CN108293981A - A kind of attached tumor cells cryopreservation methods and improvement freeze formula of liquid - Google Patents
A kind of attached tumor cells cryopreservation methods and improvement freeze formula of liquid Download PDFInfo
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- CN108293981A CN108293981A CN201810188547.7A CN201810188547A CN108293981A CN 108293981 A CN108293981 A CN 108293981A CN 201810188547 A CN201810188547 A CN 201810188547A CN 108293981 A CN108293981 A CN 108293981A
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- 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
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- 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
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Abstract
A kind of attached tumor cells cryopreservation methods of present invention offer and improvement freeze formula of liquid, by configuring improvement cells frozen storing liquid, eliminate intermediate centrifugation link, it can greatly save the experimental implementation time, save experiment consumptive material, improve conventional efficient, it is longer to solve traditional attached tumor cells cryopreservation methods experimental implementation time, and need to remove culture solution by centrifuging, operating process is cumbersome and also larger, the problems such as cell activity after recovery is relatively low is injured caused by cell.Belong to biotechnology.
Description
Technical field
The present invention provides a kind of attached tumor cells cryopreservation methods and freezes formula of liquid, belongs to biotechnology.
Technical background
Cell culture Seedling selection secondary culture, the aging of finite cell lines and hereditary shakiness in continuous cell line
The factors such as qualitative can promote cell line to have variation to be inclined in continuous culture.In addition, being also possible to that unexpected feelings occur in laboratory
Condition, if cell incubator powers off suddenly, the cross contamination between the human factor pollution of cell even in operation and cell line
Deng, therefore it is significant to carry out freezen protective to cell in an experiment.
Ultra-cryofreezing preservation be cell long-period preserve a kind of effective ways, mainly by reduce cell metabolic rate come
It plays a protective role.It is the most common type cell long-period preserving type that (- 196 DEG C) of liquid nitrogen, which preserves cell, adjusts and control is thin
The effect of the enzyme classes of intracellular growth metabolism is greatly suppressed, and keeps the biochemical reaction of cell interior very slow, or even is stopped, from
And the change of cytogenetics character is avoided, cell and tissue will not lose morphogenetic potential.But frostbite, which is ultralow temperature, protects
Maximum negative reaction is protected, the reason is that the mechanical injuries caused by ice crystal and permeability damage.But it if does not take properly
Cryopreservation methods and suitable cryoprotector, cell frost bitten is easy during freezing.Improve frozen stock solution and cryopreservation methods
It is to improve the important channel for preserving cell, many researchs at present are all to improve frozen stock solution and cryopreservation methods to protect cell to exempt from as possible
The case where being frozen process frostbite.
Traditional attached tumor cells cryopreservation methods experimental implementation time is longer, and needs to remove culture by centrifuging
Liquid, operating process is cumbersome and is injured caused by cell also larger, and the cell activity after recovery is relatively low.
Invention content
It is an object of the invention to：A kind of attached tumor cells cryopreservation methods are provided and improvement freezes formula of liquid, to solve
Traditional attached tumor cells cryopreservation methods experimental implementation time is longer, and needs to remove culture solution, operating process by centrifuging
It is cumbersome and also larger, the problems such as cell activity after recovery is relatively low is injured caused by cell.
To solve the above problems, quasi- use such a attached tumor cells cryopreservation methods：
First, configuration improvement cells frozen storing liquid include 30% fetal calf serum (FBS) by volume, 20% dimethyl
Sulfoxide (DMSO) and 50% DMEM culture mediums, after the completion of preparation, be distributed into 1ml/ pipe, freeze in -20 DEG C of refrigerators；
Old culture medium is first abandoned when freeze-stored cell, sterile PBS, which is added, along cell offside cleans 2 times, adds 1ml tryptoses
Enzyme, cross gently shake Tissue Culture Flask 5 seconds, then, exhaust trypsase with Pasteur's moral suction pipe, are placed in 37 DEG C of incubators
3 minutes vitellophags are incubated, add 2ml fresh cultures, fresh culture culture medium containing 90%DMEM by volume
And 10%FBS, it blows and beats cell 30 times with Pasteur's moral suction pipe, single cell suspension is made, counts；
Three cell cryopreservation tubes are taken, take 0.5ml cell suspensions to be added in each cell cryopreservation tube respectively with pipettor suction, then
The above-mentioned improvement cells frozen storing liquids of 0.5ml are respectively added into cryopreservation tube, tighten cryopreservation tube lid, tag-related is put into program drop
It in warm box, is put into -80 DEG C of refrigerators, is transferred in liquid nitrogen preserves for a long time afterwards for 24 hours, can periodically take out a cell recovery culture, see
Examine cell viability.
In preceding method, configuration improvement cells frozen storing liquid specifically refers to, and a sterile centrifugation tube (50ml) is taken, with shifting
25ml DMEM culture mediums are added into centrifuge tube for liquid device, then are slowly added to 10ml DMSO when shaking, and are eventually adding 15ml
FBS；
In preceding method, the sterile PBS is：Weigh 1.6g sodium chloride, 0.04g potassium chloride, 0.04g potassium dihydrogen phosphates
And seven hypophosphite monohydrate disodium hydrogens of 0.44g or 0.588g disodium hydrogen phosphate dodecahydrates, addition ultra-pure water are dissolved and are settled to
200ml, high pressure sterilization save backup at 4 DEG C；
In preceding method, the product or voluntarily prepare that trypsase is produced using hyclone companies, preparation method is：Claim
0.25g trypsase powder is added in PBS, with volumetric flask constant volume to 100ml, waits for that powder is completely dissolved, the filter membrane mistake of 0.22um
Filter is distributed into 1ml/ pipes, and -20 DEG C save backup.
The present invention also provides a kind of improvement to freeze formula of liquid, includes by weight：30% fetal calf serum, 20% diformazan
Base sulfoxide and 50% DMEM culture mediums.
Compared with prior art, the cell freezing method that the present invention uses is easy to operate, eliminates intermediate centrifugation link, can
The experimental implementation time is greatly saved, experiment consumptive material is saved, improves conventional efficient, simultaneously as need not centrifuge, is avoided thin
Born of the same parents are mechanically damaged under centrifugal condition, shorten time and reduce pollution probability, energy that cell operates in external environment
Cell viability is utmostly kept, in addition, the cell frozen using this method shows cell through recovery, MTT experiment testing result
Survival rate is significantly higher than the cell survival rate that conventional method is frozen.Ultra-cryofreezing preservation, which is one kind that cell long-period preserves, to be had
The cell cryopreservation liquid making method of efficacious prescriptions method, the improvement is simple, and cell freezing method is easy, easy to operate, is ground in antitumor drug
Hair field will be widely used.
Specific implementation mode
It is apparent to make the present invention state, the present invention will be described in further details below.
Embodiment：
It present embodiments provides a kind of improve and freezes formula of liquid, include by weight：30% fetal calf serum, the two of 20%
Methyl sulfoxide and 50% DMEM culture mediums.
It is as follows that the method that attached tumor cells freeze is carried out using above-mentioned formula：
First, configuration improvement cells frozen storing liquid, takes a sterile centrifugation tube (50ml), is added into centrifuge tube with pipettor
25ml DMEM culture mediums, then 10ml DMSO are slowly added to when shaking, 15ml FBS are eventually adding, after the completion of preparation, packing
It manages, is frozen in -20 DEG C of refrigerators at 1ml/.
Old culture medium is first abandoned when freeze-stored cell, sterile PBS, which is added, along cell offside cleans 2 times, adds 1ml tryptoses
Enzyme, cross gently shake Tissue Culture Flask 5 seconds, then, exhaust trypsase with Pasteur's moral suction pipe, are placed in 37 DEG C of incubators
3 minutes vitellophags are incubated, 2ml fresh cultures, the fresh culture culture medium containing 90%DMEM and 10% are added
FBS blows and beats cell 30 times with Pasteur's moral suction pipe, single cell suspension is made, counts.
The preparation method of sterile PBS is：Weigh 1.6g sodium chloride, 0.04g potassium chloride, 0.04g potassium dihydrogen phosphates and
Seven hypophosphite monohydrate disodium hydrogens of 0.44g or 0.588g disodium hydrogen phosphate dodecahydrates are added ultra-pure water, dissolve and be settled to 200ml,
High pressure sterilization saves backup at 4 DEG C.
The product or voluntarily prepare that trypsase is produced using hyclone companies, preparation method is：Claim 0.25g tryptoses
Enzyme powder is added in PBS, with volumetric flask constant volume to 100ml, waits for that powder is completely dissolved, the membrane filtration of 0.22um is distributed into
1ml/ is managed, and -20 DEG C save backup.
Three cell cryopreservation tubes are taken, take 0.5ml cell suspensions to be added in each cell cryopreservation tube respectively with pipettor suction, then
The above-mentioned improvement cells frozen storing liquids of 0.5ml are respectively added into cryopreservation tube, tighten cryopreservation tube lid, tag-related is put into program drop
It in warm box, is put into -80 DEG C of refrigerators, is transferred in liquid nitrogen preserves for a long time afterwards for 24 hours, can periodically take out a cell recovery culture, see
Examine cell viability.
Claims (5)
1. a kind of attached tumor cells cryopreservation methods, it is characterised in that：
First, configuration improvement cells frozen storing liquid includes 30% fetal calf serum by volume, 20% dimethyl sulfoxide (DMSO) and
50% DMEM culture mediums after the completion of preparation, are distributed into 1ml/ pipes, freeze in -20 DEG C of refrigerators；
Old culture medium is first abandoned when freeze-stored cell, sterile PBS, which is added, along cell offside cleans 2 times, adds 1ml trypsase, and ten
Font gently shakes Tissue Culture Flask 5 seconds, then, exhausts trypsase with Pasteur's moral suction pipe, is placed in 37 DEG C of incubators and is incubated 3
Minute vitellophag, adds 2ml fresh cultures, fresh culture culture medium containing 90%DMEM and 10% by volume
FBS blows and beats cell 30 times with Pasteur's moral suction pipe, single cell suspension is made, counts；
Three cell cryopreservation tubes are taken, take 0.5ml cell suspensions to be added in each cell cryopreservation tube respectively with pipettor suction, then to jelly
It deposits and the above-mentioned improvement cells frozen storing liquids of 0.5ml is respectively added in pipe, tighten cryopreservation tube lid, tag-related is put into program temperature reduction box
In, it is put into -80 DEG C of refrigerators, is transferred in liquid nitrogen preserves for a long time afterwards for 24 hours.
2. a kind of attached tumor cells cryopreservation methods according to claim 1, which is characterized in that the configuration improves cell
Frozen stock solution specifically refers to：A sterile centrifugation tube (50ml) is taken, 25ml DMEM culture mediums are added into centrifuge tube with pipettor, then
10ml DMSO are slowly added to when shaking, are eventually adding 15ml FBS.
3. a kind of attached tumor cells cryopreservation methods according to claim 1, which is characterized in that the sterile PBS is：Claim
Take 1.6g sodium chloride, 0.04g potassium chloride, 0.04g potassium dihydrogen phosphates and seven hypophosphite monohydrate disodium hydrogens of 0.44g or 0.588g 12
Hypophosphite monohydrate disodium hydrogen is added ultra-pure water, dissolves and be settled to 200ml, high pressure sterilization saves backup at 4 DEG C.
4. a kind of attached tumor cells cryopreservation methods according to claim 1, it is characterised in that：Trypsase uses
The product of hyclone companies production is voluntarily prepared, and preparation method is：0.25g trypsase powder is claimed to be added in PBS, with appearance
Measuring bottle is settled to 100ml, waits for that powder is completely dissolved, the membrane filtration of 0.22um, is distributed into 1ml/ pipes, -20 DEG C save backup.
5. a kind of improvement freezes formula of liquid, it is characterised in that：Include by weight：30% fetal calf serum, 20% dimethyl
Sulfoxide and 50% DMEM culture mediums.
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| CN201810188547.7A CN108293981A (en) | 2018-03-07 | 2018-03-07 | A kind of attached tumor cells cryopreservation methods and improvement freeze formula of liquid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109601529A (en) * | 2019-02-25 | 2019-04-12 | 安徽古井贡酒股份有限公司 | A kind of cell cryopreservation and method for resuscitation of simplicity |
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2018
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| CN102212460A (en) * | 2011-04-27 | 2011-10-12 | 中国人民解放军第三军医大学第二附属医院 | Stem cell screening system, preparation method thereof and screening method of stem cell |
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Application publication date: 20180720 |