CN108244096B - A kind of preservation liquid of the endothelial progenitor cell of long-term preservation overexpression VEGF - Google Patents
A kind of preservation liquid of the endothelial progenitor cell of long-term preservation overexpression VEGF Download PDFInfo
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 28
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- 238000000034 method Methods 0.000 claims abstract description 16
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Classifications
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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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
-
- 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
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- 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
本发明提供了一种长期保存过表达VEGF的血管内皮祖细胞的保存液，包括以下组分：白蛋白、葡萄糖、乙二醇、二甲基亚砜、表皮生长因子、硫酸镁、L‑谷氨酰胺和倍他米松。本发明还提供了该保存液的使用方法，通过本发明提供的使用方法，应用该保存液来保存过表达VEGF的血管内皮祖细胞，能够长期保存过表达VEGF的血管内皮祖细胞，并使复苏后的细胞很好地维持增殖功能和血管内皮祖细胞的生物学特性。
The present invention provides a preservation solution for long-term preservation of vascular endothelial progenitor cells overexpressing VEGF, comprising the following components: albumin, glucose, ethylene glycol, dimethyl sulfoxide, epidermal growth factor, magnesium sulfate, L-glutamyl aminoamide and betamethasone. The present invention also provides a method of using the preservation solution. By using the method of use provided by the present invention, the preservation solution is used to preserve the VEGF-overexpressing vascular endothelial progenitor cells, so that the VEGF-overexpressing vascular endothelial progenitor cells can be preserved for a long time, and the vascular endothelial progenitor cells overexpressing VEGF can be preserved for a long time. The latter cells well maintained the proliferative function and biological properties of vascular endothelial progenitor cells.
Description
Technical field
The present invention relates to field of cell culture, and in particular to a kind of long-term preservation is overexpressed the endothelial progenitor cell of VEGF
Preservation liquid.
Background technique
Various types bone defect caused by due to wound, infection, tumour etc. is the FAQs clinically encountered.According to
The recent statistics in the U.S., the surgical operation for being related to bone defect healing every year is more than 1,000,000 person-times；And in China, because of traffic, work
Accidental injury caused by wound and sports etc. is with annual 7.3% speed increase.In recent years, bone tissue engineer technology is gradually
It rises and provides effective solution strategy for the reparation of bone defect, early stage research has shown that, is lacked with organizational project Bone Defect Repari segment bone
When damage, early stage skeletonization and later period knitting effect are obvious.But in application bulk tissue engineering Bone Defect Repari large segmental bone defect,
Core part tends to occur ischemic necrosis, leads to repairing failure, and main cause, which is that, not can solve organizational project
The vascularization problem of bone, vascularization problem, which has become, restricts the bottleneck that tissue engineered bone is applied to clinic.To various rush blood vessels
The research of growth factor is one of the hot spot of current vascularization of tissue engineered bone research.
" angiogenesis " is one of the main mechanism that new vessels are formed, and embryonic period, embryonic phase is taken place mostly in, before endothelium
Somatic differentiation is at endothelial cell, and in mesoderm, endothelial cell differentiation, proliferation, migration, connection simultaneously form original capillary
Clump, this process are controlled by vascular endothelial growth factor (vascular endothelial growth factor, VEGF).
VEGF is the direct inducible factor of strong blood vessel, is a kind of with Heparin-binding dimerization glycoprotein, has induction of vascular
Endothelial cell proliferation promotes angiogenesis, increases the effects of capillary permeability, leaks intravascular constituents, is intravascular
Endothelial cell migration and vascularization offer matrix, while inhibition apoptosis of vascular endothelial cell, induction of vascular smooth muscle cell migration,
Promote vascular smooth muscle cells synthesis and secretion of MMPs, and accelerates substrate degradation and inflammation chemotactic etc., it is final to promote
Form new capillary vessel.VEGF family includes VEGF A, B, C, D and the placentation factor, wherein VEGF A be research compared with
The factor of concentration, there are four types of isomers for tool, are made of respectively 121,165,189,206 amino acid, wherein VEGF-165 activity
It is most strong, it has a very wide distribution, is the principal mode to play a role in vivo.However recombination VEGF-165 albumen is expensive, in vivo
It can be diluted quickly by blood, tissue fluid, half-life short, be difficult locally reaching lasting useful effect concentration, and part is big
Amount is serious using side effect, easily leads to local vascular tumor.
Endothelial progenitor cell (Endothelial Progenitor Cells, EPC) is equal to 1997 by Asahara earliest
It reports in year, it is that a group has migration characteristic, not yet expresses mature vascular endothelial cell phenotype, can be divided into vascular endothelial cell
Directly participate in vascularization.It exists in marrow, Cord blood and peripheral blood, and can be in angiogenic process after birth
There is very strong angiogenesispromoting effect, and forms new vessels in a manner of angiogenesis.This discovery, has updated in traditional sense
Birth after angiogenesis, injury of blood vessel reparation theory, also to solve the problems, such as that vascularization of tissue engineered bone provides new think of
Road.In recent years, ischemic myocardium, ischemic limb, damage are concentrated mainly in relation to Effect study of the EPCs in adult blood is formed
Cornea, the reparation for damaging skin etc., and its research in bone tissue engineer is at home and abroad then in just getting started.
However since the cell quantity of EPCs is limited, obtain difficult, when in vitro culture needs to expand, and cultivation cycle is long, and needs through FN
Adherent growth is mediated just to be conducive to its survival, these deficiencies limit its application in zoopery and clinical research.
In recent years, with the development of technique for gene engineering, gene therapy has become the hot spot of numerous scholars' research, transgenosis
Tissue engineering technique new idea and method is provided for vascularization.VEGF gene modification EPC can make up its lazy weight
Disadvantage, VEGF gene transfect the new vessels characteristic that EPC can be remarkably reinforced, can optimize the angiogenesis promoting effect of EPC significantly.
It is newborn that the EPC of the reports application VEGF-165 gene such as She transfection can be obviously promoted ischemic myocardium medium vessels, to be obviously improved big
Heart function after mouse acute myocardial infarction AMI.Easily at EPC in rigid equal employments bleeding of the umbilicus, implanted after transfection outside VEGF-165 genosome is utilized
In nude mice Survival of Random Flap, the angiogenesis of ischemic skin flap can promote, improve the survival rate of flap, and transfect VEGF-165 gene
EPC has the function of more powerful angiogenesis promoting.
It is one of to need what is solved to ask in order to which preferably further the EPC for being overexpressed VEGF is studied and applied
Topic is exactly to be overexpressed the preservation problem of the EPC of VEGF.
Common zooblast freezen protective liquid is made of 20% blood serum medium and 10% dimethyl sulfoxide (DMSO),
Freezen protective liquid can reduce the formation of intracellular ice crystal molecule in cooling, in order to avoid to cell damage, when preservation first will
Freezen protective liquid containing cell slowly freezes, and then stores for a long time in liquid nitrogen.But the preservation liquid ingredient containing serum is unknown
Really, not only containing ingredients such as cell factor, growth factor, hormones, there are also unknown ingredients.Inventor sends out in the course of the research
It is existing, the endothelial progenitor cells for being overexpressed VEGF are saved using above-mentioned common freezen protective liquid, will cause thin after thawing recovery
The problems such as decline of cellular expression VEGF ability, can not keep characteristics of cell biology well, and cell Proliferation function is poor.
Therefore, this field needs one kind that can maintain the endothelial progenitor cells for being overexpressed VEGF well in a long time
Expanding capacity and biological characteristics and the specific cell-preservation liquid of component.
Summary of the invention
Technical problem to be solved by the present invention lies in view of the above shortcomings of the prior art, provide a kind of preservation overexpression
The preservation liquid of the endothelial progenitor cells of VEGF, the preservation liquid component is clear, being capable of long-term preservation overexpression by using the preservation liquid
The endothelial progenitor cells of VEGF, and the cell after recovery is made to maintain the biological characteristics of expanding capacity and endothelial progenitor cells well.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides the preservation liquid that a kind of long-term preservation is overexpressed the endothelial progenitor cells of VEGF, comprising: albumin, Portugal
Grape sugar, ethylene glycol, dimethyl sulfoxide, epidermal growth factor, magnesium sulfate, L-Glutamine and betamethasone.
Preferably, the long-term preservation is overexpressed the preservation liquid of the endothelial progenitor cells of VEGF, comprising: the white egg of 0.1-20%
White, 0.1-10% glucose, 0.1-5% ethylene glycol, 5-20% dimethyl sulfoxide, 5-15ng/ml epidermal growth factor, 0.15-
0.3g/l magnesium sulfate, 2-5mM L-Glutamine and 80-100ng/ml betamethasone.
In a preferred embodiment, the long-term preservation is overexpressed the preservation liquid of the endothelial progenitor cells of VEGF,
It include: 0.5-15% albumin, 0.5-10% glucose, 2-5% ethylene glycol, 5-15% dimethyl sulfoxide, 10-15ng/ml table
Skin growth factor, 0.2-0.3g/l magnesium sulfate, 2-5mM L-Glutamine and 80-100ng/ml betamethasone.
In another preferred embodiment, the long-term preservation is overexpressed the preservation of the endothelial progenitor cells of VEGF
Liquid, comprising: 2-15% albumin, 1-10% glucose, 2-5% ethylene glycol, 5-12% dimethyl sulfoxide, 12-15ng/ml epidermis
Growth factor, 0.2-0.3g/l magnesium sulfate, 2.5-5mM L-Glutamine and 80-95ng/ml betamethasone.
In another preferred embodiment, the long-term preservation is overexpressed the preservation of the endothelial progenitor cells of VEGF
Liquid, comprising: 6-15% albumin, 4-10% glucose, 2-5% ethylene glycol, 5-12% dimethyl sulfoxide, 12-15ng/ml epidermis
Growth factor, 0.2-0.3g/l magnesium sulfate, 2.5-5mM L-Glutamine and 80-95ng/ml betamethasone.
In a specific embodiment, the long-term preservation is overexpressed the preservation liquid of the endothelial progenitor cells of VEGF,
It include: 10% albumin, 8% glucose, 5% ethylene glycol, 10% dimethyl sulfoxide, 15ng/ml epidermal growth factor, 0.25g/
L magnesium sulfate, 3mM L-Glutamine and 80ng/ml betamethasone.
The present invention also provides a kind of methods that long-term preservation is overexpressed the endothelial progenitor cells of VEGF, which comprises
(1) endothelial progenitor cell of the overexpression VEGF of logarithmic growth phase, removes old culture medium, is cleaned with PBS；
(2) PBS is removed, appropriate trypsase is added, it is made to cover culture dish surface, it will be under the cell dissociation of monolayer growth
Come；
(3) the resulting cell liquid of step (2) is centrifuged, removes supernatant；
(4) freezen protective liquid is added in the cell obtained to step (3), gently blowing and beating cell makes cell suspend uniformly, meter
Counting and adjusting the cell concentration in freezen protective liquid is 1 × 106-1×107A/ml；
(5) cell-preservation liquid that step (4) obtains is dispensed into cryopreservation tube；
(6) cryopreservation tube is frozen.
Preferably, in step (4), adjusting the cell concentration in freezen protective liquid is 5 × 106-1×107A/ml.
Preferably, in step (5), the cell-preservation liquid volume in each cryopreservation tube is 1-1.5ml.
Preferably, in step (6), cryopreservation tube is frozen using Slow-rate freezing instrument.
In a preferred embodiment, in step (6), the cooling rate of setting program frigorimeter is -1 to -2
℃/min；When temperature reaches -25 DEG C or less, -5 to -10 DEG C/min is increased to；When temperature reaches -100 DEG C, it is transferred to liquid nitrogen
Middle storage.
The present invention has developed a kind of long-term preservation overexpression for the characteristic for the endothelial progenitor cell for being overexpressed VEGF
The preservation liquid of the endothelial progenitor cells of VEGF.The preservation liquid ingredient is explicit, is capable of the endothelium ancestral of long-term preservation overexpression VEGF
Cell, and the cell after recovery is made to maintain the biological characteristics of expanding capacity and endothelial progenitor cells well.Wherein, dimethyl is sub-
Sulfone is a kind of permeability protective agent, can rapidly permeate into cell, reduces the freezing point of cell, promotes intracellular water exudation extracellular, reduces
The formation of intracellular ice crystal, to reduce damage of the ice crystal to cell；Ethylene glycol can further help in reduce cell due to
Physical damnification caused by ice crystal formation and osmotic pressure change；Albumin, glucose, epidermal growth factor, magnesium sulfate, L- paddy ammonia
The conditions such as osmotic pressure and buffer system needed for amide etc. can provide freezen protective for cell, and helped carefully in cell recovery
The fast quick-recovery of born of the same parents；Inventor has found during studying the endothelial progenitor cells for being overexpressed VEGF for a long period of time, by adding into preservation liquid
Add epidermal growth factor and betamethasone, the expanding capacity of the cell after can be further improved recovery can also be tieed up preferably
The biological characteristics of its interior progenitor cells are held, specific mechanism is still not very clear.
In conclusion compared with the prior art, the invention has the following advantages:
(1) preservation liquid ingredient provided by the invention is explicit, is free of serum.
(2) endothelial progenitor cells for being overexpressed VEGF are frozen by using preservation liquid provided by the invention, it can be very
The expanding capacity and biological characteristics that are overexpressed the endothelial progenitor cells of VEGF are maintained well.
Detailed description of the invention
The endothelial progenitor cell expanding capacity detection of the overexpression VEGF of Fig. 1 recovery；
Now in conjunction with drawings and examples, the invention will be further described:
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
The building of embodiment 1 is overexpressed the endothelial progenitor cell of VEGF
Experimental method:
1) separation, culture and identification of endothelial progenitor cell: 3-4 week old Vistar rat takes off neck and puts to death, and alcohol impregnates
Disinfection, takes long bone, cuts off, and rinses marrow, rat lymphocyte separating liquid surface is added in cell suspension, centrifugation takes two layers of liquid
EGM-2MV culture solution, CO2 cell incubator culture is added in cloud cell between body, washing.Take 0,4,7,10 days adherent
Cell, is marked CD34, FLK-1, CD133, the immunohistochemistry detection of vWF, and the 14th day cell carries out projection Electronic Speculum inspection
It can be seen that the W-P corpusculum of specificity.
2) building of PcDNA3.0-hVEGF165 carrier for expression of eukaryon: according to the VEGF165 sequent synthesis of GENEBANK
The cDNA of VEGF165 simultaneously introduces Kpn-1 restriction enzyme site at its 5 ' end, introduces EcoRV restriction enzyme site (Jin Sirui biology at its 3 ' end
Scientific & technical corporation's synthesis), Kpn-1 and EcoRV double digestion, T4 connection are carried out to the VEGF165 segment of PcDNA3.0 plasmid and synthesis
Enzyme connection.Connection product is transformed into DH-5 α competent cell, ampicillin plate screening is chosen monoclonal progress digestion and tested
Card and sequencing, obtain correct Pc DNA3.0-hVEGF165 plasmid cloning.
3) PcDNA3.0-hVEGF165 transfects endothelial progenitor cell: cell grows into vitellophag connection in the tenth day,
The EBM-2 culture medium of 5%FCS is made into 1 × 105/ml cell suspension and is inoculated in 24 orifice plates for having overlay coverslip by the hole 1ml/.Training
It supports overnight cell growth and is fused to 50%-70%, according to the operational manual of liposome Lipofectamine, by 2 μ l of liposome
Endothelial progenitor cell is transfected with 2 μ l of PcDNA3.0-hVEGF165 plasmid.
Embodiment 2-3 and comparative example 1-3
After endothelial progenitor cell transfects VEGF165, for the endothelial progenitor cell of obtained overexpression VEGF, the is taken
10 generation cells carry out cryo-conservation.Embodiment 2-3 and comparative example 1-3 uses different preservation liquid respectively.Embodiment 2-3 and comparison
The preservation liquid ingredient that example 1-2 is used is as shown in the table, and it is 20% N containing 10% dimethyl sulfoxide that comparative example 3 used, which saves liquid,
Blood serum medium.Wherein albumin is human serum albumin, is purchased from ThermoFisher company (article No. 191297010)；Cow's serum
Purchased from ThermoFisher company (article No. 10099158).
Embodiment 2-3 and comparative example 1-3 carries out the freezen protective of cell in accordance with the following methods:
(1) endothelial progenitor cell of the overexpression VEGF of logarithmic growth phase, removes old culture medium, is cleaned with PBS；
(2) PBS is removed, appropriate trypsase is added, it is made to cover culture dish surface, it will be under the cell dissociation of monolayer growth
Come；
(3) the resulting cell liquid of step (2) is centrifuged, removes supernatant；
(4) freezen protective liquid is added in the cell obtained to step (3), gently blowing and beating cell makes cell suspend uniformly, meter
Counting and adjusting the cell concentration in freezen protective liquid is 5 × 106A/ml；
(5) cell-preservation liquid that step (4) obtains is dispensed into cryopreservation tube, every pipe 1ml；
(6) cryopreservation tube being frozen using Slow-rate freezing instrument, the cooling rate of setting program frigorimeter is -1 to -2 DEG C/
min；When temperature reaches -25 DEG C or less, -5 to -10 DEG C/min is increased to；When temperature reaches -100 DEG C, it is transferred in liquid nitrogen and stores up
It deposits.
The endothelial progenitor cell expanding capacity detection for the overexpression VEGF that embodiment 4 is recovered
Detection is after 6 months freeze, the expanding capacity for the cell that embodiment 2-3 and comparative example 1-3 are saved:
(1) it is removed from liquid nitrogen cryovial, is put into rapidly in 38 DEG C of water-baths, and shake frequently, it is made in 1 minute completely
Melt, cell is then taken out in gnotobasis；
(2) it is centrifuged 5-10 minutes, discards supernatant under 1000r/min speed, appropriate culture solution (DMEM+20%FBS is added
+ bFGF) after be inoculated in culture bottle, inoculum density 1 × 109/ L sets 37 DEG C of cell incubator stationary cultures.
(3) after for 24 hours, the cell in culture bottle is pressed 1.0 × 104The density in/hole is inoculated in training in 24 hole culture dish of standard
It supports.3 hole cell dissociations are taken daily within 1-7 days after inoculation, carry out cell count, carry out the identification of endothelial progenitor cell expanding capacity,
Steps are as follows:
1) culture medium in hole is exhausted under sterile working, 1mlPBS standing 3min × 2 time are added, and (liquid sufficiently connects with ware bottom
Touching)；
2) addition pancreatin 0.2ml shakes up after abandoning PBS liquid；
3) flushable when seeing cell rounding under mirror, floating on a liquid, piping and druming (rushing attached cell into liquid)；
4) cell count under an optical microscope, counting statistics the result is shown in Figure 1.
By Fig. 1 result it is found that with the preservation liquid phase ratio that uses comparative example 1-3, by using the preservation liquid of embodiment 2-3,
The endothelial progenitor cell expanding capacity of the overexpression VEGF of recovery is more preferable.
The endothelial progenitor cell biological characteristics detection for the overexpression VEGF that embodiment 5 is recovered
Detection is after 6 months freeze, the biological characteristics for the cell that embodiment 2-3 and comparative example 1-3 are saved:
(1) it is removed from liquid nitrogen cryovial, is put into rapidly in 38 DEG C of water-baths, and shake frequently, it is made in 1 minute completely
Melt, cell is then taken out in gnotobasis；
(2) it is centrifuged 5-10 minutes, discards supernatant under 1000r/min speed, appropriate culture solution (DMEM+20%FBS is added
+ bFGF) after be inoculated in culture bottle, 1 × 109/L of inoculum density sets 37 DEG C of cell incubator stationary cultures.
(3) culture carries out attached cell digestion afterwards for 24 hours, takes 1 × 10 respectively6A cell, the CD34 and CD133 marked with PE
Antibody labeled cells, flow cytometer (FACA Calibur type) detection, the above fluorescent labeled antibody are purchased from BD company.Through flowing
The detection of formula immunophenotype, the statistical result such as following table institute of each antibody positive rate of cell saved through embodiment 2-3 and comparative example 1-3
Show:
| Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| CD34PE | 99.93% | 99.68% | 92.52% | 94.58% | 94.22% |
| CD133PE | 99.31% | 99.26% | 90.45% | 92.46% | 91.37% |
By testing result it is found that with the preservation liquid phase ratio that uses comparative example 1-3, by using the preservation liquid of embodiment 2-3,
The biological characteristics of the endothelial progenitor cell of the overexpression VEGF of recovery are preferably kept.
The technical means disclosed in the embodiments of the present invention is not limited to the technical means disclosed in the above technical means, and further includes
Technical solution consisting of any combination of the above technical features.The foregoing is a specific embodiment of the present invention, should refer to
Out, for those skilled in the art, without departing from the principle of the present invention, can also make several
Improvements and modifications, these modifications and embellishments are also considered to be within the scope of the present invention.
Claims (8)
1. the preservation liquid that a kind of long-term preservation is overexpressed the endothelial progenitor cells of VEGF, characterized by comprising: albumin, grape
Sugar, ethylene glycol, dimethyl sulfoxide, epidermal growth factor, magnesium sulfate, L-Glutamine and betamethasone, wherein 0.1-20% is white
Albumen, 0.1-10% glucose, 0.1-5% ethylene glycol, 5-20% dimethyl sulfoxide, 5-15ng/ml epidermal growth factor,
0.15-0.3g/l magnesium sulfate, 2-5mM L-Glutamine and 80-100ng/ml betamethasone.
2. the preservation liquid that long-term preservation described in accordance with the claim 1 is overexpressed the endothelial progenitor cells of VEGF, it is characterised in that packet
It includes: 0.5-15% albumin, 0.5-10% glucose, 2-5% ethylene glycol, 5-15% dimethyl sulfoxide, 10-15ng/ml epidermis
Growth factor, 0.2-0.3g/l magnesium sulfate, 2-5mM L-Glutamine and 80-100ng/ml betamethasone.
3. the preservation liquid that long-term preservation described in accordance with the claim 1 is overexpressed the endothelial progenitor cells of VEGF, it is characterised in that packet
It includes: 10% albumin, 8% glucose, 5% ethylene glycol, 10% dimethyl sulfoxide, 15ng/ml epidermal growth factor, 0.25g/l
Magnesium sulfate, 3mM L-Glutamine and 80ng/ml betamethasone.
4. being overexpressed the use of the preservation liquid of the endothelial progenitor cells of VEGF according to the described in any item long-term preservations of claim 1-3
Method, it is characterised in that the application method includes:
(1) endothelial progenitor cell of the overexpression VEGF of logarithmic growth phase, removes old culture medium, is cleaned with PBS；
(2) PBS is removed, appropriate trypsase is added, so that it is covered culture dish surface, the cell dissociation of monolayer growth is got off；
(3) the resulting cell liquid of step (2) is centrifuged, removes supernatant；
(4) the preservation liquid that long-term preservation is overexpressed the endothelial progenitor cells of VEGF is added in the cell obtained to step (3), gently blows
Beating cell makes cell suspend uniformly, and counting and adjusting the cell concentration in freezen protective liquid is 1 × 106-1×107A/ml；
(5) cell-preservation liquid that step (4) obtains is dispensed into cryopreservation tube；
(6) cryopreservation tube is frozen.
5. application method according to claim 4, which is characterized in that in step (4), adjust thin in freezen protective liquid
Born of the same parents' concentration is 5 × 106-1×107A/ml.
6. application method according to claim 4, which is characterized in that in step (5), the cell in each cryopreservation tube is protected
Liquid storage volume is 1-1.5ml.
7. application method according to claim 4, which is characterized in that in step (6), using Slow-rate freezing instrument to freezing
Pipe is frozen.
8. application method according to claim 7, which is characterized in that in step (6), the cooling of setting program frigorimeter
Speed is -1 to -2 DEG C/min；When temperature reaches -25 DEG C or less, -5 to -10 DEG C/min is increased to；When temperature reaches -100 DEG C
It is transferred in liquid nitrogen and stores.
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