CN107006453A - A kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method - Google Patents
A kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method Download PDFInfo
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- CN107006453A CN107006453A CN201710423232.1A CN201710423232A CN107006453A CN 107006453 A CN107006453 A CN 107006453A CN 201710423232 A CN201710423232 A CN 201710423232A CN 107006453 A CN107006453 A CN 107006453A
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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 invention discloses a kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method.The frozen stock solution is to contain dimethyl sulfoxide (DMSO), basic fibroblast growth factor, EGF, Ultroser in DMEM/F 12TMG serum substitutes, L glutamine, Dextran 40.Not only cost is low for the frozen stock solution, and solves the problem of anabiosis rate is low after mesenchymal stem cell cryopreserving, improves the cryopreservation resuscitation rate of mescenchymal stem cell.
Description
Technical field
The invention belongs to cell biology, field of biomedicine technology, and in particular to a kind of umbilical cord mesenchymal stem cells freeze
Liquid storage and its application method.
Background technology
Umbilical cord is the cord structures being connected between embryo's umbilical region and placenta, be cladded with amnion, include vein, arteria umbilicalis and is come from
The glue sample mucus connective tissue of germinal layer is fertile Dun Shi glue.Numerous studies confirm that umbilical cord is irrigated in Dun Shi glue rich in abundant mesenchyma
Stem cell.
Umbilical cord mesenchymal stem cells (MSCs) have higher differentiation potential, can be broken up to multiple directions.It bone,
There is wide potential applicability in clinical practice in terms of the organizational projects such as cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle；Tool
There is stronger immunoregulation effect, available for the treatment autoimmune disease such as lupus erythematosus and chorionitis, reduction cell or device
Immunological rejection after official's transplanting, improves the success rate of cell or organ transplant；Radiation in jury function can be promoted, made with single
Hemocytoblast transplanting is compared, and mescenchymal stem cell and candidate stem cell co-transplantation can significantly improve leukaemia and refractory anemia etc.
The therapeutic effect of disease；The histoorgan of damage or lesion can be repaired, for treating bone and muscle degenerative disease, cardiovascular and cerebrovascular
Disease, hepatopathy, brain and neurologic defict and senile dementia etc..
Cell cryopreservation is in direct freeze-stored cell under being not added with protective condition, and the water in intracellular and external environment can form ice
Crystalline substance, can cause the intracellular a series of change of generation, and such as rise of mechanical damage, electrolyte concentration, osmotic pressure change, be dehydrated, pH
It is worth change, protein denaturation etc., cell death can be caused.But protective agent (glycerine or DMSO) is added such as into culture medium, can be made
Freezing point is reduced, and under slow conditions of cryopreservation, ICW can be made to be appeared before freezing extracellular.It is stored in below -130 degree
Can add water the formation of ice crystal in low temperature.When dissolving cell, speed is fast, is allowed to run through cell most easily damaged -5~0 and takes the photograph
After family name's degree, cell remains to growth, and vigor is not by any infringement.Currently used protective agent is DMSO and glycerine, and they are to cell
Nontoxicity, molecular weight is small, and solubility is big, easy penetration cell, and concentration scope commonly uses 10% between 5~10%.
For protection cell maximum keep alive rate, generally with cell culture medium as solvent, in addition to dimethyl sulfoxide (DMSO), also add each
Cytoprotection and nutriment are planted, to improve Cell viability；To obtain the umbilical cord mesenchymal stem cells of convenient sources, cell is improved
Motility rate, carries out effective stem cell transplantation and sets up stem cell bank, improve stem cell cryopreserving resuscitation technique, a kind of long-term to set up
The research and application of the method for Cord blood stem cell are significant.
The content of the invention
It is an object of the invention to provide a kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method, carried using the present invention
Cell recovery motility rate after the cells frozen storing liquid of confession freezes for a long time is higher.
A kind of umbilical cord mesenchymal stem cells frozen stock solution, is contained in DMEM/F-12 culture mediums
Described umbilical cord mesenchymal stem cells frozen stock solution, preferably contains in DMEM/F-12 culture mediums
The application method of described umbilical cord mesenchymal stem cells frozen stock solution, is to take the culture well-grown cells of 12-15d
Bottle, nutrient solution is poured into centrifuge tube, as terminate liquid, then cell is suspended with pancreatin digestion 5min, is added terminate liquid
Digestion is terminated, supernatant discarding after centrifugation is added after brine, centrifuged again, obtain described umbilical cord mesenchyma and do
Cells frozen storing liquid freezes the cell of acquisition, and the concentration as needed for freeze-stored cell number is 2 × 107Individual cell/ml frozen stock solutions, is quantitatively adding
The described NSC frozen stock solution of precooling, after fully mixing, this cell suspension is transferred in cryopreservation tube, often pipe 1ml, is sealed
Mouthful, mark freezes the date, is put in program temperature reduction box；It is classified frozen cell：4 DEG C, 40min；- 20 DEG C, 30min；- 80 DEG C, mistake
Night；Next day, slow liquid nitrogen surface of being down to was up to input Liquid nitrogen storage.
The application method of above-mentioned umbilical cord mesenchymal stem cells frozen stock solution, is that the pancreas enzyme concentration used contains for 0.125%
EDTA 37 DEG C of digestion 5min of pancreatin.
The present invention uses UltroserTMG serum substitutes replace hyclone as the source of nutrition of cell,
UltroserTMG includes nutriment necessary to the growth of 6 class eukaryotic cells, including adhesion factor, associated proteins, growth
The factor, trace quantity mineral substance element, hormone, vitamin etc.；Compared with hyclone, its bioactivity is 5 times of hyclone,
Nutritional ingredient more horn of plenty, improves mesenchymal stem cell cryopreserving anabiosis rate, and concentration is low, has saved cost；And dextran-
40 can be very good to maintain osmotic pressure, during temperature drop, remain to maintain good osmotic pressure environment, it is to avoid cell because
Temperature drop, osmotic pressure changes and the phenomenon of cell death occurs, so as to improve mesenchymal stem cell cryopreserving anabiosis rate.
Brief description of the drawings
Fig. 1 is the growth conditions of cell after the cell recovery frozen using different umbilical cord mesenchymal stem cells frozen stock solutions.
Fig. 2 is the FCM analysis knot after the cell recovery frozen using umbilical cord mesenchymal stem cells frozen stock solution of the present invention
Really.
Embodiment
The present invention is intended to further illustrate below in conjunction with embodiment, is not intended to limit the present invention.
1) preparation of umbilical cord mesenchymal stem cells frozen stock solution：
Contain following components in 10ml mesenchymal stem cell cryopreserving liquids：
Basic fibroblast growth factor bFGF (mother liquid concentration is 50ug/ml) 10ul (final concentration 50ng/ml),
EGF EGF (mother liquid concentration is 50ug/ml) 10ul (final concentration 50ng/ml),
Mother liquor is first prepared, -20 DEG C of preservations are then added to final concentration when then preparation every time.
2) umbilical cord mesenchymal stem cells freeze
P1 passes the 3rd day after P2, when cell fusion reaches 85%-90%, blake bottle is taken out from incubator and is put into cleaning
Workbench, nutrient solution in blake bottle is poured into new T175 blake bottles, is used as terminate liquid.
10ml physiological saline is added into each blake bottle and gently rinses cell bottle one time, the physiology after washing is then outwelled
Salt solution.
Pancreatin working solution 5ml is added in each blake bottle, digestion is shaken up, observation is shown in most of adherent under inverted microscope
Spindle cell start shedding off it is rounded adrift digest about 5min, then toward adding 7ml terminate liquids in each blake bottle,
Digestion is terminated, then postdigestive cell suspension poured out in the 50ml centrifuge tubes of sky (general 4 bottles are poured into 1 50ml centrifugation
In pipe).
With time blake bottle of about 50ml brines, the physiological saline after washing is also poured into sky centrifuge tube,
210g centrifuges 10min.
After centrifugation terminates, supernatant is outwelled, cell number pipe (general 5 pipe unification is managed) is merged into, with haemostatic clamp screen cloth
Slant setting on centrifuge tube, then with 5ml pipettes by cell suspension by screen filtration into new 50ml centrifuge tubes.
After centrifugation terminates, supernatant is outwelled, cell is resuspended and is merged into a 50ml centrifuge tube, 45ml is settled to, is sampled
1.5ml censorships detection of mycoplasma, cell count, Cell viability detection, fluidic cell Phenotypic examination.Then centrifuged again with 210g
10min。
After centrifugation terminates, collect 30ml supernatants and send Sterility testing.
According to the required aim cell for freezing different densities, add appropriate complete culture solution and cell is resuspended, finally make
Cell density is adjusted to freeze 2 times of purpose density.
Plus working solution is frozen, isometric cell cryopreservation working solution is slowly added to along tube wall into cell suspension, is gently blown
Beat uniform, be that cell density requires consistent with specifying.
It is dispensed on request in cryopreservation tube, and sticks corresponding cell label.
The cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, 4 DEG C, 40min；- 20 DEG C, 30min；-80
DEG C, overnight；Next day, slow liquid nitrogen surface of being down to was up to input Liquid nitrogen storage.
3) umbilical cord mesenchymal stem cells are recovered
Freeze-stored cell pipe is rapid from liquid nitrogen is transferred to 40 DEG C of thermostat water baths, be maintained at the top of cryovial the water surface with
On avoid pollution.Rapid rewarming in 1min.Centrifuge tube is moved into after thawing immediately, 5ml4 DEG C of precooling mescenchymal stem cell is added complete
Nutrient solution pressure-vaccum is uniform.
4) umbilical cord mesenchymal stem cells are counted
Using Countess II automatic cytological calculating instruments, according to mescenchymal stem cell size, circularity, brightness gating is led to
Cross automatic focus on and draw Cell viability and quantity with counting algorithm.Take 10ul cell suspending liquids and 10ull trypan blue liquid isometric
It is well mixed in the small centrifuge tubes of 1.5ml, then takes 10ul to be placed in specific slide, in insertion calculating instrument, by sets in advance
Threshold counts cell, draws cell recovery rate.
5) umbilical cord mesenchymal stem cells flow cytometer detection
Prepare the umbilical cord mesenchymal stem cells after recovery, carry out antibody labeling, labelled antibody difference after PBS washings to cell
It is PE-CD73, PE-CD105, PE-CD90, PE-CD34, FITC-CD45, FITC-HLA-Dr, PE-CD44, PE-Mouse
IgG1 and FITC-Mouse IgG1.After being incubated washing, upper machine testing,
As a result Fig. 2 is seen.Wherein CD73, CD105, CD90, CD44 and CD29 positive rate be more than 95%, CD34, CD45 and
HLA-Dr positive rates are less than 2%, and from the point of view of streaming result, the isolated cell of this method meets MSC standards of perfection.
6) it is the checking present invention and the cryopreservation resuscitation of traditional frozen stock solution (hyclone 10%, DMSO10%, DMEM/F12)
Then the difference of rate, spy's culture is frozen carefully with a collection of cell using frozen stock solution freeze-stored cell of the present invention and using traditional frozen stock solution
Born of the same parents；Recovery frozen stock solution freeze-stored cell of the present invention and using traditional frozen stock solution freeze-stored cell simultaneously, two groups of cells are cultivated, culture
Observe and take pictures under the microscope after 3 days.
As a result：
The cell recovery rate frozen using umbilical cord mesenchymal stem cells frozen stock solution of the present invention is apparently higher than traditional frozen stock solution (tire
Cow's serum 10%, DMSO10%, DMEM/F12) cryopreservation resuscitation rate, living cells quantity increases, and can preferably maintain the work of cell
Property, cell state is more preferable.Figure 1A is using the cell after umbilical cord mesenchymal stem cells frozen stock solution cryopreservation resuscitation culture of the present invention 3 days
Growth conditions；Figure 1B is then the cell growth state after traditional frozen stock solution recovery is cultivated 3 days.
7) clearly to be described to the technical scheme in present example, experiment of single factor is used to the present invention.
Different component is respectively adopted freezes protection liquid freezing and storing umbilical mesenchymal stem cells, and relatively more different freezes protection liquid
Cell viability situation after cryopreservation resuscitation.
Table 1 freezes protection liquid and cryopreservation resuscitation rate for the protection liquid freezing and storing umbilical mesenchymal stem cells that freeze of different component.
Table 1
Frozen stock solution component is DMEM/F12+1%Ultroser as shown in Table 1TMDuring G+1% glutamine, anabiosis rate is only
50.43 ± 4.21, then UltroserTMWhen G concentration is changed to 2%, it is found that viable count rises after cell recovery, but recovery
Rate still only has 60.35 ± 3.24.Then in DMEM/F12+2%UltroserTMAdded on the basis of G+1% glutamine
50ng/ml EGF, cell recovery rate brings up to 72.56 ± 2.37；Then change 50ng/ml EGF into 50ng/ml bEGF, send out
Existing cell recovery rate is very nearly the same, is 73.69 ± 2.89；50ng/ml EGF and 50ng/ml bEGF are added simultaneously again
DMEM/F12+2%UltroserTMG+1% glutamine, it is found that cell recovery rate brings up to 85.42 ± 3.41, finally exist
DMEM/F12+2%UltroserTM0.5% is added on the basis of G+1% glutamine+50ng/ml bEGF+50ng/ml EGF
Dextran-40, it is found that anabiosis rate brings up to 92.45 ± 2.49, and the concentration of dextran-40 then is brought up into 1%, discovery
Cell recovery rate brings up to 98.34 ± 4.47.Answered as can be seen here using the cell of umbilical cord mesenchymal stem cells frozen stock solution of the present invention
Soviet Union's rate is apparently higher than the cryopreservation resuscitation rate of traditional frozen stock solution, and uniformly, cell outline complete display, diopter is good for cell size.
Claims (4)
1. a kind of umbilical cord mesenchymal stem cells frozen stock solution, it is characterised in that contained in DMEM/F-12 culture mediums
2. umbilical cord mesenchymal stem cells frozen stock solution according to claim 1, it is characterised in that be in DMEM/F-12 cultures
Contain in base
3. the application method of umbilical cord mesenchymal stem cells frozen stock solution according to claim 2, it is characterised in that take culture
The well-grown cell bottles of 12-15d, nutrient solution is poured into centrifuge tube, as terminate liquid, is then made carefully with pancreatin digestion 5min
Born of the same parents suspend, and add terminate liquid and terminate digestion, supernatant discarding after centrifugation is added after brine, centrifuged again, obtains
Described umbilical cord mesenchymal stem cells frozen stock solution freezes the cell of acquisition, and the concentration as needed for freeze-stored cell number is 2 × 107It is individual thin
Born of the same parents/ml frozen stock solutions, are quantitatively adding the described NSC frozen stock solution of precooling, and after fully mixing, this cell suspension is shifted
Into cryopreservation tube, often pipe 1ml, sealing, mark and freeze the date, be put in program temperature reduction box；It is classified frozen cell：4 DEG C,
40min；- 20 DEG C, 30min；- 80 DEG C, overnight；Next day, slow liquid nitrogen surface of being down to was up to input Liquid nitrogen storage.
4. the application method of umbilical cord mesenchymal stem cells frozen stock solution according to claim 3, it is characterised in that the pancreas used
Enzyme concentration is 0.125% pancreatin containing EDTA, 37 DEG C of digestion 5min.
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| CN107494517A (en) * | 2017-08-07 | 2017-12-22 | 章毅 | Serum-free frozen stock solution and its application in mescenchymal stem cell is frozen |
| CN107810948A (en) * | 2017-10-04 | 2018-03-20 | 刘劼 | A kind of serum-free frozen stock solution of human umbilical cord mesenchymal stem cells |
| CN108812645A (en) * | 2018-07-20 | 2018-11-16 | 吉林济惠生物科技有限公司 | A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells |
| CN109090102A (en) * | 2018-09-05 | 2018-12-28 | 成都汇欣生命科技有限公司 | A kind of general serum-free frozen stock solution of mescenchymal stem cell and preparation method thereof |
| CN109430252A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof |
| CN109456937A (en) * | 2018-12-27 | 2019-03-12 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium preparing Endometrial stem cell and preparation method |
| CN109479871A (en) * | 2017-09-12 | 2019-03-19 | 深圳华云生物科技发展有限公司 | Frozen stock solution, preparation method, application and China's Tong Shi glue tissue cryopreservation methods |
| CN110317782A (en) * | 2019-07-18 | 2019-10-11 | 天津博雅秀岩生物技术有限公司 | Improve the method survived after mescenchymal stem cell recovery and frozen stock solution used |
| CN111838132A (en) * | 2020-07-20 | 2020-10-30 | 江苏瑞思坦生物科技有限公司 | Stem cell cryopreservation method and cell cryopreservation liquid used by same |
| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
| CN113303323A (en) * | 2020-02-27 | 2021-08-27 | 东莞市恩联干细胞生物科技研究院 | Non-freezing preservation method for umbilical cord tissue |
| CN114586769A (en) * | 2020-12-07 | 2022-06-07 | 精准生技股份有限公司 | Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof |
| EP4021180A4 (en) * | 2019-08-29 | 2023-11-08 | Board of Regents, The University of Texas System | Cell cryopreservation medium |
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| CN116076489A (en) * | 2017-08-07 | 2023-05-09 | 中国干细胞集团上海生物科技有限公司 | Freezing method of mesenchymal stem cells |
| CN107494517A (en) * | 2017-08-07 | 2017-12-22 | 章毅 | Serum-free frozen stock solution and its application in mescenchymal stem cell is frozen |
| CN116250526B (en) * | 2017-08-07 | 2024-01-30 | 中国干细胞集团上海生物科技有限公司 | Frozen stock solution for mesenchymal stem cells |
| CN116076489B (en) * | 2017-08-07 | 2024-01-30 | 中国干细胞集团上海生物科技有限公司 | Freezing method of mesenchymal stem cells |
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| CN107810948A (en) * | 2017-10-04 | 2018-03-20 | 刘劼 | A kind of serum-free frozen stock solution of human umbilical cord mesenchymal stem cells |
| CN107810948B (en) * | 2017-10-04 | 2020-07-31 | 刘劼 | Serum-free freezing medium for human umbilical cord mesenchymal stem cells |
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| CN109430252A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof |
| CN109456937A (en) * | 2018-12-27 | 2019-03-12 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium preparing Endometrial stem cell and preparation method |
| CN110317782B (en) * | 2019-07-18 | 2023-06-13 | 天津瑞博斯生物技术有限公司 | Method for improving survival of mesenchymal stem cells after resuscitation and frozen stock solution used by method |
| CN110317782A (en) * | 2019-07-18 | 2019-10-11 | 天津博雅秀岩生物技术有限公司 | Improve the method survived after mescenchymal stem cell recovery and frozen stock solution used |
| EP4021180A4 (en) * | 2019-08-29 | 2023-11-08 | Board of Regents, The University of Texas System | Cell cryopreservation medium |
| CN113303323A (en) * | 2020-02-27 | 2021-08-27 | 东莞市恩联干细胞生物科技研究院 | Non-freezing preservation method for umbilical cord tissue |
| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
| CN111838132A (en) * | 2020-07-20 | 2020-10-30 | 江苏瑞思坦生物科技有限公司 | Stem cell cryopreservation method and cell cryopreservation liquid used by same |
| CN114586769A (en) * | 2020-12-07 | 2022-06-07 | 精准生技股份有限公司 | Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof |
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