이 연구는 교육부의 지원을 받는 한국국립연구재단(NRF)을 통해 기초과학 연구 프로그램에 의해 지원됩니다(NRF-2018R1D1A1A02049346)

모든 동물 연구 절차는 실험실 동물 복지법, 실험실 동물의 치료 및 사용 안내서 및 가톨릭 대학교 의과 대학의 기관 동물 관리 및 사용위원회 (IACUC)가 제공하는 설치류 실험지침 및 정책에 따라 제공되었습니다.
1. MSC 및 주사용 젤라틴 하이드로겔 의 준비
<ol><li>37°C및 5% CO2에서100mm 배양 접시에 있는 MsC 배양. MSC 의 성장이 80 %에 도달하면 DPBS로 접시를 두 번 씻고 3 7 °C에서 트립신 대체 1 mL을 3 분 동안 추가하십시오. 참고: MSP는 기존의 절차에 따라 뮤린 골수로부터 격리되었다16,덜벡코의 수정 독수리의 매체 (DMEM)에서 배양 10% 태아 소 혈청을 포함 (FBS) 및 1% 항생제-항 mycotic 솔루션, 이 연구를 위해 통과 사이 사용 7\u20129.</li>
	<li>배양 배지 와 원심분리기 9mL를 500 x g에서 3분 간 추가합니다. 다음으로, 결과 상체를 버리고, PBS의 1 mL에서 세포를 재일시 중지하고, 얼음에 세포 현탁액을 유지한다.</li>
	<li>트라이판 블루의 10 μL로 셀 서스펜션 10 μL을 희석하고 자동화된 세포 카운터를 사용하여 세포 농도를 얻습니다.</li>
	<li>1 x 107 셀/mL의 밀도로 MSC를 1mL 튜브로 다시 일시 중단하고 전송합니다.</li>
	<li>PBS에서 GH 컨쥬게이트 용액의 6.25 wt%를 준비하고 2개의 바이알로 분리합니다. 다음으로 GH 솔루션을 HRP(GH 솔루션 A)의 6μg/mL 또는 H2O2(GH 솔루션 B)의 0.07 wt%와 혼합합니다. 참고: 공지된프로토콜(12,15)에따라 젤라틴-하이드록시페닐 프로피오닉산(GH) 컨쥬게이트를 준비한다.<ol>
<li>HRP(GH 솔루션 A) 및 GH 컨쥬게이트 용액에 대한 GH 컨쥬게이트 용액의 9:1 체적 비율을 각각H2O2(GH 솔루션 B)에 보관하십시오.</li>
	</ol>
</li>
	<li>MSC를 GH 솔루션 A와 혼합하기 전에 셀 서스펜션을 1,000 x g로 잠시 원심 분리하고 결과 슈퍼나티를 조심스럽게 흡인시합니다. 이어서, MSC를 함유하는 펠릿을 GH 솔루션 A와 혼합한다.</li>
</ol>2. 시투 MSC 로딩 및 체외 문화에서 입체적 입체
<ol><li>듀얼 주사기의 양쪽에 GH 솔루션 A(MSC 포함) 및 GH 솔루션 B를 로드합니다. 8웰 챔버 슬라이드에 5 x 106 셀/mL의 최종 밀도에서 MSC와 결합된 GH 솔루션의 플레이트 300 μL.</li>
	<li>시투 하이드로겔 형성 및 효소 교차 연결을 통한 후속 MSC 캡슐화 후, 10% FBS 및 1% 항생제-항진용액을 함유한 700 μL의 DMEM을 추가합니다.</li>
	<li>슬라이드를 37°C 및 5% CO2에서 배양하고 2\u20123일마다 배양 배지를 교체한다.</li>
</ol>3. GH 하이드로겔 내의 체외 증식 및 생존 확인
<ol><li>GH 하이드로겔 내에서 3D 배양 된 MSC의 생존가능성을 확인하려면 미리 정해진 인큐베이션 시간 후에 라이브/죽은 세포 염색 분석체를 사용하십시오.</li>
	<li>3, 5, 7 또는 14 일 동안 GH 하이드로겔에서 캡슐화된 MSC의 배양에 따라 매체를 흡면하고 PBS로 두 번 잘 씻는다.</li>
	<li>DPBS의 10mL에서 칼세인 AM 5 μL 및 에디듐 호모디머-1(EthD-1)의 20 μL을 포함하는 염색 용액을 준비한다.</li>
	<li>스테인링 용액 200μL을 우물에 넣고 실온에서 어둠 속에서 30분 동안 배양합니다.</li>
	<li>염색 용액을 흡인하고 PBS로 우물을 두 번 씻으세요.</li>
	<li>조심스럽게 슬라이드에서 챔버를 분리하고 GH 하이드로 겔 위에 전체 커버 슬립을 배치합니다. 캡슐화된 MSC의 증식 및 형태학적 변화의 정도를 시각화하기 위해 공초점 현미경 검사를 사용합니다. 참고: 형광 화상은 200배율 하에서 획득되었고, 칼세인의 경우 470/540 nm의 여기/방출 파장에서, EthD-1의 경우 516/607 nm로 이미지되었습니다.</li>
</ol>4. 마우스에서 심근 경색의 유도
<ol><li>7주 된 남성 C57BL/6 마우스(20\u20122g)를 식염수의 졸레틸(30 mg/kg)과 롬펀(10 mg/kg)의 혼합물을 인트라보네주사로 마취한다.</li>
	<li>수술 전에는 제모 크림을 사용하여 마우스 가슴을 뒤풀이고 요오드로 피부를 살균하십시오.</li>
	<li>수술대에 마우스를 놓고 카테터를 기관체에 삽입하여 기계 환기를 통해 산소를 보충합니다.</li>
	<li>외과 가위를 사용하여 피부를 부드럽게 잘라낸 다음 마이크로 가위로 늑간 근육을 관통합니다. 2차 및 3번째 왼쪽 갈비뼈를 5-0 실크 봉합사를 사용하여 분리하여 가슴 구멍을 유지합니다.</li>
	<li>8-0바늘 홀더를 사용하여 왼쪽 전방 내림차순(LAD) 관상 동맥을 조심스럽게 리게이트합니다. 폴리 프로필렌 봉합사 및 전기 카우테리를 사용하여 봉합사를 잘라.</li>
	<li>전방 좌심실 벽의 즉각적인 색상 변화를 관찰하십시오.</li>
</ol>5. MSC 로딩 GH 하이드로겔의 인트라미카르탈 이식
<ol><li>LAD 결찰에 의한 심근 경색을 유도한 후, 26G 바늘을 장착한 이중 주사기를 사용하여 경색 국경 영역(총 2 x 105 MSC/20 μL)의 두 가지 지점에 MSC 로딩 GH 용액 10μL을 주입합니다.<ol>
<li>1단계에서 설명된 동일한 절차에 따라 MSC 로딩 GH 솔루션을 듀얼 주사기로 준비하고 전송합니다. 참고: 경질 지역 내에서 MSC 로딩 GH 하이드로겔의 이식을 평가하기 위해 MSC와 GH 컨쥬게이트는 각각 PHK26 및 형광제 이소티오카네이트(FITC)로 사전 표지되었다.</li>
	</ol>
</li>
	<li>열린 가슴 구멍을 복원하고 5-0 봉합사를 사용하여 근육과 피부를 닫습니다. 참고: 가슴 이닫기 전에 카테터 주사기를 사용하여 공기를 제거합니다.</li>
	<li>기관관을 제거하고 복구 하는 동안 적외선 램프 아래 케이지에 마우스를 배치 합니다.</li>
	<li>수술 후 진통의 경우 피하 케토프로펜 주사(하루 5 mg/kg)를 최소 72시간 동안 투여하십시오. 모든 마우스는 수술 후 적절한 회복뿐만 아니라 적절한 통증 치료를 보장하기 위해 적절한 시간 동안 면밀히 모니터링해야합니다.</li>
</ol>6. 에코카르디그래피
<ol><li>이식 후 4주 후, 처음에는 5%의 이소플루란으로 마우스를 마취한 다음 이소플루란 농도를 1%로 조절한다.</li>
	<li>제모 크림을 사용하여 가슴을 뒤처질시키고 마우스를 가열 패드에 놓습니다. 가슴에 초음파 트랜스듀서 젤을 바치면 됩니다.</li>
	<li>2차원 파라스테랄 짧은 축 뷰를 획득하고 유두 근육 수준에서 M 모드 추적을 기록합니다. 참고: 왼쪽 파라스테럴 라인에 선형 어레이 변환기(7\u201215 MHz)를 배치하고 해부학 구조를 봅니다.</li>
	<li>LVAW, LVID 및 LVPW에 대한 해당 라인을 측정하여 심장 벽 두께, 챔버 치수 및 분수 단축을 얻습니다. 참고: 동일한 해부학 적 위치에서 적절한 평가를 보장하기 위해 유두 근육의 수준에서 배출 분수 (EF), 분수 단축 (FS), 및 최종 수축부(ESV)를 포함한 심장 기능을 비교합니다.</li>
</ol>7. 조직학적 평가
<ol><li>MSC-로딩 GH 하이드로겔을 경각심에 이식한 후 소정의 시간에,CO2 챔버에서 마우스를 안락사시키고 조직학적 분석을 위해 심장을 수집한다15.</li>
	<li>헤마톡시린과 에오신(H&E) 및 마슨의 트리크롬(MT) 염색의 경우, 해부된 심장 조직을 4% 파라포름알데히드(PFA)로 수정하고 파라핀에 포함시한다. 다음으로, 표준프로토콜(17)에따라 MT 얼룩으로 미세토메를 사용하여 파라핀 임베디드 심장 블록을 4 μm 직렬 섹션으로 잘라냅니다.</li>
	<li>20배 배율로 슬라이드 스캐너에서 이미지를 획득하고 치료 그룹의 경색 크기를 계산합니다. 광원 크기 (%) = 총 경색 주위 / 총 LV 둘레 × 100</li>
	<li>중간 선 길이 측정하여 둘 다 둘다 계산합니다. LV 미드라인 둘레의 경우 내막과 상골 표면 사이의 중심선 길이를 측정합니다. 미드라인 경외도의 경우, 심근18의전체 두께의 50% 이상을 포함하는 경색의 길이를 측정한다. 참고: ImageJ 소프트웨어를 사용하여 모든 이미지 분석을 수행했습니다.</li>
	<li>유두 근육 수준에서 흉터의 벽 두께를 측정합니다.</li>
	<li>콜라겐 영역의 일부를 계산합니다. 콜라겐 영역 (%) = 간질 섬유증 /심근 세포 영역 x 100의 총 면적</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+marrow-derived+mesenchymal+stem+cell:+isolation,+in+vitro+expansion,+and+characterization.">Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization.</a> Br J Haematol. 123 (4), 702-711 (2003).</li><li>Ojha, 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=Characterization+of+the+structural+and+functional+changes+in+the+myocardium+following+focal+ischemia-reperfusion+injury.">Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury.</a> Am J Physiol Heart Circ Physiol. 294 (6), 2435-2443 (2008).</li><li>Takagawa, 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> Journal of Applied Physiology. 102 (6), 2104-2111 (2007).</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> Journal of the American College of Cardiology. 54 (17), 1619-1626 (2009).</li><li>Dib, N., Khawaja, H., Varner, S., McCarthy, M., Campbell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Therapy+for+Cardiovascular+Disease:+A+Comparison+of+Methods+of+Delivery.">Cell Therapy for Cardiovascular Disease: A Comparison of Methods of Delivery.</a> Journal of Cardiovascular Translational Research. 4 (2), 177-181 (2011).</li></ol>

저자는이 작품으로 선언 하는 이해의 충돌이 없습니다.

주사용 GH 하이드로겔은 상균하게 시상에 다양한 치료제를 통합하는 능력 때문에 생체 내 응용 분야에서 큰 잠재력을 가지고 있습니다. 또한, 그들의 물리적 및 생화확적인 속성은 질병 의존적 요구 사항에 따라 쉽게 조작될 수 있습니다. 이러한 점에서, 주사용 하이드로겔은 현재 심장 줄기 세포 치료의 주요 한계를 해결하기 위해 제안된 생존 및 세포 보존(즉, 24시간 이식 후 10% 이내)에서<sup class...

심장 줄기 세포 치료는 심근 수리 및 재생1,2에대한 잠재적 인 접근법으로 부상하고있다. 최근 동물모델 및 임상시험에서 양성결과에도 불구하고 심근수리를 위한 줄기세포 기반 요법의 적용은 극각심장조직에서 주입된 세포의 생존율이 낮고 생존율이낮아서3,4. 그 결과, 주사용 생체재료5,심장 패치6,세포시트7을포함한 세포 기반 조직 공학의 사용은 숙주 심근 내의 세포 보존 및 통합을 개선하기 위해 집중적으로 연구되고 있다.
생체 공학 심장 조직 수리에 대한 다양한 잠재적 접근법 중, 관해 줄기 세포 (MSC), 배아 줄기 세포 (ESC), 유도 만능 줄기 세포 (iPSC)와 같은 적절한 세포 유형과 결합 된 주사용 하이드로겔은 심근 부위에 세포를 효과적으로 전달하는 매력적인옵션입니다 8,9. 잘 알려진 천연 폴리머인 젤라틴은 생체 적합성, 상당한 생분해성 및 생체 의학 응용 분야에서 사용되는 광범위한 생체 재료와 비교할 때 면역원성을 감소시키기 때문에 주사용 매트릭스로 사용할 수 있습니다. 젤라틴 기반 주 사용 플랫폼은 큰 잠재력을 가지고 있지만, 생체 내의 적용성은 낮은 기계적 강성과 생리 환경에서 쉽게 저하성에 따라 제한된 남아있다.
이러한 한계를 극복하기 위해 하이드록시페닐 프로피온산으로 구성된 젤라틴 기반 하이드로겔의 참신하고 간단한 디자인이 생체 내 적용을 위해 제안되었습니다. 젤라틴-하이드록시페닐 프로피오닉산(GH) 컨쥬게이트는 효소, 고추냉이 과산화효소(HRP)의 존재 시정에서 교차연결될 수 있으며, 그 후 하이드로겔 내의 다양한 약물, 생체분자또는 세포를 캡슐화하여 조직 공학 응용 분야에서 큰 잠재력을시사한다. 또한, 최근 캡슐화된 MSC를 함유한 GH 하이드로겔의 치료 효과를 조사하고, 뮤린모델(15)에서MI 후 성공적인 심장 수리 및 재생에 대한 사용을 입증하였다. 이 프로토콜에서, 우리는 GH 하이드로겔 내의 캡슐화 및 체외 3차원 (3D) 증식을 위한 간단한 기술을 설명합니다. 또한 관상 동맥 결찰및 MSC 로딩 GH 하이드로겔의 자궁 내 이식을 통해 뮤린 MI 모델을 생성하도록 설계된 외과 적 수술을 원각 심장으로 소개합니다.

<table>
	<tbody>
		<tr>
			<td>4 % paraformaldehyde (PFA)</td>
			<td>Intron</td>
			<td>IBS-BP031-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>AILEE</td>
			<td>SK534</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>ETHICON</td>
			<td>M8732H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Nunc LAB-TEK</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocath Plus (22GA) catheter</td>
			<td>BD Angiocath Plus</td>
			<td>REF382423</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimyocotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>GYROGEN</td>
			<td>1582MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slipe</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Deluxe High Temperature Cautery kit</td>
			<td>Bovie</td>
			<td>QTY1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EOSIN</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>K49350783 739</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fechtner conjunctiva forceps titanium</td>
			<td>WORLD PRECISISON INSTRUMENTS</td>
			<td>WP1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>HEBU</td>
			<td>HB0458</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Ildong Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>50300</td>
			<td>Homeothermic Blanket System</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>50301</td>
			<td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>SIGMA-ALDRICH</td>
			<td>HHS80</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxide (HRP; 250-330 U/mg)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2; 30 wt % in H2O)</td>
			<td>SIGMA-ALDRICH</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Green Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD cell staining kit</td>
			<td>Thermo Fisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>HANIL</td>
			<td>Micro 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>KASCO</td>
			<td>37-1452</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>HEBU</td>
			<td>HB7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>MT staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1079-1SET</td>
			<td>Weigert&#8217;s iron hematoxylin solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HT15-1KT</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>LK LABKOREA</td>
			<td>H06-660-107</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PHK26 staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>HEBU</td>
			<td>HB7454</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M micopore</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue cassette</td>
			<td>Scilab Korea</td>
			<td>Cas3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer gel</td>
			<td>SUNGHEUNG</td>
			<td>SH102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trout-Barraquer needle holder curved</td>
			<td>KASCO</td>
			<td>50-3710c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips</td>
			<td>Affiniti 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>JUNSEI</td>
			<td>25175-0430</td>
			<td></td>
		</tr>
	</tbody>
</table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

포스트인파르트 심부전을 예방하기 위한 현재 심장 줄기 세포 치료법에 직면한 주요 문제 중 하나는 부상당한 심근 내의 이식된 세포의 낮은 유지율과 생존율이 치료 효능을 제한하는 것입니다. 최근에는 스캐폴딩 생체 재료의 사용이 줄기 세포 치료제를 개선하고 극대화하는 데 주목받고 있습니다. 이 프로토콜의 목적은 주사용 하이드록시페닐 프로피온산(GH) 하이드로겔을 사용하여 골수 유래 중간엽 줄기 세포(MSC)를 이식하는 간단하고 간단한 기술을 도입하는 것입니다. 하이드로겔은 시투에서 교차 연결되는 능력과 높은 생체 적합성으로 인해 심장 조직 엔지니어링 응용 을 위한 세포 전달 플랫폼으로서 유리합니다. 우리는 MSC 로딩 GH 하이드로겔 (MSC/하이드로겔)을 제조하고 체외 배양에서 3차원 (3D)에서 생존과 증식을 평가하는 간단한 방법을 제시합니다. 또한, 마우스에서 MSC/하이드로겔의 자궁내 이식 기술을 시연하여 왼쪽 전방 내림차순(LAD) 관상 동맥 결찰 및 후속 MSC/하이드로겔 이식을 통해 심근 경색(MI)을 유도하는 수술 절차를 설명하고 있습니다.

광원심근에 효과적으로 MSC를 전달하기 위해 도 1에 설명된 시투 교차 연결 하이드로겔에서 MSC 로딩이 이 프로토콜에 사용되었다. 생체 내 이식 이전에, GH 하이드로겔에서 MSC의 증식 및 생존은 체외 라이브/죽은 세포 염색 분석서에서 3D에 의해 확인되었다(라이브: 녹색; 죽은: 빨강). 그림 2에도시된 바와 같이, 대표적인 이미지는 충분한 MSC 확산을 나타?...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

줄기 세포 기지를 둔 치료는 심근 경색 후에 부상당한 심장 조직을 복구하는 능률적인 전략으로 부상했습니다. 우리는 효소적으로 교차 연결 될 수있는 젤라틴 하이드로 겔을 사용하여 줄기 세포 이식에 대한 최적의 생체 내 응용 프로그램을 제공합니다.

Murine 모델에서 심근 경색 후 MSC 로딩 주사용 하이드로겔의 자궁 내 이식

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

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4.3K 조회수.  The Catholic University of Korea. 줄기 세포 기지를 둔 치료는 심근 경색 후에 부상당한 심장 조직을 복구하는 능률적인 전략으로 부상했습니다. 우리는 효소적으로 교차 연결 될 수있는 젤라틴 하이드로 겔을 사용하여 줄기 세포 이식에 대한 최적의 생체 내 응용 프로그램을 제공합니다.

비디오: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

고양이에서 급성 심근 경색

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

연구

의학

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

경색의 Murine 모델에서 관상 동맥 내고과 Intramyocardial 사출

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

돼지의 심근 경색 및 기능적 결과의 평가

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

하이드로 겔은 구축 및 쥐 심근 경색 모델에서 치료를 제공하기 위해 접착제 접근을 섬유소 기반.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

급성 심근 경색의 돼지 모델의 주요 결과 평가

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

신생아 쥐의 심근 경색, 심장 재생의 모델

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

왼쪽 전방 내림차순 관상 동맥의 영구 결찰을 사용하는 뮤린 심근 경색 모델

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

마우스에서 심근 경색을 연구하는 극저온 손상 모델

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

뮤린 모델에서 허혈혈 재관류 부상 후 줄기 세포의 지연 된 자궁 내 납품

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

생쥐의 심근경색 유도를 위한 수정된 간단한 방법

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...