CN105907704A - Method for isolated culture of umbilical vein endothelial cell - Google Patents
Method for isolated culture of umbilical vein endothelial cell Download PDFInfo
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- CN105907704A CN105907704A CN201610336519.6A CN201610336519A CN105907704A CN 105907704 A CN105907704 A CN 105907704A CN 201610336519 A CN201610336519 A CN 201610336519A CN 105907704 A CN105907704 A CN 105907704A
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Abstract
The invention relates to the field of isolated culture of adult cells in the technical field of biology, in particular to a method for isolated culture of umbilical vein endothelial cells. The method comprises the following steps: completely stripping an umbilical vein from wharton jelly; longitudinally cutting off the umbilical vein along one side so as to change the umbilical vein into a rectangular blood vessel wall surface from a tube shape and rinsing; adding II-type collagenase for digestion; and taking out the blood vessel and rinsing after digestion, mixing rinsing liquid with enzymatic hydrolysate, centrifuging, removing supernatant and carrying out culture by using a culture medium. The cell separation method is simple to operate, and can be independently carried out by a single person; red cell contamination is relatively low; liquid exchange can be carried out 72 hours after cell inoculation, so that the cell adhesion efficiency is relatively high. A large amount of endothelial cells obtained by primary generation are obtained; CD31 positive expression of P3 cells can reach 95% or above; preparation of umbilical cord mesenchymal stem cells is not affected when the endothelial cells are separately prepared; and the experiment material can be fully utilized.
Description
Technical field
The present invention relates to the separation and Culture field of the adult cell of biological technical field, a kind of method particularly relating to separation and Culture umbilical vein vascular endothelial cells.
Background technology
Blood vessel is the main body of human body internal circulation system, and the inner surface of whole blood vessel is all covered by one layer of endotheliocyte.Due to endotheliocyte can itself directly and contacting blood, so they can be by number of ways in blood pressure regulating, blood coagulation and fibrinolytic, the sticking and migrate of inflammatory cell, and physiology, the pathological process such as vascularization play a significant role.
In cell research, vascular endothelial cell is conventional experiment in vitro cell, and the research great majority that the vascular endothelial cell utilizing people to originate is carried out are to be that material is carried out with Human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC).Mainly select umbilical cord as the source of drawing material of endotheliocyte, on the one hand, to be owing to umbilical cord is as the garbage in birth process, abundance, easily obtain；On the other hand, cord vessels does not has branch, it is simple to operation, and has preferable length, it is possible to ensure enough collections of cell.
The separation method of classical umbilical vein vascular endothelial cells is collagenase perfusion digestion method, is [the Jaffe that put forward in 1973 such as Jaffe EA the earliest
EA, Nachman RL, Becker CG, et al.J Clin Invest, 1973,52 (11): 2745-2756], it is widely used by people so far, the most also correlation techniques such as following the example of scraped by machinery.
The success rate being obtained umbilical vein vascular endothelial cells by perfusion digestion method is higher, but perfusion operation has some shortcomings: first, in perfusion carries out the separation process of umbilical vein vascular endothelial cells, use mosquito forceps or other method to be fixed in needing sterile tube is inserted umbilical vein tube chamber and in this one end, then use PBS and use enzyme to be irrigated digestion to obtain cell.But owing to umbilical vein tube chamber is less and relatively more glutinous sliding, single to carry out intubation relatively difficult；If loosely cleaning and easily cause leakage in filling process the most fixing, thus affecting experiment effect and cause reagent waste, impact is cleaned and digestion effect；In the case of having congestion or sterile tube to fix loosely in the blood vessel, again cannot completely rinse well when rinsing, the blood being easily mixed in blood vessel, cause erythrocyte residual too much, affect the adherent effect of vascular endothelial cell；Additionally during carrying out enzymic digestion, because more difficult closed-loop operation, so being difficulty with 37 DEG C to carry out aseptic digestion process, have impact on the digestive efficiency of enzyme.
Being scraped by machinery and follow the example of acquisition umbilical vein vascular endothelial cells, mainly make cell detachment by mechanicals efforts, easily cause cell mechanical damage, therefore employing degree is the highest.
Summary of the invention
In order to solve in above prior art one man operation's difficulty present in the perfusion digestion method of tradition umbilical vein vascular endothelial cells compared with big, easily cause leakage, easily by other cell contaminations, problem that enzyme dosage is big, inventor considers, work out a kind of applicable one man operation, operation relative ease, be difficult to be contaminated, the method for separation and Culture umbilical vein vascular endothelial cells that enzyme dosage is little.
The present invention is obtained through the following steps:
A kind of method of separation and Culture umbilical vein vascular endothelial cells, comprises the following steps:
(1) after umbilical cord cleans, umbilical vein is led to glue from China and be completely exfoliated out；
(2) umbilical vein is longitudinally cut off along side so that it is become rectangle blood tube wall face from tubulose, then use physiological saline solution that blood vessel wall is rinsed until rinsing liquid clear, colorless；
(3) add II Collagenase Type to digest, II Collagenase Type mass volume ratio concentration be the proportioning of 0.05%-0.1%, enzyme and umbilical vein be the long blood vessel of 0.25-0.5mL:1cm, digestion time is 15-30min, digestion is carried out in constant-temperature table, and digestion temperature is 37 DEG C, and shaking speed is 120r/min；
(4) being taken out by blood vessel after having digested and use α-MEM culture medium to rinse blood vessel, rinsing liquid mixes with enzymolysis solution, discards supernatant after being centrifuged, and uses culture medium to cultivate.
Described method, discards supernatant after 1500r/min is centrifuged 10min in preferred steps (4) after being centrifuged, use culture medium A re-suspended cell precipitation, cultivate, replace medium to culture medium B after 3 days, and cell degrees of fusion passes on after reaching 80%；
Described culture medium A and B add hyclone FBS, basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF in α-MEM culture medium and obtain,
Wherein, in culture medium A final concentration of the 20% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 20ng/mL,
In culture medium B final concentration of the 10% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 10ng/mL.
Described method, for reducing the pollution of other heteroproteose cells such as vascular smooth muscle cell in preferred steps (3), the mass volume ratio concentration of II Collagenase Type is 0.05%；Enzyme is the long blood vessel of 0.5mL:1cm with the proportioning of umbilical vein；Digestion time is 25min.
Described method, preferred steps (1) uses Sterile ophthalmic cut and about 0.5cm osculum is cut along throat aspirate tube chamber in umbilical cord one end, then use aseptic dressing tweezer along the strip off of umbilical vein direction and umbilical vein to be fully exposed by umbilical cord, umbilical vein is led to glue from China and is completely exfoliated out.
Described method, is preferably used after culture medium A re-suspended cell precipitation counts with 1 × 104/cm2Density proceed to 75cm2In Tissue Culture Flask, put into 37 DEG C, 5%CO2, saturated humidity is cultivated.
Described method, in preferred steps (1), cell can reach 8-10 generation.
Described method, in preferred steps (3), digestion uses container to be 50mL sterile centrifugation tube.
Described method, gathers in preferred steps (1), umbilical cord is 24h.
Beneficial effect:
The method separating endotheliocyte of the present invention is simple to operate, single can independently carry out, and red blood cell contamination is less, can carry out changing liquid again by 72h after cell inoculation, and cell attachment is in hgher efficiency；Vascular endothelial cell quantity obtained by primary is many, and 10cm umbilical cord can obtain cell 2 × 106, P3 cell CD31 positive expression is up to more than 95%, and prepares vascular endothelial cell in separation and do not affect the preparation of umbilical cord mesenchymal stem cells simultaneously, can make full use of experiment material.
Accompanying drawing explanation
Fig. 1
After umbilical vein vascular endothelial cells Isolation and culture, form under optical microscope, wherein A is initial adhered state, and B is for covering with rear cobblestone-appearance；
Fig. 2
For umbilical vein vascular endothelial cells external one-tenth pipe ability testing result；
Fig. 3 is umbilical vein vascular endothelial cells flow cytometer detection result, and wherein, A is CD31 testing result, and B is CD34 testing result；
Fig. 4 is umbilical vein vascular endothelial cells blood vessel VIII factor Immunofluorescence test result: figure A is test results, and figure B is negative control result.
Detailed description of the invention
Below in conjunction with being embodied as example, the present invention is described further, but protection scope of the present invention is not limited to that:
Embodiment 1 carries out the extraction of umbilical vein vascular endothelial cells cell according to this programme
This example uses multipara to agree to the neonatal umbilical cord source as umbilical vein vascular endothelial cells of mandate.
The reagent that this experiment is used is as follows: II Collagenase Type (Sigma), α-MEM culture medium (Hyclone, Cat.No.SH30265.01B), VEGF VEGF(Peprotech), fibroblast growth factor FGF(Peprotech), hyclone FBS(Gibco), trypsin Hyclone).
(1) neonatal umbilical cord (about 10cm) is washed three times in the normal saline dual anti-containing 1%, remove the blood stains on surface, the osculum that umbilical cord is cut about 0.5cm is cut along umbilical vein direction with Sterile ophthalmic, then aseptic dressing tweezer is used to be pushed aside by umbilical cord along incision site along umbilical vein longitudinal direction, so that umbilical vein is fully exposed, aseptic dressing tweezer is used umbilical vein to be stripped down；(2) use eye scissors that longitudinally blood vessel is cut off tube wall, thus it is cut to rectangle blood tube wall face by tubulose.Blood vessel wall is fully washed 3-5 time with the normal saline dual anti-containing 1%；
(3) then proceeding in 50mL sterile centrifugation tube and add II Collagenase Type that mass body volume concentrations is 0.05%, 10cm blood vessel uses II Collagenase Type volume to be 5mL, and in being finally putting into 37 DEG C of constant-temperature tables, 120r/min digests 25min；
(4) having digested rear blood vessel and used 20mL α-MEM culture medium (without FBS, b-FGF and VEGF) rinsing, after collecting collagenase and rinsing liquid merging, 1500r/min is centrifuged 10min, uses culture medium A re-suspended cell precipitation, with 5 × 10 after counting4/ hole proceeds in 6 orifice plates, puts into 37 DEG C, 5%CO2Middle cultivation.Replacing medium to culture medium B after 3 days, cell degrees of fusion passes on after reaching 80%, and follow-up incubation all uses culture medium B.In culture medium A final concentration of the 20% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 20ng/mL, in culture medium B final concentration of the 10% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 10ng/mL.
In embodiment 1, it is 2 × 10 that 10cm umbilical cord obtains initial cell quantity5, when changing liquid after 3d, floating cells is less, and cell grows to 80% degrees of fusion needs 120h.
Embodiment 2 collagenase perfusion method carries out the extraction of umbilical vein vascular endothelial cells cell
The reagent that this experiment uses is identical with embodiment 1.
Carry out the separation of umbilical vein vascular endothelial cells according to tradition collagenase perfusion method, because method is the most ripe, this place repeats no more.In digestion process, the II Collagenase Type digestion 25min using mass body volume concentrations to be 0.05%, with 5 × 10 after collection cell4/ hole proceeds in 6 orifice plates, puts into 37 DEG C, 5%CO2Middle cultivation, changes liquid and removes floating erythrocyte, replace medium to culture medium B after 3 days after 24h, cell degrees of fusion passes on after reaching 80%, and follow-up incubation all uses culture medium B.In culture medium A final concentration of the 20% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 20ng/mL, in culture medium B final concentration of the 10% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 10ng/mL.
In embodiment 2, it is 1.6 × 10 that 10cm umbilical cord obtains initial cell quantity5, changing erythrocyte floating during liquid after 3d and be more than embodiment 1, cell grows to 80% degrees of fusion needs 168h.
Embodiment 3
Compared with embodiment 1, in this embodiment step (3), 10cm blood vessel uses II Collagenase Type mass body volume concentrations to be 0.1%, and digestion time is 15min, and remaining is identical with embodiment 1.
In embodiment 3, it is 1.8 × 10 that 10cm umbilical cord obtains initial cell quantity5, cell grows to 80% degrees of fusion needs 144h.
Embodiment 4
Compared with embodiment 1, in this embodiment step (3), 10cm blood vessel uses II Collagenase Type mass body volume concentrations to be 0.1%, and digestion time is 25min, and remaining is identical with embodiment 1.
In embodiment 4, it is 2.2 × 10 that 10cm umbilical cord obtains initial cell quantity5, cell grows to 80% degrees of fusion needs 120h, but occurs the vascular smooth muscle cell of part spindle shape in attached cell, and endotheliocyte purity is less than 90%.
Embodiment 5 umbilical vein vascular endothelial cells is identified
Take the cell reaching eighth generation in embodiment 1, carry out relevant identification experiment.
1, cellular morphology is observed
Take eighth generation umbilical vein vascular endothelial cells according to 1 × 105/ hole is inoculated in 6 orifice plates, observation of cell form under optical microscope, and time initial, cell is fusiformis, and in cobblestone-appearance after being paved with, such as the umbilical vein vascular endothelial cells cultivated in embodiment 1 is as it is shown in figure 1, meet the morphological characteristic of endotheliocyte.
2, vascularization experiment
1) the 20-200 μ l rifle head of 96 well culture plate plates and sterilizing is placed in-20 DEG C of pre-coolings, Matrigel(BD) it is placed in 4 DEG C of fridge overnight thawings；
2) Matrigel using the pre-every hole of sniper's shot head to add 20 μ l spreads glue, is swift in motion, it is to avoid bubble formation, hatches 30min in being subsequently placed in 37 DEG C of incubators；
3) cell suspension adjusts to 2 × 105/ mL, with 104/ hole is inoculated in culture plate, be placed in 37 DEG C, cultivate 4-8 hour in 5%CO2 saturated humidity after observation experiment result；Test result indicate that, in this method, the umbilical vein vascular endothelial cells of isolated has external one-tenth vessel patency (Fig. 2).
3, flow cytomery cell phenotype
Be separately added into 5 μ l streaming IgG antibody 1-PE in three loading pipes, CD31-PE, CD34-PE(are BD company and produce), then take eighth generation umbilical vein vascular endothelial cells in embodiment 1 and adjust to 2 × 106/ mL, adds 100 μ l cell suspension in each loading pipe, after mixing, lucifuge hatches 15min, adds upper machine testing after 200 μ l PBS mixings the most again.Experiment duplicate detection three times, result show CD31 positive rate all more than 90%, CD34 positive rate, all below 10%, meets endotheliocyte standard (Fig. 3).
4, Immunofluorescence test blood vessel VIII factor antigen expression
1) take on the coverslip after eighth generation cell is inoculated in sterilization, take out after cell climbs full slide, rinse 3 times with PBS, then use 4% paraformaldehyde to fix 20min；
2)
PBS rinses 3 times, each 5min, and the lowlenthal serum (containing 0.5%TritonX-100) adding 5% is closed, room temperature 20min；
3) mouse-anti human Factor Ⅷ-related antigen monoclonal antibody 50ul(santa cruz is added), 4 DEG C are overnight；
4)
PBS solution rinses 3 times, each 3min, uses the anti-50ul(santa cruz of Alexa Flour 555 labelling goat anti rat fluorescence two), under room temperature, lucifuge hatches 45min；
5)
PBS solution rinses 3 times, and each 5min is subsequently adding 50 ul DAPI(Sigma), under room temperature, lucifuge hatches 5min；
6) after PBS solution rinses 3 times, mounting is observed, and has graininess red fluorescence immune deposits, it was demonstrated that blood vessel VIII factor Ⅷ related antigen positive expression (Fig. 4) in result visible cell slurry.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention also should not be limited by the examples; the change made under other any spirit without departing from the present invention and principle, modify, combine, substitute, simplify and all should be equivalence substitute mode, within being included in protection scope of the present invention.
Claims (8)
1. the method for a separation and Culture umbilical vein vascular endothelial cells, it is characterised in that comprise the following steps:
(1) after umbilical cord cleans, umbilical vein is led to glue from China and be completely exfoliated out；
(2) umbilical vein is longitudinally cut off along side so that it is become rectangle blood tube wall face from tubulose, then use physiological saline solution that blood vessel wall is rinsed until rinsing liquid clear, colorless；
(3) add II Collagenase Type to digest, II Collagenase Type mass volume ratio concentration be the proportioning of 0.05%-0.1%, enzyme and umbilical vein be the long blood vessel of 0.25-0.5mL:1cm, digestion time is 15-30min, digestion is carried out in constant-temperature table, and digestion temperature is 37 DEG C, and shaking speed is 120r/min；
(4) being taken out by blood vessel after having digested and use α-MEM culture medium to rinse blood vessel, rinsing liquid mixes with enzymolysis solution, discards supernatant after being centrifuged, and uses culture medium to cultivate.
Method the most according to claim 1, it is characterised in that discard supernatant after centrifugal in step (4), uses culture medium A re-suspended cell precipitation, cultivates, replace medium to culture medium B after 3 days, and cell degrees of fusion passes on after reaching 80%；
Described culture medium A and B add hyclone FBS, basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF in α-MEM culture medium and obtain,
Wherein, in culture medium A final concentration of the 20% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 20ng/mL,
In culture medium B final concentration of the 10% of hyclone FBS, the final concentration of basic fibroblast growth factor b-FGF and blood vessel endothelial cell growth factor VEGF is respectively 10ng/mL.
Method the most according to claim 1, it is characterised in that in step (3), the mass volume ratio concentration of II Collagenase Type is 0.05%；Enzyme is the long blood vessel of 0.5mL:1cm with the proportioning of umbilical vein；Digestion time is 25min.
Method the most according to claim 1, it is characterized in that using Sterile ophthalmic to cut in step (1) cuts about 0.5cm osculum by umbilical cord one end along throat aspirate tube chamber, then use aseptic dressing tweezer along the strip off of umbilical vein direction and umbilical vein to be fully exposed by umbilical cord, umbilical vein is led to glue from China and is completely exfoliated out.
Method the most according to claim 2, it is characterised in that with 1 × 10 after using culture medium A re-suspended cell precipitation to count4/cm2Density proceed to 75cm2In Tissue Culture Flask, put into 37 DEG C, 5%CO2, saturated humidity is cultivated.
Method the most according to claim 1, it is characterised in that in step (1), cell can reach 8-10 generation.
Method the most according to claim 1, it is characterised in that in step (3), digestion uses container to be 50mL sterile centrifugation tube.
Method the most according to claim 1, it is characterised in that gather in umbilical cord is 24h in step (1).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308434A (en) * | 2021-05-31 | 2021-08-27 | 中山大学孙逸仙纪念医院 | Construction method of endothelial cell and pericyte co-culture model for researching tube formation |
| CN115322949A (en) * | 2022-07-26 | 2022-11-11 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
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| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
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2016
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308434A (en) * | 2021-05-31 | 2021-08-27 | 中山大学孙逸仙纪念医院 | Construction method of endothelial cell and pericyte co-culture model for researching tube formation |
| CN115322949A (en) * | 2022-07-26 | 2022-11-11 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
| CN115322949B (en) * | 2022-07-26 | 2024-03-15 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
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