승모판 07CVD04 :이 연구는 하트웰 재단, 미국 심장 협회 (과학자 개발 권한 부여 # 0830384N) 및 재단 Leducq에 의해 지원됩니다.

1. 준비 <ol><li> 비옥한 메추라기 또는 단계 14 닭 알을 품어 - 25 (2 일간) (4.5 일) 60 % 습도와 37 ° C.에 </li><li> 무균 얼 밸​​런스드 소금 솔루션을 준비 (EBSS) <ol><li> 600 ML 18 MΩ 물 100 ML 10X EBSS 추가 </li><li> 2.2 g의 나트륨 중탄산염 추가 </li><li> 7.2 솔루션의 산도를 조절 </li><li> 최대 1000 ML에 대한 해결책을 가지고 </li><li> 0.2 μm의 필터를 통과하여 솔루션을 소독 </li></ol></li><li> 무균 M199 문화 매체를 준비합니다. <ol><li> 700 ML 18 MΩ 물이 M199 분말 1 패키지를 추가합니다. </li><li> 2.2 g의 나트륨 중탄산염 추가 </li><li> 7.2 솔루션의 산도를 조정합니다. </li><li> 10 ML (1 %) 페니실린 - 스트렙토 마이신 추가 </li><li> 최대 1000 ML에 대한 해결책을 가지고 </li><li> 0.2 μm의 필터를 통과하여 솔루션을 소독 </li><li> 1 ML 무균 100X 인슐린 트랜스페린 - 셀레늄 - G 보충 추가 </li><li> 10 ML (1 %) 무균 닭 혈청 추가 </li></ol></li><li> 1.5 MG / ML의 콜라겐 농도에서 입체 콜라겐 젤을 준비합니다. 우리가 실험실에서 이전 작품보다 1.5 MG / ML의 콜라겐 농도는 세포가 이동할 수 있도록 너무 biomechanically 딱딱하게되는 매트릭스를 제공하는 것으로 나타났습니다. MG / ML은 세포 덩어리가 세포의 섬을 생산, 로컬 섬유는 분쇄기에 넣어 수 있도록 몇 가지 콜라겐 섬유가 1.5보다 콜라겐 겔 농도 낮아집니다. <ol><li> 위해서는 멸균 15 ML의 원심 관에 다음 얼음처럼 차가운 시약 추가 : <ul><li> 배 M199 : 볼륨 = 총 판매량 필요한 / 3 </li><li> 무균 18 MΩ 물 : 볼륨 필요 = 총 부피 (M199 볼륨 + 닭 혈청 볼륨 + 0.1 M NaOH 볼륨 + 콜라겐 볼륨) </li><li> 무균 닭 혈청 : 볼륨 = 총 볼륨 필요 * 0.01 </li><li> 쥐 꼬리 콜라겐 I : 볼륨 = (콜라겐 겔 농도가 * 총 판매량 필요) / 콜라겐 재고 농도 </li><li> 무균 0.1 M NaOH : 볼륨 = 콜라겐 볼륨 * 0.2 참고 : 물의 양을 0.1 M NaOH, 그리고 콜라겐은 콜라겐 주식 솔루션의 농도에 따라 달라집니다 밝혔다. </li></ul></li><li> 멸균 피펫 잘 콜라겐 솔루션을 섞는다. </li><li> 즉시 1.9 cm이 성장 지역 우물에 콜라겐 솔루션의 0.3 ML를 전송합니다. 이것은 완전히 우물을 충당하기 위해 약 3mm 두께 젤을 제공하기 위해 충분한 콜라겐 솔루션입니다. 적어도 250 μm의의 겔 두께 세포 침략 연구 필요합니다. </li><li> 37 최소한 1 시간 동안 젤로 콜라겐 솔루션을 허용 ° C 5 % CO 2, 100 % 습도. </li></ol></li></ol> 2. 사전 EMT Endocardial 전지 격리와 문화 <ol><li> 배아 절연 <ol><li> 계란의 큰 끝을 통해 실험실 테이프 중 하나 인치 긴 조각을 놓으십시오. 공기 전지가있는 곳입니다. 테이프는 깨지기 쉬운 퀘일 알 껍질을 안정화. </li><li> 부드럽게 찔린 멸균 곡선 가위 끝에있는 공기 세포에있는 계란의 녹화 영역입니다. </li><li> 노른자가 통과할 수 있도록 충분히 큰 계란의 녹화 부분에 구멍을 잘라 버릴거야. 찔린에 구멍을 시작하고 나선 모양으로 바깥쪽으로 했네요. 외부 세포막을 통해 상처지만, 노른자를 관통하지 않도록해야합니다. </li><li> 쉘에있는 구멍이 완료되면, 메추라기 배아를 시각화. 태아는 일반적으로 노른자의 상단 측면에 위치해 있습니다. </li><li> 한손으로 계란에있는 구멍을 삭감을 통해 노른자를 부어 시작합니다. 반면에 # 55 트위터 잡아. 난황의 상단 측면에있는 배 (胚)가 종료 계란으로 부드럽게 배아에서 # 55 핀셋을 슬라이드와 노른자에서 제거합니다. 참고 : 닭고기 계란은 배아 격리가 선명하고 깨끗한 표면에 닭 계란을 균열 및 100mm 페트리 접시에 계란을 깨는 포함됩니다. 배아는 노른자의 상단 측면에 위치해야합니다. 부드럽게 배아에 따라 # 55 핀셋을 슬라이드와 노른자에서 제거합니다. </li><li> 얼음처럼 차가운, 살균 EBSS를 포함 100mm 페트리 접시에 배아를 놓습니다. </li></ol></li><li> 햄버거와 해밀턴 1 기준에 따라 배아를 무대. 단계 14에서 태아가 - 과거 무대 13아르지만, 아직 20-21 somites와 꼬리 버드와 함께 단계 15에서. 단계 14 주요 특징 - 태아의 몸에 90 °보다 약간 큰 머리 각도입니다. </li><li> 단계 14에서 메추라기 배아의 마음을 제거 - # 55 핀셋으로 유명 운하와 OFT는 흉부 벽과의 접촉을 어디 transversely 절단하여. 페트리 접시 한쪽에 함께 마음을 놓으십시오. </li><li> 멸균 전송 피펫으로 격리 마음을 수집하고 살균, 얼음처럼 차가운 EBSS 가득한 새로운 100mm 페트리 접시에 그들을 놓으십시오. </li><html><li> # 55 핀셋으로 심실에서 transversely OFTs와 AV 운하 컷. </li><li> 섹션은 나머지 AV 운하 및 / 또는 길이 방향 유출 트렉츠 (OFTs). </li><li> 20 UL의 볼륨에 피펫을 설정합니다. AV 운하와 OFTs을 길이 방향 - 컷 여섯 대기음이 피펫을 사용합니다. </li><li> 콜라겐 젤로 유명 운하와 OFTs을 추방. 젤의 표면에서 초과 EBSS을 대기음. </li><li> 그들이 평평하므로 방향 explants에 # 55 핀셋을 사용하고 있으므로 루멘 사이드가 콜라겐 젤을 얼굴. 핀셋과 펑크 콜라겐 젤을하지보십시오. </li><li> 37 2 시간 후에 ° C와 5% CO 2, # 55 핀셋으로 explants에서 myocardium를 제거합니다. endocardial 세포는 콜라겐 젤의 표면에 남아 있어야합니다. 또는, myocardium가 explant에 남아있을 수 있습니다. myocardium 제거와 함께가 아닌 함께 endocardial 세포가 개입없이 EMT을 받고있다. myocardium 존재로 endocardial 세포의 일부가 mesenchyme로 변환되지만, 변화의 정도는 외부 요인과 변조된 수 있습니다. 참고 : 중간이 추가되기 전에 myocardium가에 남아있을 것입니다하더라도 explant 적어도 2 시간 겔에 첨부할 수 있어야합니다. </li><li> 각 잘 포함 explants 0.4 ML M199 매체를 추가합니다. </li><li> 문화 37 세포 ° C 5 % CO 2, 100 % 습도. </li></ol> 3. 포스트 EMT AV 쿠션 Mesenchymal 세포 분리 및 문화 <ol><li> 날카로운, 깨끗한 표면에 닭 계란을 깨서 100mm 페트리 접시에 계란을 깰. 배아는 노른자의 상단 측면에 위치해야합니다. 배아를 둘러싼 세포막을 잡아서 # 5 핀셋으로 배아를 선택하고 얼음처럼 차가운, 살균 EBSS 가득한 100mm 페트리 접시에 배아를 놓습니다. </li><li> 햄버거와 해밀턴 1 기준에 따라 배아를 무대. 무대 25 배 (胚)의 특징은 독특한 팔꿈치와 무릎 관절과 약간의 눈 착색되어 있습니다. </li><li> 모든 HH25 배아의 마음을 분리하여 배양 접시 한쪽에 함께 마음을 놓으십시오. </li><li> 멸균 전송 피펫으로 격리 마음을 수집하고 살균, 얼음처럼 차가운 EBSS 가득한 새로운 100mm 페트리 접시에 그들을 놓으십시오. </li><li> 한번에 모든 마음에서 유명 지역을 격리. 페트리 접시 한쪽에 함께 AVS를 놓​​습니다. 부드럽게 세포 생존에 영향을 미칠 수있는 모든 혈액을 제거하는 AV 지역을 선동하다. </li><li> 멸균 전송 피펫으로 격리 AVS를 수집하고 살균, 얼음처럼 차가운 EBSS 가득한 새로운 100mm 페트리 접시에 그들을 놓으십시오. </li><li> 순차적으로 AV에서 방석을 분리. AV 쿠션에는 myocardium 선물이 없습니다 있는지 확인합니다. </li><li> 멸균 전송 피펫으로 격리 방석을 수집하고 살균 15 ML의 원심 관에 넣어. </li><li> 3 분 15 XG에서 원심 분리기 튜브의 바닥에 방석을 스핀. </li><li> 쿠션을 방해하지 않고 가능한 한 EBSS 뜨는의 많은를 제거하는 살균 1,000 ML 팁을 사용합니다. </li><li> 37 미리 예열되어 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 1 ML ° C. 추가 그들이 완전히 해소되기 전까지는 쿠션이 트립신에 부화 허용하는 3-5 분 정도 정상적으로됩니다. </li><li> 트립신을 담금질하기 위해 원심 관에 닭 혈청 100 μL를 추가합니다. </li><li> 5 분 160 XG에 펠렛은 세포. 멸균, 1000 μL 팁으로 뜨는을 제거합니다. </li><li> M199 매체와 pipetting으로 혼합 2 ML에있는 세포를 Resuspend. hemocytometer 의지하고 고립되었습니다 세포의 총 수를 결정하기 위해 세포 현탁액 10 μL을 가지고 </li><li> 콜라겐 젤은 절연 세포의 수를 기반으로 이루어집니다 얼마나 많은 결정합니다. 세포 250 μL 젤 볼륨 2 × 10 5 세포 / ML의 밀도에 도금해야합니다. </li><li> 5 분 160 XG에 펠렛은 세포. 멸균, 1000 μL 팁으로 뜨는을 제거합니다. </li><li> 위에 나열된 프로토콜을 사용하여, 세포 펠렛으로, 그 순서에 배 M199, 물, 닭 혈청 주사, 콜라겐, 그리고 0.1 M NaOH를 추가합니다. 부드러운 pipetting하여 섞는다. </li><li> 물론 각각 1.9 cm이 성장 지역에 250 μL를 추가합니다. </li><li> 37 최소한 1 시간 동안 젤로 콜라겐 솔루션을 허용 ° C 5 % CO 2, 100 % 습도. </li><li> 문화 미디어 0.4 ML를 추가하고 밤새 품어. </li><li> 스트레스없는 조건에서 culturing 젤을 계속하려면, 잘 살균 200 μL 피펫 팁을 사용하여 문화의 측면에서 젤을 해방. 기계 제한된 문화가 잘 문화의 측면에 연결된 상태 젤 있습니다. </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="그림 1" /> 그림 1 :. 배아의 예 (A) HH14 - 퀘일 배아, 그리고 (B) HH25 닭 배아가 여기에 표시됩니다. fig2.jpg &quot;고도 =&quot;그림 2 &quot;/&gt; 그림 2 : 문화의 2 시간 후에 HH14 밸브 endocardial explants (A) myocardium 선물로, 또는 (B) myocardium 제거와 함께.. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="그림 3" /> 그림 3 :. HH14 밸브 endocardial explants가 (A) myocardium 선물로 세포의 많은 mesenchymal 변화에 endocardial 받아야과 문화 48 시간 후 스핀들 모양, mesenchymal 표현형 있습니다. (B) myocardium가 세포의 제거되면 문화의 48 시간 후 조약돌 모양, endocardial 표현형를 유지합니다. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="그림 4"/> 그림 4 :. 콜라겐 젤의 HH25 밸브 mesenchymal 세포 세포 즉시 시딩 후 반올림됩니다. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="그림 5"/> 그림 5 :. 문화 HH25 mesenchymal 세포 (A) 제약 콜라겐 젤 문화 48 시간 후 HH25 mesenchymal 세포가 확산 시작됩니다. (B) 칠일 해방 후 시딩 및 6 일 후에 스트레스없는 콜라겐 젤의 HH25 세포는 원래 면적의 약 50 % 젤 압축된있다.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

무대 HH14에서 endocardial 세포 분리를위한 방법 - 원래 런얀 및 Markwald 개발한 마음은 시작과 배아 EMT 2 조절 요인을 연구하기 위해 체외 환경에서 제어를 제공합니다. HH14 - myocardium하지 않고 교양 endocardial explants가 biomechanical 또는 생화 학적 개입없이 EMT를 받아야하지 않습니다. myocardium가 explant에 남아있다면 그것은 mesenchymal 변화를 받아야하는 endocardial 세포의 하위 ?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
<tr>
<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of avian embryonic valvular progenitor cells

배아 심장 밸브의 올바른 형성과 기능은 개발 진행을 위해 중요합니다. 초기 배아 심장은 myocardium 둘러싸인 endocardium의 U - 모양의 튜브입니다. myocardium secretes 심장 젤리, 방실 (AV) 접합 및 유출 트랙트 (OFT) 루멘으로 hyaluronan 풍부한 젤라틴 매트릭스. 단계에서 HH14 valvulogenesis는 endocardial 세포의 일부가 mesenchymal 변환 (EMT)에 endocardial myocardium의 신호를 받아야 받게하고, 심장 젤리를 침공 때 시작됩니다. HH25 단계에서 밸브 모양의 쿠션을 완전히 mesenchymalized, 그리고 그것은 결국 심장의 밸브 모양의 장치와 septal을 형성하고이 mesenchyme집니다. EMT 및 세포 분화의 과정을 시작하고 조절하는 메커니즘을 이해하는 것은 심각한 선천성 심장 때문에 결함들의 연결 중요합니다. 본 연구에서는 우리는 prevalvular 쿠션의 두 가지 세포 phenotypes 아르 사전 EMT endocardial 및 사후 응급 mesenchymal 세포를 분리하는 방법을 제시한다. 사전 EMT endocardial 세포 또는 myocardium없이 교양 수 있습니다. 포스트 EMT 유명 쿠션 mesenchymal 세포 내부 기계 제약이나 스트레스없는 콜라겐 젤을 배양해 수 있습니다. 체외 모델에서 이러한 3D 주요 밸브 모양의 morphogenic 이벤트를 모방하고 초기 늦게 무대 valvulogenesis의 메커니즘을 deconstructing하는 데 유용합니다.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

이 문서는 분리를위한 방법과 culturing 퀘일 또는 닭고기 HH14를 제공합니다<sup&gt; -</sup&gt; 밸브 endocardial 세포와 HH25 밸브 쿠션 mesenchymal 세포.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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JoVE Journal

Biology

10.1K 조회수.  Cornell University. 이 문서는 분리를위한 방법과 culturing 퀘일 또는 닭고기 HH14를 제공합니다 - 밸브 endocardial 세포와 HH25 밸브 쿠션 mesenchymal 세포.

비디오: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

포스트 - 나탈 마우스 소뇌 과립 신경 세포의 전구 세포 뉴런의 분리 및 문화

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

연구

생물학

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

인간 배아 줄기 세포의 문화 및 유지 관리

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

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 and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

마우스 기본 췌장 포상 세포의 분리 및 문화

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...