from 4 ℃ to-60 ℃ within 30min, freezing for 5h, drying a sample at-60 ℃ for 8 h, raising the temperature to-30 ℃ within 2 h, incubating for 3 h, and then incubating for 3 h from-30 ℃ to room temperature within 1h for later use.
Comparative example 4
The comparative example differs from example 1 in that:
the method comprises the steps of filling a sealing solution in a centrifugal tube, removing the sealing solution after soaking for 30min, cooling the centrifugal tube from 4 ℃ to-60 ℃ within 30min, freezing for 5h, applying vacuum (0.15bar), drying a sample at-60 ℃ for 8 h, raising the temperature to room temperature within 2 h, and incubating for 3 h for later use.
Comparative example 5
This comparative example differs from example 1 in that no freeze-drying is used, specifically:
and (3) filling a sealing liquid in the centrifuge tube, soaking for 30min, removing the sealing liquid, and drying the centrifuge tube at a constant temperature of 37 ℃ for 24 h.
Comparative example 6
Adopts a common centrifugal tube
A commercial 15mL centrifuge tube was purchased and used for cell-related experiments without confining liquid and freeze-drying.
Performance testing
K562 cell recovery assay
Examples 1-4 and comparative examples 1-4 were used to perform cell recovery experiments in which centrifuge tubes were used (corning, cat # 430791).
The test method comprises the following steps:
1. preparing a fluorescent dye DiO (Shanghai Biyuntian biotechnology, Inc., C1038): dissolving in dimethyl sulfoxide to obtain solution with concentration of 1 mg/mL;
2. centrifuging 2mL of cultured K562 cells (ATCC, CCL-243) at 200g for 3min, removing the culture medium, adding 1mL of PBS to resuspend cell precipitates, centrifuging at 200g for 3min, removing the supernatant, and repeating the steps once;
3. adding 100 μ L paraformaldehyde (4%, Shanghai Biyun biotechnology, Inc., P0099) solution, fixing cells at room temperature for 15min, adding 1mL PBS, resuspending the cell precipitate, centrifuging at 200g for 3min, removing supernatant, and repeating;
resuspending cells in 4.1mL of PBS solution, adding 10 μ L of DiO solution, shaking the cells gently at 150r/min, staining at room temperature for 30min, centrifuging at 200g for 3min, removing supernatant, resuspending cell precipitate in 1mL of PBS solution, and repeating centrifugation and washing once;
5. the cells stained with green fluorescence were counted and adjusted to a concentration of about 1X 10 5 /mL；
6. Taking 1 microliter of cells on a glass slide, manually and accurately counting under a fluorescence microscope, and transferring all the counted cells into a centrifuge tube (corning, cat # 430791);
description 1: observing through a microscope to ensure that all cells are transferred into the centrifuge tube, and if individual residues exist on the glass slide, subtracting the number of the residual cells from the total number of the counted cells to accurately count the cells in the centrifuge tube;
description 2: the centrifuge tubes were those treated differently in the examples and comparative examples.
7.150r/min, incubating the centrifuge tube with cells at room temperature for 30min, centrifuging the cells for 3min at 200g, discarding the supernatant, taking 100 μ L PBS to resuspend the cells, transferring to a 96-well plate (corning, cat # 3599), cleaning the centrifuge tube for 3 times, and transferring the cleaning solution to the 96-well plate;
8. standing the 96-well plate for 1h in a dark place, then counting cells in the 96-well plate under a fluorescence microscope, and calculating the cell recovery rate and the process loss rate.
And (3) test results:
TABLE 2
(II) recovery experiment of tumor cells by simulating whole blood
1. Preparing a fluorescent dye DiO (Shanghai Biyuntian biotechnology, Inc., C1038): dissolving in dimethyl sulfoxide at a concentration of 1 mg/mL;
2. centrifuging 2mL of cultured MCF-7 cells (ATCC, HTB-22) at 200g for 3min, removing the culture medium, adding 1mL of PBS to resuspend cell precipitates, centrifuging at 200g for 3min, removing supernatant, and repeating the steps once;
3. adding 100 μ L paraformaldehyde (4%, Shanghai Bin Yuntian biotechnology, Inc., P0099) solution, fixing cells at room temperature for 15min, adding 1mL PBS to resuspend cell precipitate, centrifuging at 200g for 3min, removing supernatant, and repeating once;
resuspending cells in 4.1mL of PBS solution, adding 10 μ L of DiO solution, shaking the cells gently at 150r/min, staining at room temperature for 30min, centrifuging at 200g for 3min, removing supernatant, resuspending cell precipitate in 1mL of PBS solution, and repeating centrifugation and washing once;
5. the cells stained with green fluorescence were counted and adjusted to a concentration of about 1X 10 5 /mL；
6. 1 microliter of cells are taken out and placed on a glass slide, the cells are manually and accurately counted under a fluorescence microscope, and the counted cells are all transferred into a centrifuge tube (corning, cat 430791) processed in the example 1;
description 1: observing through a microscope to ensure that all cells are transferred into the centrifuge tube, and if individual residues exist on the glass slide, subtracting the number of the residual cells from the total number of the counted cells to accurately count the cells in the centrifuge tube;
7. centrifuging 10mL of cultured THP-1(ATCC, TIB-202) at 200g for 3min, removing the culture medium, adding 1mL of PBS to resuspend the cell precipitate, centrifuging at 200g for 3min, removing the supernatant, and repeating the steps once;
8. adding 100 μ L paraformaldehyde (4%, Shanghai Biyun biotechnology, Inc., P0099) solution, fixing cells at room temperature for 15min, adding 1mL PBS, resuspending the cell precipitate, centrifuging at 200g for 3min, removing supernatant, repeating the above steps, and adjusting the concentration to 1 × 106/mL;
9. adding 1mL of THP-1 cell solution into a centrifuge tube, adding 5 μ L of streptavidin magnetic beads (Thermo, cat # 65001), incubating the centrifuge tube with cells at room temperature for 30min at 150r/min, and DynaMag TM 15 magnet (Thermo, cat # 12301D) magnetically separating the solution for 3min, resuspending the magnetic beads in 100. mu.L PBS, transferring to 96-well plate (corning, cat # 3599), centrifuging cells for 3min at 200g of the solution after magnetic separation, discarding the supernatant, taking 100. mu.L PBS to resuspend the cells and transferring to 96-well plate, washing the centrifuge tube for 3 times, and transferring the washing solution to the 96-well plate;
10. standing the 96-well plate for 1h in a dark place, then counting cells in the 96-well plate under a fluorescence microscope, and calculating the recovery rate and the process loss rate of the MCF-7 cells.
And (3) test results:
TABLE 3
The invention combines sealing and defoaming to act on an experimental device, can obviously reduce cell loss in the experimental process, can be transported and stored at room temperature by freeze-drying treatment, is convenient for users to use, and provides assistance for clinical rare cell detection.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. A method of reducing cell loss during cell collection comprising the steps of:
1) filling a sealing liquid in a centrifugal container, soaking for 30-60min, and removing the sealing liquid;
2) cooling the centrifugal container treated in the step 1) from 4-8 ℃ to-80-60 ℃ within 30-60 minutes, and freezing for 5-6 hours;
3) applying vacuum (0.15-0.2bar) to the centrifugal container treated in the step 2), drying the centrifugal container for 8-10 hours at-80 to-60 ℃, raising the temperature to-30 to-20 ℃ within 1-2 hours, keeping the temperature for 3-5 hours, and then incubating for 3-4 hours at the temperature of-30 to-20 ℃ to 20-30 ℃ within 0.5-1 hour.
2. The method of claim 1, wherein the blocking fluid comprises fetal bovine serum, calcium chloride, magnesium chloride, an anti-foaming agent.
3. The method of claim 2, wherein the antifoaming agent is one or more selected from the group consisting of silicone emulsion, cyclic polydimethylsiloxane, higher alcohol fatty acid ester complex, polyoxyethylene polyoxypropylene pentaerythritol ether, polyoxyethylene polyoxypropylene amine ether, polyoxypropylene glycerol ether, polyoxypropylene polyoxyethylene glycerol ether, and polydimethylsiloxane.
4. The method of reducing cell loss during cell collection according to claim 2, wherein the concentration of the antifoaming agent is 0.1-50 ppm.
5. The method of reducing cell loss during cell collection according to claim 2, wherein the pH of the blocking solution is from 7.0 to 7.4.
6. The method of claim 2, wherein the blocking solution is prepared by a method comprising the steps of: and respectively adding fetal calf serum, calcium chloride, magnesium chloride and a defoaming agent into the serum-free cell culture medium to prepare the sealing liquid, and adjusting the pH of the sealing liquid to 7.0-7.4.
7. The method of claim 2, wherein the fetal bovine serum is present at a concentration of 0.1% to 10% by volume.
8. The method of reducing cell loss during cell collection according to claim 2, wherein the calcium chloride is present at a concentration of 0.5 to 10 mM; the concentration of the magnesium chloride is 1-10 mM.
9. A centrifuge vessel produced by the production method according to any one of claims 1 to 8.
10. Use of a centrifugation container according to claim 9 for cell collection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210654949.8A CN115011559A (en) | 2022-06-10 | 2022-06-10 | Method for reducing cell loss in cell collection process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210654949.8A CN115011559A (en) | 2022-06-10 | 2022-06-10 | Method for reducing cell loss in cell collection process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115011559A true CN115011559A (en) | 2022-09-06 |
Family
ID=83074672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210654949.8A Pending CN115011559A (en) | 2022-06-10 | 2022-06-10 | Method for reducing cell loss in cell collection process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115011559A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5232828A (en) * | 1992-03-09 | 1993-08-03 | Becton, Dickinson And Company | Coating agents for cell recovery |
| US6140123A (en) * | 1998-10-07 | 2000-10-31 | Cedars-Sinai Medical Center | Method for conditioning and cryopreserving cells |
| WO2012098358A1 (en) * | 2011-01-20 | 2012-07-26 | Biopharma Technology Ltd | Freeze drying method |
| CN110095599A (en) * | 2019-04-22 | 2019-08-06 | 浙江大学 | The Microimmunofluorescence test method of cell-free loss |
| CN113652468A (en) * | 2021-07-19 | 2021-11-16 | 江苏硕世生物科技股份有限公司 | Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof |
-
2022
- 2022-06-10 CN CN202210654949.8A patent/CN115011559A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5232828A (en) * | 1992-03-09 | 1993-08-03 | Becton, Dickinson And Company | Coating agents for cell recovery |
| US5324629A (en) * | 1992-03-09 | 1994-06-28 | Becton, Dickinson And Company | Coating agents for cell recovery |
| US6140123A (en) * | 1998-10-07 | 2000-10-31 | Cedars-Sinai Medical Center | Method for conditioning and cryopreserving cells |
| WO2012098358A1 (en) * | 2011-01-20 | 2012-07-26 | Biopharma Technology Ltd | Freeze drying method |
| CN110095599A (en) * | 2019-04-22 | 2019-08-06 | 浙江大学 | The Microimmunofluorescence test method of cell-free loss |
| CN113652468A (en) * | 2021-07-19 | 2021-11-16 | 江苏硕世生物科技股份有限公司 | Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nguyen et al. | Extracellular vesicles from bone marrow-derived mesenchymal stromal cells support ex vivo survival of human antibody secreting cells | |
| Chilvers et al. | Development of a novel protocol for isolation and purification of human granulosa cells | |
| Lara et al. | Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle | |
| Cobbs et al. | Methods for the detection of cytomegalovirus in glioblastoma cells and tissues | |
| Hong et al. | Serum proteins are extracted along with monolayer cells in plasticware and interfere with protein analysis | |
| CN110951673A (en) | Method for inducing transdifferentiation of somatic cells into mammary epithelial cells in vitro by using small molecular compound | |
| WO2019129178A1 (en) | Composition containing anti-cd45 monoclonal antibody, and method for using same | |
| NZ211675A (en) | Immunocytochemical microscope control slides and their preparation | |
| Gomes-Alves et al. | In vitro expansion of human cardiac progenitor cells: exploring'omics tools for characterization of cell-based allogeneic products | |
| CN104833805A (en) | Circulating tumor cell detection and identification kit and application thereof | |
| Qian et al. | [Retracted] Exploring Purification Methods of Exosomes from Different Biological Samples | |
| CN112852725A (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN115011559A (en) | Method for reducing cell loss in cell collection process | |
| CN116116385B (en) | Extraction of exosomes in blood and proteomic analysis method thereof | |
| CN115651076B (en) | Surface marker of human bone marrow mesenchymal stem cell-derived apoptosis vesicle and application thereof | |
| CN110864939A (en) | Protein extraction method and application | |
| Vlasov et al. | FRTL‐5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid | |
| CN115044639A (en) | Screening method of anti-aging drug | |
| CN108872603B (en) | Method for identifying liver cancer stem cells | |
| WO2021088847A1 (en) | Method for culturing gynaecological tumour primary cells and matching culture medium | |
| RU2762489C1 (en) | Method for isolating spermatozoa from aspiration and/or biopsy material from an appendage and/or testicle for use in in vitro fertilization and/or cryopreservation programs | |
| CN102174465A (en) | Method for separating enriched target cells from tissues | |
| CN112980006A (en) | Protein cross-linked nano affinity microsphere, preparation method and application | |
| CN103728171B (en) | Extraction method of earthworm total protein suitable for two-dimensional electrophoresis experiment | |
| CN111909235B (en) | Method for separating sperm releasing protein by utilizing Trans Well chamber |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220906 |
|
| RJ01 | Rejection of invention patent application after publication |