CN108450458A - A kind of serum-free cell frozen stock solution - Google Patents
A kind of serum-free cell frozen stock solution Download PDFInfo
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- CN108450458A CN108450458A CN201810257683.7A CN201810257683A CN108450458A CN 108450458 A CN108450458 A CN 108450458A CN 201810257683 A CN201810257683 A CN 201810257683A CN 108450458 A CN108450458 A CN 108450458A
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- serum
- dmso
- stock solution
- frozen stock
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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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- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of serum-free cell frozen stock solutions, belong to field of cell culture.Serum-free frozen stock solution group of the present invention becomes：CD culture mediums (chemical composition defined medium, Chemically Defined Media) 90 99%, cell culture grade dimethyl sulfoxide (DMSO) (DMSO) 1 10%.This frozen stock solution does not contain animal sources serum, foreign protein will not be introduced, reduce the possibility of animal pathogeny pollution, activity can reach 80% 90% after freeze-stored cell recovery, cell phenotype does not change substantially, to maintain the physiological function after cell recovery and biological function, it can be used for continuing culture or clinical direct feedback.This serum-free frozen stock solution is safe and effective, and preparation method is simple, and cost is controllable, has good potential applicability in clinical practice.
Description
Technical field
The present invention relates to technical field of cell culture, and in particular, to a kind of serum-free cell frozen stock solution.
Background technology
Cell cryopreservation and recovery are the important technologies in cell culture technology, and cell cryopreservation is the important of cell long-period preservation
Means.Cells frozen storing liquid, a kind of solution that must be used when as cell cryopreservation, effect are that the cell that will need to freeze suspends
The nutriment necessary to frozen stock solution, supply cell life metabolism, while freezing ice crystal can be prevented or reduced to cell
Damaging action.In the prior art, the side for generally using culture medium, serum and dimethyl sulfoxide (DMSO) to mix according to a certain percentage
Method carrys out freeze-stored cell, and DMSO dosages are 10%, and too low DMSO concentration can cause to freeze less effective, but the DMSO of high concentration has
Larger toxicity can damage cell body；And the serum used in traditional frozen stock solution formula is of high cost, increases animal pathogenic
The possibility of pollution.It can be to the fetal calf serum in solution medium in addition, some researches show that the cells contacted for a long time with fetal calf serum
Endocytosis occurs, the mescenchymal stem cell after endocytosis fetal calf serum is likely to occur the variation of certain protein expressions, is applied to human body
After immune response caused by heterogenous animal albumen can occur, cannot be directly used to clinical infusion.Therefore, the jelly containing serum is utilized
The cell that liquid storage freezes can bring certain risk to Clinical practice.
And serum used in presently commercially available serum-free frozen stock solution and DMSO substitutes include glycerine, chitosan and seaweed
Acid, institute include adhesion factor, binding protein, growth factor, trace quantity mineral substance element, hormone, dimension life using serum free medium
Element, additive complicated component prepare trouble；The used culture medium of cell non-serum frozen stock solution provided by the present invention is cultivated for CD
Base, and DMSO contents in frozen stock solution are greatly reduced on this basis, make the reduction of its toxicity, and without using complicated component
DMSO subs.
Invention content
It is an object of the invention to：In view of the above problems, a kind of serum-free cell frozen stock solution, the present invention gram are provided
The defect that traditional serum frozen stock solution contains animal sources serum or high concentration DMSO, economical and effective have been taken, and external source dirt will not be introduced
Dye.The mescenchymal stem cell and other kinds tumour cell frozen with the frozen stock solution of the present invention, cellular morphology is intact, has
Very strong survival rate and adherent ability.
The present invention realizes as follows by the following technical programs：
A kind of serum-free cell frozen stock solution, including following components：CD culture mediums and dimethyl sulfoxide (DMSO) (DMSO), the CD trainings
It includes amino acid, vitamin, inorganic salts and water to support base；The vitamin include in vitamin B, vitamin C and vitamin D extremely
Few one kind；The specific preparation method of the CD culture mediums is：It weighs and does not contain any serum, the chemically defined training of animal derived protein
It supports in based powders to sterile glass container, high pressure sterilization RO water is added, is sufficiently stirred, utilize after complete solubilization sterile
It is 7.0-7.5 that the NaOH of 0.5-2.0mol/L, which adjusts PH, and CD culture mediums are obtained after 0.22 μm of filter filters.
Further, the mass volume ratio of the CD culture mediums is 90-99%, the volume of the dimethyl sulfoxide (DMSO) (DMSO)
Percentage 1-10% is prepared by following volumes deal：
100 parts of CD culture mediums；
1-10 parts of dimethyl sulfoxide (DMSO).
Specifically, 100 parts of the CD culture mediums of 4 DEG C of preservations are taken in Biohazard Safety Equipment gnotobasis, are heated up under water bath
To room temperature；1-10 parts of dimethyl sulfoxide (DMSO) is taken in Biohazard Safety Equipment gnotobasis；CD is mixed in room temperature, safety cabinet gnotobasis
Culture medium and dimethyl sulfoxide (DMSO) are configured to the frozen stock solution of final concentration of culture medium containing 90-99%CD and 1-10% dimethyl sulfoxide (DMSO)s.
Further, the amino acid includes lysine, tryptophan, phenylalanine, methionine, threonine, different bright ammonia
Acid, leucine, valine, alanine, arginine, asparatate, cystine, proline, tyrosine, glycine, dried meat ammonia
At least one of acid, serine, cysteine, asparagine, glutamine, aspartic acid and glutamic acid.
Further, the inorganic salts include calcium, phosphorus, potassium, sodium, chlorine, magnesium, the phosphate of sulphur, nitrate, sulfate, carbon
At least one of hydrochlorate and hydrochloride.
Further, the dimethyl sulfoxide (DMSO) (DMSO) is clinical grade cell cryopreservation permeability protective agent.
Specifically, the mixed method of cell and above-mentioned frozen stock solution is：5 × 1061.5 parts of serum-frees are added in a cell to freeze
Then liquid storage carries out cell resuspension, cell suspension is added in cryopreservation tube, cryopreservation tube is placed in the program for being in normal temperature state
In cooling box, then program temperature reduction box is positioned over -80 DEG C of ultra low temperature freezers and is stayed overnight, is transferred to liquid nitrogen container within second day and freeze in frame and protect
It deposits, it is spare.
In conclusion by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
A. survival rate is high, dirty after the present invention provides a kind of preparation methods simple, of low cost, hypotoxicity, cell recovery
Contaminate the low serum-free frozen stock solution of risk.
B. the frozen stock solution needs now with the current, without being further cultured for after outwelling frozen stock solution after cell recovery, can directly be added into
Into culture solution, until pass on or use after covering with, it is convenient and efficient；
C. the cells frozen storing liquid will not introduce foreign protein, reduce the possibility of animal pathogenic pollution, frozen using this
Cell of the liquid after cryopreservation resuscitation, survival rate up to 80-90%, adherent growth is good, purity is high, differentiation capability is strong, can be used for continuing
Culture or clinical direct feedback safely and effectively clinically have a good application prospect.
Description of the drawings
Examples of the present invention will be described by way of reference to the accompanying drawings, wherein：
Fig. 1 is that mouse compact bone mescenchymal stem cell and mouse mastopathy cell freeze 1,3 month respectively in frozen stock solution,
24 hours growing states after cell recovery under microscope.
Fig. 2 is that mouse compact bone mescenchymal stem cell and mouse mastopathy cell freeze 1,3 respectively in frozen stock solution
Month, utilize 24 hours after cell recovery under the microscope of violet staining cell colony generative capacities.
Specific implementation mode
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive
Feature and/or step other than, can combine in any way.
Any feature disclosed in this specification (including any accessory claim, abstract), unless specifically stated,
It is replaced by other equivalent or with similar purpose alternative features.That is, unless specifically stated, each feature is a series of
An example in equivalent or similar characteristics.
Embodiment 1
Mouse compact bone mescenchymal stem cell (CBMSCs) freezes：
It is separately cultured mouse compact bone mescenchymal stem cell (CBMSCs) using tissue block adherent method, by mouse femur and the upper arm
It is affixed on after bone shears is broken in 10cm culture dishes, DME/F12 culture solutions is added and are cultivated, wait for that one timing of cell density disappears to it
Change, collect cell for freezing.
By volume, by the DMSO mixings of the CD culture mediums of 90-99% and 1-10%, cells frozen storing liquid is made, now matches existing
With.
By the mescenchymal stem cell of above-mentioned amplification with 5 × 106It is a to be resuspended in 1.5 parts of serum-free frozen stock solutions, it then will be thin
Born of the same parents' suspension is added in cryopreservation tube, and cryopreservation tube is placed in the program temperature reduction box in normal temperature state, then program temperature reduction box is placed
It is stayed overnight in -80 DEG C of ultra low temperature freezers, is transferred to liquid nitrogen container within second day and freezes in frame and preserve.
Behind 1 month, 3 months after freezing, 37 DEG C of water-bath recoveries calculate cell survival rate by Trypan Blue, and
By cell culture in 6 orifice plates, the adherent ability and growth ability of cell are detected.Specific testing result is in table 1, Fig. 1 and Fig. 2
It shows.
Embodiment 2
Mouse breast cancer (4T1) cell cryopreservation：
The 4T1 cells for taking the exponential phase that 1 ware is within 40 generations abandon supernatant, and sulfate buffer cleaning is added
Once, pancreatin is added to be digested, adds the culture medium containing serum and carry out termination digestion, postdigestive cell suspension will be terminated
It is added in centrifuge tube, supernatant is abandoned after centrifugation.
By volume, by the DMSO mixings of the CD culture mediums of 90-99% and 1-10%, cells frozen storing liquid is made, now matches existing
With.
Take 5 × 106A cell is added to progress cell resuspension in 1.5 parts of serum-free frozen stock solutions, then adds cell suspension
Enter into cryopreservation tube, cryopreservation tube is placed in the program temperature reduction box in normal temperature state, then program temperature reduction box is positioned over -80 DEG C
Ultra low temperature freezer is stayed overnight, and is transferred to liquid nitrogen container within second day and is frozen in frame and preserves.
After freezing 1 month, 3 months, water-bath recovery is carried out with 37 DEG C of water, cell survival is calculated by Trypan Blue
Rate, and by cell culture in 6 orifice plates, detect the adherent ability and growth ability of cell.Specific testing result table 1, Fig. 1 and
It is shown in Fig. 2.
Table 1
The invention is not limited in specific implementation modes above-mentioned.The present invention, which expands to, any in the present specification to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.It is other
Aspect and improvement in the scope of the invention will be it will be apparent that according to the present invention to those skilled in the art in the invention
The change that information is made, it is feasible to substitute etc., therefore it is also within the scope of the present invention.
Claims (5)
1. a kind of serum-free cell frozen stock solution, which is characterized in that including following components：CD culture mediums and dimethyl sulfoxide (DMSO)
(DMSO), the CD culture mediums include to include amino acid, vitamin, inorganic salts and water；The vitamin includes vitamin B, dimension life
At least one of plain C and vitamin D；The specific preparation method of the CD culture mediums is：It weighs and does not contain any serum, animal
In the chemically defined culture medium powder to sterile glass container of source protein, high pressure sterilization RO water is added, is sufficiently stirred, waits for that powder is complete
It is 7.0-7.5 to adjust PH using the NaOH of sterile 0.5-2.0mol/L after fully dissolved, is made after 0.22 μm of filter filters
Obtain CD culture mediums.
2. a kind of serum-free cell frozen stock solution as described in claim 1, which is characterized in that the quality volume of the CD culture mediums
Than being prepared by following volumes deal for 90-99%, the percentage by volume 1-10% of the dimethyl sulfoxide (DMSO) (DMSO)：
100 parts of CD culture mediums；
1-10 parts of dimethyl sulfoxide (DMSO).
3. a kind of serum-free cell frozen stock solution as described in claim 1, which is characterized in that the amino acid include lysine,
Tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, alanine, arginine, asparagine
Acid, cystine, proline, tyrosine, glycine, proline, serine, cysteine, asparagine, glutamine, asparagus fern
At least one of propylhomoserin and glutamic acid.
4. a kind of serum-free cell frozen stock solution as described in claim 1, which is characterized in that the inorganic salts include calcium, phosphorus,
At least one of potassium, sodium, chlorine, magnesium, the phosphate of sulphur, nitrate, sulfate, carbonate and hydrochloride.
5. a kind of serum-free cell frozen stock solution as described in claim 1, which is characterized in that the dimethyl sulfoxide (DMSO) (DMSO)
For clinical grade cell cryopreservation permeability protective agent.
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| CN201810257683.7A CN108450458A (en) | 2018-03-27 | 2018-03-27 | A kind of serum-free cell frozen stock solution |
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| CN201810257683.7A CN108450458A (en) | 2018-03-27 | 2018-03-27 | A kind of serum-free cell frozen stock solution |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109362712A (en) * | 2018-11-29 | 2019-02-22 | 广州润虹医药科技股份有限公司 | A kind of adipose tissue frozen stock solution, cryopreservation methods and its application |
| CN110250162A (en) * | 2019-07-05 | 2019-09-20 | 北京太东生物科技有限公司 | Frozen Biological protective agent and its application |
| CN111296412A (en) * | 2020-04-17 | 2020-06-19 | 赛尔瑞成(北京)生命科学技术有限公司 | Serum-free low-DMSO (dimethyl sulfoxide) cell cryopreservation solution and application thereof |
| CN111838139A (en) * | 2020-09-08 | 2020-10-30 | 依科赛生物科技（太仓）有限公司 | Protein-free cell cryopreservation liquid and application thereof |
| CN115281180A (en) * | 2022-07-04 | 2022-11-04 | 四川大学华西医院 | mRNA-platelet compound frozen stock solution and preparation method thereof |
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| CN109362712A (en) * | 2018-11-29 | 2019-02-22 | 广州润虹医药科技股份有限公司 | A kind of adipose tissue frozen stock solution, cryopreservation methods and its application |
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| CN111296412B (en) * | 2020-04-17 | 2022-10-04 | 赛尔瑞成(北京)生命科学技术有限公司 | Serum-free low-DMSO (dimethyl sulfoxide) cell cryopreservation solution and application thereof |
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| CN115281180A (en) * | 2022-07-04 | 2022-11-04 | 四川大学华西医院 | mRNA-platelet compound frozen stock solution and preparation method thereof |
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Application publication date: 20180828 |