Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

마우스에서 대동맥 1. 분리 여기에 설명 된 모든 절차가 웨인 주립 대학의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. <ol><li> 마취 기계의 유도 챔버에 마우스를 넣습니다. 25 % 신선한 공기와 75 % O 2와 함께 4 %의 이소 플루 란 흐름을 설정합니다. 전용 코 콘 뒤에 유도 챔버를 통해 25 %의 신선한 공기와 75 % O 2와 함께 이소 플루 란 (유도 4 %가 유지 보수를 위해 ± 1.0 %)의 흡입에 의해 마우스를 마취. 주 : 이소 플루 란 마취 75 % 산소 / 25 % 공기, 100 % 산소로 전달 될 수있다. </li><li> 마취의 적절한 수준 (발가락 핀치에 철수의 부족)에 도달 할 때까지 마우스마다 5 분을 확인합니다. </li><li> 유도 챔버에서 마우스를 가지고 마취 기계에 연결되어있는 전용 코 콘하지만 이소 플루 란 흡입을 계속합니다. 스위치이소 플루 란 마취를 유지하기 위해 1 % 흐른다. </li><li> 이 마취이지만, 그 뒷면에 위치하는 수술 패드에 마우스를 넣어. 사지를 확보하기 위해 실험실 테이프를 사용합니다. </li><li> 마우스 따뜻하게 유지하기 위해 가열 램프를 사용합니다. 마우스를하지 않습니다 과열 있도록 마우스에서 적절한 거리에 램프를 유지합니다. </li><li> 70 % 에탄올로 가슴 스프레이. </li><li> 정중선에서 복부를 열고 복부 대동맥을 노출 해부 가위를 사용합니다. 흉강을 열고 심장과 폐를 노출합니다. </li><li> 피를 해제 해부 가위로 중간에서 복부 대동맥을 잘라. </li><li> 헤파린 1000 U / mL로 포함하는 PBS 1 mL를 (25 G 바늘) 1 ML의 주사기를 입력합니다. 좌심실에 헤파린 1000 U / mL로 포함 된 PBS를 주입하고 대동맥을 관류. 흉부 대동맥을 노출 마우스의 오른쪽에 큰 동맥에서 집게로 심장과 폐를 밀어 넣습니다. </li><li> 신속 마이크로 다이를 사용하여 대동맥을 제거ssection 집게와 얼음처럼 차가운 1X PBS (멸균)에 넣어은 다음 층류 후드에 컨테이너를 운반 할 것. </li><li> 대동맥의 한쪽 끝으로 25 G 바늘 장착 된 1 ML의 주사기를 삽입하고 부드럽게 혈액을 제거하는 얼음처럼 차가운 PBS와 대동맥을 세척하십시오. </li><li> 가능한 한 첨부 된 지방 조직의 훨씬 작은 측면 혈관을 제거하기 위해 마이크로 해부 집게를 사용합니다. </li><li> 즉시 성장 배지 내피 대동맥 옮긴다. </li><li> 멸균 메스 블레이드를 사용하여 1mm 반지로 대동맥을 잘라. 수확 약 8 - 대동맥 당 10 반지입니다. </li><li> 마이크로 해부 가위를 사용하여 각 대동맥 고리를 엽니 다. </li></ol> 2. 종자 매트릭스의 대동맥 세그먼트 <ol><li> 사용하지 않을 응고 방지 -20 ° C에서 성장 인자 - 감소 된 매트릭스를 유지하는 경우. 완전한 해빙을 허용하기 위해 적어도 하룻밤 4 ° C에서 성장 인자 감소 행렬을 넣습니다. </li><li> 적어도 -20 ℃로 6 웰 플레이트 및 피펫 팁을 사전 냉각10 분. 공기 방울을 도입하지 않고 행렬의 1 mL를 플레이트의 얼음과 코트에 잘 하나를 6 웰 플레이트를 넣습니다. 20 분 행렬이 응고 할 수 있도록하기위한 37 ° C 배양기에서 접시를 놓습니다. </li><li> 멸균 미세 해부 집게를 사용하여 응고 매트릭스 상 대동맥 개 임플란트. 내피를 건드리지 않고 루멘 측 다운 행렬에이 조각을 놓습니다. 매트릭스에 서로에 가까운 4 대동맥 세그먼트 - 3를 배치합니다. </li><li> (~ 200 μL) 젖은 세그먼트를 유지하기 위해 충분한 내피 세포 성장 매체를 추가합니다. </li><li> 4 ~ 6 시간 동안 5 % CO 2에서 37 ° C에서 접시를 품어. 하루가 끝날 때, 대동맥 세그먼트를 커버하도록 충분한 매체를 추가한다. </li><li> 위상 대비 현미경으로 주기적으로 돋아 대동맥 세그먼트를 관찰한다. 중간 수준 및 세포 성장 매일 확인합니다. 적용 대동맥 세그먼트를 유지하기 위해 필요한 경우 매체를 추가합니다. </li><li> 4 번째 날, 부드럽게 매체를 제거하고 대동맥 세그먼트 (F)을 제거성장 내피 세포의 중단없이 멸균 바늘을 사용하여 매트릭스 ROM. </li><li> 새로운 내피 세포 성장 배지 2 mL를 넣고과 내피 세포가 2 매트릭스 증식을 계속 허용 - 삼일. </li></ol> 마우스 대동맥 내피 세포의 3. 초기과 Passaging <ol><li> 젤라틴 코트 (0.1 %)로 새로운 T12.5 플라스크를 30 분 동안 37 ℃에서 배양한다. </li><li> 멸균 37 ° C PBS로 조심스럽게 매트릭스 판을 씻으십시오. </li><li> 매트릭스 판에 중립 단백질 분해 효소 (50 U / ㎖)의 2 mL를 넣고 가끔 흔들어와 플랫폼 로커에 실온에서 품어. 세포의 대부분이 분리되어 있는지 확인하기 위해 위상차 현미경으로 세포를 확인합니다. </li><li> 중립 단백질 분해 효소를 불 활성화 D-발 2 ML을 추가합니다. </li><li> 15 mL의 원심 분리기 튜브에 조심스럽게 상층 액을 수집합니다. 2 mL의 D-발과 접시를 씻고 뜨는를 수집합니다. </li><li> 타나에서 5 분 동안 900 XG에 세포 현탁액을 원심m 온도. </li><li> 젤라틴으로 코팅 T12.5 플라스크 내피 세포 성장 배지 플레이트 4 ㎖의 세포 펠렛을 다시 일시. 2 시간 동안 37 ° C에서 5 % CO 2에서 세포를 인큐베이션. </li><li> 매체를 교체합니다. 90 % 합류 -가 85 %가 될 때까지 37 ° C, 5 % CO 2에서 세포를 인큐베이션. </li></ol> 마우스 대동맥 내피 세포의 (4)과 Passaging <ol><li> 코트 두 개의 새로운 T12.5 플라스크는 젤라틴 (0.1 %)로, 30 분 동안 37 ° C에서 품어. 약 15 분 동안 37 ℃에서 예열 트립신 - EDTA (0.25 % 트립신, PBS 0.02 EDTA) 및 멸균 PBS. </li><li> 멸균 37 ° C PBS로 조심스럽게 세포를 씻으십시오. </li><li> 트립신 - EDTA (0.25 % 트립신, PBS 0.02 EDTA) 0.5 mL를 추가하고 ~ 1 분 또는 세포의 대부분이 라운드를 설정할 때까지 37 ° C에서 품어. </li><li> 소화를 중지 내피 세포 성장 매체의 2 ML을 추가합니다. 위아래로 수회 플라스크 내의 세포 현탁액 피펫. 필요한 경우, 셀을 사용세포를 수집하는 스크레이퍼. </li><li> 15 mL의 원심 분리 튜브에 세포 현탁액을 수집한다. 실온에서 5 분 동안 900 XG에 세포 현탁액을 원심 분리기. </li><li> 젤라틴으로 코팅이 T12.5 플라스크 내피 세포 성장 배지 플레이트 4 ㎖의 세포 펠렛을 다시 일시. 2 시간 동안 37 ° C에서 5 % CO 2에서 세포를 인큐베이션. </li><li> 매체를 교체합니다. 90 % 합류 -가 85 %가 될 때까지 37 ° C, 5 % CO 2에서 세포를 인큐베이션. </li><li> 3 통로 -이 후 마우스 대동맥 내피 세포를 사용합니다. </li></ol> 마우스 대동맥 내피 세포의 5. 특성 <ol><li> 다시 플레이트 세포. <ol><li> 젤라틴 코트 (0.1 %)을 6- 웰 플레이트는 30 분 동안 37 ℃에서 배양한다. PBS와 함께 접시를 씻어. </li><li> 사전 가열 약 15 분 동안 37 ° C에 트립신 - EDTA 및 멸균 PBS를. </li><li> 멸균 PBS 3 회 세포를 씻으십시오. 세포에 0.5 트립신의 용액이 / EDTA를 추가, 37 ° C 오에 ~ 1 분 동안 품어세포의 대부분까지 r은 라운드를 켭니다. </li><li> 소화를 중지 내피 세포 성장 매체의 2 ML을 추가합니다. 위아래로 수회 플라스크 내의 세포 현탁액 피펫. 필요한 경우, 세포를 수집하고 세포 스크레이퍼를 사용한다. </li><li> 15 mL의 원심 분리 튜브에 세포 현탁액을 수집한다. 실온에서 5 분 동안 900 XG에 세포 현탁액을 원심 분리기. </li><li> 내피 세포 성장 배지에서 세포를 다시 일시 중지, 상층 액을 버린다. 잘 3 × 10 5 /의 밀도에 젤라틴으로 코팅 된 6 웰 플레이트에서 세포를 시드. </li><li> 세포 배양 표면에 부착 할 수 있도록 밤새 37 ° C, 5 % CO 2에서 세포를 인큐베이션. </li></ol></li><li> DIL-AC-LDL 바인딩 -lectin uptaken 및 Ulex. <ol><li> 의 1,1&#39;- 디 옥타 데실 3,3,3- &#39;를 저장 3&#39;- tetramethylindo - 카보시 과염소산 염 표지 -20 ° C에서 아세틸 화 LDL (DIL-AC-LDL). 염색하기 전에 얼음에 DIL-AC-LDL을 해동. 주식 DIL-AC-LDL의 SOLU기는 / mL의 1 밀리그램이다. 4 ° C에서 FITC 표지 Ulex의 europaeus의 응집소 (Ulex -Lectin, 1 μg의 / ㎖)를 저장합니다. 주식 Ulex -Lectin 솔루션은 1 ㎎ / ㎖이다. 저장 4 ° C에서의 Hoechst 33342. 주식의 Hoechst 33342 솔루션은 100 μg의 / mL로한다. </li><li> 10 μg / ML의 최종 농도를 만들어 배지 1 ㎖에 DIL-AC-LDL 스톡 용액 10 μL를 추가한다. </li><li> 염색하기 전에, 구 배지를 제거하고 새로운 배지를 각 웰에 세포를 세척 하였다. </li><li> 새로운 매체를 제거하고 DIL-AC-LDL 염색 매체를 추가 할 수 있습니다. </li><li> 1 시간 동안 37 ℃로, 5 % CO 2에서 세포를 인큐베이션. </li><li> 멸균 1X PBS 3 회 세포를 씻으십시오. </li><li> 세포를 해결하기 위해 10 % 포르말린 1 ML을 추가합니다. 약 30 분 동안 실온에서 접시를 넣습니다. </li><li> 멸균 1X PBS 3 회 세포를 씻으십시오. 빛을 피하십시오. </li><li> Ulex -Lectin 10 μL를 포함 1 mL의 멸균 1X PBS를 추가합니다. </li><li> 객실 temperatu에서 세포를 품어1 시간 동안 어둠 속에서 재. </li><li> 멸균 1X PBS 3 회 세포를 세척하여 Ulex-렉틴을 제거합니다. </li><li> 1 μg의 / ㎖의 최종 농도 훽스트 33342를 추가 어둠 속에서 10 분을 품어. </li><li> DIL-AC-LDL (빨강, 576 나노 미터의 여기 파장), Ulex -Lectin (녹색, 519 나노 미터의 여기 파장) 및 훽스트의 형광보기 (파란색, 361 나노 미터의 여기 파장) 거꾸로 형광 현미경. </li></ol></li><li> CD31, VEGFR2, eNOS에 대한 형광 염색, VE-cadherin의 및 Calponin합니다. <ol><li> 스토어 FITC 접합 항 - 마우스 CD31 항체, eNOS의 항체, VE-cadherin의의 4 ° C에서 항체, FITC 접합 항 - 토끼 IgG를을 어두운. -20 ° C에 보관 VEGFR2 항체. </li><li> 멸균 1X PBS 3 회 세포를 씻으십시오. </li><li> 세포를 해결하기 위해 10 % 포르말린 1 ML을 추가합니다. 약 30 분 동안 실온에서 접시를 넣습니다. </li><li> 멸균 1X PBS 3 회 세포를 씻으십시오. </li><li> CD31 얼룩하려면 1 mL의 멸균 1 배를 추가CD31-FITC 항체의 10 μL를 포함 PBS. 각각-VE cadherin의 또는 차 항체 calponin, 얼룩 하나 VEGFR2은 이노스는-VE cadherin의 또는 calponin, VEGFR2, eNOS의 4 μL를 포함 멸균 1X PBS의 1 mL를 넣어합니다. </li><li> 어둠 속에서 1 시간 동안 얼음에 세포를 품어. </li><li> 멸균 PBS 3 회 세포를 세척하여 항체를 제거합니다. </li><li> VEGFR2의 염색을 위해 이노스는, VE-cadherin의 또는 calponin, FITC 접합 된 항 - 토끼 IgG를 10 μL를 포함 멸균 1X PBS의 1 mL를 넣어. 어둠 속에서 1 시간 동안 얼음에 세포를 품어. 멸균 PBS 3 회 세포를 세척하여 IgG를 제거합니다. </li><li> 1 μg의 / ㎖의 최종 농도 훽스트 33342를 추가 어둠 속에서 10 분을 품어. </li><li> CD31, VEGFR2 이노스의 형광보기, VE-cadherin의 반전 형광 현미경, calponin (녹색, 518-535 나노 미터의 여기 파장) 및 훽스트 (파란색, 361 나노 미터의 여기 파장은). </li></ol></li></ol>

<ol>
	<li>Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+early+structural-functional+changes+in+the+endothelium+for+vascular+disease.">Implications of early structural-functional changes in the endothelium for vascular disease.</a> Arteriosclerosis, thrombosis, and vascular biology. 27, 266-274 (2007).</li><li>Cid, M. C., Segarra, M., Garcia-Martinez, A., Hernandez-Rodriguez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells,+antineutrophil+cytoplasmic+antibodies,+and+cytokines+in+the+pathogenesis+of+systemic+vasculitis.">Endothelial cells, antineutrophil cytoplasmic antibodies, and cytokines in the pathogenesis of systemic vasculitis.</a> Current rheumatology reports. 6, 184-194 (2004).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Reactive+protein-induced+endothelial+microparticle+generation+in+HUVECs+is+related+to+BH4-dependent+NO+formation.">C-Reactive protein-induced endothelial microparticle generation in HUVECs is related to BH4-dependent NO formation.</a> Journal of vascular research. 44, 241-248 (2007).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+circulating+CD31+/CD42-+microparticles+are+associated+with+impaired+systemic+artery+elasticity+in+healthy+subjects.">Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects.</a> American journal of hypertension. 20, 957-964 (2007).</li><li>Mackay, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema.">Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema.</a> Respiratory research. 14, 23 (2013).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> Journal of immunological methods. 244, 205-215 (2000).</li><li>Bowden, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+alpha4+integrin+and+VCAM-1+in+CD18-independent+neutrophil+migration+across+mouse+cardiac+endothelium.">Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.</a> Circulation research. 90, 562-569 (2002).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+induces+apoptosis+in+mouse+aortic+endothelial+cells+and+endothelial+dysfunction+in+mice+fed+high-calorie+and+high-cholesterol+diets.">Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets.</a> Life sciences. 92, 1165-1173 (2013).</li><li>Atkins, G. B., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+differentiation:+molecular+mechanisms+of+specification+and+heterogeneity.">Endothelial differentiation: molecular mechanisms of specification and heterogeneity.</a> Arteriosclerosis, thrombosis, and vascular biology. 31, 1476-1484 (2011).</li><li>Aitsebaomo, J., Portbury, A. L., Schisler, J. C., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brothers+and+sisters:+molecular+insights+into+arterial-venous+heterogeneity.">Brothers and sisters: molecular insights into arterial-venous heterogeneity.</a> Circulation research. 108, 929-939 (2008).</li><li>Shimamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+and+foods+on+contraction+of+endothelial+cells+as+a+key+mechanism+in+atherogenesis+and+treatment+of+atherosclerosis+with+endothelial-cell+relaxants+(cyclic+AMP+phosphodiesterase+inhibitors).">Drugs and foods on contraction of endothelial cells as a key mechanism in atherogenesis and treatment of atherosclerosis with endothelial-cell relaxants (cyclic AMP phosphodiesterase inhibitors).</a> Advances in experimental medicine and biology. 60, 77-105 (1975).</li><li>Chiu, J. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+disturbed+flow+on+vascular+endothelium:+pathophysiological+basis+and+clinical+perspectives.">Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives.</a> Physiological reviews. 91, 327-387 (2011).</li><li>Gimbrone, M. A., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction,+hemodynamic+forces,+and+atherogenesis.">Endothelial dysfunction, hemodynamic forces, and atherogenesis.</a> Annals of the New York Academy of Sciences. 902, 239-240 (2000).</li><li>Rostgaard, J., Qvortrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+demonstrations+of+filamentous+molecular+sieve+plugs+in+capillary+fenestrae.">Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.</a> Microvascular research. 53, 1-13 (1997).</li><li>Brutsaert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial-myocardial+signaling:+its+role+in+cardiac+growth,+contractile+performance,+and+rhythmicity.">Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity.</a> Physiological reviews. 83, 59-115 (2003).</li></ol>

The authors have nothing to disclose.

이 연구는 특별한 장비없이 마우스 대동맥에서 세포를 분리 및 배양 내피하는 간단한 방법을 보여줍니다. 면역 염색 세포의 대부분이 제 2 통로 후 내피 세포이었던 것으로 나타났다. 제 3 통로에 두 번째의 세포는 시험 관내 및 내피 세포 생물학을 연구하는 생체 실험에 적합한 것이 좋습니다. 이 의정서에서 키 노트 절차에 오...

혈관 내피 세포뿐만 아니라 혈액이나 조직을 분리하는 장벽 층, 그것은 400m2 (1)의 표면적 전체에 걸쳐 뻗어 혈관 트리 광대 내분비선 여겨진다. 내피의 복지는 혈관 항상성에 필수적이다. 역기능 내피는 동맥 경화증, 혈관염 및 허혈 / 재관류 손상 등 2-4 등 다양한 심혈관 질환에 참여하고 있습니다. 현재까지이 질환의 설정에 관련된 특정 세포 및 분자 메커니즘은 잘 의한 내피 세포의 확산 해부학 적 특성으로 이해되지 않습니다. 유전자 조작 기술은 다른 어떤 포유 동물 종에 비해 생쥐에서 개발되어 있기 때문에 마우스 연구의 중요한 모델이다. 대동맥의 작은 크기 enzymat하게 때문에 기본 쥐 대동맥 내피 세포의 분리는 특히 어려운 간주비현실적 내피 세포의 IC 소화. 일부는 분리 및 내장 컴퓨터가 5-7을 필요로 정화하는 과정을보고했다. 이 프로토콜의 목적은 특수한 장치를 사용하지 않고 마우스 대동맥 내피 세포를 분리하고 확장하는 간단한 방법을 사용하는 것이다. 이 프로토콜에서 갓 격리 대동맥 작은 세그먼트로 절단 및 내피 세포 발아 수 있도록 아래로 향하게 내피 세포와 매트릭스에 시드. 세그먼트가 제거 된 후, 내피 세포는 내피 세포 선호 배지에서 확장되고 두 개 또는 세 개의 통로 후 실험에 대한 준비가되어 있습니다. 기술 된 방법의 이점은 그 1) 단일 대동맥 내피 세포 수확의 상당히 높은 수치; 2) 세포 생존 능력은 잘 보존된다 3) 특별한 장비 나 기술이 필요하지 않습니다. 이는 내피 세포의 병태 생리의 특정 세포와 분자 메카니즘을 식별하는 효과적인 수단을 제공한다. 홍보를 공부에 관심이있는 사람들을 위해어느 유전자 녹아웃 마우스에서 imary 배양 내피 세포, 유전자 녹아웃 생쥐 나 쥐 질병 모델이 프로토콜은 매우 유용하고 실천하기 쉽습니다.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and primary culture of mouse aortic endothelial cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

내피 세포 돋 자연 내피 세포는 마우스 대동맥 세그먼트에서 시작 발아. 마우스 대동맥 세그먼트는 4 일 동안 내피 세포 성장 배지에서 성장 인자 - 감소 된 매트릭스에서 성장시켰다. 4 일 - 내피 세포의 발아는 보통 2에 나타납니다. 현미경은 4 일 (그림 1)에 촬영했다. 사진에 나타낸 바와 같이, 다수의 내피 세포 떨어진 부분에서 이동한다. 새로 형성...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

isolation-and-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.5K 조회수.  Wayne State University. The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

비디오: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

차 설치류 뇌 미세 혈관 내피 세포의 분리

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

연구

신경과학

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

생물학

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

종양 특이 내피 세포의 분리 및 문화 확장

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

의학

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

기본 Murine 골격 근육 Microvascular 내 피 세포의 고립

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

배아 심실 내피 세포의 분리

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

일차 마우스 폐 내피 세포의 분리

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

면역학 및 감염병학

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

쥐 관상 동맥 혈관 평활근 세포의 분리

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

마우스 관상 내피 세포의 분리

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

소형 돼지에서 1 차 대동맥 내피 세포의 격리 및 배양

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

마우스 일차 간 정현파 내피 세포의 분리 및 특성화

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...