Name: isolation and culture of primary endothelial cells from canine arteries and veins
Uploaded: 2016-11-18T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

윤리 문 : 본 연구에 사용 된 혈관은 신선한 개 사체에서 얻은 잉여 재료로 수확 (N = 4) 기타 관련이없는 연구 (대학 3R 정책)에 대한 안락사 건강한 개에서. 비정상적인 혈관 (인트라과 간외 문맥 정맥 션트, N = 1 각) 위트레흐트 대학의 반려 동물에 대한 대학 병원에 제출 개에서 소유자의 동의 후 사후을 수확했다. 1. 분리 및 문화 차 개 내피 세포의 <ol><li> 2 ㎖ /도 0.5 % w / v를 젤라틴과와 사전 코트 6 웰 플레이트는 37 ° C에서 5 % CO 2의 가습 분위기 인큐베이터에서 2 시간 동안 둡니다. 이전의 기본 세포를 파종을 초과 젤라틴 용액을 제거합니다. </li><li> 무균 신선한 개 사체 (그림 1A)에서 혈관 (들)의 관심 (예를 들어, 대동맥, 대정맥, 대정 간이)를 제거합니다. 용기 시스템의 해부학 유지관심있는 선박의 양단에 직선 집게를 배치하여. 내피 세포의 손상을 방지하기 위해 집게로 조직의 불필요한 조작을하지 마십시오. <ol><li> 수술 가위로 양쪽 끝에있는 고정 용기를 잘라 시신에서 제거합니다. 얼음에 행크의 균형 소금 솔루션 (HBSS)에 전송. 참고 : 약 5cm의 혈관 길이는이 절차 바람직하지만 1cm를 측정하는 것과도 문화에 충분한 세포를 제공 할 것입니다. </li></ol></li><li> 얼음처럼 차가운 HBSS 가득 페트리 접시에 혈관을 전송합니다. 수술 가위를 사용하여 용기 자체의 손상 (그림 1B)를 유지, 용기의 외부에서 모든 부착 조직과 지방을 제거합니다. 최적의 뷰의 클램프 또는 집게로 옆으로 주변 조직을 당겨 : 절단시 반드시 선박이 항상 명확하게 볼 수 있습니다합니다. <ol><li> 합자로 선박의 어떤 지점을 닫습니다 (예를 들면, 3-0 polyglactin)이어서 제거수술 가위 나 메스와. </li></ol></li><li> 주의 깊게 만곡 홀스 테드 모기 집게 용기 단부 입력 용기의 타 단부에 나이프의 선단부를 클램핑하고 외부 (도 1C-G)를 완전히 안쪽까지 서서히 상기 용기를 반전 후퇴. 외부는 이제 내피 세포 층으로 구성되어 있습니다. 어떤 어려움이 용기 반전시에 발생되는 경우, 마찰을 줄이기 위해 다시 HBSS에 잠기. </li><li> 완전히 폐쇄하고 소화 절차 (그림 1H)에 비 내피 혈관 조직의 노출을 방지하기 위해 선박의 양쪽 끝에서 지갑 문자열 봉합을 놓습니다. 반전 용기를 조작 할 수 합자 끝을 사용합니다. </li><li> 소화 배양 후에 수행되면, 소화 프로토콜 (단계 170) 전에 반전 용기 cryopreserve. <ol><li> cryovial에 반전 용기를 놓고 세포 배양 매체를 동결 채우기. 동결 공동 사용 아래 -80 ° C에 고정의 ntainer. -80 ℃에서 저장은 선박 1 주일 이내에 사용하는 경우. 장기 보관, -180 ° C에서 장소 크리오 바이알 (cryovial)하십시오. EC의 격리를 수행 할 때, 수조 (37 ℃)에서 급속 해동 크리오 바이알 (cryovial)를 즉시 얼음처럼 차가운 HBSS에 놓는다. 1.7에 표시된대로 진행합니다. 참고 : 분리 후 내장 컴퓨터의 총 생산량이 때문에 냉동 과정에서 생존 능력의 손실에 낮은 것을 고려. </li></ol></li><li> 50 ML 튜브에 용기를 전송하고 (그림 1I) (해동의 경우 또는 잔류 동결 매체) 적혈구를 제거 HBSS로 두 번 씻어. </li><li> 간헐 부드러운 37 ° C에서 1 시간 동안 개 내피 세포에서 30 ㎖의 콜라게나 제 유형 II (0.15 U / ml) 및 디스 파제 (0.15 U / ㎖)의 용액 성장 중간 (CECGM)와 50ml의 튜브 용기 다이제스트 동요. </li><li> g 튜브로부터 상기 용기를 분리하고, X (250)에서 5 분 동안 세포 현탁액을 원심 분리. </li><li> CECGM A의 세포 펠렛을 재현 탁ND 시드 2ml의 세포 현탁액은 웰 당 (1 - 웰 용기의 크기에 따라). 문화 공기에서 5 % CO 2의 가습 분위기 37 ℃에서 세포는 일주일에 두 번 배지를 변경. </li><li> 80 % 도달 - 70의 포화 상태가 통로 셀. <ol><li> 죽은 또는 비 부착 세포를 제거하기 위해 미리 예열 HBSS로 세포를 씻으십시오. 각 웰에 200 ㎕의 재조합 세포 분해 효소 (1 배)를 추가하고 5 분 동안 또는 모든 셀이 분리 될 때까지 다시 인큐베이터에 배치합니다. </li><li> 15 ㎖의 튜브에 세포를 옮기고, 10 % 소 태아 혈청 (FCS)을 포함한 배지 10 ㎖ (CEGM)을 첨가함으로써 트립신을 멈춘다. 250 × g에서 5 분 동안 원심 분리기. 젤라틴 미리 코팅 된 플레이트 또는 플라스크에 문화를 계속합니다. </li></ol></li></ol> 2. 특성 <ol><li> 문화 개 대동맥 내피 세포 (CnAOECs)와 현미경으로 두 번 주간 기본 및 상업 세포주의 형태를 비교합니다. 참고 : T그 대표적인&gt; 70 %의 컨 플루 언시에 도달 할 때 가장 잘 관찰 될 수있는 패치의 ECS에 패턴을 성장. <ol><li> 0.5 % W / V 젤라틴은 미리 코팅 T75 플라스크에 CECGM 문화 CnAOECs. 일주일에 한번 통과 셀 생장이 70 - 80 %. <ol><li> 계대를 들어, 미리 예열 HBSS 한 번 세포를 씻어 1 ml의 재조합 세포 분해 효소 (1 배)를 추가합니다. 5 분 동안 또는 모든 셀이 분리 될 때까지 다시 인큐베이터에서 플라스크를 놓습니다. 10 % FCS를 포함한 10 ㎖ 배지 (CEGM)을 첨가함으로써 트립신을 비활성화한다. 250 XG에서 5 분 동안 세포 현탁액을 원심 분리하여 상등액을 버리고 1 ml의 배양 배지에서 세포를 재현 탁. </li><li> 세포 현탁액의 10 μL 나누어지는을 가지고 1 희석 : 0.4 % 트리 판 블루로 1. 자동 세포 계수기를 사용하여 세포 수 및 새로운 T75 플라스크에서 4.0 × 10 5 생세포를 접시. 5 % CO 2와 가습 분위기에서 37 ° C에서 플라스크와 문화에 미리 예열 CECGM의 10 ML을 추가합니다. </li></ol></li></ol></li><li> 배양 CaPECs 및 CnAOECs의 통과 일로부터 RNA를 분리합니다. <ol><li> , 펠릿 적어도 1 × 103 세포를 수집 시료 전처리 시약 (SPR)의 20 μL를 추가하고, 세포를 용균 1 분 동안 배양한다. 배양 후, 조심스럽게 -70 ℃에서 총 RNA 및 저장을 포함하는 세포 용 해물을 모은다. </li></ol></li><li> 제조업체의 지시에 따르는 cDNA 합성 키트 (예를 들어, iScript)를 사용하여 cDNA를에 mRNA의 변환합니다. qPCR에를 수행 할 준비가 될 때까지 오랜 기간 동안 4 ° C 일주까지, 또는 -20 ° C에서의 저장소의 cDNA. </li><li> 내피 세포 원점을 확인하기 위해 내피 세포 마커 CD31의 유전자 발현을 측정한다. MIQE의 PRECIS 가이드 라인 (10)을 사용하여 실험 장치 및 정량화를 수행합니다. 정규화, 참조 GAPDH 유전자, RPS19 및 B2MG 11의 발현을 측정한다. <ol><li> 뉴 클레아없는 물에서 얻어진 cDNA를 10 배 희석을 준비합니다. </li><li> 준비표준 라인 풀링 된 cDNA 샘플로부터 4 배 희석하고 비 템플릿 대조군 클레아없는 물을 사용한다. qPCR의 반응에 대한 50 배의 총 희석 도달 샘플 다섯 배 희석. 4 ㎕의 cDNA를 역방향 및 정방향 프라이머 (표 1) 20 pmol의 혼합 형광 6 μl를 이용하여 384 웰 포맷에서 중복 피펫 10 μL 반응. </li><li> 변성 95 ° C에서 10 초 39주기 다음 Taq 중합 효소 활성화를위한 5 분, 어닐링 및 신장을위한 Tm은 30 초 동안 95 ° C에 프로그램을 설정합니다. 각 프라이머 세트에 대한 Tm은 표 1에 나타낸다. </li><li> 곡선을 용융 수행하는 증폭 단 하나의 제품을 보장하기 위해 모든 실행에 따라 분석한다. 참조 유전자의 상대적인 양을 평균하여 샘플의 발현 수준을 정상화하고, 반응 효율은 95 %와 105 % 사이 인 경우 ΔCT을 계산한다. </li></ol></li><li> ANGI와 EC의 기능을 평가ogenesis 분석. <ol><li> 미리 냉각 된 혈관 슬라이드의 각 웰에 세포 외 기질의 10 μl를 첨가하고 잘의 표면을 덮도록 피펫 팁으로 확산. 얼음에 세포 외 매트릭스를 유지하고, 겔에 기포의 도입을 피한다. 30 분 동안 5 % CO 2, 37 ℃에서 항온 물로 적신 페이퍼 타월로 페트리 접시에 슬라이드를 배치하여 겔 고화. </li><li> 1.0 × 10 4 차 추가 되는 EC 50 μl의 내피 성장 중간마다 잘. 5 % CO 2와 37 ° C에서 6 시간 동안 슬라이드를 품어. </li><li> 6 시간 후 이미지의 전체도를 포함해야하고, 20 배 배율로 사진을 찍을. </li></ol></li></ol>

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The authors have no competing financial interests.

송곳니되는 EC에 초점을 맞춘 연구에서 CnAOEC 기본 회선은 개 3, 12, 13의 내피 계통을 모델링하는 데 사용됩니다. 인간의 연구에서, HUVEC 문화는 여전히 황금 표준으로 간주됩니다. 분명히, 탯줄에서 파생되는 EC에 단지 집중하는 것은 심장 혈관 연구에서 확고한 제한입니다. 내피 세포는 동정맥 사양을 결정하는 특정 유전자의 발현 패턴을 갖는다. 생후 혈관 이러한 차이를 고려하기 위해, 우...

개는 심혈관 질환의 연구에 큰 동물 모델로서 사용하고 또한 선천적 (유전 적), 혈관 이상 (1, 2)으로 고생 할 수있다. 상업적 내피 세포주 종종 내피 세포 (EC)의 기능을 평가하기 위해 사용되는 이들 질병을 연구. 개를위한 개 대동맥에서 파생 된 (CnAOEC) 가능한 한 상업 내피 세포 라인이있다. 이 세포주는 주로 제어되는 EC 통상 3-5과 같은 연구에 사용된다. 인간 심장 연구에서 가장 일반적으로 사용되는 내피 세포주 각각 인간 제대 정맥 및 동맥 유래의 인간 제대 정맥 내피 세포 (HUVEC를) 및 인간 배꼽 동맥 내피 세포 (HUAECs)가있다. HUVECs를 1980 년대 6 이후 혈관 연구의 황금 표준으로 사용되어왔다. 그들은 내피 세포의 기능과 질병 적응을 연구하는 고전적인 모델 시스템으로 간주됩니다. 다른 혈관 내피 세포로부터 분리는 외관 창출 다양전자 및 미세 7 유전 적 배경과 노출로 인한 기능을 제공합니다. 또한, HUVECs를하고 HUAECs은 탯줄에서 조건에 대해 완벽하게 모방 성인 혈관 그들이에 질병에 대응하여 노출되지 않을 수 있습니다 발달 혈관 구조를 유도된다. 따라서, 일반적으로 심혈관 질환에 HUVECs를 발견 결과와 HUAECs을 번역하는 것은 부적절하다. 적응과 성인되는 EC의 행동을 연구 할 때, 관심있는 선박에서 주되는 ​​EC는보다 직접적인 방법으로 사용되어야한다. 이들 세포를 분리하기 위해 여러 가지 방법이보고되어있다. 또한 HUVECs를 위해 사용되는 광범위하게 설명 된 방법은, 효소 적 소화 용액 (8)과 용기를 세척한다. 이것은 종종 평활 근육 세포 및 섬유 아세포 9 비되는 EC의 오염을 초래한다. 분리를위한 또 다른 자주 사용되는 방법은 다음 fluorescence- 다진 혈관 조직의 소화 효소 인분화 (CD) (31) (7, 8). FACS 분류의 내피 세포 마커 클러스터 이후 세포 배양에 기초한 (FACS)를 정렬 활성화 전지 셀의 비교적 대용량을 필요로하며, 따라서 작은 혈관의 내피 세포의 분리에는 적합하지 않다. 우리는 따라서, 고순도의 각종 송곳니 혈관으로부터 순수한 내피 세포 집단을 분리하기위한 새로운 강력한 방법을 개발 목표로. 새로운 분리 방법의 효율성을 테스트하기 위해, 우리는 고립과 크고 작은 여러 개 동맥과 정맥에서 순수한 개 주 내피 세포 (CaPEC) 문화를 획득했습니다. 이 방법은 또한 선천적 인트라 또는 여분 간 문맥 정맥 션트, 개 2에서 일반적인 질환과 같은 질병 및 / 또는 비정상적인 혈관에서 발생하는 내피 세포의 배양을 할 수 있습니다. 용기의 대부분이 남아 INT 이후 방법은 혈관 평활근 세포와 같은 용기의 추가적인 중요한 세포 유형의 분리를 허용절차를 수행하는 동안 역할을합니다.

<table><tbody><tr><td>Collagenase type II</td><td>Life Technologies</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Life Technologies</td><td>31966-021</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-050</td><td></td></tr><tr><td>Canine Endothelial Cells Growth Medium&amp;nbsp;</td><td>Cell Applications</td><td>Cn211-500</td><td></td></tr><tr><td>CnAOECs</td><td>Cell Applications</td><td>Cn304-05</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)&amp;nbsp;</td><td>GE Healthcare</td><td>16000-044</td><td></td></tr><tr><td>TrypLE Express</td><td>Life Technologies</td><td>12604-013</td><td></td></tr><tr><td>SPR</td><td>Bio-Rad</td><td>170-8898</td><td></td></tr><tr><td>iScript synthesis kit</td><td>Bio-Rad</td><td>170-8891</td><td></td></tr><tr><td>SYBR green super mix</td><td>Bio-Rad</td><td>170-8886</td><td></td></tr><tr><td>Recovery Cell Freezing Medium</td><td>Gibco/Life Technologies</td><td>12648-010</td><td>Keep on ice prior to use</td></tr><tr><td>Freezing container, Nalgene Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Surgical scissors (Mayo or Metzenbaum)</td><td>B. Braun Medical</td><td>BC555R</td><td></td></tr><tr><td>Mosquito forceps&amp;nbsp;</td><td>B. Braun Medical</td><td>FB440R</td><td></td></tr><tr><td>Mosquito forceps curved</td><td>B. Braun Medical</td><td>FB441R</td><td></td></tr><tr><td>polyglactin 3-0</td><td>Ethicon</td><td>VCP311H</td><td></td></tr><tr><td>Trypan blue</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Automated counting chamber</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Counting Slides, Dual Chamber</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>BD356231</td><td>Slowly thaw on ice</td></tr><tr><td>&amp;micro;-Slide Angiogenesis</td><td>Ibidi</td><td>81501</td><td></td></tr><tr><td>Endothelial Growth Medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EGM-2 SingleQuot Kit&amp;nbsp;</td><td>Lonza</td><td>CC-4176</td><td></td></tr></tbody></table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

다른 혈관 성공적 설명 분리 프로토콜 (도 2)을 행 하였다. 해부하고 건강한 개에서 대동맥, 대정맥, 대정 간이 및 관상 동맥 반전 할 수 있었다 (각 개 모든 혈관을, N = 4). 같은 접근되는 EC는이 선천성 문맥 정맥 션트 (간외 및 간내, N = 1 각)에서 분리 하였다으로. 대동맥 쉽게 반전되었지만 대동맥 세그먼트 복부 대동맥 이상 도전했다. 흉부 세그먼트에서 대동?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

개 동맥과 정맥에서 차 내피 세포의 분리 및 문화

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

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JoVE Journal

Medicine

11.2K 조회수.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

비디오: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

판막병 내피 세포의 분리

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

연구

생물학

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 ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

마우스 저항 동맥의 미세 혈관 내피 튜브의 분리

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

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 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.

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 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 ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

인간 복재 정맥 내피 세포 분리 및 제어된 수준의 전단 응력 및 스트레칭에 대한 노출

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

의학

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 Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

다운스트림 적용을 위한 별개의 지방 저장소에서 혈관 내피 세포의 분리 및 식별

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://www.jove.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

1차 뇌 주피세포의 분리 및 배양: 내피 세포 오염 없이 소 뇌 모세 세포에서 주피 세포를 분리하고 배양하는 절차

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...