저자는 교수 헨드릭 Tevaearai 및 연구의 초기 부분에 대한 재정 지원에 대한 외과학 교실을 주셔서 감사합니다.

참고 : 루이스 남성과 여성 쥐, 200~220그램이 표준 실험실 조건 (12 시간 빛과 어둠 사이클, 광고 무제한으로 물과 음식, IVC 케이지)에서 보관 하였다. 모든 동물은 동물 보호에 FELASA와 스위스 법의 권고 사항을 준수하여 처리 하였다. 1 셀 준비 : 골수에서 중간 엽 줄기 세포 분리 <ol><li> 5 분 동안 유도 챔버 내에 5 % 이소 플루 란 및 2 O 5 L / 분으로 래트를 마취. 마취 시스템에 연결 코 콘으로 동물의 주둥이를 놓습니다. 진정 작용을 확인하기 위해 발가락이나 꼬리 핀치를 수행합니다. </li><li> 엉덩이에 발목으로부터 피부를 절단하여 가위로 다리에서 피부를 제거합니다. 근육을 제거하고 동물 일어난 과다 출혈에 대한 대퇴 동맥을 잘라. 고관절을 노출하고, 대퇴골의 머리를 탈구. 또한 대퇴골 머리를 손상하지 않는 것이 중요하다. 50 ML 플라스틱 튜브에 멸균 PBS에서 뼈를 놓습니다. </li><li> 모든 뮤 제거을 깨지 않고 뼈 scles과 인대. 참고 : 뼈가 에탄올로 세척 용액 또는 오염의 결합을 피하기 위해 그대로 유지하는 것이 중요합니다. 5 분 동안 70 % 에탄올에서 박리 뼈를 헹군다. </li><li> 멸균 층류 후드에서 가위로 뼈의 양쪽 끝을 잘라. 뼈 말단에서 멸균 PBS를 주입하여 골수 플러시. 15 ML 튜브에 골수를 수집합니다. 300 × g으로 원심 분리기 7 분. </li><li> 상층 액을 제거합니다. 적혈구 용해 버퍼의 3 ML에서 펠렛을 일시 중단합니다. 300의 X g에서 7 분 동안 다시 회전하기 전에 실온에서 1 분간 정지를 품어. 150ml의 배양 플라스크에서, 무균 세포 배양 배지 (395 ml의 IMDM 배지, 5 ㎖ 펜 / 패 혈성, 100 ㎖의 FBS)를 추가하고 세포를 시드. </li><li> 부착되지 않은 세포를 제거하기 위해 두번째 일 매체 변경합니다. <ol><li> 에 원하는 수량이 얻어 질 때까지 2~3주마다 초 일 매체 변경을 수행합니다. </li><li> 또한, 씨앗에 의해 biograft 준비필요한 3 곳 바와 같은 고체 기질 상에 세포를 보내고. </li></ol></li><li> 심장 이식의 날, 세포를 준비하거나 심 외막 응용 프로그램 전에 biograft을 수확. <ol><li> Accutase 세포 분리 용액을 사용하여 세포를 수집한다. 세포를 계산합니다. 원하는 세포 량을 얻기 위해 계산 된 세포 현탁액의 부피가 1.5 ml의 원심 분리 관을 채운다. 300 XG에 7 분 동안 원심 분리하고 상층 액을 제거합니다. </li><li> 또한, 멸균 PBS 린스, 세포 배양 매체로부터 biograft을 수확, 혈청없이 신선한 문화 매체에 보관하십시오. </li></ol></li></ol> 2 첫 번째 개흉술 및 LAD 내고 <ol><li> 쥐의 무게를 측정. 37 ° C에서 가열 패드의 전원을 켭니다. 5-7 분 동안 유도 챔버 내에 5 % 이소 플루 란과 5 L / 분으로 100 % O 2를 가진 쥐를 마취. 진정 작용을 확인하기 위해 발가락이나 꼬리 핀치를 수행합니다. 가열 패드에 쥐를 놓습니다. </li><li> 14 동물 삽관G 정맥 카테터. 2.5 L / 분의 산소, 2.5 %의 이소 플루 란, 2 ML의 호흡량, / 분 90 호흡 호흡 주파수에 대해 프로그래밍 설치류 인공 호흡기에 관을 삽입 카테터를 연결합니다. </li><li> 0.1 ㎎ / ㎏에서 부 프레 노르 핀 용액을 준비합니다. 피하보기 용액의 3 분의 1을 주입. 가슴의 왼쪽 부분을 면도. 1 % Betadine 용액으로 소독. </li><li> 네 번째 늑간에서 흉골에 수직으로 피부를 절개. 3 흉부 근육의 레이어 (pectoralis 주요 오름차순 pectoralis 및 외부 경사의 pectoralis)를 분리합니다. (갈비뼈 4와 5 사이) 네 번째 늑간 공간을 엽니 다. </li><li> 갈비뼈를 확산하고 마음을 노출하는 작은 트랙터를 사용합니다. 조심스럽게 심낭을 엽니 다. 찾아 아트리움 아래 7.0 봉합 4mm와 관상 동맥 (LAD)를 좌전 하행를 결찰. </li><li> 3.0 봉합사를 사용하여 두 개의 바늘로 늑간 공간을 닫습니다. 흉골에서 근위 및 원위이 봉합을 배치합니다. 첫째, t말단 봉합을 ighten. 가슴을 부풀게하고 기흉을 피하기 위해 2 초 동안 호흡기 배기 호스 클램프. 두번째 봉합사를 조입니다. (더 봉합이 필요하지 않습니다) 다시 제자리에 근육 층을 배치합니다. 5.0 봉합과 피부를 닫습니다. </li><li> 1 % Betadine 용액으로 소독 봉합사. 부 프레 노르 핀 용액을 나머지 주입. 마취 시스템의 전원을 끄십시오. 삽관 카테터를 제거합니다. </li><li> 1-2 시간 동안 따뜻한 램프 아래에있는 케이지의 쥐를 유지합니다. 케이지 온도계를 유지하고, 가열을 통해 피하기 위하여 따뜻한 램프와 케이지 사이의 거리를 제어한다. IVC 단위로 쥐를 돌려줍니다. 피하 프레 놀핀 0.05-0.1 ㎎ / ㎏ 포스트에게 주사 -operatively 48 시간마다 6-12 시간. </li></ol> 두 번째 개흉술 치료 비아의 3 심 외막 관리 <ol><li> 반복 2.1-2.3 단계를 반복합니다. </li><li> 다섯 번째 늑간에서 흉골에 수직으로 피부를 절개. 3 층을 분리흉부 근육의 (pectoralis 주요 오름차순 pectoralis 및 외부 경사의 pectoralis). (갈비뼈 5와 6 사이) 다섯 번째 늑간을 엽니 다. </li><li> 갈비뼈를 확산하고 마음을 노출하는 작은 트랙터를 사용합니다. 일부 고수가 있다면, 신중하게 미세 집게로 제거합니다. </li><li> 합자 아래 창백한 영역으로 나타납니다 경색 영역을 찾습니다. 참고 : 필요한 경우, 더 나은 좌심실를 시각화 정점에 삽입 봉합 10cm 조각 (7.0)를 사용합니다. 마음을 노출 클램프에 의해 느슨하게 유지 봉합에 부드럽게 잡아 당깁니다. </li><li> 다음 처리 중 하나를 적용 : <ol><li> 심장의 표면에 biograft를 놓습니다. 10 분 동안 실온에서 데워진 미리 채워진 피브린 실란트 주사기를 사용한다. 25 G 루어 바늘을 사용하여 biograft에서 피브린 실란트의 드롭 (50 ~ 100 μL)를 적용합니다. biograft가 완벽하게 밀봉되어 있는지 확인합니다. </li><li> 대안 적으로, 표면에 O 피펫 팁과 수집 된 세포 펠렛을 적용합자 아래 창백한 영역으로 마음을 바. 세포 펠렛에 피브린 실란트의 드롭 (50 ~ 100 μL)를 적용합니다. </li></ol></li><li> 정점에서 봉합의 조각을 제거합니다. </li><li> 반복 2.6-2.8 단계를 반복합니다. </li></ol>

<ol>
	<li>Gjesdal, O., Bluemke, D. A., Lima, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+remodeling+at+the+population+level--risk+factors,+screening,+and+outcomes.">Cardiac remodeling at the population level--risk factors, screening, and outcomes.</a> Nat. Rev. Cardiol. 8, 673-685 (2011).</li><li>Alcon, A., Cagavi Bozkulak, E., Qyang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+functional+heart+tissue+for+myocardial+repair.">Regenerating functional heart tissue for myocardial repair.</a> Cell Mol. Life Sci. 69, 2635-2656 (2012).</li><li>Guex, A. G., Fortunato, G., Hegemann, D., Tevaearai, H. T., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Protocol+for+the+Culture+of+Cells+on+Plasma-Coated+Electrospun+Scaffolds.">General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds.</a> Methods Mol. Biol. 1058, 119-131 (2013).</li><li>Selye, H., Bajusz, E., Grasso, S., Mendell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+techniques+for+the+surgical+occlusion+of+coronary+vessels+in+the+rat.">Simple techniques for the surgical occlusion of coronary vessels in the rat.</a> Angiology. 11, 398-407 (1960).</li><li>Gomes, A. C., Falcao-Pires, I., Pires, A. L., Bras-Silva, C., Leite-Moreira, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+heart+failure:+an+updated+review.">Rodent models of heart failure: an updated review.</a> Heart Fail. Rev. 18, 219-249 (2013).</li><li>Horstick, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+procedure+affects+physiological+parameters+in+rat+myocardial+ischemia:+need+for+mechanical+ventilation.">Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.</a> Am. J. Physiol. 276, 472-479 (1999).</li><li>Duchatelle, J. P., Vivet, P., Cortes, M., Groussard, O., Pocidalo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+Hemodynamic-Effects+of+Lateral+Thoracotomy+or+Sternotomy+in+Mechanically+Ventilated+Rats.">Respiratory and Hemodynamic-Effects of Lateral Thoracotomy or Sternotomy in Mechanically Ventilated Rats.</a> Eur. Surg. Res. 17, 10-16 (1985).</li><li>Giraud, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-based+engineered+skeletal+muscle+grafts+normalize+heart+function+early+after+myocardial+infarction.">Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction.</a> Artif. Organs. 32, 692-700 (2008).</li><li>Siepe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast-seeded+biodegradable+scaffolds+to+prevent+post-myocardial+infarction+evolution+toward+heart+failure.">Myoblast-seeded biodegradable scaffolds to prevent post-myocardial infarction evolution toward heart failure.</a> J. Thorac. Cardiov. Sur. 132, 124-131 (2006).</li><li>Guex, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, (2014).</li><li>Terrovitis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+Quantification+and+Optimization+of+Acute+Cell+Retention+by+In+Vivo+Positron+Emission+Tomography+After+Intramyocardial+Cardiac-Derived+Stem+Cell+Delivery.">Noninvasive Quantification and Optimization of Acute Cell Retention by In Vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem Cell Delivery.</a> J. Am. Coll. Cardiol. 54, 1619-1626 (2009).</li><li>Guo, H. D., Wang, H. J., Tan, Y. Z., Wu, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+marrow-derived+cardiac+stem+cells+carried+in+fibrin+improves+cardiac+function+after+myocardial+infarction.">Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction.</a> Tissue Eng. Part A. 17, 45-58 (2011).</li><li>Qiao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+and+Proliferation+Time+Course+of+Stem+Cells+Transplanted+in+the+Myocardium.">Death and Proliferation Time Course of Stem Cells Transplanted in the Myocardium.</a> Mol. Imaging Biol. 11, 408-414 (2009).</li><li>Conradi, L., Pahrmann, C., Schmidt, S., Deuse, T., Hansen, A., Eder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+for+Assessment+of+Immune+Responses+Following+Implantation+of+Engineered+Heart+Tissue+(EHT).">Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT).</a> J. Vis. Exp. 52 (e2605), (2011).</li></ol>

The authors have nothing to disclose.

LAD의 영구 결찰은 돌이킬 수없는 심근 손상이 발생합니다. 먼저 동물 모델은 그 이후 만성 MI에 대한 표준과 적합한 모델로 간주되어왔다. 1960 4에 기재 하였다. 그 안정성과 재현성 MI 5에 대한 치료의 실험 평가를 허용했다. 초기 설명을 다음과 같은 개선 된 절차는 35-13% 6의 수술 사망률을보고 하였다. 예상대로, LAD 결찰은 이주 내에 관찰 장애인 심장...

19 세기 말부터, 심혈관 질환은 선진국에서 번호를 하나의 연쇄 살인범을 유지하고있다. 그 중에서도, 관상 동맥 질환은 주요 원인을 나타낸다. 심근 경색 (MI)과의 급성기 결과는 점진적으로 만성 위상 및 중증 심부전으로 개발 부적응 리모델링옵니다. 최근 상당한 기술과 치료의 발전에도 불구하고, 때문에 심장 마비의 진행에 이환율과 사망률은 여전히 하나를 성장하고있다. 이러한 맥락에서, 세포 요법은 심부전 향해 질병의 진행을 정지하고 심근 최근에 식별 된 재생 능력을 자극하는 새로운 치료 옵션으로 증가 관심을 얻고있다. 실험 및 임상 연구는 다양한 세포 유형의 심장 이식 후 얻은 유익한 효과의 강력한 증거를 제공하고 있습니다. 주요 결과는 개선 cardi 포함교류 수축 기능, 좌심실 재 형성을 감소 경색 크기를 감소하고, 경색 지역에 혈관 밀도를 증가했다. 그러나, 세포 주입 후 세포 수가 낮은 유지율은 중요한 단점이 남아 있었다. 셀 전달이의 향상을 biomatrix와 세포의 협회는 최근 연구자 및 임상 이익을 육성했다. 관상 동맥 (LAD)를 좌전 하행의 결찰 전층 경색과 성숙한 흉터 결과 작은 동물 모델에서 MI에 대한 기준 방법입니다. MI의 만성 단계에 적용 세포 치료는 두번째 수술 적 치료가 필요합니다. 중간 흉골 보통 세포 또는 biografts의 외막 주입 심근 내 주입을 허용하도록 수행된다. 이러한 침습적 수술은 사망률, 수술 후 회복 시간, 통증, 감염의 위험을 증가시킨다. 여기에 제시된 최소 침습 접근 방식뿐만 아니라 그러한 편견을 방지 할뿐만 아니라 O를 제공합니다치료 응용 프로그램에 대한 마음의 ptimal 접근성. MI 및 젤 타입 biomatrix과 관련된 세포의 심 외막 이식 왼쪽 늑간 thoracotomies를 통해 심장 박동 상태에서 수행됩니다.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:104px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

심장 세포 치료는 세포와 관련된 바이오 물질의 관심과 주입을 증가 얻고있다 심근 세포의 전달을 최적화하는 중요한 문제가되고있다. 심근 경색의 설치류 모델 (MI) 동맥 (LAD) 결찰은 일반적으로 개흉술을 통해 수행 된 내림차순 좌전 구성된; 흉골 절개를 통해 초 오픈 심장 수술은 전통적 치료의 심 외막 응용 프로그램에 대한 수행되었습니다. LAD 결찰 모델의 설명 때문에, 수술 후 사망률은 그러나 두 번째 수술이 중요한 남아있다, 35~13% 떨어졌다. 통증과 감염을 수술 후 회복을 개선하고 줄이기 위해 최소 침습 수술이되게됩니다. 두 thoracotomies는, LAD 결찰에 대한 초기 치료 한 외막 투여 제를 실시 하였다. 고체 또는 젤 타입의 행렬과 관련된 세포로 구성된 Biografts는 경색 아칸소에 적용되었다개. 이 6 주 후에 실시 심 초음파 검사에 의해 확인으로 LAD 결찰은 심장 기능의 손실이 발생했습니다. Goldner 심장 섹션에서 수행 트리 크롬 염색 전층 흉터 형성을 확인했다. 제 1 및 제 2 수술은 덜이 10 %, 수술 후 사망률되었습니다.

모든 동물은 thoracotomies 후 1 시간 이내에 회복되었다. 상처 치유가 빠른이었다. 어떤 감염이나 부종은 관찰되지 않았다. 이중 좌측 개흉술은 심장에 최적의 액세스에게 (그림 1)을 허용했다. 통증 및 수술 후 사망률이 낮았다. 동물은 수술에서 빠르게 회복하고 (그림 2) 체중이 증가. 카플란 마이어 생존 %가 첫번째 개흉술에 대한 96 %이었다. (104) 위에 4...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

설치류 모델에서 LAD 결찰에 의한 심근 경색을 치료하는 biograft의 주입은 통상적으로 두 개의 심장 절개 수술을 요구하고있다. 사망률 감소 및 세포와 연관된 고체 및 젤라틴 biomatrices의 정착을위한 최적의 조건을 제공하기 위해, 최소 침습 절차가 개발되었다.

쥐에서 심장 마비에 대한 세포 기반 치료 : 심근 경색에 대한 두 개흉술 및 세포 및 Biomatrix의 심 외막 이식

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K 조회수.  University of Fribourg.  설치류 모델에서 LAD 결찰에 의한 심근 경색을 치료하는 biograft의 주입은 통상적으로 두 개의 심장 절개 수술을 요구하고있다. 사망률 감소 및 세포와 연관된 고체 및 젤라틴 biomatrices의 정착을위한 최적의 조건을 제공하기 위해, 최소 침습 절차가 개발되었다. 

비디오: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

엔지니어링된 심장 조직 이식 다음과 같은 면역 반응의 평가 (EHT)에 대한 Bioluminescence 이미징

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

연구

생체공학

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

심방과 심실에서 심장 근육 세포에 관형 막 네트워크 분석

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

시드 및 마우스 모델에서 생 합성 조직 설계 Tracheal 이식의 이식

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinocyte 확산에 2-및 3 차원의 평가 유형 I 교원 질 기판

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

자기 고열증 및 포토열 조합 암 치료를 위한 자기, 음향 및 광학 삼중 반응 마이크로 버블

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

생체 내 심근 수리를 위한 인간 iPS 세포에서 파생된 메쉬 모양의 조작된 심장 조직의 준비

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

심혈관 자기 공명 영상에 의한 마우스 심장 좌심실 기능, 심근 균주 및 혈역학력의 정량화

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D 인간 줄기 세포 유래 심근 세포에서 심장 수축성 조절 요법의 평가

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...