CN109652361A - A kind of the primary of human umbilical vein endothelial cell is separately cultured and identification method - Google Patents

A kind of the primary of human umbilical vein endothelial cell is separately cultured and identification method Download PDF

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CN109652361A
CN109652361A CN201710946120.4A CN201710946120A CN109652361A CN 109652361 A CN109652361 A CN 109652361A CN 201710946120 A CN201710946120 A CN 201710946120A CN 109652361 A CN109652361 A CN 109652361A
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umbilical cord
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曹延粉
赵燕芳
黄庆雷
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Beijing Hongrun Tianyuan Gene Biotechnology Co Ltd
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract

The present invention is directed primarily to the separation of field of biotechnology cell, culture and identification method, more particularly to one kind is separately cultured umbilical vein vascular endothelial cells (human umbilical vein endothelial cell, HUVEC method), content is as follows: using healthy puerpera's neonatal umbilical cord, in Biohazard Safety Equipment, the collagenase type I of preheating is fed into umbilical vein blood vessel, and it is immersed in the physiological saline of preheating and digests, vein blood vessel is rinsed after the completion of digestion, supernatant is abandoned after merging centrifugation, cell precipitation, which is resuspended, in culture medium can start to cultivate, operation of the present invention is simple, entire digestion process is in station, digestion temperature is slightly below 37 DEG C, it is appropriate to increase digestion time, excessive digestion not will cause for relatively fine vascular tissue, simultaneously Avoid the pollution risk of environment and other heteroproteose cells, obtained endothelial cell morphology, the immunophenotype on surface and be typical endothelial cell characteristics at pipe characteristic, and remaining umbilical cord tissue still can be used for being separately cultured for the separating treatment such as umbilical cord mesenchymal stem cells of the other cells of umbilical cord tissue.

Description

A kind of the primary of human umbilical vein endothelial cell is separately cultured and identification method
Technical field
The present invention is the primary isolation and culture method of human umbilical vein endothelial cell HUVEC, belongs to biotechnology neck Domain.
Background technique
Vascular endothelial cell is the cell of blood vessel monolayer alignment, and crucial work is played in the mass exchange of blood and tissue With, participate in maintain angiokinesis, anticoagulation, and inflammation in terms of play an important role, internal endothelial cell Missing and dysfunction will lead to blood vessel scleratheroma, the generation of the diseases such as thrombus, cerebrovascular disease, diabetes, therefore to blood Endothelial cell research will provide important help for the treatment of ischemic disease and intravascular tissue engineering.
In the basic research of endothelial cell, neonatal umbilical cord materials are convenient, do not limited by ethics, from wherein isolated Human umbilical vein endothelial's cell (human umbilical vein endothelial cell, HUVEC) can be widely used for reality The vascular endothelial cell for testing research, can also be used as the model for studying certain pathological states, this experimental applications modified enzyme TrypLE disappears Change method carries out primary separation, in vitro culture and the amplification identification of HUVECs, the endothelial cell of high quality high-purity is obtained, to be used for The correlative study of vascular endothelial cell.
The separation of endothelial cell is evolved all in accordance with the method for Jaffe at present, using enzyme in 37 DEG C of water-bath perfusion digestions, But this digestion process is semi-enclosed environment, is easy pollution using water-bath, and need biggish container, 37 DEG C at a temperature of, Digestion time more difficult to control be easy to cause digestion excessively particularly with tiny vascular tissue, to influence the purity of cell.
Summary of the invention
The present invention studies a kind of isolation and culture method of simple umbilical vein vascular endothelial cells.
What the present invention was obtained through the following steps.
(1) umbilical cord for taking newborn 15-20cm long cuts off the part that there are clamp trace and hemotoncus in both ends, and vein blood vessel is discharged In crimson blood blood remove syringe needle with 50ml syringe suction part physiological saline, be inserted at umbilical cord vein blood vessel Single port, punching Umbilical cord vein blood vessel is washed, up to no blood flows out.
(2) umbilical cord seals one end with haemostatic clamp, and pouring into 0.1% Type I collagen enzyme of about 5mL from the other end with syringe fills it It is full of entire umbilical cord, clamps both ends port with umbilical cord clamps.
(3) umbilical cord is placed in 15cm culture dish, the physiological saline of 37 DEG C of preheatings is added, cover whole umbilical cord, digestion 35 ~ 45min, digestion finish opening haemostatic clamp, and digestive juice is flowed into preprepared centrifuge tube, is then rushed with physiological saline It washes lumen 3 times or more, efflux merging is collected in centrifuge tube, and 400g is centrifuged 8min.
(4) it uses 4ml endothelial cell complete medium culture medium that precipitating is resuspended from after the complete heart, is inoculated in 1 T25, sets In 5% CO2Stationary culture in 37 DEG C of incubators of saturation degree, full dose is carried out after cell culture 12-24h, and to change liquid not adherent to remove Cell and remnant tissue converge to 80% or more until cell is grown, and carry out cell passage.
(5) cell passes on: culture medium is outwelled, is cleaned 2 times with physiological saline, the trypsin digestion cell of 37 DEG C of preheatings is added, It observes under the microscope, once most cells are rounded, that is, 3 times of normal saline dilution reaction is added；With pipette by cell Piping and druming is got off, and is transferred in a 50ml sterile centrifugation tube, 400g, and 8min is centrifuged, and outwells supernatant, and fresh culture is resuspended, According to 1.1 × 104/cm2Inoculation passage.It is tested using the pervious cell of P3.
(6) it cellular identification: morphological observation: is both needed to carry out observing cellular morphology under inverted microscope before changing liquid passage And state, the growth of HUVEC adherent monolayers inlay arrangement in short fusiform or paving stone sample, cell is flat polygonal；Cell surface Immune labeled analyte detection: during skin progenitor cells are gradually divided into mature endothelial cell in the blood vessels, early stage stem cell The expression of surface marker CD133 declines, and CD34 and VEGFR-2 expression increases, and starts to express CD31, VIII factor Ⅷ related antigen The mark of the mature endothelial cells such as vWF (von Willebrand factor), attached cell are washed 2 times, 0.25% pancreatin/EDTA Cell suspension is made after digestion, filters, density is 1 × 106/ ml, every part of cell suspension take 150 μ L × 2 to dispense 2 test tubes, It is separately added into FITC-KDR, each 10 μ L of FITC-CD31, FITC-vWF, is protected from light 30 min, PBS washs 2 measurements, upper machine 10000 cells are collected afterwards, are as a result indicated with various antigen presentation positive percentages, at the detection of pipe ability: the every hole of 96 orifice plates After 400 μ lMatrigel, 37 DEG C of solidification 1h are added, HUVEC is inoculated with by the density in 5000/ hole, 10000/ hole, 20000/ hole (preferably 20000/ hole) is placed after incubator is incubated for 3 ~ 6h and is observed.
Separation HUVEC method of the invention is easy to operate, and entire digestion process is in station, and digestion temperature is slightly below It is 37 DEG C, appropriate to increase digestion time, excessive digestion not will cause for relatively fine vascular tissue, while avoiding environment With the pollution risk of other heteroproteose cells.Obtained endothelial cell morphology, the immunophenotype on surface and be typical case at pipe characteristic Endothelial cell characteristics, and remaining umbilical cord tissue still can be used for other cells such as umbilical cord mesenchymal stem cells separation training It supports.
Detailed description of the invention
Fig. 1 is the primary morphological observation A for being separately cultured Human umbilical vein endothelial cells: inoculation has a small amount of cell to paste afterwards for 24 hours Wall；B: cell Proliferation to 90% or more is converged.
Fig. 2 is the testing result of HUVEC vascularization ability.
Fig. 3 is umbilical vein vascular endothelial cells flow cytometer detection result.
Specific embodiment
Below with reference to specific implementation example, the present invention is described further, but protection scope of the present invention and not only limits In this.
(1) separation of HUVEC: taking the umbilical cord of newborn 15-20cm long, cuts off the part of clamp trace and hemotoncus, squeezes Blood in umbilical cord out repairs neat two section with scissors, is put into 15cm culture dish；Umbilical vein pipe is inserted into a 50ml syringe In, with normal saline flushing 3 times of 37 DEG C of preheatings, until being flowed out without blood；One end is sealed with haemostatic clamp, with syringe from the other end Pouring into about 5mL Type I collagen enzyme makes it fill entire umbilical cord, clamps port, umbilical cord is placed in 15cm culture dish, and preheating is added Physiological saline, cover whole umbilical cord, digest 30min, umbilical cord is often stirred during digestion, flows enzyme solutions in the blood vessels, Endothelial cell is promoted uniformly to contact with enzyme, digestion finishes opening haemostatic clamp, digestive juice flowed into preprepared centrifuge tube, Then it uses normal saline flushing lumen 3 times or more, efflux merging is collected in centrifuge tube, and 400g is centrifuged 8min, from weight after the complete heart Outstanding precipitating, is inoculated in 1 T25, is placed in stationary culture in 37 DEG C of incubators of 5% CO2 saturation degree, cell culture 12-24h is laggard Row full dose changes liquid to remove not adherent cell and remnant tissue, and culture supernatant is sucked out with 10ml pipette and abandons, separately takes one The 10ml pipette of Zhi Xin take fresh cultured be based on culture bottle, 4ml/ bottles.A subculture is changed within later 3 days, until cell is grown 80% or more is converged to, cell passage is carried out.
(2) passage of HUVEC: outwelling culture medium, is cleaned 2 times with physiological saline, and 37 DEG C of 1.5ml preheatings are added 0.25% trypsin digestion cell, is observed under the microscope, once most cells are rounded, that is, it is whole that 3 times of physiological saline is added Only react；Cell is blown and beaten with pipette, and is transferred in a 50ml sterile centrifugation tube, 400g, 8min is centrifuged, outwells Supernatant, fresh culture are resuspended, and are inoculated with and pass on according to 1:3, are tested using the pervious cell of P3.
(3) identification of HUVEC:
1) morphological observation: the growth of cell adherent monolayers inlays arrangement in short fusiform or paving stone sample, and cell is flat polygonal Shape is shown in Fig. 1；
2) detection of cell surface immune marker: attached cell is washed 2 times, and it is outstanding that cell is made after 0.25% pancreatin/EDTA digestion Liquid, filtering, density are 1 × 106/ ml, every part of cell suspension take 150 μ L × 2 to dispense 2 test tubes, are separately added into FITC- Each 10 μ L of KDR, FITC-CD31, FITC-vWF, is protected from light 30 min, and PBS washs 2 measurements, collects 10000 after upper machine As a result cell is indicated with various antigen presentation positive percentages, sees Fig. 3；
3) at the detection of pipe ability: after 400 μ lMatrigel, 37 DEG C of solidification 1h are added in the every hole of 96 orifice plates, by 5000/ hole, Density inoculation HUVEC (preferably 20000/ hole) in 10000/ hole, 20000/ hole is placed after incubator is incubated for 3 ~ 6h and is observed, sees figure 2。

Claims (10)

1. the technical solution present invention is mainly a kind of umbilical vein vascular endothelial cells (human umbilical vein Endothelial cell, HUVEC) primary separation, culture and identification method, comprise the steps that
(1) umbilical cord for taking newborn 15-20cm long cuts off the part that there are clamp trace and hemotoncus in both ends, and venous blood is discharged with tweezers Blood in pipe removes syringe needle with a 50ml syringe suction part physiological saline, is inserted at umbilical cord vein blood vessel Single port, Umbilical cord vein blood vessel is rinsed, up to no blood flows out；
(2) umbilical cord seals one end umbilical vein with haemostatic clamp, and pouring into 0.1% Type I collagen enzyme of about 5mL from the other end with syringe makes it Entire umbilical vein is filled, clamps both ends port with umbilical cord clamps；
(3) it is placed in 15cm culture dish, the physiological saline of 37 DEG C of preheatings is added, cover whole umbilical cord, digest 35 ~ 45min, disappear Change finishes opening umbilical cord clamps, and digestive juice is flowed into preprepared centrifuge tube, then uses normal saline flushing lumen at least 3 Time, efflux merging is collected in centrifuge tube, and 400g is centrifuged 8min；
(4) it uses 4ml vascular endothelial cell complete medium that precipitating is resuspended from after the complete heart, is inoculated in 1 T25, is placed in 5% CO2Stationary culture in 37 DEG C of incubators of saturation degree carries out full dose after cell culture 12-24h and changes liquid to remove not adherent cell And remnant tissue converges to 80% or more until cell is grown, and carries out cell passage；
(5) cell passes on: culture medium is outwelled, is cleaned 2 times with physiological saline, the TrypLE vitellophag of 37 DEG C of preheatings is added, 3 times of normal saline dilution reaction is added once most cells are rounded in microscopically observation；Cell is blown with pipette It lays and, and be transferred in a 50ml sterile centrifugation tube, 400g, be centrifuged 8min, outwell supernatant, fresh culture is resuspended, presses According to 1.1 × 104/cm2Inoculation passage, is tested using the pervious cell of P3；
(6) identification of HUVEC:
1) it morphological observation: is being both needed to carry out before changing liquid passage to observe cellular morphology and state, HUVEC under inverted microscope Adherent monolayers growth inlays arrangement in short fusiform or paving stone sample, and cell is flat polygonal；
2) the immune labeled analyte detection of cell surface: collecting the P3 cell of growth logarithmic phase, and attached cell is washed 2 times, 0.25% pancreas Cell suspension is made after enzyme/EDTA digestion, filters, density is 1 × 106/ ml, every part of cell suspension take 150 μ L × 2 to dispense 2 A test tube is separately added into FITC-KDR, each 10 μ L of FITC-CD31, FITC-vWF, is protected from light 30 min, and PBS washs 2 surveys It is fixed, 10000 cells are collected after upper machine, are as a result indicated with various antigen presentation positive percentages；
3) at the detection of pipe ability: after 400 μ lMatrigel, 37 DEG C of solidification 1h are added in the every hole of 96 orifice plates, by 5000/ hole, Density inoculation HUVEC (preferably 20000/ hole) in 10000/ hole, 20000/ hole is placed after incubator is incubated for 3 ~ 6h and is observed.
2. according to the method described in claim 1, it is characterized in that step (4) vascular endothelial cell complete medium, the culture Base be by DMEM/F12, low concentration fetal calf serum (2%), hEGF, cortisol, gentamicin and amphotericin B, hVEGF, HbFGF, R3-IGF-1, ascorbic acid and heparin sodium are formulated.
3. according to the method described in claim 1, it is characterized in that Type I collagen enzyme concentration is 0.1% in step (2)；Use volume For 0.4ml/cm long blood vessel；Digestion time is 40min.
4. according to the method described in claim 1, it is characterized in that step (3) digestion operating process in Biohazard Safety Equipment into Row, required Type I collagen enzyme and physiological saline need to be preheated to 37 DEG C in advance.
5. making it fill entire navel according to the method described in claim 4, pouring into Type I collagen enzyme from umbilical cord one end with syringe Band clamps both ends port with umbilical cord clamps, umbilical cord is placed in 15cm culture dish, and the physiological saline of 37 DEG C of preheatings, covering is added Whole umbilical cord.
6. according to the method described in claim 1, it is characterized in that step (5), is digested after obtaining HUVEC using pancreatin, with 1.1 ×104/cm2Density inoculation be passaged to 175cm2It is interior, it is put into 37 DEG C, 5%CO2, cultivated in saturated humidity.
7. according to the method described in claim 1, it is characterized in that it is 15cm culture dish that digestion, which uses container, in step (3).
8. according to the method described in claim 1, it is characterized in that umbilical cord is interior acquisition for 24 hours in step (1).
9. according to the method described in claim 1, it is characterized in that the identification method in step (6) is cellular morphology state, stream Formula detects cell surface marker FITC-KDR, and the detection of the vascularization ability of FITC-CD31, FITC-vWF and cell is identified Umbilical vein vascular endothelial cells.
10. the every hole of 96 orifice plates is added 400 according to according to the method described in claim 9, the process of vascularization ability detection is After μ lMatrigel, 37 DEG C of solidification 1h, it is inoculated with HUVEC by the density in 5000 ~ 20000/ holes (preferably 20000/ hole), places culture Case is incubated for the preferred 4h of 3 ~ 6h() after, it takes pictures under the microscope.
CN201710946120.4A 2017-10-12 2017-10-12 A kind of the primary of human umbilical vein endothelial cell is separately cultured and identification method Pending CN109652361A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110195037A (en) * 2019-05-29 2019-09-03 西安交通大学 A kind of human umblilical vein endothelial primary separation and culture method
CN116179475A (en) * 2023-04-23 2023-05-30 北京国卫生物科技有限公司 Isolated culture method of human umbilical vein vascular endothelial cells

Citations (2)

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WO2008110570A1 (en) * 2007-03-13 2008-09-18 Medizinische Universität Graz Method to study proliferation of endothelial progenitor cells and the potential influence of compounds on their proliferation behaviour
CN105969720A (en) * 2016-07-29 2016-09-28 上海瑞鹿生物技术有限公司 Human vascular endothelial cell culture solution and culture method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008110570A1 (en) * 2007-03-13 2008-09-18 Medizinische Universität Graz Method to study proliferation of endothelial progenitor cells and the potential influence of compounds on their proliferation behaviour
CN105969720A (en) * 2016-07-29 2016-09-28 上海瑞鹿生物技术有限公司 Human vascular endothelial cell culture solution and culture method

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110195037A (en) * 2019-05-29 2019-09-03 西安交通大学 A kind of human umblilical vein endothelial primary separation and culture method
CN116179475A (en) * 2023-04-23 2023-05-30 北京国卫生物科技有限公司 Isolated culture method of human umbilical vein vascular endothelial cells

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