CN104371968A - Method for separating and purifying mouse lung microvascular endothelial cell - Google Patents
Method for separating and purifying mouse lung microvascular endothelial cell Download PDFInfo
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- CN104371968A CN104371968A CN201410285772.4A CN201410285772A CN104371968A CN 104371968 A CN104371968 A CN 104371968A CN 201410285772 A CN201410285772 A CN 201410285772A CN 104371968 A CN104371968 A CN 104371968A
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Abstract
A method for separating and purifying mouse lung microvascular endothelial cell is characterized by comprising using an enzyme to perform digestive treatment on subpleural lung marginal tissue to obtain a single-cell suspension, and performing CD31 immunomagnetic bead sorting to obtain the target cell. Concretely, the method comprises getting subpleural lung marginal tissue of a large suckling mouse, firstly using a mixed solution of collagenase II and neutral protease for digestion to prepare the single-cell suspension, then performing incubation combination with CD31 immunomagnetic bead, then utilizing a magnetic bead sorting purification system to screen CD31+ cell, and removing magnetic beads combined with CD31+ cell by using a magnetic-bead release solution, so as to obtain high-purity mouse lung microvascular endothelial cell. The advantages comprise that the high-purity mouse lung microvascular endothelial cell can be obtained from mouse lung tissue through separation extraction by using enzyme digestion to prepare the single-cell suspension and performing CD31 immunomagnetic bead sorting.
Description
Technical field
The present invention relates to a kind of method of separation and Extraction high purity capillary endothelium from lung tissue of rats.
Background technology
Capillary endothelium is positioned at capillary blood vessel internal surface, nutritive substance, virulence factor, cytokine and medicine etc. by blood circulation intravasation organize outward must through barrier, function has diversity, is the crucial point of adjustment of the basic pathology changes such as blood coagulation, hyperemia, hemostasis, oedema, inflammation, immunity, tumour generation.The vitro culture of capillary endothelium provides important channel for understanding its biological characteristics in depth, significant to the approach and drug disease researching and developing disease caused by protection and treatment Function of Capillary Endothelial Cells obstacle.
The capillary endothelium phenotype of different tissues organ and function have remarkable heterogeneity, and the research of a certain visceral relationship physiological and pathological mechanism needs to cultivate corresponding capillary endothelium.Although the report that the histoorgan microvascular endothelial cell in vitros such as the current existing many animals heart, lung, skin, brain are cultivated, but because microvascular caliber is little, be scattered in histoorgan inside, can not direct perfusion digestion liquid or effectively isolate capillary blood vessel section, the separating and purifying technology of capillary endothelium is still a difficult problem, is difficult to the pollution avoiding non-capillary endothelium.
The vitro culture of PMEC (Pulmonary Microvascular Endothelial Cells) is current mainly to be taked enzyme digestion and organizes Explant culture, enzyme digestion easily obtains a large amount of capillary endothelium in a short time, but non-capillary endothelium can evenly mix wherein, simple manual operations is difficult to carry out purifying; Organize Explant culture also because the difference of tissue sample and treatment process cannot be determined best to go the block time, be difficult to avoid swimming out of of PMEC (Pulmonary Microvascular Endothelial Cells) and pollute.
Enzyme digestion and immunomagnetic bead technique combine by the present invention, can be separated fast, be purified to highly purified capillary endothelium from lung tissue of rats.
Summary of the invention
The object of this invention is to provide a kind of method of separation and Culture high purity Pulmonary Microvascular Endothelial Cells in Rats.
The object of the invention is to be achieved through the following technical solutions.
From a method for lung tissue of rats separation and Culture high purity capillary endothelium, its step is as follows.
A. the neck that broken by large suckling mouse is put to death, and cuts thoracic cavity open, right ventricle perfusion Hank ' s liquid, and aseptic taking-up lungs, reject visceral pleura, clip margo border of the lung tissue, remove great vessels, shred as fritter; Described right ventricle perfusion Hank ' s liquid, in 4oC precooling, pours into lungs and turns white; The described volume shredded as fritter is about 1 mm
3.
B. by the rinsing of described lung tissue fritter Hank ' s liquid, add in Digestive system and digest; Described Digestive system is 0.2% collagenase
with 0.1% neutral protease mixing solutions, digestion temperature is 37oC, and digestion time is 20 ~ 30 min.Described postdigestive solution is crossed 200 order cell sieves, collection filtrate is centrifugal, and sedimentation cell group is resuspended with 0.01 M PBS is single cell suspension; Described centrifugal treating, rotating speed is 800 ~ 1200 rpm, and the time is 5 ~ 10 min; Described 0.01 M PBS contains 0.1% bovine serum albumin, 2 mM EDTA; Described single cell suspension density is 1 × 10
7individual/mL.
C. add CD31 immunomagnetic beads by described single cell suspension, hatch and obtain cell-antibody-bead complexes.Described CD31 immunomagnetic beads is that CD31 monoclonal antibody and magnetic bead are hatched to combine and obtained, and temperature is 4oC, and the time, density was 4 × 10 in order to spend the night
8individual/mL, every milliliter of single cell suspension adds CD31 immunomagnetic beads 25 μ L; Described hatches process, is placed in rotary shaker, and temperature is 15 ~ 25 oC, and the time is 30 ~ 60 min.
D. described cell-antibody-bead complexes is never separated in conjunction with in magnetic bead and cell; Described separating treatment uses KingFisher 96 magnetic bead sorting purification system, and programming is: mix at a slow speed 30 s, collects 2 times, each 5 s; Repeat 4 times, cell-antibody-bead complexes is released to the DMEM substratum containing 5% foetal calf serum, 1 mM calcium chloride and 4 mM magnesium chlorides.
E. the magnetic bead of buffer release liquid removal cell surface combination will be added in described cell-antibody-bead complexes suspension; Described buffer release liquid is DNase
solution, every ml cells suspension adds 4 μ L, and temperature is room temperature, and action time is 10 ~ 20 min; Described removal magnetic bead KingFisher 96 magnetic bead sorting purification system, programming is: middling speed mixes 1 min, and collect 2 times, each 1 s, middling speed discharges 3 s; Repeat 1 time.
F. by the CD31+ cell centrifugation of described removal magnetic bead, resuspended with perfect medium, cultivation is hatched in inoculation; Described centrifugal treating, rotating speed is 800 ~ 1200 rpm, and the time is 5 ~ 10 min; Described perfect medium is DMEM substratum, containing 20% foetal calf serum, 2 mM L-glutaminate, 100 IU penicillin and 100 μ g/mL Streptomycin sulphates; Described inoculation culture cell density is 1 × 10
5individual/mL.
G. described inoculation culture cell changes a perfect medium in every 3 days, to Secondary Culture during subconfluent state; The 0.01 M PBS solution digestion de-wall of described Secondary Culture containing 0.05% trypsinase and 0.005% EDTA, the density that goes down to posterity is 1 × 10
5individual/mL.
Embodiment
Embodiment one.
1. major experimental material.
(1) laboratory animal: the large suckling mouse of 7 age in days SD.
(2) Hank ' s liquid: purchased from GIBCO company, article No. 14170-112.
(3) 0.01 M PBS: get NaCl 8.0 g, Na
2hPO
42.9 g, KH
2pO
40.2 g, KCl 0.2 g, is dissolved in 1000 mL ultrapure waters, 0.22 μm of filtration sterilization, in 4 DEG C of freezer storages.
(4) Digestive system: prepare, containing 0.2% collagenase with 0.01 M PBS
(purchased from Worthington company, article No. LS004176) and 0.1% neutral protease
(available from Sigma, article No. D4693-1G), 0.22 μm of sterile filters filtration sterilization.
(5) perfect medium: DMEM basic medium is (purchased from GIBCO company, article No. 11960-044), containing 20% foetal calf serum (purchased from GIBCO company, article No. 10099-141), 2 mM L-glutaminate are (purchased from GIBCO company, article No. 25030-081), 1% dual anti-solution (100 IU/ml penicillin and 100 μ g/ml Streptomycin sulphates, purchased from GIBCO company, article No. 15140-122).
(6) little anti-rat CD31 monoclonal antibody: purchased from AbD Serotec company, article No. MCA 1334G, specification is that 200 μ l(are containing 200 μ g), every 25 μ l magnetic beads use 0.5 μ g antibody.
(7) mouse IgG magnetic bead kit: comprising 5 ml density is 4 × 10
8individual/mL bag is by the magnetic bead of anti-mouse IgG, and 3 pipe buffer release liquid composition 1(often pipe are 15000 ~ 20000 U freeze-drying DNase
), and 2 ml buffer release liquid compositions 2.The often pipe composition 1 that is formulated as of buffer release liquid adds 0.3ml composition 2 and dissolves, and packing is frozen, and every ml sample adds 4 μ l.
(8) sample buffer: prepare with 0.01 M PBS, containing 0.1% bovine serum albumin and 2 mM EDTA, 0.22 μm of sterile filters filtration sterilization.
(9) magnetic bead scavenging solution: prepare with 0.01 M PBS, containing 0.1% bovine serum albumin, 0.22 μm of sterile filters filtration sterilization.
(10) EDTA-trypsin solution: prepare, containing 0.005% with 0.01 M PBS.
2. operation steps.
The large suckling mouse of 7 age in days 5, disconnected neck is put to death, and cuts thoracic cavity open, and right ventricle perfusion 4oC precooling Hank ' s liquid turns white to lungs, and aseptic taking-up lungs, reject visceral pleura, clip margo border of the lung tissue, and remove great vessels, shredding is 1 mm
3fritter, the rinsing of Hank ' s liquid, adds Digestive system 37oC and digests 20 min, extremely without shaping lung tissue fritter.Digestive system crosses 200 order cell sieves, and collect centrifugal 10 min of filtrate 1000 rpm, sedimentation cell group is resuspended with 10 ml sample buffers, and density is about 1 × 10
7individual/mL.
Get 250 μ l to wrap by the magnetic bead of anti-mouse IgG, wash with reference to specification sheets magnetic bead scavenging solution, be resuspended in 250 μ l magnetic bead scavenging solutions, add 5 μ l little anti-rat CD31 monoclonal antibody, be placed in rotary shaker in 4oC overnight incubation, non-binding antibody is washed away with magnetic bead scavenging solution, the magnetic bead being combined with CD31 antibody is resuspended in 250 μ l magnetic bead scavenging solutions, add 10 described ml sample buffers, be placed in rotary shaker, hatch 30 min in 20oC, make cell and CD31 immunomagnetic beads be combined into cell-antibody-bead complexes.
Utilize KingFisher 96 magnetic bead sorting purification system by cell-antibody-bead complexes and unconjugated cell and Beads enrichment, programming is: mix at a slow speed 30 s, collects 2 times, each 5 s; Repeat 4 times, cell-antibody-bead complexes is released to the DMEM substratum containing 5% foetal calf serum, 1 mM calcium chloride and 4 mM magnesium chlorides.4 μ L buffer release liquid are added in every 1 ml ml cells-antibody-bead complexes, incubated at room 15 min, utilize KingFisher 96 magnetic bead sorting purification system to remove the magnetic bead of release, programming is: middling speed mixes 1 min, collect 2 times, each 1 s, middling speed discharges 3 s; Repeat 1 time, obtain the CD31+ cell removing magnetic bead, centrifugal 10 min of 1000 rpm, it is resuspended that sedimentation cell group is resuspended in perfect medium, by 1 × 10
5individual/mL density inoculation culture.
Inoculation culture cell changes a perfect medium in every 3 days, and to subconfluent state, take off wall with the digestion of EDTA-trypsin solution and go down to posterity, the density that goes down to posterity is 1 × 10
5individual/mL.
3. cultivation results.
The high-purity C D31+ PMEC (Pulmonary Microvascular Endothelial Cells) of original cuiture grows to converging state in inoculation after 7 days, and cell is short fusiformis or polygon, and nucleus is obvious, and in typical " pebbles sample " outward appearance after converging, individual layer zyklopisch aligned growth, is shown in Fig. 1.After Secondary Culture, within 5-7 days, regrow to converging state, there is good growth performance.
The PMEC (Pulmonary Microvascular Endothelial Cells) (scale is 200 μm) of Fig. 1 vitro culture growth subconfluent state
Claims (2)
1. the method for separation, purifying capillary endothelium from a lung tissue of rats, it is characterized in that: described method relates generally to the preparation of the single cell suspension of animal lung tissue and the immunological magnetic bead sorting of CD31+ cell, compare with traditional lung tissue capillary endothelium separation purification method and there is the high feature of purity.
2. the method for a kind of separation, purifying capillary endothelium from animal lung tissue according to claim 1, is characterized in that the method comprises the following steps:
A. the neck that broken by large suckling mouse is put to death, and cuts thoracic cavity open, right ventricle perfusion Hank ' s liquid, and aseptic taking-up lungs, reject visceral pleura, clip margo border of the lung tissue, remove great vessels, shred as fritter; Described right ventricle perfusion Hank ' s liquid, in 4oC precooling, pours into lungs and turns white; The described volume shredded as fritter is about 1 mm
3;
B. by the rinsing of described lung tissue fritter Hank ' s liquid, add in Digestive system and digest; Described Digestive system is 0.2% collagenase
with 0.1% neutral protease mixing solutions, digestion temperature is 37oC, and digestion time is 20 ~ 30 min;
Described postdigestive solution is crossed 200 order cell sieves, collection filtrate is centrifugal, and sedimentation cell group is resuspended with 0.01 M PBS is single cell suspension; Described centrifugal treating, rotating speed is 800 ~ 1200 rpm, and the time is 5 ~ 10 min; Described 0.01 M PBS contains 0.1% bovine serum albumin, 2 mM EDTA; Described single cell suspension density is 1 × 10
7individual/mL;
C. add CD31 immunomagnetic beads by described single cell suspension, hatch and obtain cell-antibody-bead complexes;
Described CD31 immunomagnetic beads is that CD31 monoclonal antibody and magnetic bead are hatched to combine and obtained, and temperature is 4oC, and the time, density was 4 × 10 in order to spend the night
8individual/mL, every milliliter of single cell suspension adds CD31 immunomagnetic beads 25 μ L; Described hatches process, is placed in rotary shaker, and temperature is 15 ~ 25oC, and the time is 30 ~ 60 min;
D. described cell-antibody-bead complexes is never separated in conjunction with in magnetic bead and cell; Described separating treatment uses KingFisher 96 magnetic bead sorting purification system, and programming is: mix at a slow speed 30 s, collects 2 times, each 5 s; Repeat 4 times, cell-antibody-bead complexes is released to the DMEM substratum containing 5% foetal calf serum, 1 mM calcium chloride and 4 mM magnesium chlorides;
E. the magnetic bead of buffer release liquid removal cell surface combination will be added in described cell-antibody-bead complexes suspension; Described buffer release liquid is DNase
solution, every ml cells suspension adds 4 μ L, and temperature is room temperature, and action time is 10 ~ 20 min; Described removal magnetic bead KingFisher 96 magnetic bead sorting purification system, programming is: middling speed mixes 1 min, and collect 2 times, each 1 s, middling speed discharges 3 s; Repeat 1 time;
F. by the CD31+ cell centrifugation of described removal magnetic bead, resuspended with perfect medium, cultivation is hatched in inoculation; Described centrifugal treating, rotating speed is 800 ~ 1200 rpm, and the time is 5 ~ 10 min; Described perfect medium is DMEM substratum, containing 20% foetal calf serum, 2 mM L-glutaminate, 100 IU penicillin and 100 μ g/mL Streptomycin sulphates; Described inoculation culture cell density is 1 × 10
5individual/mL;
G. described inoculation culture cell changes a perfect medium in every 3 days, to Secondary Culture during subconfluent state; The 0.01 M PBS solution digestion de-wall of described Secondary Culture containing 0.05% trypsinase and 0.005% EDTA, the density that goes down to posterity is 1 × 10
5individual/mL.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673744A (en) * | 2015-03-10 | 2015-06-03 | 王俊力 | In-vitro culturing method for vascular endothelial cells of rat |
| CN104877955A (en) * | 2015-06-12 | 2015-09-02 | 北京农学院 | Method for separating and purifying rat dermal microvascular endothelium cells |
| CN104946580A (en) * | 2015-07-06 | 2015-09-30 | 中国人民解放军第四军医大学 | Isolated culturing and identifying method for lung microvascular endothelial cells of rat |
| CN107034181A (en) * | 2017-03-06 | 2017-08-11 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method of efficient people's buccal fat pad fat stem cell |
| CN109055301A (en) * | 2018-08-17 | 2018-12-21 | 湖南师范大学 | A kind of brain fine vascular process for separation and purification |
| CN109852575A (en) * | 2018-12-27 | 2019-06-07 | 河北生命原点生物科技有限公司 | A kind of separation method of alveolar type II cells |
| CN110357964A (en) * | 2019-08-09 | 2019-10-22 | 无锡傲锐东源生物科技有限公司 | 1 protein monoclonal antibody of AntiCD3 McAb and application thereof |
| CN113564100A (en) * | 2021-07-30 | 2021-10-29 | 广东省第二人民医院（广东省卫生应急医院） | Method for extracting and purifying brain microvascular endothelial cells |
| CN114908033A (en) * | 2022-04-18 | 2022-08-16 | 广东莱迪生物医药研究院有限公司 | Method for sorting cells by virtue of large-batch magnetic beads |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101437937A (en) * | 2006-03-07 | 2009-05-20 | 哈玛赛特公司 | Method for endothelial cell extraction from adipose tissues |
-
2014
- 2014-06-25 CN CN201410285772.4A patent/CN104371968A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101437937A (en) * | 2006-03-07 | 2009-05-20 | 哈玛赛特公司 | Method for endothelial cell extraction from adipose tissues |
Non-Patent Citations (4)
| Title |
|---|
| KATHARINA SCHELLER等: "Upcyte Microvascular Endothelial Cells Repopulate Decellularized Scaffold", 《TISSUE ENGINEERING: PART C》 * |
| 刘勇军等: "改良大鼠肺微血管内皮细胞原代培养技术及细胞鉴定", 《中华损伤与修复杂志( 电子版)》 * |
| 胡国栋等: "一种改良的肺微血管内皮细胞培养方法", 《南方医科大学学报》 * |
| 颜春晓等: "免疫磁珠法原代分离培养人子宫内膜癌微血管内皮细胞及鉴定", 《中华肿瘤防治杂志》 * |
Cited By (11)
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| CN104673744A (en) * | 2015-03-10 | 2015-06-03 | 王俊力 | In-vitro culturing method for vascular endothelial cells of rat |
| CN104673744B (en) * | 2015-03-10 | 2018-05-25 | 武汉市中西医结合医院 | A kind of vascular endothelial cells extracorporeal culturing method |
| CN104877955A (en) * | 2015-06-12 | 2015-09-02 | 北京农学院 | Method for separating and purifying rat dermal microvascular endothelium cells |
| CN104946580A (en) * | 2015-07-06 | 2015-09-30 | 中国人民解放军第四军医大学 | Isolated culturing and identifying method for lung microvascular endothelial cells of rat |
| CN107034181A (en) * | 2017-03-06 | 2017-08-11 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method of efficient people's buccal fat pad fat stem cell |
| CN109055301A (en) * | 2018-08-17 | 2018-12-21 | 湖南师范大学 | A kind of brain fine vascular process for separation and purification |
| CN109055301B (en) * | 2018-08-17 | 2021-09-10 | 湖南师范大学 | Method for separating and purifying cerebral micro-blood vessels |
| CN109852575A (en) * | 2018-12-27 | 2019-06-07 | 河北生命原点生物科技有限公司 | A kind of separation method of alveolar type II cells |
| CN110357964A (en) * | 2019-08-09 | 2019-10-22 | 无锡傲锐东源生物科技有限公司 | 1 protein monoclonal antibody of AntiCD3 McAb and application thereof |
| CN113564100A (en) * | 2021-07-30 | 2021-10-29 | 广东省第二人民医院（广东省卫生应急医院） | Method for extracting and purifying brain microvascular endothelial cells |
| CN114908033A (en) * | 2022-04-18 | 2022-08-16 | 广东莱迪生物医药研究院有限公司 | Method for sorting cells by virtue of large-batch magnetic beads |
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