CN109380214A - A kind of serum-free cell freezes agent and preparation method thereof, application method, frozen stock solution - Google Patents
A kind of serum-free cell freezes agent and preparation method thereof, application method, frozen stock solution Download PDFInfo
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- CN109380214A CN109380214A CN201811618889.4A CN201811618889A CN109380214A CN 109380214 A CN109380214 A CN 109380214A CN 201811618889 A CN201811618889 A CN 201811618889A CN 109380214 A CN109380214 A CN 109380214A
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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
The present invention provides a kind of serum-free cells to freeze agent and preparation method thereof, application method, and freezing agent includes DMSO with for serum composition, and the generation serum composition includes serum substitute and trehalose.Of the invention freezes agent without containing any serum composition, and trehalose is compounded simultaneously to adjust the character of solution, it is set to be more conducive to the preservation of cell, it is also more excellent to freeze effect, compared with tradition freezes agent, cell after recovery is significantly improved in survival rate, therefore should be widely promoted and applied.
Description
Technical field
The present invention relates to cell cryopreservation field, in particular to a kind of serum-free cell freeze agent and preparation method thereof,
Application method, frozen stock solution.
Background technique
Cell cryopreservation is that cell is placed on to low temperature environment, cell metabolism is reduced, so as to a kind of technology of long term storage.Cell
Freezing is one of main method that cell saves, plays the role of cell conservation.
Cell cryopreservation is one of the main method that cell saves, and cell can be made to be temporarily disengaged from growth shape using Cryopreservation Technology
State and its cell characteristics is saved, so when needed again recovery cell for testing.
In currently available technology, the main component in cells frozen storing liquid have 10% dimethyl sulfoxide (DMSO), 20%~
90% fetal calf serum (FBS), remaining ingredient are complete medium.Culture medium and FBS can provide necessary nutrients for cell
Matter.DMSO is a kind of permeability protective agent, can reduce cell freezing point, reduces the formation of ice crystal, so that reaching reduces cell damage
The protecting effect of wound.But FBS belongs to animal derived substance, and comparison of ingredients is complicated, there are potential risks in clinical application,
Increase the probability of pollution, and due to containing animal blood serum in frozen stock solution, will lead to the ingredient of frozen stock solution, there are unstable
Factor.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of serum-free cell and freezes agent, this freeze agent without containing any serum at
Point, avoid frozen stock solution can contaminated potential drawback presence, ingredient is also more stable, and has compounded trehalose simultaneously to adjust
The character of whole solution makes it be more conducive to the preservation of cell, and it is also more excellent to freeze effect, compared with tradition freezes agent, after recovery
Cell be significantly improved in survival rate, therefore should be widely promoted and applied.
The second object of the present invention is to provide the above-mentioned preparation method for freezing agent, and preparation method itself is easy to operate, preceding
Step linking is close afterwards, and operating condition is mild, and no three wastes generates, and whole operation process is environmentally protective.
The third object of the present invention is to provide the above-mentioned application method for freezing agent, and application method is simple, easy to operate, to ring
Border requirement is low, suitable for more widely being applied.
The fourth object of the present invention is to provide comprising the above-mentioned frozen stock solution for freezing agent, further comprises and is used in the frozen stock solution
The cell suspensions of suspension cell, this like cell can form uniform dispersion liquid, be more conducive to freeze going on smoothly for operation.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
Freeze agent the present invention provides a kind of serum-free cell, including DMSO with for serum composition, the generation serum composition
Including serum substitute and trehalose.
In the prior art, the main component in cells frozen storing liquid has 10% dimethyl sulfoxide (DMSO), 20%~90% tire
Cow's serum (FBS), remaining ingredient are incomplete culture medium.Culture medium and FBS can provide necessary nutriment for cell.
DMSO is a kind of permeability protective agent, can reduce cell freezing point, reduces the formation of ice crystal, so that reaching reduces cellular damage
Protecting effect.But FBS belongs to animal derived substance, and comparison of ingredients is complicated, and there are potential risks in clinical application, also increase
The probability of pollution, and due in frozen stock solution containing animal blood serum, will lead to the ingredient of frozen stock solution, there are the factors of instability.
In order to solve the above technical problems, The present invention provides a kind of cells frozen storing liquid without containing serum, which freezes
The comparison of ingredients of liquid storage is simple, in addition to conventional use of dimethyl sulfoxide (DMSO), also contains for serum composition, for serum composition
In mainly contain serum substitute and trehalose, this freezes in agent not only without containing serum composition in this way, also further addition
Trehalose, trehalose can be formed under the severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration in cell surface
Unique protective film is effectively protected protein molecule invariance inactivation, so that the life process for the body that sustains life and biology are special
Sign, and the addition of trehalose can also be played the role of adjusting solution character, improve the stability of solution, be allowed to more conducively cell
It freezes.
Preferably as further enforceable scheme, the volume ratio of the DMSO and the generation serum composition is 1:(9-
15) between, DMSO with for serum composition control in than convenient proportional region, just can guarantee freeze operation it is smooth into
Row.
Preferably, the volume ratio of the DMSO and the generation serum composition is 1:10.
Preferably as further enforceable scheme, in the generation serum composition, the concentration of trehalose is 0.1-
The concentration of 0.2mol/L, trehalose also should not be too large, if the stability of the too big possible solution of additive amount will receive destruction, also can
Influence the effect frozen.
Preferably as further enforceable scheme, the model Gibco KNOCKOUT of the serum substituteTMBlood
Clear substitute 10828-028, this serum substitute is relatively common, is easy to get.
The embodiment of the invention also provides the preparation methods that serum-free cell freezes agent, include the following steps:
Vortex in serum substitute is added in trehalose powder to dissolve to obtain for serum composition；
By DMSO be added in serum composition mix to get；
Preferably, vortex is dissolved in centrifuge tube and carries out.
It can carry out in accordance with the following steps when specific operation: take trehalose powder, be placed in 50ml centrifuge tube；Serum is added
Substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.1-0.2mol/L。
The embodiment of the invention also provides the frozen stock solutions that agent is frozen comprising above-mentioned serum-free cell；In the frozen stock solution preferably
Ground further includes the cell suspensions that serum free medium is constituted.
The embodiment of the invention also provides the application methods that above-mentioned serum-free cell freezes agent, include the following steps:
After mixing by cell and cell suspensions, the cell cryopreservation agent is added dropwise, -80 DEG C or less freeze.
The above-mentioned temperature frozen can be adjusted according to actual needs.
Preferably as further enforceable scheme, cell cryopreservation agent is first pre-chilled in mixture of ice and water before being added,
It can reinforce the effect of subsequent freeze-stored cell by the operation of pre-cooling.
Preferably, cell and cell suspensions after mixing, final concentration of (1-6) × 10 of cell6A/ml.
Preferably as further enforceable scheme, the volume ratio of cell suspensions and cell cryopreservation agent is controlled 1:
Between (3-5), it is therefore preferable to 1:4.
Preferably as further enforceable scheme, freeze by the way of Programmed cryopreservation, rate of temperature fall 0.5-
Between 2 DEG C/min, it is therefore preferable to 1 DEG C/min.
Programmed cryopreservation instrument control temperature rate is more accurate, and principle is to adjust liquid nitrogen by microcomputer program to be pumped into rate cooling；
Cooling box content is isopropanol, and the substance rate of temperature fall is about 1 DEG C/min or so after being placed in -80 DEG C of refrigerators below.In fact
It can be selected according to actual needs, if exact science is required to study, Programmed cryopreservation can be used, if it is general section
Learning research can be used Programmed cryopreservation box, either Programmed cryopreservation instrument or cooling box, all be commercially available commodity, cooling box is
Laboratory ordinary articles.
Compared with prior art, the invention has the benefit that
(1) serum-free cell of the invention freezes agent, does not contain any serum composition, avoiding frozen stock solution can be contaminated
The presence of potential drawback, ingredient is also more stable, and has compounded trehalose simultaneously to adjust the character of solution, keeps it more conducively thin
The preservation of born of the same parents, freezes that effect is also more excellent, and compared with tradition freezes agent, the cell after recovery has in survival rate significantly to be mentioned
Height, therefore should be widely promoted and applied；
(2) the above-mentioned preparation method for freezing agent of the present invention, itself is easy to operate, and step linking in front and back is close, operating condition
Mildly, no three wastes generates, and whole operation process is environmentally protective；
(3) the above-mentioned application method for freezing agent of the present invention is easy to operate, not high to environmental requirement, be suitable for more widely into
Row application.
(4) thin for being suspended the present invention also provides being further comprised in the frozen stock solution comprising the above-mentioned frozen stock solution for freezing agent
The cell suspensions of born of the same parents, this like cell can form uniform dispersion liquid, be more conducive to freeze going on smoothly for operation；
(5) cryopreservation methods of the invention, which can significantly improve, freezes rear cell recovery rate, and reduces different batches cell jelly
It is unstable and avoid by the mycoplasma in serum, virus to deposit anabiosis rate, the pollution that prion and other virions cause.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the side of freezing of the cell recovery rate and comparative example 1 after being frozen using the cryopreservation methods of the embodiment of the present invention 5
Method freeze after cell recovery rate comparison.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The DMSO's and 90% that cell cryopreservation agent is 10% by percent by volume is constituted for serum composition.
For serum composition by serum substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), trehalose structure
At.
2, preparation method:
1) it is configured for serum composition: under aseptic condition, taking trehalose powder, be placed in 50ml centrifuge tube；Serum is added to replace
For object (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.1mol/L。
2) DMSO is added to for the DMSO and percent by volume in serum composition, forming percent by volume as 10%
For 90% for serum composition.
3, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 1 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation agent is added dropwise
The volume ratio of 600 μ l, cell suspensions and cell cryopreservation agent is controlled in 1:3.
2) opening program cools down, and rate of temperature fall is 0.5 DEG C/min, is finally cooled to -80 DEG C or less and is frozen.
Embodiment 2
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The DMSO's and 95% that cell cryopreservation agent is 5% by percent by volume is constituted for serum composition.
For serum composition by serum substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), trehalose structure
At.
2, preparation method:
1) it is configured for serum composition: under aseptic condition, taking trehalose powder, be placed in 50ml centrifuge tube；Serum is added to replace
For object (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.2mol/L。
2) DMSO is added in serum composition, forming DMSO that percent by volume is 5% and percent by volume is
95% for serum composition.
3, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 6 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation agent is added dropwise
The volume ratio of 1000 μ l, cell suspensions and cell cryopreservation agent is controlled in 1:5.
2) opening program cools down, and rate of temperature fall is 2 DEG C/min, is finally cooled to -85 DEG C or less and is frozen.
Embodiment 3
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The DMSO's and 91% that cell cryopreservation agent is 9% by percent by volume is constituted for serum composition.
For serum composition by serum substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), trehalose structure
At.
2, preparation method:
1) it is configured for serum composition: under aseptic condition, taking trehalose powder, be placed in 50ml centrifuge tube；Serum is added to replace
For object (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.2mol/L。
2) DMSO is added in serum composition, forming DMSO that percent by volume is 9% and percent by volume is
91% for serum composition.
3, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 5 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation agent is added dropwise
The volume ratio of 800 μ l, cell suspensions and cell cryopreservation agent is controlled in 1:4.
2) opening program cools down, and rate of temperature fall is 1 DEG C/min, is finally cooled to -80 DEG C or less and is frozen.
Embodiment 4
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The DMSO's and 90% that cell cryopreservation agent is 10% by percent by volume is constituted for serum composition.
For serum composition by serum substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), trehalose structure
At.
2, preparation method:
1) it is configured for serum composition: under aseptic condition, taking trehalose powder, be placed in 50ml centrifuge tube；Serum is added to replace
For object (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.15mol/L。
2) DMSO is added to for the DMSO and percent by volume in serum composition, forming percent by volume as 10%
For 90% for serum composition.
3, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 5.5 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation is added dropwise
The volume ratio of 800 μ l of agent, cell suspensions and cell cryopreservation agent is controlled in 1:4.
2) opening program cools down, and rate of temperature fall is 1.5 DEG C/min, is finally cooled to -90 DEG C or less and is frozen.
Embodiment 5
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The DMSO's and 90% that cell cryopreservation agent is 10% by percent by volume is constituted for serum composition.
For serum composition by serum substitute (Gibco KNOCKOUTTMSerum substitute 10828-028), trehalose structure
At.
2, preparation method:
1) it is configured for serum composition: under aseptic condition, taking trehalose powder, be placed in 50ml centrifuge tube；Serum is added to replace
For object (Gibco KNOCKOUTTMSerum substitute 10828-028), be vortexed dissolution trehalose powder, until trehalose concentration is
0.1mol/L。
2) DMSO is added to for the DMSO and percent by volume in serum composition, forming percent by volume as 10%
For 90% for serum composition.
3, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 1 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation agent is added dropwise
The volume ratio of 800 μ l, cell suspensions and cell cryopreservation agent is controlled in 1:4.
2) opening program cools down, and rate of temperature fall is 1 DEG C/min, is finally cooled to -80 DEG C or less and is frozen.
Comparative example 1
1, main component: cell suspensions and cell cryopreservation agent.
Cell suspensions are made of serum free medium 100%.
The fetal calf serum that the DMSO and percent by volume that cell cryopreservation agent is 10% by percent by volume are 90%
(FBS)。
2, application method:
1) cell super-clean bench gnotobasis is added cell suspensions in 1.5mlEP pipe and is uniformly suspended cell, final concentration of cells
It is 5 × 106A/ml, suspension volume are 200 μ l.It is pre-chilled in cell cryopreservation agent mixture of ice and water, cell cryopreservation agent is added dropwise
The volume ratio of 800 μ l, cell suspensions and cell cryopreservation agent is controlled in 1:4.
2) opening program cools down, and rate of temperature fall is 1 DEG C/min, is finally cooled to -80 DEG C or less and is frozen.
The anabiosis rate that the anabiosis rate that comparative example 1 freezes rear cell freezes rear cell with embodiment 5 is compared, it is specific to tie
Fruit is as shown in Fig. 1, it is seen that the anabiosis rate of cell can be significantly improved using cryopreservation methods of the invention.
Other embodiments also obtain test result identical with attached drawing 1 by being compared with comparative example 1.
Cryopreservation methods of the invention, which can not only significantly improve, freezes rear cell recovery rate, while can also reduce different batches
Cryopreservation rate is unstable and avoids by the mycoplasma in serum, virus, what prion and other virions caused
Pollution.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of serum-free cell freezes agent, which is characterized in that including DMSO with for serum composition, the generation serum composition includes
Serum substitute and trehalose.
2. serum-free cell frozen stock solution according to claim 1, which is characterized in that the DMSO and the generation serum composition
Volume ratio be 1:(9-15) between；
Preferably, the volume ratio of the DMSO and the generation serum composition is 1:10.
3. serum-free cell according to claim 1 freezes agent, which is characterized in that in the generation serum composition, trehalose
Concentration be 0.1-0.2mol/L.
4. serum-free cell according to claim 1 freezes agent, which is characterized in that the model of the serum substitute
Gibco KNOCKOUTTMSerum substitute 10828-028.
5. freezing the frozen stock solution of agent comprising the described in any item serum-free cells of claim 1-4；
It preferably, further include the cell suspensions of serum free medium composition in the frozen stock solution.
6. the preparation method that the described in any item serum-free cells of claim 1-4 freeze agent, which is characterized in that including walking as follows
It is rapid:
Vortex in serum substitute is added in trehalose powder to dissolve to obtain for serum composition；
By DMSO be added in serum composition mix to get；
Preferably, vortex is dissolved in centrifuge tube and carries out.
7. the application method that the described in any item serum-free cells of claim 1-4 freeze agent, which is characterized in that including walking as follows
It is rapid:
After mixing by cell and cell suspensions, the cell cryopreservation agent is added dropwise, -80 DEG C or less freeze.
8. application method according to claim 7, which is characterized in that cell cryopreservation agent is first in mixture of ice and water before being added
Middle pre-cooling；
Preferably, cell and cell suspensions after mixing, final concentration of (1-6) × 10 of cell6A/ml.
9. application method according to claim 7, which is characterized in that the volume ratio control of cell suspensions and cell cryopreservation agent
System is in 1:(3-5) between, it is therefore preferable to 1:4.
10. application method according to claim 7, which is characterized in that freeze by the way of Programmed cryopreservation, cooling speed
Rate is between 0.5-2 DEG C/min, it is therefore preferable to 1 DEG C/min.
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| KR20120055934A (en) * | 2010-11-24 | 2012-06-01 | 중앙대학교 산학협력단 | Method of cryopreservation of spermatogonial stem cells using trehalose |
| CN102550542A (en) * | 2011-08-09 | 2012-07-11 | 臻景生物技术（上海）有限公司 | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells |
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Application publication date: 20190226 |