수술 부 I. 준비 <ol><li> 쥐의 수술 전날, 모든 솔루션 만들어진 특정하게하지만, 아직 효소를 추가하지 마십시오. 버퍼 A, B 및 세척 버퍼는 모든 저장하기 전에 불임에 대한 필터링해야합니다. 버퍼는 모든 4 ° C.에 보관해야 </li><li> 6 - 잘 조직 문화 플레이트는 10-20 μg / 2 ML ML 1XSFM에서 laminin뿐만 아니라 전날과 도금해야합니다, 37 CO 2 배양기에서 ° C에서 저장해야합니다. </li><li> 수술 당일, 70 % 에탄올로 클램프, 가위, 그리고 집게를 살균하여 수술 악기를 준비하고 그들을 종이 타월에 말리면. </li><li> 0.5 ML의 헤파린 솔루션 (90 U / ML)로 가득 한 ML의 주사기는 준비가 이루어집니다. </li><li> 이제는 반대의 연동 펌프를 사용하여 70 % 에탄올을 통해 실행하여 재관류 장치를 세척하는 것이 좋습니다. 일단 에탄올과 rinsing 완료를 두 번 증류수로기구를 린스, 마침내 37 ° C 물에 목욕에 배치 비커에 수집을 통해 버퍼 A.와 흐름을 씻어. </li><li> 청소 재관류 장치를 준비하려면, 버퍼 B. 40 ML과 버퍼 및 왼쪽 주사기 튜브 40 ML과 오른쪽 주사기 튜브를 채울 </li><li> 버퍼 장치를 통해 흐름을 허용하여 카테 테르의 끝에으로 국무 재관류 튜빙. 버퍼 40 ML 수준에 주사기로 리필. 이 단계 후 튜브에 갇혀 아무 공기 방울이 없습니다 있는지 확인합니다. </li><li> 자, 재관류 튜빙 이제 버퍼 B.와 함께 준비하는 것을 버퍼에게 주사기 꼭지 밸브를 닫고 같은 버퍼 B와 과정을 반복 </li><li> 마지막으로, 재관류 장치는 버퍼 B 꼭지 밸브 개방과 왼쪽지만, 흐름과 IV 라인에있는 조절기를 사용하여 중지됩니다. </li><li> 수집 비커의 흐름 불구하고 버퍼를 폐기하십시오. </li><li> 수술하기 전에 할 마지막 것은 다른 솔루션은 전날 필터링 것처럼 효소 솔루션을 귀하의 재고 솔루션에 필요한 효소를 추가한 다음 필터를하는 것입니다. 일단 필터링이 솔루션은 실내 온도에서 남아있을 수 있습니다. </li></ol> 부 II. 랫 심장 해부 <ol><li> 다음과 같은 표준 프로토콜은 가스실에서 Isoflurane과 쥐를 안락사. </li><li> 70 % 에탄올로 흉부의 절개 영역을 소독. </li><li> 오픈 가슴은 가위를 사용하고 마음을 쉽게받을 수 있습니다. </li><li> 0.5 ML의 헤파린 솔루션 (90 U / ML)과 좌심실을 주사. </li><li> 얼음 차가운 버퍼 B.에서 대동맥 그대로, 장소의 많은 부분과 함께 마음을 제거 </li></ol> 부 III. Myocytes 절연 <ol><li> 다음 절차가 제대로 붙어있다면 일반적인 쥐의 심장은 막대 모양 myocytes 높은 분수를 (같은 죽은 둥근 세포의 반대 의미) 양보해야합니다. </li><li> 시작하려면, 카테터에 대동맥을 겸자. 카테 테르의 끝에은 관상 동맥을 통해 심장의 좋은 재관류을 보장하기 위해 심장에 너무 많이 강요해서는 안됩니다. </li><li> 일단 채워, 봉합으로 카테 테르에 대동맥을 묶는. </li><li> 다음, 빠른 드립 속도 (~ 60 방울 / 분) 허용하고 마음을 씻어 버퍼 B을 허용하도록 IV 라인 조절기를 엽니다. </li><li> 버퍼 B 세척 동안 심방과 마음 기대며 어떤 지방이나 폐 조직을 제거하는 작은 가위와 집게를 사용합니다. </li><li> 버퍼 B가 완료되면, 버퍼 A.으로 흐름을 전환 </li><li> 버퍼가 5 분 유량 수 있지만, 여러 작은 작업을 신속하게 수행하여야한다. </li><li> 첫째, 37 ° C의 물을 욕조에있는 효소 솔루션을 따뜻하게. </li><li> 마지막으로, 버퍼는 주사기 (하지만 튜브에있는 사람들)에서 소진되었을 때, 주사기의 효소 솔루션의 50 ML을 로드할 재관류 장치의 </li><li> 이것은 효소 솔루션 perfuses 마음까지, 지금 모든 물 목욕 (pipetting)에서 회수 비커의 흐름을 통해 삭제하는 것이 필요합니다. </li><li> 즉시 효소 솔루션 마음을 perfuses로 주사기 A를 보충하기 위해 수집하는 비커에서 효소 솔루션을 전송하도록 설정됩니다 연동 펌프를 활성화 </li><li> 10 분 대한 마음을 통해 흐름에 효소 솔루션을 허용합니다. 심장이 소화되면서, 그일은 부풀어보고 시작합니다. </li><li> 주사기에있는 효소 솔루션에 0.1 M CaCl 2 37.5 μL를 추가 0.1 MM 칼슘 2 효과적인 농도를 제공 +. </li><li> 10 분 주사기에 0.1 M CaCl 2 추가 50 μL를 추가하여 0.2 mm까지 칼슘의 농도를 증가하고 재관류가 추가로 10 분 정도 진행하게되면. </li><li> 심실을 잘라 20 ML 효소 솔루션을 포함하는 작은 살균 비커로 전송할 수 있습니다. </li><li> 비커에 0.1 M CaCl 2 40보다 μL를 추가하여 0.4 mm까지 칼슘 농도를 높일 수 있습니다. </li><li> 부드럽게 작은 멸균 가위 10 개 이상의 조각으로 마음을 말하다. </li><li> 37 5 분 비커를 품어 ° C 부드러운 락을 함께. </li><li> 0.1 M CaCl 2 40 ML 추가0.6 MM 칼슘 2 +의 효과적인 농도를주고 부드럽게 플라스틱 전송 pipet 3-5 시간으로 심장 조각을 씹다. </li><li> 37 추가 5 분 잠복기 후에 ° C가 0.1 M CaCl 2 40 ML을 추가하고 부드럽게으로 이전 씹다. </li><li> 에서 별도의 소화 단일 myocytes 아닌 소화 멸균 500 μm의 메쉬를 통해 여과를 사용하여 결합 조직합니다. </li><li> 세포가 실온에서 10 분 동안 50 ML 튜브에 정착하도록 허용합니다. </li><li> 이전 pipet과 함께 뜨는 폐기하십시오. </li><li> 부드럽게 씻어 버퍼 # 1 세포를 resuspend하고 세포가 실온에서 10-20 분 정착하실 수 있습니다. </li><li> 세포가 적응하는 동안, 거꾸로 현미경 정지 세포의 품질과 경쟁력을 평가할 수있는 기회로 이번 작은 나누어지는을하고 사용합니다. 좋은 준비가 선명 강선과 같은 막대 세포의 높은 비율 (&gt; 80 %)이 포함됩니다. </li><li> 세포가 정착되면, 워시 버퍼 # 2에 뜨는 부드럽게 resuspend 세포를 폐기하십시오. 세포가 상온에서 해결하자, 이것은 다시 10-20분을하고 뜨는을 폐기해야합니다. </li><li> 세탁 과정을 반복 한 번 씻으 버퍼 # 3, 그리고 세포로 더 많은 문화를 준비합니다. </li></ol> 부 IV. Myocyte 세포 배양 <ol><li> 부드럽게 5% 세럼 미디어 세포를 resuspend. 조직 배양 플레이트에 세포를 전송하기 전에, 1X SFM과 접시를 씻으십시오. 3-5시간에 대한 CO 2 조직 문화 인큐베이터에서 원하는 밀도와 부화에있는 세포를 전송합니다. </li><li> 1X SFM에 미디어를 전환합니다. </li><li> 셀은 4 일까지에 교양과 같은 실험에 필요한 사용할 수 있습니다. </li></ol> 부품 V. 대표 결과 / 성과 프로토콜이 올바르게 이루어집니다 거꾸로 현미경보다 70 % 라이브 심장 myocytes이 있어야합니다. <img src="/files/ftp_upload/1308/fig1.jpg" alt="그림 1" /> 표 1 : 1 L에 대한 솔루션 요리법 10X 칼슘 2 + 무료 KH 버퍼 : <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> 질량 </td><td> 최종 농도 </td></tr><tr><td> NaCl </td><td> 68.96 g </td><td> 1,180 MM </td></tr><tr><td> KCl </td><td> 3.58 g </td><td> 48 MM </td></tr><tr><td> HEPES </td><td> 59.58 g </td><td> 250 MM </td></tr><tr><td> K 2 HPO 4 </td><td> 2.85 g </td><td> 12.5 MM </td></tr><tr><td> MgSO 4 </td><td> 3.08 g </td><td> 12.5 MM </td></tr></tbody></table> 표 2 : 1 L 버퍼에 대한 솔루션 요리법 : 100 ML 10X KH 버퍼에 1.98 g의 포도당을 추가하고 포도당과 함께 근처 생리 상태 (~ 300)에 osmolarity를​​ 조정 한 L.의 최종 볼륨을 가져. NaOH와 산도 7.4을 가져와. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> 질량 </td><td> 최종 농도 </td></tr><tr><td> 10X KH 버퍼 </td><td> 100 ML </td><td> 100 ML </td></tr><tr><td> 포도당 </td><td> 1.98 g </td><td> 11 MM </td></tr><tr><td> 1 L 가져오기 </td><td></td><td></td></tr><tr><td> NaOH와 산도 7.4 가져오기 </td><td></td><td></td></tr><tr><td colspan="3"> 포도당과 함께 근처의 생리적 조건 (~ 300)에 osmolarity를​​ 조정합니다. </td></tr></tbody></table> 표 3 : 효소 버퍼에 대한 솔루션 요리법 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> 질량 </td><td> 최종 농도 </td></tr><tr><td> Collagenase 유형 II </td><td> 25 MG </td><td> 0.05 % (W / V) </td></tr><tr><td> 프로 테아제 XIV </td><td> 10 MG </td><td> 0.02 % (W / V) </td></tr><tr><td> BDM </td><td> 0.025 g </td><td> 5 MM </td></tr><tr><td> Carnitine </td><td> 0.020 g </td><td> 2 MM </td></tr><tr><td> 타우린 </td><td> 0.031 g </td><td> 5 MM </td></tr><tr><td> 글루탐산 </td><td> 0.020 g </td><td> 2 MM </td></tr><tr><td> 0.1 M CaCl 2 </td><td> 12.5 UL </td><td> 25 UM </td></tr><tr><td> 버퍼 </td><td></td><td> 50 ML에 작성 </td></tr></tbody></table> 표 4 : 25X BDM / 타우린 / BSA (B / T / B)를위한 솔루션 요리법 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> 질량 </td><td> 최종 농도 </td></tr><tr><td> BDM </td><td> 0.076 g </td><td> 125 MM </td></tr><tr><td> 타우린 </td><td> 0.094 g </td><td> 125 MM </td></tr><tr><td> BSA </td><td></td><td> 0.1 % (W / V) </td></tr><tr><td> 버퍼 </td><td></td><td> 6 ML에 작성 </td></tr></tbody></table> 표 5 : 버퍼 B 및 씻으 버퍼에 대한 솔루션 요리법 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 버퍼 : </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> 주소 : </td><td></td><td></td><td></td><td></td></tr><tr><td> 0.5 M CaCl 2 </td><td> 0.4 ML </td><td> 0.1 ML </td><td> 0.125 ML </td><td> 0.15 ML </td></tr><tr><td> CaCl 2 농도 최종 </td><td> 1 ㎜ </td><td> 1 ㎜ </td><td> 1.25 MM </td><td> 1.5 MM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 ML </td><td> 2 ML </td><td> 2 ML </td></tr><tr><td> 로 최종 볼륨을 버퍼 </td><td> 200 ML </td><td> 50 ML </td><td> 50 ML </td><td> 50 ML </td></tr></tbody></table> 표 6 : Culturing 미디어를위한 솔루션 레시피 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X 세럼 - 무료 미디어 (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> 최종 농도 </td></tr><tr><td> (희석 이후) </td></tr><tr><td> Carnitine </td><td> 0.099 g </td><td> 10 MM (5 ㎜) </td></tr><tr><td> 타우린 </td><td> 0.063 g </td><td> 10 MM (5 ㎜) </td></tr><tr><td> 크레아틴 </td><td> 0.075 g </td><td> 10 MM (5 ㎜) </td></tr><tr><td> Antimycotic / 항생제 </td><td> 0.5 ML </td><td> 1퍼센트 (0.5 %) (V / V) </td></tr><tr><td> 미디어 199 </td><td> 45 ML </td><td> 50 ML에 작성 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> 최종 농도 </td></tr><tr><td> 2X SFM </td><td> 25 ML </td><td> 1X </td></tr><tr><td> 미디어 199 </td><td></td><td> 50 ML에 작성 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X 5% 세럼 미디어 </td><td></td><td></td></tr><tr><td></td><td></td><td> 최종 농도 </td></tr><tr><td> 2X SFM </td><td> 25 ML </td><td> 1X </td></tr><tr><td> FBS </td><td> 2.5 ML </td><td> 5 % (V / V) </td></tr><tr><td> 미디어 199 </td><td></td><td> 50 ML에 작성 </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

중요한 단계는 고립된 마음이 재관류 시스템에 걸려있는 속도입니다. 효소 소화 기간의 길이는 각 쥐에 대해 약간 다를 수 있습니다. 조정은 마음이 소화의 일반 기간이 지난 후된다 얼마나 부드러 운지에 따라 달라집니다. 천천히 소화 효소가 + - 관용 건강한 세포 칼슘이를위한 필수 + 후 칼슘 2 복구. hyaluronidase는 단백 분해 효소 XIV 대신 사?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
<tr>
<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of adult rat heart myocytes

교양 차 성인 쥐 심장 세포는 심장 혈관 연구에 중요한 모델 시스템입니다. 그럼에도 불구하고, 강력하고 실용적 교양 성인 myocytes의 설립은 많은 연구자에 대한 기술적 도전, 속도 - 제한 단계하실 수 있습니다. 여기 며칠 동안 문화에 가능한 남아 성인 쥐 심장 myocytes 높은 수율을 얻을 수있는 프로토콜을 설명했다. 마음은 낮은 칼슘 아래의 고립과 collagenase와 프로 테아제와 perfused입니다<sup&gt; 2 +</sup&gt; 조건은 단일 myocytes를 복구할 수 있습니다. CA<sup&gt; 2 +</sup&gt; - 관용 세포는 세포외 칼슘의 stepwise 증가를 얻을 수있다<sup&gt; 2 +</sup세 이후 세척 단계에&gt; 집중. 세포는 필터링된 문화 매체 resuspended 및 laminin 코팅 전표에 도금입니다. 이 프로토콜을 사용하여 얻은 교양 myocytes은 최대 4 일 동안 실용적이며, 전기 생리학, 생화학, 이미징 및 분자 생물학을 포함하여 대부분의 실험에 적합합니다.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

본 논문에서 우리는 성인 쥐 심장 myocytes를 분리하고 문화에 전형 방법을 설명했다. Collagenase와 단백 분해 효소는 단일 myocytes을 소화하고 격리하는 데 사용됩니다. Myocytes는 대부분의 실험 요구 사항을 충족이 프로토콜을 따라 교양.

성인 쥐 심장 Myocytes의 차 문화

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.1K 조회수.  Columbia University. 본 논문에서 우리는 성인 쥐 심장 myocytes를 분리하고 문화에 전형 방법을 설명했다. Collagenase와 단백 분해 효소는 단일 myocytes을 소화하고 격리하는 데 사용됩니다. Myocytes는 대부분의 실험 요구 사항을 충족이 프로토콜을 따라 교양.

비디오: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

어른 토끼 망막의 Organotypic 문화

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

연구

생물학

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

랫 중심에 엔지니어링된 조직의 주입

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

공촛점 이미징에 대한 분리 및 성인 심장 Myocytes의 유전자 조작

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Mitochondria 높은 결합 랫 마음의 준비

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

성인 마우스에서 동방 노드 근육 세포의 분리, 문화 및 기능 특성화하는 방법

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

성인 쥐 심장에서 고품질 Cardiomyocytes, 내피 세포 및 섬유 아세포의 동시 격리

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...