CN108029679A - A kind of frozen stock solution for being used to freeze mononuclearcell - Google Patents
A kind of frozen stock solution for being used to freeze mononuclearcell Download PDFInfo
- Publication number
- CN108029679A CN108029679A CN201810082345.4A CN201810082345A CN108029679A CN 108029679 A CN108029679 A CN 108029679A CN 201810082345 A CN201810082345 A CN 201810082345A CN 108029679 A CN108029679 A CN 108029679A
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- mononuclearcell
- dextran
- stock solution
- frozen stock
- lysate
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- 210000005087 mononuclear cell Anatomy 0.000 title claims abstract description 50
- 239000011550 stock solution Substances 0.000 title claims abstract description 40
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 87
- 210000004027 cell Anatomy 0.000 claims abstract description 45
- 229920002307 Dextran Polymers 0.000 claims abstract description 33
- 239000006166 lysate Substances 0.000 claims abstract description 21
- 210000002966 serum Anatomy 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 10
- 238000005138 cryopreservation Methods 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000001681 protective effect Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 230000009467 reduction Effects 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 150000008065 acid anhydrides Chemical class 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 claims 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 2
- 239000008121 dextrose Substances 0.000 claims 2
- -1 dextrose Acid anhydride Chemical class 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 19
- 230000004069 differentiation Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 238000007710 freezing Methods 0.000 description 8
- 230000008014 freezing Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004899 motility Effects 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 150000003625 trehaloses Chemical class 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 244000037640 animal pathogen Species 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000002790 cross-validation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000001113 umbilicus Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of frozen stock solution for being used to freeze mononuclearcell, it is made of hyclone, dimethyl sulfoxide (DMSO) and dextran lysate, wherein, hyclone accounts for 45~95%, and dextran lysate accounts for 10~50%, and dimethyl sulfoxide (DMSO) accounts for 5~10%.The dextran, refers to low molecular weight dextran, its molecular weight is 40,000.The present invention substitutes Some Animals serum using the dextran of low molecular weight, can effectively reduce the introduction volume of foreign protein.The frozen stock solution of the present invention can be effectively kept cytoactive when directly being frozen after mononuclearcell extracts.After mononuclearcell freezes processing using the frozen stock solution of the present invention, if the recovery culture of compartment time, cell recovery rate can be maintained at high levels, and the differentiation capability of cell is still very strong.
Description
Technical field
The present invention relates to a kind of frozen stock solution for being used to freeze mononuclearcell, belong to biotechnology cryopreservation field.
Background technology
Whether health has close pass for immunocompetence possessed by mononuclearcell and personal age and body in human body
System.Mononuclearcell how could more permanent preservation be a problem, if cell cryopreservation is got up, if can be thin
It is also a challenge that it can also be allowed to keep original activity after born of the same parents' recovery.
Mononuclearcell deep-bed drying can keep its vigor and function, no matter to the Fiber differentiation of later stage cell or freeze
All it is of great significance to directly treating for patient after depositing cell recovery.Freezen protective mononuclearcell can be not only solved to suffering from
Person repeatedly blood sampling and the problem of repeatedly inducing cell during culture, can also be most strong single of fighting capacity under health state
Nucleus preserves, and oncotherapy is used for after recovery culture or anti-ageing healthcare is treated.
Influence mononuclearcell freeze, recover after the principal element of cytoactive freeze including mononuclearcell, recover
The factor of the formula of mode and mononuclearcell frozen stock solution, wherein frozen stock solution is in the highest flight.If single core is preserved for a long time
Cell, then in order to avoid cell is damaged, have to mononuclearcell to deposit in liquid nitrogen environment.Mononuclearcell passes through
Cross its cellular morphology after freezing and recovering, the quality of phenotype is mainly dependent on whether using a kind of effective reagent and appropriate of freezing
Cooling, rewarming program.Dimethyl sulfoxide (DMSO) is that the current country is frozen in formula using most optimal cell-protectings, carefully
Born of the same parents' cryoprotector can protect cell membrane and organelle from damage to avoid the formation of intracellular ice crystal during freezing.Tire ox blood
Clear is also the regular guest in cell cryopreservation formula, because it is a kind of very complicated mixing by being formed after plasma removing fibrin
Thing, wherein containing various plasma proteins, polypeptide, fat, carbohydrate, growth factor, hormone, inorganic matter etc., these materials
All be conducive to protect cell.The shortcomings that hyclone has advantage also to have shortcoming, it is maximum is just the introduction of animal protein, adds
By the possibility of animal pathogen contamination, certain influence can be produced to human body adoptive immunity cell therapy.
The content of the invention
For the above-mentioned prior art, the present invention provides a kind of frozen stock solution for being used to freeze mononuclearcell.The present invention has
Directly freezing after solving the problems, such as peripheral blood mononuclear cells extraction is imitated, mononuclearcell can be effectively protected from freezing
Damage, keeps physiological function and biological characteristics after mononuclearcell recovery, it is ensured that mononuclearcell has cell increasing after freezing
Grow and differentiation capability.
The present invention is achieved by the following technical solutions：
A kind of frozen stock solution for being used to freeze mononuclearcell, by hyclone, dimethyl sulfoxide (DMSO) and dextran lysate
Composition, wherein, hyclone accounts for 45~95%, and dextran lysate accounts for 10~50%, and dimethyl sulfoxide (DMSO) accounts for 5~10%, presses
Volume percentage；
The dextran lysate refers to the PBS solution of dextran, and the concentration of dextran is 0.1~2g/ml.
Preferably, hyclone accounts for 70%, and dextran lysate accounts for 20%, and dimethyl sulfoxide (DMSO) accounts for 10%.
The dextran, refers to low molecular weight dextran, its molecular weight is 40,000 or so.
The present invention replaces part hyclone using dextran lysate, reduces the introduction volume of foreign protein, reduces
The possibility of animal pathogenic pollution；Dextran is introduced, can be good at the function instead of blood plasma, and hinders the poly- of red blood cell
Collection.
The preparation method of the frozen stock solution for freezing mononuclearcell is：Dextran is dissolved with 1 × PBS buffer
(solid granular), makes its final concentration of 0.1~2g/ml (preferably 0.1g/ml), obtains dextran lysate；Then, by tire ox
Serum, dimethyl sulfoxide (DMSO) mix mixing with dextran lysate, to obtain the final product.
Application of the frozen stock solution for being used to freeze mononuclearcell in mononuclearcell is frozen.
A kind of cryopreservation methods of mononuclearcell：Freshly extd mononuclearcell is subjected to weight using above-mentioned frozen stock solution
It is outstanding；Cell after resuspension uses program temperature reduction box (NALGENE program temperature reduction boxes, article No.：5100-0001) be placed in -80 DEG C it is ultralow
In temperature refrigerator 24 it is small when, be then transferred to freezen protective in liquid nitrogen.
Further, the mononuclearcell is cord blood mononuclear cells, is that the single core of peripheral blood is thin further
Born of the same parents.
The frozen stock solution of the present invention can be effectively kept cytoactive when directly being frozen after mononuclearcell extracts.It is single
After nucleus freezes processing using the frozen stock solution of the present invention, if the recovery culture of compartment time, cell recovery rate can be maintained at
High levels, and the differentiation capability of cell is still very strong.
Brief description of the drawings
Fig. 1：Next day motility rate comparison diagram after the recovery of mononuclearcell frozen stock solution 1-12 freeze-stored cells.
Fig. 2：14 days amplification times comparison diagrams after the recovery of mononuclearcell frozen stock solution 1-12 freeze-stored cells.
Embodiment
With reference to embodiment, the present invention is further illustrated.
Involved instrument, reagent, material etc. in following embodiments, are existing in the prior art unless otherwise noted
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection
Survey method etc., is existing normal experiment method, detection method etc. in the prior art unless otherwise noted.
Instant cells frozen storing liquid is sold in market, is exclusively used in the long-time Cord blood of cell under serum-free culturing conditions.
In order to make cell recovery rate higher, frozen stock solution series suggests using exponential phase of growth cell.The experiment purpose of the present invention is to make
Cell mortality is lower after freshly extd mononuclearcell cryopreservation resuscitation, and culture being capable of normal growth and cell table after recovery
Type matches with expection.So the present invention determines to do using several instant cells frozen storing liquids in test to compare.
In addition, the common mononuclearcell of the present inventor freezes formula of liquid i.e. by volume 90%FBS+10%
DMSO, the correlation which is suitable for after mononuclearcell extraction in vitro after the completion of amplification cultivation freeze.The formula is for list
Directly freezing if appropriate for not yet obtaining experimental verification after the extraction of a nucleus.
Inspection information also found that trehalose has various bioactivators non-specific protective effect, it can be in low temperature
The protective film of uniqueness is formed under environment in cell surface, is effectively protected protein molecule consistency inactivation.So the present invention
Trehalose is also added thereto in contrast screening experiment.
First, a collection of instant cells frozen storing liquid is chosen in market, it is numbered is：No. 1 superior serum-free cell freezes
Liquid (article No.：DWK3CFM0100), No. 2 the same as Li Hai sources serum-free cell frozen stock solution (article No.：TL-501), No. 3 friendly health (article No.s：
NC1001), No. 4 LiveCyte (article No.s：LC-1601), No. 5 ScienceCell CFM (article No.s：0133), No. 6 classical formulas
90%FBS+10%DMSO, No. 7 II (article No.s of Cryopreservation Medium KM Banker：88-702-CB).
Secondly, after reading up the literature, inventor, which is configured with No. 8-12 5 kinds and may be more suitable for freshly extd mononuclearcell, to be frozen
Formula.Wherein, No. 8 formulas are 95%FBS+5%DMSO by volume, and No. 9 formulas are 40%FBS+10% by volume
DMSO+50% culture mediums, No. 10 formulas are by volume 70%FBS+20% dextran lysates+10%DMSO, and No. 11 are matched somebody with somebody
Side is 70%FBS+20% trehaloses+10%DMSO by volume, No. 12 formulas for 75%FBS+20% trehaloses by volume+
5%DMSO.Pass through the indices (increasing of cell recovery motility rate, cell of cell growth after mononuclearcell cryopreservation resuscitation culture
Value multiple etc.) satisfactory frozen stock solution agent prescription is filtered out using the methods of contrast, rejecting to them.Pass through experimental result
Found after (such as Fig. 1,2) comparative analysis, of the invention No. 10 mononuclearcell frozen stock solutions are (right for 70%FBS+20% by volume
The sugared acid anhydride lysate+10%DMSO of rotation) according to there is very big superiority.After the use of this formula, the freeze-stored cell motility rate of recovery is higher, lures
The cell phenotype led after culture is relatively best, and cell growth rate is fast.
Cell culture medium used in the present invention is Corning 88-581-CM lymphocyte serums.
Embodiment 1：Prepare dextran lysate reagent
The dextran 2g of 40,000 molecular weight is weighed with assay balance, is put in 50ml centrifuge tubes, 20ml is drawn with pipettor
1 × PBS is instilled in above-mentioned centrifuge tube, is put in after mixing spare in 4 DEG C of refrigerators.
Embodiment 2：Prepare mononuclearcell frozen stock solution 6 (hyclone, dimethyl sulfoxide (DMSO))
Take 90% hyclone that 10% dimethyl sulfoxide (DMSO) is then slowly added dropwise by percent by volume, finally shake up prepare it is single
Nucleus frozen stock solution.Saved backup in 4 DEG C.
Embodiment 3：Prepare mononuclearcell frozen stock solution 9 (hyclone, dimethyl sulfoxide (DMSO), cell culture medium)
Take 40% hyclone that 10% dimethyl sulfoxide (DMSO) is then slowly added dropwise by percent by volume, be eventually adding 50% cell
Culture medium is prepared into mononuclearcell frozen stock solution after shaking up.Saved backup in 4 DEG C.
Embodiment 4：Prepare mononuclearcell frozen stock solution 10 (hyclone, dimethyl sulfoxide (DMSO), dextran)
70% hyclone, 20% dextran lysate (are configured into standby according to 1 method of embodiment by percent by volume
With) mix, 10% dimethyl sulfoxide (DMSO) is then slowly added dropwise, finally shakes up and prepares mononuclearcell frozen stock solution.It is standby in 4 DEG C of preservations
With.
Embodiment 5
It is right after fresh single core that a bleeding of the umbilicus is extracted using the fresh single core extraction SOP flows of company where applicant
Cell count, the cell of equivalent is taken out according to culture demand 2*10^7/ml, and the direct fresh cultured of a copy of it cell is received for 14 days
Collect data, other cell using mononuclearcell frozen stock solution not of the same race freeze when being placed in preserving one section under liquid nitrogen environment
Between after recovery culture 14 days.All frozen stock solution formulation operations and cell cryopreservation operation are both needed to ensure to carry out under 0 degree of environment.It is logical
The amplification times for crossing repeatedly cell to next day Cell viability after recovery i.e. cell recovery rate and after 14 days carry out cross validation, select
No. 10 frozen stock solutions of the present invention.
The data entirely tested are arranged after screening experiment, in order to more intuitively show that the present invention is i.e. No. 10 thin
The advantages of born of the same parents' frozen stock solution (+10% dimethyl sulfoxide (DMSO) of+70% hyclone of 20% dextran lysate), done following two figures：
Fig. 1, Fig. 2.
It can be seen from the above results using cells frozen storing liquid No. 1-12 carry out freeze-stored cell respectively, recover after many experiments
The Cell viability of next day.No. 3 acellular survivals of frozen stock solution cell, the cell of No. 1, No. 2, No. 4, No. 5 frozen stock solution cryopreservation resuscitation are answered
The average value for the rate that revives is less than other groups, and Fig. 1 shows the cell recovery motility rate average value highest of No. 10 frozen stock solutions.Fig. 2 recovery trainings
Support 14 days amplification times average value of cell and see that the amplification times of No. 10 cells are higher than other groups.
* * * *
Above-described embodiment is provided to those skilled in the art, how to be implemented with full disclosure and description and uses what is advocated
Embodiment, rather than for limiting scope disclosed herein.Obvious modification will to those skilled in the art
Within the scope of the appended claims.The all publications, patents and patent applications of this specification citation are incorporated by reference into this
Text, as these publications, patents and patent applications each especially and individually show to be incorporated herein by reference.
Claims (6)
- A kind of 1. frozen stock solution for being used to freeze mononuclearcell, it is characterised in that：By hyclone, dimethyl sulfoxide (DMSO) and dextrose Acid anhydride lysate forms, wherein, hyclone accounts for 45~95%, and dextran lysate accounts for 10~50%, and dimethyl sulfoxide (DMSO) accounts for 5~ 10%, by volume percentage；The dextran lysate refers to the PBS solution of dextran, and the concentration of dextran is 0.1~2g/ml.
- 2. the frozen stock solution according to claim 1 for being used to freeze mononuclearcell, it is characterised in that：By hyclone, two Methyl sulfoxide and dextran lysate composition, wherein, hyclone accounts for 70%, and dextran lysate accounts for 20%, dimethyl Sulfoxide accounts for 10%.
- 3. the frozen stock solution according to claim 1 or 2 for being used to freeze mononuclearcell, it is characterised in that：The dextrose The molecular weight of acid anhydride is 40,000.
- 4. the preparation method for being used to freeze the frozen stock solution of mononuclearcell described in claim 1 or 2 or 3, it is characterised in that：With 1 × PBS buffer dissolves dextran, makes its final concentration of 0.1~2g/ml, obtains dextran lysate；Then, by tire ox Serum, dimethyl sulfoxide (DMSO) mix mixing with dextran lysate, to obtain the final product.
- 5. the frozen stock solution for being used to freeze mononuclearcell the answering in mononuclearcell is frozen described in claim 1 or 2 or 3 With.
- A kind of 6. cryopreservation methods of mononuclearcell, it is characterised in that：By freshly extd mononuclearcell usage right requirement 1 Or the frozen stock solution described in 2 or 3 is resuspended；Cell after resuspension is placed in 24 in -80 DEG C of ultra low temperature freezers using program temperature reduction box Hour, it is then transferred to freezen protective in liquid nitrogen.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810082345.4A CN108029679B (en) | 2018-01-29 | 2018-01-29 | Freezing medium for freezing mononuclear cells |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810082345.4A CN108029679B (en) | 2018-01-29 | 2018-01-29 | Freezing medium for freezing mononuclear cells |
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| CN108029679B CN108029679B (en) | 2020-09-22 |
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| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
| CN110140716A (en) * | 2019-07-08 | 2019-08-20 | 方艳秋 | A kind of peripheral blood mononuclear cells improvement frozen stock solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108849854A (en) * | 2018-07-13 | 2018-11-23 | 深圳市润科生物科技有限公司 | A kind of peripheral blood mononuclear cells cryopreservation methods |
| CN108849854B (en) * | 2018-07-13 | 2021-02-09 | 深圳市润科生物科技有限公司 | Peripheral blood mononuclear cell cryopreservation method |
| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
| CN110140716A (en) * | 2019-07-08 | 2019-08-20 | 方艳秋 | A kind of peripheral blood mononuclear cells improvement frozen stock solution |
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