이 연구는 (심장 혈관 연구, db 위한 독일 센터), DZHK에 의해 지원 되었다 EKFS (Else Kröner Fresenius 재단, F.H.), 고 (독일 교육 및 연구), BMBF BIH Charité 임상 과학자 프로그램 투자에 의해 의해 Charité-Universitätsmedizin 베를린, 베를린 건강 연구소 (F.H.).

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</ol>

저자는 공개 없다.

여기, 우리는 먼저 메츠 관련 HFpEF 보여 주는 심 방 개장22표시의 쥐 모델에서 단일 셀 Acm의 격리에 대 한 프로토콜을 설명 합니다. 절차는 수술 준비로 점점 어려운 대동맥의 cannulation 과도 한 지방 조직을 만들 수 있는 유일 하 게 도전. 표 2 주소 격리 절차의 가장 일반적인 문제 문제 해결 가이드 제공.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>문제</td>
			<td>가능한 원인</td>
			<td>솔루션</td>
		</tr>
<tr>
<td>나비 호스를 통해 아무 흐름</td>
			<td>Cannulation 노트와 대동맥의 부적당 한 폐쇄</td>
			<td>강화 하기 전에 지방 조직 제거</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>각 들여쓰기 3 cannulation 매듭을 추가</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>증가 힘에 대 한 손으로 매듭을 당겨</td>
		</tr>
<tr>
<td></td>
			<td>차단 된 바늘</td>
			<td>루멘으로 위치 재조정</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>주사기와 부드러운 풀에 의해 차단</td>
		</tr>
<tr>
<td>오른쪽 아 트리 움 / 관상 공동 하지 비정상적</td>
			<td>심장 자료의 부적당 한 폐쇄</td>
			<td>추가 노트와 블록 흐름</td>
		</tr>
<tr>
<td></td>
			<td>준비 하는 동안 손상 된 오른쪽 아 트리 움</td>
			<td>클립/매듭을 가진 가까운 누설</td>
		</tr>
<tr>
<td>불 쌍 한 소화 / 아 트리 움 부드럽게 하지 않습니다</td>
			<td>잘못 된 온도 통해 감소 된 효소 활동</td>
			<td>37 ° C에 온도를 조정합니다</td>
		</tr>
<tr>
<td></td>
			<td>비활성/타락 효소</td>
			<td>바꾸기</td>
		</tr>
<tr>
<td></td>
			<td>오래 된/지나치게 거리 아 트리 움</td>
			<td>소화 시간을 증가 (+ 50의 단계에서 %)</td>
		</tr>
<tr>
<td></td>
			<td>대동맥 밸브 손상</td>
			<td>Cannulation 동안 얕은 침투</td>
		</tr>
<tr>
<td>불 쌍 한 셀 수율</td>
			<td>아 트리 움-소화</td>
			<td>소화 시간을 단축 (+ 25의 단계에서 %)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>나비 호스를 낮추어 intraluminal 압력 감소</td>
		</tr>
<tr>
<td></td>
			<td>아 트리 움 소화</td>
			<td>증가 소화 시간 (25%의 단계)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>나비 호스를 올려서 intraluminal 압력 증가</td>
		</tr>
<tr>
<td></td>
			<td>너무 적극적인 셀 분산</td>
			<td>더 부드러운</td>
		</tr>
</tbody></table>표 2: 문제 해결 가이드. 이 테이블은 격리 절차 및 해당 솔루션의 일반적인 문제를 표시합니다.
최근 연구에서 우리는 ZFS-1 비만 쥐 모델 증가 심 방 크기22로 광범위 한 심 방 개장 전시 나타났습니다. 왼쪽된 심 방 크기 증가 확장기 부전26 에 대 한 중요 한 전조 표식으로 인정을 받고 있다 고 하면 조직의 상대적인 microvascular 혈관의 rarefication. 이것은 특히 도전 하는이 모델에서 단일 셀 격리 렌더링 격리 절차 동안 대동맥의 역행 관류를 통해 소화 버퍼의 감소 분포. 개장 하는 심 방, 심 방 섬유 증, 및 증가 된 섬유 증의 다른 품질 증명 특징에서 쥐를 위해 표시 되었습니다-대사 당뇨병과는 ZFS1의 부모 스트레인에 대 한 쥐 모델27쥐. 콜라겐 예금 하려면 cardiomyocytes는 기질에서 해방, 따라서, 더 세포 격리 절차28의 소화 효소의 효능을 저하.
압력 장치를 용이 하 게 개장 하는 심 방의이 모델에서 향상 된 절연 결과 메커니즘 왼쪽된 아 트리 움의 관상 동맥 혈액 흐름의 지역화 된 감쇠와 관련 된 가장 가능성이 높습니다. 관상 동맥 혈액 흐름의 한 주요 구성 요소는 관상 동맥 관류 압력, 관상 동맥 압력 및 각 구멍29의 끝 확장기 혈압 사이 기온 변화도로 정의 됩니다. 설명된 절차 동안 심장 기지의 막힌 관상 동맥 압력 및 심장에 걸쳐 intraluminal 압력에 세계적인 증가 이끌어 낸다. 왼쪽된 아 트리 움의 후속 펑크 왼쪽된 심 방 구멍에서 intraluminal 압력의 지역, 선택적 드롭을 수 있습니다. 따라서, 큰 관상 동맥 관류 압력 기울기 설정 뿐만 아니라 관류 볼륨도 남기도록된 소화 솔루션 혼잡 한 좌 심 실에서 좌 심을 증가.
또한, 소화 효소의 선택은 ACM 격리에 대 한 중요 한: I 및 II 보였다 collagenases30처럼 적은 타겟이 명확 하 고 덜 순수한 효소를 우수 하 콜라의 효소 혼합을 정화. 이 효소는 형태학 상으로 그리고 기능적으로 그대로 cardiomyocytes의 높은 수익률만 허용 하지 않습니다 하지만 또한 어떤 그들의 격리31후 단일 세포의 응집을 최소화 한다. 정제 효소 추가 높은 dispase와 collagenases의 혼합 또는 중간 thermolysin 콘텐츠 쥐 cardiomyocyte 격리 가장 일반적으로 사용 됩니다. 심 방 myocytes의 최상의 결과 인수 했다 VCMs는 높은 농도에서 가장 고립 된, 0.195의 농도와 Wünsch 단위/mL이를 사용 하 여32프로토콜.
메츠 관련 HFpEF의 보급은 상승 하 고 있다2 및 심 방 cardiomyopathies 심 방 리 모델링을 선도 하 고 심 방 세 동 관련성이 높은 임상,이 분야에서 연구의 중추적인 관심은 있습니다. HFpEF에 대 한 많은 새로운 동물 모델33,34 떠오르고 있다 그리고 심 방 심 리듬 장애의 발생률이 증가 따라 개장 하는 질병의 특징 기능. 설명된 방법 매우 높은 수확량을 가진 추가 연구에 대 한 심 방 개장을 가진 쥐 모델에서 가능한 단일 cardiomyocytes 분리 연구자 있으며 기계 및 전기 기능을 보존.

메츠 당뇨병 mellitus 타입-2와 심혈 관 질환 위험 요인의 클러스터를 설명 하 고 포함 하는 증가 동맥 혈압, dyslipidemia (트리 글리세라이드를 제기 하 고 고밀도 지 단백 콜레스테롤을 낮 췄 다), 금식 증가 포도 당, 그리고 중앙 비만1. 메츠의 전세계 보급 되며 지속적으로 상승2, 25-30%로 추정 된다. HFpEF은 메츠와 자주 관련 된 이기종 임상 증후군. HFpEF 및 그것의 이전 단계 (즉, 고혈압 성 심장 질환) 중 심장 개장은 또한 동반 atria3을 개장 하는. 감소 된 수축 성 기능 및 구조적인 변화는 왼쪽된 아 트리 움의 사망률 증가, 심 방 세 동, 및 새로운 발병 심장 마비4와 연결 되었습니다. 개장 하는 심 방 이온 채널 기능, 캘리포니아2 + 항상성, 심 방 구조, 섬유 아 세포 활성화 및 조직 섬유 증5에 변화에 의해 특징입니다. 왼쪽에 메츠 관련 HFpEF의 기본 병 적인 메커니즘 아직도 제대로 이해 하 고 더 깊이 있는 조사를 필요로 개장 하는 심 방. 동물 모델은 유용한 도구가 될 고 심 방 cardiomyopathies6,7,,89의 분야에서 많은 발전을 입증 했다.
격리 된 단일 셀 cardiomyocytes와 연구 자주 확증 vivo에서 조사 결과 보완 하는 데 사용 됩니다. 절연 및 잠재적인 후속 세포 배양, 신호 경로, 이온 채널 전류, 및 흥분-수축-커플링의 조사에 대 한 수 있습니다. 생리 적인 조건 하에서 cardiomyocytes 증식 하지 않습니다. Atrial natriuretic 요소의 transcriptional 규제 시퀀스와는 유인원 바이러스 40 큰 T 항 원 유전자 변형 쥐에서 사이의 융합 AT-110라는 첫 번째 불멸 하 게 Acm의 창조로 이끌어 냈다. AT-1 셀의 추가 개발 HL-1 셀만 순차적으로 passaged 수 없습니다 하지만 또한 저절로11계약에 상승을 했다. 그러나 그들은,, 구조와 기능 차이 덜 조직 된 열 대권 외의, hyperpolarization-활성화 안쪽으로 현재12myofibrils11, 개발의 높은 발생 등 갓 고립 된 세포에 비해 표시. 쥐와 쥐 모델의 다양 한에서 심 실 cardiomyocytes (VCM)의 절연은 잘 설립된13,14,15,16,,1718 , 19. 일반적으로, 삭제는 Langendorff 장치에 탑재 마음과 retrogradely 끼얹는다는 캘리포니아2 +-collagenases 및 프로 테아 제와 같은 소화 효소를 포함 하는 무료 버퍼. 칼슘은 다음 생리 적 조건에 단계적 방식으로 재. 그러나, Acm의 고립에 전용 프로토콜 사용할 수 있는20,21, 증가 증과 차이 압력 관련 있더라도 개장 하는 심 질환 모델에서 그들의 유용성은 제한 됩니다.
이 문서에서는, 우리는 심 방 개장 (즉, 특히 메츠 관련 HFpEF의 ZFS1 쥐 모델에 대 한)22를 나타내는 동물에서 심 방 단일 셀 cardiomyocytes의 격리에 대 한 프로토콜을 실행 했다. 기존 격리 프로토콜 최적화 하 고 제어 하 고 형태학 상으로 그리고 기능적으로 그대로 cardiomyocytes의 높은 수율으로 이어지는 심장 충 치의 intraluminal 압력 수정 간단 하 고, 주문 품 장치에 의해 보완 했다. 다음 프로토콜 단계별 가이드와 함께 연구원, 주문 품 장비, 솔루션의 목록 뿐만 아니라 포괄적인 문제 해결 가이드에 대 한 자세한 설명을 제공합니다.

<table><tbody><tr><td>ZSF-1 Obese rat</td><td>Charles River Laboratories, Inc.</td><td></td><td>21 weeks old</td></tr><tr><td>Fine Iris Scissors</td><td>Fine Science Tools GmbH</td><td>14094-11</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools GmbH</td><td>14001-18</td><td></td></tr><tr><td>Micro Dressing Forceps (curved, serrated)</td><td>Aesculap, Inc.</td><td>BD312R</td><td></td></tr><tr><td>Tissue Forceps (straight, 1 x 2 teeth)</td><td>Aesculap, Inc.</td><td>BD537R</td><td></td></tr><tr><td>Tying Forceps (angled)</td><td>Aesculap, Inc.</td><td>MA624R</td><td></td></tr><tr><td>Rodent and Small Animal Guillotine</td><td>Kent Scientific Corp.</td><td>DCAP</td><td></td></tr><tr><td>Low Cost Induction Chamber 3.0 L</td><td>Kent Scientific Corp.</td><td>SOMNO-0730&amp;nbsp;</td><td></td></tr><tr><td>Butterfly Winged Infusion Set 21 G</td><td>Hospira, Inc.</td><td>181106101</td><td></td></tr><tr><td>Abbocath 16 G</td><td>Hospira, Inc.</td><td>0G7149702</td><td></td></tr><tr><td>Microlance Hypodermic Needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td>modify needle to make cannula</td></tr><tr><td>Braun Original Perfusor Syringe 50 ml</td><td>B. Braun Melsungen AG</td><td>8728810F</td><td></td></tr><tr><td>Braun Inject Solo Syringe 10 ml</td><td>B. Braun Melsungen AG</td><td>2057926</td><td></td></tr><tr><td>Beaker 50ml</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 106 17</td><td></td></tr><tr><td>Duroplan petri dish (100 x 20 mm)</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 755 48</td><td></td></tr><tr><td>Seraflex Suture USP 3/0</td><td>SERAG-WIESSNER GmbH &amp;amp; Co. KG</td><td>IC208000</td><td></td></tr><tr><td>VWR disposable Square Weighin Boats 100ml</td><td>VWR, Inc.</td><td>10803-148</td><td></td></tr><tr><td>Styrofoam surface</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Inc.</td><td>71380</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich, Inc.</td><td>P4504</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich, Inc.</td><td>P5379</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich, Inc.</td><td>S0876</td><td></td></tr><tr><td>Magensium sulfate heptahydrate</td><td>Sigma-Aldrich, Inc.</td><td>230391</td><td></td></tr><tr><td>Magensium chloride</td><td>Sigma-Aldrich, Inc.</td><td>M8266</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich, Inc.</td><td>T0625</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Inc.</td><td>G7528</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich, Inc.</td><td>B0753</td><td></td></tr><tr><td>Calcium chloride solution (1 M)</td><td>Sigma-Aldrich, Inc.</td><td>21115</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich, Inc.</td><td>A9647</td><td></td></tr><tr><td>Liberase</td><td>Roche (Sigma-Aldrich, Inc.)</td><td>LIBTM-RO</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica GmbH</td><td>3862357</td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie Deutschland GmbH &amp;amp; Co. KG</td><td>10182054</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich, Inc.</td><td>L2020</td><td></td></tr><tr><td>WillCo glass-bottom dish 500&amp;micro;l 0.005mm</td><td>WillCo Wells B.V.</td><td>HBST-3522</td><td></td></tr><tr><td>Fluo4 AM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>F14201</td><td>5&amp;micro;M for 20min at RT</td></tr><tr><td>Di-8-ANNEPS</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>D3167</td><td>10&amp;micro;M for 45 min at 37&amp;deg; C&amp;nbsp;</td></tr><tr><td>Mitotracker RED FM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>M22425</td><td>20nM for 30 min at 37&amp;deg; C</td></tr><tr><td>Jacketed reaction vessel 500 ml</td><td>Gebr. Rettberg GmbH</td><td>107024414</td><td></td></tr><tr><td>Jacketed reaction vessel 1000 ml</td><td>Gebr. Rettberg GmbH</td><td>107025414</td><td></td></tr><tr><td>Jacketed bubble trap</td><td>Gebr. Rettberg GmbH</td><td>134720001</td><td></td></tr><tr><td>ED heating immersion circulator</td><td>Julabo GmbH</td><td>9116000</td><td></td></tr><tr><td>Reglo Digital MS-2/6 peristaltic pump</td><td>Ismatec (Cole-Parmer Gmbh)</td><td>ISM 831</td><td></td></tr><tr><td>Voltcraft Thermometer 302 K/J</td><td>Conrad Electronic SE</td><td>030300546</td><td></td></tr><tr><td>Tubing</td><td></td><td></td><td></td></tr><tr><td>LSM 700 microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>ZEN 2.3 imaging software</td><td>Carl Zeiss, Inc.</td><td>410135-1011-240&amp;nbsp;</td><td></td></tr><tr><td>Single channel heater controller TC-324B</td><td>Warner Instruments, LLC</td><td>64-2400</td><td></td></tr><tr><td>8 channel perfusion system</td><td>Warner Instruments, LLC</td><td>64-0185</td><td></td></tr><tr><td>8 channel Multi-Line In-Line Solution Heaters</td><td>Warner Instruments, LLC</td><td>64-0105</td><td></td></tr></tbody></table>

isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction

이 문서에서는, 우리는 변화 증후군 (메츠)의 쥐 모델에서 단일 셀 심 방 cardiomyocytes (Acm)의 분리에 대 한 최적화, Langendorff 기반 절차 설명-관련 보존된 방출 분수 (HFpEF)와 함께 심장 마비. 메츠 관련 HFpEF의 보급은 상승 하 고, 그리고 심 cardiomyopathies 개장 하는 심 방 및 심 방 세 동와 관련 된 관련성이 높은 임상으로 심 방 개장 사망률의 독립적인 예측 인자. 격리 된 단일 셀 cardiomyocytes와 연구 자주 확증 vivo에서 조사 결과 보완 하는 데 사용 됩니다. 순환 선박 rarefication 및 조직 간 질 성 섬유 증이이 질환의 동물 모델에서 Acm의 성공적인 단일 셀 격리에 대 한 잠재적으로 제한 요소를 포즈.
우리가 실질적으로 형태학 상으로 그리고 기능적으로 그대로 Acm의 수확량을 증가 수동으로 격리 절차 동안 심장 구멍의 intraluminal 압력을 조절 하는 장치를 사용 하 여이 문제를 해결 했습니다. 인수 세포 세포 배양 및 기능성 칼슘 이미징 (즉, 흥분-수축-커플링) 등 다양 한 실험의 다양 한에서 사용할 수 있습니다.
우리 연구원 단계별 프로토콜, 최적화 된 솔루션, 필요한 장비 및 포괄적인 문제 해결 가이드를 준비 하는 철저 한 지침의 목록 제공 한다. 절차의 초기 구현을 오히려 어려울 수도 있습니다, 그러나 성공적인 적응 메츠 관련 광범위 한 실험에 대 한 HFpEF의 쥐 모델에서 최신의 ACM 격리를 수행 하는 독자 수 있게 됩니다.

나이의 21 주에 가능한 Acm (6.1 단계에 설명 된 대로 추정), 칼슘 다시 적응 (단계 5.4-5.7), 후의 60-90% (그림 4A)이 메서드에서 ZSF-1 비만 쥐에서 분리 수 있습니다. 쥐, Acm 다르고 VCMs24,25에 비해 더 많은 이기종 표현 형 특징입니다. 그림 4B 와 보존된 막 sarcomere 구조를 기능적으로 통합 셀의 두 강한 지표는 개별 ACM 보여줍니다.
인수 Acm는 다양 한 방법으로 처리할 수 있습니다. 그림5에서 exemplified, 세포 형태 및 기능을 연구 하는 데 사용 하는 형광 염료와 로드 수 있습니다. 예를 들어, 디-8-ANEPPS 심 방 쥐 셀 (그림 5A) 관 시스템의 윤곽을 그리 다 사용 되었다. 실험의 또 다른 세트, 미토 콘 드리 아 얼룩까지 빨간색 형광 염료 (예를 들어, mitotracker-레드-FM) 심 방 개장 cytosolic 미토 콘 드리 아 (그림 5B) 감지 하 채택 된다. 그림 5C같이 절연이 프로토콜을 Acm 라이브 셀 캘리포니아2 + 영상에 적합 있으며 표시 그대로 흥분 수축에 결합해 1 Hz 필드 자극. 모든 이미지는 다양 한 연구원6,7 (그림 5D)에 사용할 수 있는 알고리즘을 사용 하 여 추가 분석을 위해 사용할 수 있습니다.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57953/57953fig4.jpg"> 그림 4: 가능한, 단일 셀 이에요의 각각 수익률 (A)이이 패널 표시 1mm Ca에 Acm의 재 적응 후 격리의 수익률2 + NT. (B)이이 패널 절연, 단일 셀 심 방 cardiomyocyte 보여줍니다. Sarcomere 구조, 세포 막, 그리고 핵 명확 하 게 볼 수 있습니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57953/57953fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57953/57953fig5.jpg"> 그림 5: 형광 염료와 쥐 Acm의 얼룩. (A)이이 패널 표시는 세포 막 및 형광 염료 Di8ANNEPS와 관 네트워크의 얼룩. (B)이이 패널 표시는 캘리포니아2 +의 횡단 라인 스캔을 보여줍니다이 패널 형광 염료 mitotracker-레드-변조가 (C)와 미토 콘 드리 아의 얼룩-단일 셀에 형광 염료 Fluo4 오전와 여기. 화살표에서 1 Hz. (D)이이 패널 표시 longitudenal Ca2 + 과도 패널 C에서 파생 된 전기 자극을 나타냅니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57953/57953fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

심 방 Cardiomyocytes 보존된 방출 분수와 함께 대사 증후군 관련 심장 마비의 쥐 모델에서의 격리

여기, 우리가 보존된 방출 분수와 함께 대사 증후군 관련 심장 마비의 쥐 모델에서 단일 셀 심 방 cardiomyocytes의 격리에 대 한 최적화, Langendorff 기반 절차를 설명합니다. 심장 구멍의 intraluminal 압력의 수동 조절 기능 그대로 myocytes 흥분-수축-결합 연구에 대 한 적합 한를 구현 됩니다.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

isolation-atrial-cardiomyocytes-from-rat-model-metabolic-syndrome

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.1K 조회수.  Charité University Medicine. 여기, 우리가 보존된 방출 분수와 함께 대사 증후군 관련 심장 마비의 쥐 모델에서 단일 셀 심 방 cardiomyocytes의 격리에 대 한 최적화, Langendorff 기반 절차를 설명합니다. 심장 구멍의 intraluminal 압력의 수동 조절 기능 그대로 myocytes 흥분-수축-결합 연구에 대 한 적합 한를 구현 됩니다.

비디오: Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

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 ...

연구

생물학

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

의학

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

칼슘의 동시 측정을위한 인간의 심방 근육 세포의 분리<sup&gt; 2 +</sup&gt; 과도하고 막 전류

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

성인 마우스에서 심방 근세포의 격리

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

성인 마우스에서 심방 및 심실 근세포의 동시 격리를위한 랑엔도르프 방법의 수정

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

마우스 심근 세포를 일관되게 분리하는 간단하고 효과적인 방법

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

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 of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

진동절단 심근슬라이스에서 인간 심실 심근세포의 분리

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

성인용 마우스 심장에서 심방 심근 세포, 심실 심근 세포 및 비근구의 동시 격리 및 문화

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.