Name: culturing mouse cardiac valves in the miniature tissue culture system
Uploaded: 2015-10-19T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

이 프로토콜은 동물 연구 윤리위원회의 LUMC 지침을 따른다. 악기 1. 준비, 문화 매체, 및 MTCS 참고 : 층류 후드에있는 모든 준비를 수행합니다. MTCS 관류 실, 버블 트랩 및 스탠드 리버 등. 2010 년 (11)에 설명되어 있습니다. <ol><li> 70 % 에탄올로 포셉 및 마이크로 가위를 소독. 멸균 타이로드의 버퍼 (130 mM의 염화나트륨, 5.4 밀리미터의 KCl, 6.0 mM의 헤 페스, 1.2 mM의 황산, 1.2 21G 바늘 5 ML의 주사기를 준비 mM의 KH 2 PO 4, 20 mM의 포도당, 1.5 mM의 염화칼슘 2 · 2H 2 O, 의 pH = 7.2). 무균의 KCl 용액 (100 mM의)와 21G 바늘 5 ML의 주사기를 준비합니다. </li><li> 65 심장 당 매체 ml의 준비 : (A /; 페니실린의 100 단위, 100 스트렙토 마이신의 μg의, 및 암포 테리 신 B의 0.25 UG / ㎖) DMEM 10 % 태아 송아지 혈청, 안티 곰팡이 / 항생제로 보충을, 인슐린 Transferrin-셀레늄 (ITS / ml의 인슐린 10 μg의 5.5 μg의 / ㎖ 트랜스페린, 6.7 NG / ㎖ 셀렌 산 나트륨) 및 필터 소독. </li><li> 70 % 에탄올 및 종이 타월 건조와 함께 분사하여 관류 챔버와 버블 트랩을 소독. 초기 관류 챔버없이 그러나 MTCS (도 1D)을 조립한다. 스탠드에 거품 트랩을 배치합니다. 쉬운로드 펌프 헤드와 연동 롤러 펌프를 사용합니다. <ol><li> 실리콘 튜브는 저수지 (50 ML 튜브) 및 펌프, 펌프와 버블 트랩 버블 트랩과 저수지 사이의 연결 (그림 1D 참조)를 만들어 사용. 매체는 가스 투과성 실리콘 호스 (1m)과 가능한 한 짧게 인큐베이터 외부 튜브를 통해 가스 배양기에서 가스와 평형에 도달 할 수 있도록 충분한 시간 동안 인큐베이터 안에 튜브를 만든다. </li></ol></li><li> (적절한 순환을 허용 / 1 ml의 주위 분의 흐름 속도를) 펌프를 켜, 70 % 에탄올로 50 ML 튜브를 입력하고 에탄올 CIR하자모든 튜브를 살균 30 분 동안 culate. 탱크를 비우고 시스템에서 에탄올을 펌핑하여 에탄올을 제거합니다. </li><li> 멸균 증류수로 50 ML 튜브를 입력 펌프를 켜고 30 분 동안 물 순환 에탄올 나머지 없애 보자. 탱크를 비우고 시스템에서 물을 펌핑하여 물을 제거합니다. </li><li> 매체의 45 mL를 50 ml의 튜브를 작성, 인큐베이터에있는 스탠드를 넣어. 펌프 (1 ml / 분의 주위의 흐름 속도)를 켜고 모든 튜브를 채우기 위해 매체를 순환, 모든 기포를 제거하고 배지는 적어도 1 시간 동안 배양기에서 가스 조성에 적응하도록 허용한다. 표준 조직 배양 조건 (5 % CO 2, 37 ℃로)에서 인큐베이터를 유지한다. 기포가 튜브에 존재하지 않는 것을 확인합니다. </li></ol> 마우스 심장의 2. 분리 <ol><li> 헤파린 500 단위와 마우스를 주입한다. 이는 응고로부터 혈액을 방지하고 (B)의 제거를 허용 할 것이다마음에서들 (100d). 10 분 후, 정밀 증발기를 이용하여 4 % 이소 플루 란 유도 챔버에 마우스를 마취. 마우스가 발가락 핀치에 응답하지 않는 경우 마취는 충분하다. </li><li> 해부 보드에 마우스를 전송하고, 동축 회로에 접속 보호구 통해 마취를 유지한다. 70 % 알코올로 마우스의 모피를 소독. 가위 복강을 열고 사용하면 대정맥과 다이어프램을 노출. </li><li> 심장을 노출 마지막에서 첫 번째 갈비뼈에 시작 횡 절개를하고 마우스의 머리 흉부 케이지를 반영합니다. 마우스가 더 이상 숨을 쉬지으로 마취를 중지합니다. 심장 박동을 관찰한다. </li><li> (다이아 프램을 통해) 흉강으로 복부에서 하대 정맥에 살균 타이로드의 버퍼를 포함하는 5 ML의 주사기에 부착 된 21G 바늘을 삽입합니다. 혈액이 바늘 삽입의 대정맥에 꼬리를 종료 할 수 있는지 확인합니다. 제 관류전자 타이로드의 버퍼 부드럽게과 대정맥에 일정한 압력으로 심장이 부분적으로 붉은 색을 잃을 때까지. 참고 : 심장은 심장에서 혈액의 제거를 허용 치는 남아있다. </li><li> 무균의 KCl 솔루션을 포함하는 5 ML의 주사기에 부착 된 21G 바늘과 바늘을 교체합니다. 조심스럽게 심장이 박동을 멈출 때까지 대정맥에의 KCl 용액을 관류하고 바늘을 제거합니다. 참고 : KCl을 솔루션은 관상 시스템의 쉽게 관류 바늘의 삽입 및 관류를 허용 편안한 확장기의 마음을 유지합니다. </li><li> 약간 곡선 집게를 사용하여 마음을 들어 올려 사용하는 가위는 심장 손상과 심장에 부착하는 근위부 동맥과 정맥을 주변 조직에서 무료로 심장을 해부 그러나 둡니다. 얼음처럼 차가운 PBS는 항진균제 / 항생제 (/)로 보충이 포함 된 15 ML 튜브에 마음을 전송합니다. 최대 3 시간 동안 얼음처럼 차가운 PBS의 마음을 보관합니다. </li></ol> 3. Cannulatio관류 상공 회의소에서 마우스 마음의 N <ol><li> 층류 후드의 모든 절차를 수행합니다. 10cm 페트리 접시에 15 ML 튜브에서 격리하는 마음을 전송하고 추가 PBS는 A / 보충. </li><li> 해부 현미경, 모든 비 심장 조직을 제거하는 미세 가위와 집게를 사용하지만 상행 대동맥을 보존 마음에 팔 머리 동맥과 폐동맥 혈관, 2mm의 기부와 최소한 분기에. 좌심실 내강 (일반적으로 2mm)에 대한 액세스를 만드는 마음의 정점의 끝을 잘라. 해부 현미경 흐름 후드에서 재관류 챔버를 놓습니다. </li><li> 실리콘 튜브를 사용하여 삽입 바늘 매체 5 ML의 주사기를 연결합니다. 20 ㎖ 매체와 관류 챔버를 입력하고 관류 챔버에서 2 무딘 바늘 (그림 1) 사이에 회전 무대에서 마음을했습니다. 심장이 앞에 위치하도록 회전 스테이지의 높이를 조절바늘. </li></ol> 승모판을 배양 4. 결찰 (그림 1 참조) <ol><li> 봉합사 (실크 7.0)과 대동맥을 결찰. 봉합 좌심방을 결찰. 좌심방에 폐정맥을 통해 (그림 1. 1 NR) 바늘을 삽입하고 좌심방에서 해당 항목에 봉합 근위부와 결찰. 이 승모판 막에 손상을 하듯이 바늘이 좌심방으로 너무 멀리 있지 않은지 확인하십시오. </li><li> 좌심실로 직선 운동으로 바늘 (그림 1 nr.2)를 삽입합니다. 50 ㎖ 튜브에 관류 챔버로부터 전송 매체. 생체 접착제와 심근에 바늘을 밀봉합니다. </li><li> 접착제가 건조 후, 신중하게 nr.1 바늘과 누출이 있는지 확인하기 위해 매체 채워진 주사기를 연결하여 마음에 매체를 주입. 바늘 nr.2 마지막 남은 혈액이 심장에서 관류되어에서 해당 매체 종료를 확인합니다. </li></ol> 5. 리대동맥 판막을 배양 gation (그림 1 참조) <ol><li> 대동맥의 바늘 (그림 1 nr.1)를 삽입하고 봉합 결찰. 이 대동맥 판막을 손상하는 것처럼 바늘이 대동맥으로 너무 멀리 있지 않은지 확인하십시오. 좌심실로 직선 운동으로 바늘 (그림 1 nr.2)를 삽입합니다. 50 ㎖ 튜브에 관류 챔버로부터 전송 매체. 생체 접착제와 심근에 바늘을 밀봉합니다. </li><li> 접착제가 건조 후, 신중하게 nr.2 바늘과 누출이 있는지 확인하기 위해 매체 채워진 주사기를 연결하여 마음에 매체를 주입. 바늘 nr.1 마지막 남은 혈액이 심장에서 관류되어에서 해당 매체 종료를 확인합니다. </li></ol> 스탠드에 6. 관류 상공 회의소 <ol><li> 매체를 옮겨 20 mL를 관류 챔버를 입력합니다. 챔버에 가스켓 및 뚜껑을 놓고 세탁기와 나사로 조입니다. </li><li> incuba에서 조립 MTCS 제거터. 스탠드에 재관류 챔버를 연결합니다. 튜브를 연결합니다 (그림 1D 참조). 인큐베이터 (5 % CO 2, 37 ℃로)에 스탠드를 놓습니다. 펌프에 튜브를 연결합니다. 600 μL / 분 주위에 흐름 속도와 펌프의 전원을 켭니다. </li><li> 저장 및 / 또는 기포 트랩 매질의 레벨이 수신 매체 방울의 하강에 의해 흐름의 존재의 시각화를 허용하는지 확인. 배양 후, 심장, 시스템으로부터 제거 고정 조직 학적 검사를 위해 처리된다. </li></ol>

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The authors declare no conflicts of interest.

심장 마우스 밸브를 배양에서 중요한 단계는 흐름의 적절한 방향을 보장하기 위해 마우스에서 심장 절제 밸브에 수직 바늘의 생존과 라이 게이션을 위해 가능한 한 짧은 관류 챔버에서 결찰 사이의 시간을 포함 . 또한, 매체없이 관류 챔버에서 결찰 후 흐름을 확인하는 것은 적절한 삽입 및 바늘의 결찰을 보장합니다. 그것은 멸균 문화를 유지하고 잠재적으로 흐름을 방해 마음에 갇혀 얻을 수있?...

심장 판막 질환은 서구 세계에서 이환율과 사망률의 주요 원인이다 그 유병률은 연령에 따라 증가하며 75년 인구의 10 % 이상 및 1 이전에 영향을 미친다. 마음의 전신 부분의 밸브는, 대동맥 및 승모판 밸브는 주로 영향을받습니다. 심장 판막 질환 (2)의 기계적 특성 변화를 초래 밸브의 고도로 조직화 구조의 손실을 특징으로한다. 구조적 완전성은 밸브의 기능에 따라서 중요하다. 밸브의 전단지는 판막 간질 세포 (VIC), 판막 내피 세포 (VEC), 고도로 계층화 된 패턴 3,4으로 구성되어 세포 외 매트릭스로 구성되어있다. VIC를은 전자 재료 합성, 분해 및 조직에 대한 책임이 있습니다. 요인은 혈액, 내장 컴퓨터에서 나오는 또는 그 기능을 조율 VIC를에 ECM 행위에 거주. 또한,기계적 힘은 층류 또는 진동형 전단 응력, VIC를도 5의 동작에 영향을주는 압축 또는 인장 응력을 초래 심장주기 동안 전단에 작용. 밸브의 구조를 조절하는 방법을 이해하기 위해서는, 먼저 VIC를가 심장 사이클 동안 경험 한 자극의 다양한 세트에 응답하는 방법을 이해하여야한다. 시험 관내 연구 판막 세포의 특성 및 능력에 대한 매우 유익한왔다. 시험관 내에서 이들 세포의 반응은, 그러나 항상 정확하게 생체 반응 6을 흉내낼 수있다 예를 들면, 자극에 VIC의 반응의 EC의 존재 및 ECM의 조성 5에 따라 달라진다. 더욱이, 자극 판막 세포의 반응은 전단 7에 특정 위치에 달려있다. 생화학 적 자극에 더하여, 판막 셀의 동작은 O 작용 기계적 힘에 의해 결정된다N 밸브 (8). 밸브의 각 지역은 혈역학 적 스트레스의 고유의 세트 실시한다. 현재 생체 모델은 기계적인 힘이 판막의 구조 (5)의 중요한 결정 요인임을 보여 주었다 있지만, 관련 메커니즘은 아직 불분명하다. 생체 내 모델은 판막 개발 9,10의 기초가되는 분자 메커니즘에 대한 통찰력을 제공하고 있지만, 성인 판막 생물학에 대한 통찰력은 여전히 애매하다. 따라서, 생체 외 유동 모델되는 심장 밸브는 11 시간의 연장 된 기간 동안 심장에 자연스러운 위치에서 배양 될 수 개발되었다. 이는 밸브가 구성 천연 유지와 VIC를 최대한 자연스러운 VIC를 자극에 반응을 생체 내와 같은 환경을 경험할 수있는 장점을 갖는다. 또한, 마음에 그들의 자연적인 위치에있는 밸브의 문화는 각 쓰는 용이관련 혈역학 적 스트레스에 판막 지역. 즉,이 생체 모델에서, 소형 조직 배양 시스템 (MTCS)는, 밸브는 심장 밸브 리모델링에서의 역할의 조사를 허용하는 다른 생화학 적 및 혈역학 적 자극에 노출 될 수있다.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400-045</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-06</td><td></td></tr><tr><td>Silk 7-0</td><td>Ethicon</td><td>768G</td><td></td></tr><tr><td>100 mm culture dish</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>50 ml tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>5 ml syringe</td><td>BD</td><td>309649</td><td></td></tr><tr><td>21 G needle</td><td>BD</td><td>304432</td><td></td></tr><tr><td>Heparin</td><td>LEO</td><td>012866-08</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>Tedpella</td><td>1346</td><td></td></tr><tr><td>Silicon tubing</td><td>Thermo Scientific</td><td>8060-0020</td><td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td></tr><tr><td>Silicon tubing for pump</td><td>Masterflex</td><td>96400-13</td><td>I.D. x O.D. x Wall: 0.8 x1.59 x 0.40 mm</td></tr><tr><td>Biocompatible glue (Histoacryl)</td><td>B. Braun</td><td>1050071</td><td></td></tr><tr><td>precision vaporizer</td><td>Dr&amp;auml;ger</td><td>Vapor 200</td><td></td></tr><tr><td>peristaltic roller pump</td><td>Masterflex</td><td>7521-35</td><td></td></tr><tr><td>Easy-load pump head</td><td>Masterflex</td><td>7518-00</td><td></td></tr><tr><td>Flow chamber</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr><tr><td>Bubble trap</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr></tbody></table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

대동맥 판막 (그림 2) 또는 승모판 막 (11)은 최소 3 일 동안 배양 할 수있다. (대동맥판과 승모판 대한 확장기의 총수에 대한 수축기 위치를 나타낸다)가 개방 위치에서 배양함으로써, 판막 세포 생존 유지. TUNEL 양성 세포 (도 2H, I) 또는 절단 된 카스파 제 -3 발현의 부재에 의해 결정된 어떠한 세포 사멸이 관찰되지 않는 (도시되지 리버 외., 2010 (11)). (...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

소형 조직 배양 시스템에서 마우스 심장 밸브를 배양

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.8K 조회수.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

비디오: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

마우스 배아 심장에서 심 외막 세포의 체외 배양에서

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

연구

공학

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

이소성 심장 이식 마우스 모델을 사용하여 폐동맥 판막 이식

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

의학

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

생물학

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

분리 및 문화 성인 마우스 세포 신호 전달 용의 Cardiomyocytes과의<em&gt; 시험관</em&gt; 심장 비대

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

관상 동맥 혈관 신생의 체외 모델

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

격리, 문화 및 성인 마우스 심근 세포의 형질 도입

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

시험관 내 대동맥 판막의 석회화를 연구하기 위해 마우스 간막판 세포의 격리

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Source: Bouchareb, R. et al., <a href="https://app.jove.com/v/62419">Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.</a>&#160; J. Vis. Exp. &#160;(2021).
This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.

대동맥 판막 간질 세포의 칼슘 농도를 측정하기 위한 석회화 분석

Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.&#160; J. Vis. Exp. ...