저자는 이 출판물의 생산 비용을 충당한 Cairn Research Ltd.의 친절한 재정적 기여에 대해 인정하고 싶습니다. 또한, 이네스 뮬러 씨와 스테파니 케스텔(Stefanie Kestel) 씨의 뛰어난 기술 지원에 감사드립니다.
저자의 연구는 심장 혈관 연구를위한 독일 센터에 의해 지원됩니다 (DZHK), 도이치 포르스충스게마인샤프트 (DFG, 독일 연구 재단, VO 1568/3-1, IRTG1816 RP12, SFB1002 TPA13 및 독일의 우수 전략 - EXC 2067/1- 390729940) 및 엘스 크뢰너-프레세니우스 스티프퉁(EKFS 2016_A20).

1. 셀룰러 제제
참고: 본 프로토콜에 사용된 인간 iPSC는 건강한 기증자로부터 파생되고 이전에 설명된12,13,14와같이 WNT 시그널링 및 젖산 정제 기술의 완전히 정의된 소분자 변조를 사용하여 단층층에서 분화되었다. iPSC-CM은 아래설명된 문화 매체로 2-3일마다 유지되었습니다.
<ol><li>기저 배지(RPMI 1640) 및 2% 보충(B27)의 배양 배지를 준비한다. 4 °C에 보관하십시오. 실온(RT)에서 사용하십시오.</li>
	<li>기초 매체(RPMI 1640), 2% 보충제(B27) 및 1:2000 ROCK 억제제의 도금 매체를 준비한다. 4 °C에 보관하십시오. RT에서 사용하십시오.</li>
	<li>코팅 살균 10mm 원형 유리 #0 커버립 150 μL 1:60 인자 무료 지하 멤브레인 매트릭스와 4 h에 대 한 4°C에서 배양. 참고: 유출을 방지하기 위해 적절한 표면 장력을 유지하면서 유리 전체를 덮는 것을 보장하기 위해 커버슬립 볼륨의 최적화가 필요합니다. 10mm 둥근 커버립에 150 μL이 권장됩니다. 배치 크기, 커버 슬립 유형, 커버 슬립 볼륨 및 배양 플레이트 유형은 실험자의 요구에 적합할 수 있습니다.</li>
	<li>EDTA 기반 세포 해리 시약으로 iPSC-CM 해리를 시작합니다. 1,000 μL 파이펫으로 부드럽게 플러싱하여 단층이 완전히 분리되었는지 확인합니다.</li>
	<li>셀룰러 서스펜션을 15mL 튜브로 옮기고 이중 볼륨 도금 매체를 추가합니다. 원심분리기 10분 100 x g.</li>
	<li>도금 매체의 원하는 부피(재서스펜션 부피)로 펠릿을 다시 중단합니다. 셀을 수동으로 또는 전자적으로 계산합니다.</li>
	<li>나중에 격리된 세포 분석을 허용하는 커버슬립당 최적의 밀도(15,000)를 선택합니다.</li>
	<li>이 원하는 밀도에서 모든 커버립을 플레이트하는 데 필요한 '활성' 셀룰러 서스펜션(A)의 부피를 계산합니다. 다음 수식을 적용하고 별도의 튜브로 인출하십시오. <img alt="Equation 1" src="/files/ftp_upload/61890/61890equ01v2.jpg">
</li>
	<li>원하는 볼륨에서 각 커버슬립을 수용하는 데 필요한 추가 도금 매체(B)의 부피를 계산합니다. 다음 수식을 적용하고 활성 현탁액 튜브에 결과 볼륨을 추가합니다. <img alt="Equation 2" src="/files/ftp_upload/61890/61890equ02v2.jpg">
</li>
	<li>커버슬립에서 매트릭스를 제거하고 각 커버슬립에 셀 서스펜션(A+B)의 '커버슬립 볼륨'을 적용합니다. 세포 분포를 보장하기 위해 튜브에서 정기적으로 재중지하십시오.</li>
	<li>1 h에 대한 37 °C에서 인큐베이션. 도금 매체로 웰을 부드럽게 채웁니다.</li>
	<li>24 시간 후, 일반 문화 매체와 미디어를 교환하고 2-3 일마다 유지합니다.</li>
</ol>2. 실험 용 설정
<ol><li>40배배율, 고숫자 조리개 렌즈(N.A: > 0.75)를 사용하여 반전된 피형성 현미경을 장착하여 실험을 수행합니다.</li>
	<li>현미경의 전송 된 조명 포트에 빠른 스위칭 따뜻한 흰색 LED를 커플. 간단한 빨간색 660 nm 필터를 전송된 라이트 경로에 삽입합니다. 참고: 이 빛은 광합성 실험 전반에 걸쳐 활성화되어 녹색 형광 신호를 오염하지 않고 샘플을 관찰할 수 있습니다.</li>
	<li>포토메트리 레코딩을 위해 빠른 스위칭 470 nm LED 헤드를 탑재합니다. 현미경의 상피 형광 포트에 470/40 여기 필터를 삽입하여 LED에 의해 생성 된 빛을 청소하십시오. 참고: 최적의 신호 정량화를 위해 광학 출력의 고속 피드백 제어가 있는 조명 시스템을 권장합니다.</li>
	<li>현미경 내의 미러 유닛 회전 목마에 495 nm 길이의 패스 빔 스플리터를 포함하는 현미경 큐브를 삽입합니다.</li>
	<li>현미경 C-마운트 포트에 조정 가능한 필드 다이어프램을 포함하는 검출 암을 맞추어 관심 영역을 선택할 수 있도록 합니다.</li>
	<li>별도로 광증 검출기 (PMT)와 현미경에 USB 카메라를 결합합니다. 이것은 배출 감지 시스템의 기초를 형성합니다.</li>
	<li>565nm 길이의 패스 빔 스플리터를 포함하는 필터 큐브와 535/50 배출 필터를 PMT 포트에 삽입합니다. 이렇게 하면 두 검출기 간에 방출 광이 분할됩니다. 참고: 배기가스 감지 시스템의 전송된 포트에 부착된 카메라는 모든 실험에서 밝은 필드에서 전송된 빛을 감지할 수 있습니다.</li>
	<li>PMT를 전원 공급 장치와 PMT 앰프에 결합합니다. PMT 증폭기 출력을 데이터 수집 시스템의 아날로그 입력 핀에 연결합니다.</li>
	<li>PMT에서 아날로그 데이터를 1kHz 이상으로 필터링합니다.</li>
	<li>나이퀴스트 기준을 충족하고 별칭을 방지하기 위해 아날로그 신호(2kHz 이상)에 존재하는 가장 높은 주파수의 두 배 이상의 주파수로 데이터를 디지털화합니다.</li>
</ol>3. VF2.1Cl셀로딩
참고: 이 염료와 관련된 모든 단계는 저조도 조건에서 수행해야 합니다.
<ol><li>타이로드의 목욕 용액(mM)을 준비하십시오: 140 NaCl, 10 HEPES, 10 포도당, 4 KCl, 1 MgCl2,2 CaCl2,pH = 7.35 및 37°C에 따뜻하게.</li>
	<li>1,000x VF2.1Cl 5μL과 20% 용용 폴로사머 용액의 50 μL을 혼합하여 마이크로센트심분리기 튜브에 적재 용액의 알리쿼트를 준비합니다.</li>
	<li>20mm 페트리 접시에 5μL의 로딩 용액을 5mL의 온난티 티로드 용액(총 0.1x 염료 농도)에 적용합니다. 참고: 최종 염료 농도는 0.1배입니다. 이것은 1/10제조 업체에 의해 제안 th. 이는 자원을 절약하고, 무시할 수 있는 세포 독성을 보장하며, 중요한 것은 여전히 신호대 잡음 비율이 높은 로드된 셀에서 선명한 광학 신호를 유지한다는 것입니다.</li>
	<li>접시에 iPSC-CM 커버 슬립 을 넣고 37 °C에서 20 분 동안 배양합니다.</li>
	<li>가열 된 라이브 세포 이미징 챔버를 조립합니다. 현미경 단계에 마운트하고 신선한 Tyrode의 용액의 500 μL을 채웁니다.</li>
	<li>37°C에서 신선한 티로드용액으로 커버슬립을 씻으시다.</li>
	<li>iPSC-CM 커버슬립을 미세 한 점 집게를 사용하여 미리 데운 목욕 챔버에 조심스럽게 적용하십시오. 참고: 챔버와 그 내용물들은 항상 생리학적 온도에서 가열되도록 하십시오. 원하는 경우, 따뜻하게 된 티로드의 용액의 지속적인 관류로 시작하십시오.</li>
</ol>4. 전기장 자극
참고: iPSC-CM의 외부 트리거링은 선택 사항이지만 셀룰러 역학 및 실험 매개 변수의 표준화에 유용합니다. 그것은 분석의 용이성을 증가하고 주파수 의존적 인 효과의 조사를 할 수 있습니다.
<ol><li>두 개의 백금 전극이 5mm 간격을 가진 자극 인서트를 기록 챔버에 부착합니다.</li>
	<li>외부 자극기를 자극 삽입에 연결합니다. 0.5Hz에서 5ms 양극성 필드 펄스로 설정합니다.</li>
	<li>자극을 1V 위쪽으로 늘려 최적의 자극 전압을 결정합니다. 임계값 자극은 셀이 수축하기 시작하는 가장 낮은 전압으로 정의됩니다. 이 임계값보다 약 25% 높은 전압을 적용합니다. 일반 범위는 1 V와 30 V 사이입니다.</li>
	<li>외부 자극기로 자극 주파수를 수정하거나 획득 소프트웨어로 트리거합니다.</li>
</ol>5. 광학 액션 잠재력 획득
참고: 이 프로토콜은 획득 및 분석을 위해 상용 소프트웨어를 사용합니다.
<ol><li>전송된 라이트 패스와 USB 카메라를 사용하여 밝은 시야에서 심사이클을 시각화합니다.</li>
	<li>격리된 셀을 선택하고 관심 셀의 빛만 모니터링할 수 있도록 필드 다이어프램으로 광학 경로를 단단히 자르게 합니다.</li>
	<li>PMT 증폭기를 활성화하고 PMT 공급을 750 V로 설정합니다.</li>
	<li>획득 소프트웨어와 함께 자극 프로토콜(4단계 참조)을 실행하고 동시에 470 nm 흥분 광을 활성화합니다. 후자는 원격 패널을 통해 수행되거나 고정 강도(TTL 신호)로 자동화할 수 있습니다.</li>
	<li>PMT 증폭기의 게인 및 오프셋을 조정하여 신호가 포화되지 않고 기록 시스템의 검출 범위에 최적화되었는지 확인합니다.</li>
	<li>안정적인 작업 잠재력을 감지할 수 있도록 10개의 스윕을 기록합니다.</li>
	<li>기록을 계속하고 즉시 현미경 단계를 이동하여 세포가 없는 영역에서 배경 신호를 잠시 습득합니다. 흥분 빛을 끕니다. 참고: 이 배경 값(F오프셋)은 배경 형광을 설명하는 데 사용됩니다.</li>
	<li>원하는 경우, 약리학 조작에 대한 세포 반응을 식별하기 위해 1 μM 니페디핀과 같은 국소 적으로 관련 된 기준 약물을 사용합니다.</li>
	<li>순차적인 방식으로, 새로운 셀을 선택할 때마다 5.2-5.7 단계를 반복합니다. 원하는 경우 커버립을 교체하여 단일 좌석에서 높은 실험 회전율을 보장합니다. 참고: 로딩 및 이미지 수집 프로토콜은 그림 2에설명되어 있습니다.</li>
</ol>6. 데이터 분석
<ol><li>분석 소프트웨어로 저장된 레코딩을 열고 단일 셀에서 자극된 작업 잠재력을 포함하는 평균 10개의 스윕을 엽니다.</li>
	<li>F오프셋을 나타내는 기준 신호의 평균을 가지고 평균 추적에서 이를 뺍니다.</li>
	<li>다음 공식으로 ∆F/F0을 계산합니다(F가 형광을 측정하고 F0은 확장기 형광입니다). <img alt="Equation 3" src="/files/ftp_upload/61890/61890equ03v2.jpg">
</li>
	<li>추적 기준선(확장기) 및 관심 영역(AP)을 식별하고 원하는 심장 작용 잠재적 매개 변수를 측정합니다. 여기에는 50%(APD50)및 90%(APD90)재극화에 대한 감쇠 시간에 국한되지 않습니다.</li>
	<li>이 단일 셀에서 스프레드시트 소프트웨어로 데이터를 내보냅니다.</li>
	<li>모든 레코딩에 대해 6.1 - 6.5 단계를 반복합니다. 적절한 페어링되지 않은 테스트 또는 분산 분석으로 결과를 평가합니다.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61890/61890fig2v2.jpg"> 그림 2: 로딩 및 이미지 수집 프로토콜입니다. (A)iPSC-CM 및 네이티브 심근세포에 대한 완전한 VF2.1Cl 로딩 프로토콜의 흐름 차트. (B)막 전압의 변화에 대응하여 VF2.1Cl 방출의 자궁내포 및 검출을 위해 이 프로토콜에 사용되는 빔 스플리터(BS) 및 필터 구성의 단순화된 회로도. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig2v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<ol>
	<li>Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+fluorescent+voltage+indicators+for+studying+membrane+potential.">Small molecule fluorescent voltage indicators for studying membrane potential.</a> Current Opinion in Chemical Biology. 33, 74-80 (2016).</li><li>Liang, P., 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=Drug+screening+using+a+library+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+reveals+disease-specific+patterns+of+cardiotoxicity.">Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li><li>Horv&#225;th, 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=Low+resting+membrane+potential+and+low+inward+rectifier+potassium+currents+are+not+inherent+features+of+hiPSC-derived+cardiomyocytes.">Low resting membrane potential and low inward rectifier potassium currents are not inherent features of hiPSC-derived cardiomyocytes.</a> Stem Cell Reports. 10 (3), 822-833 (2018).</li><li>Salama, G., Morad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merocyanine+540+as+an+optical+probe+of+transmembrane+electrical+activity+in+the+heart.">Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart.</a> Science. 191 (4226), 485-487 (1976).</li><li>Hortigon-Vinagre, 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=The+use+of+ratiometric+fluorescence+measurements+of+the+voltage+sensitive+dye+Di-4-ANEPPS+to+examine+action+potential+characteristics+and+drug+effects+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes.</a> Toxicological Sciences. 154 (2), 320-331 (2016).</li><li>Blinova, K., 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=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Miller, E. W., 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=Optically+monitoring+voltage+in+neurons+by+photo-induced+electron+transfer+through+molecular+wires.">Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires.</a> Proceedings of the National Academy of Sciences. 109 (6), 2114-2119 (2012).</li><li>Bedut, S., 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=High-throughput+drug+profiling+with+voltage-+and+calcium-sensitive+fluorescent+probes+in+human+iPSC-derived+cardiomyocytes.">High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 311 (1), 44-53 (2016).</li><li>McKeithan, W. L., 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=An+automated+platform+for+assessment+of+congenital+and+drug-induced+arrhythmia+with+hiPSC-derived+cardiomyocytes.">An automated platform for assessment of congenital and drug-induced arrhythmia with hiPSC-derived cardiomyocytes.</a> Frontiers in Physiology. 8, 766 (2017).</li><li>Duncan, 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=Drug-mediated+shortening+of+action+potentials+in+LQTS2+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Drug-mediated shortening of action potentials in LQTS2 human induced pluripotent stem cell-derived cardiomyocytes.</a> Stem Cells and Development. 26 (23), 1695-1705 (2017).</li><li>Asakura, K., Hayashi, S., Ojima, A., Taniguchi, T., Miyamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+acquisition+and+analysis+methods+in+multi-electrode+array+experiments+with+iPS+cell-derived+cardiomyocytes.">Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.</a> Journal of Pharmacological and Toxicological Methods. 75, 17-26 (2015).</li><li>Lian, X., 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/&#946;-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/&#946;-catenin signaling under fully defined conditions.</a> Nature Protocols. 8 (1), 162-175 (2013).</li><li>Burridge, P. W., 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=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature methods. 11 (8), 855-860 (2014).</li><li>Kleinsorge, M., Cyganek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-directed+differentiation+of+human+iPSCs+into+atrial+and+ventricular+cardiomyocytes.">Subtype-directed differentiation of human iPSCs into atrial and ventricular cardiomyocytes.</a> STAR Protocols. , 100026 (2020).</li><li>Knollmann, B. C., Katchman, A. N., Franz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monophasic+action+potential+recordings+from+intact+mouse+heart:+validation,+regional+heterogeneity,+and+relation+to+refractoriness.">Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness.</a> Journal of Cardiovascular Electrophysiology. 12 (11), 1286-1294 (2001).</li><li>Leopold, J. A., Loscalzo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+precision+medicine+in+cardiovascular+disease.">Emerging role of precision medicine in cardiovascular disease.</a> Circulation Research. 122 (9), 1302-1315 (2018).</li><li>Voigt, N., Zhou, X. B., Dobrev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+atrial+myocytes+for+simultaneous+measurements+of+Ca2++transients+and+membrane+currents.">Isolation of human atrial myocytes for simultaneous measurements of Ca2+ transients and membrane currents.</a> Journal of Visualized Experiments. (77), e50235 (2013).</li><li>Voigt, 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=Enhanced+sarcoplasmic+reticulum+Ca2++Leak+and+increased+Na+-Ca2++exchanger+function+underlie+delayed+afterdepolarizations+in+patients+with+chronic+atrial+fibrillation.">Enhanced sarcoplasmic reticulum Ca2+ Leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation.</a> Circulation. 125 (17), 2059-2070 (2012).</li><li>Voigt, 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=Cellular+and+molecular+mechanisms+of+atrial+arrhythmogenesis+in+patients+with+paroxysmal+atrial+fibrillation.">Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation.</a> Circulation. 129 (2), 145-156 (2014).</li><li>Fakuade, F. E., 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=Altered+atrial+cytosolic+calcium+handling+contributes+to+the+development+of+postoperative+atrial+fibrillation.">Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.</a> Cardiovascular Research. , 162 (2020).</li><li>Gross, E., Bedlack, R. S., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+ratiometric+fluorescence+measurement+of+the+membrane+dipole+potential.">Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.</a> Biophysical Journal. 67 (1), 208-216 (1994).</li><li>Matiukas, 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=Near-infrared+voltage-sensitive+fluorescent+dyes+optimized+for+optical+mapping+in+blood-perfused+myocardium.">Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium.</a> Heart Rhythm. 4 (11), 1441-1451 (2007).</li><li>Mutoh, 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=Spectrally-resolved+response+properties+of+the+three+most+advanced+fret+based+fluorescent+protein+voltage+probes.">Spectrally-resolved response properties of the three most advanced fret based fluorescent protein voltage probes.</a> PLoS One. 4 (2), 4555 (2009).</li><li>Hochbaum, D. R., 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=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nature Methods. 11 (8), 825-833 (2014).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> Journal of the American Chemical Society. 137 (33), 10767-10776 (2015).</li><li>Deal, P. E., Kulkarni, R. U., Al-Abdullatif, S. H., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isomerically+pure+tetramethylrhodamine+voltage+reporters.">Isomerically pure tetramethylrhodamine voltage reporters.</a> Journal of the American Chemical Society. 138 (29), 9085-9088 (2016).</li><li>Fluhler, E., Burnham, V. G., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectra,+membrane+binding,+and+potentiometric+responses+of+new+charge+shift+probes.">Spectra, membrane binding, and potentiometric responses of new charge shift probes.</a> Biochemistry. 24 (21), 5749-5755 (1985).</li><li>Fromherz, P., Muller, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+fluorescence+of+amphiphilic+hemicyanine+dyes+in+neuron+membrane.">Voltage-sensitive fluorescence of amphiphilic hemicyanine dyes in neuron membrane.</a> Biochimica et Biophysica Acta. 1150 (2), 111-122 (1993).</li><li>Salama, 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=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> Journal of Membrane Biology. 208 (2), 125-140 (2005).</li><li>Jin, L., 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=Single+action+potentials+and+subthreshold+electrical+events+imaged+in+neurons+with+a+fluorescent+protein+voltage+probe.">Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.</a> Neuron. 75 (5), 779-785 (2012).</li><li>Kralj, J. M., Douglass, A. D., Hochbaum, D. R., MacLaurin, D., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+action+potentials+in+mammalian+neurons+using+a+microbial+rhodopsin.">Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.</a> Nature Methods. 9 (1), 90-95 (2012).</li><li>Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+membrane+voltage+measurements+using+FRET+with+new+fluorescent+proteins.">Improving membrane voltage measurements using FRET with new fluorescent proteins.</a> Nature Methods. 5 (8), 683-685 (2008).</li><li>Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W., Kn&#246;pfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+genetically+encodable+fluorescent+voltage+sensor+exploiting+fast+Ci-VSP+voltage-sensing+movements.">Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.</a> PLoS One. 3 (6), 2514 (2008).</li><li>Bradley, J., Luo, R., Otis, T. S., DiGregorio, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+optical+reporting+of+membrane+potential+in+situ+using+a+neuronal+tracer+dye.">Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.</a> The Journal of neuroscience. 29 (29), 9197-9209 (2009).</li><li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells+&amp;+tissues.">Optical imaging of voltage and calcium in cardiac cells &amp; tissues.</a> Circulation Research. 110 (4), 609-623 (2012).</li><li>Kappadan, V., 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=High-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11, 464 (2020).</li><li>Kettlewell, S., Walker, N. L., Cobbe, S. M., Burton, F. L., Smith, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrophysiological+and+mechanical+effects+of+2,3-butane-dione+monoxime+and+cytochalasin-D+in+the+Langendorff+perfused+rabbit+heart.">The electrophysiological and mechanical effects of 2,3-butane-dione monoxime and cytochalasin-D in the Langendorff perfused rabbit heart.</a> Experimental Physiology. 89 (2), 163-172 (2004).</li><li>K&#233;pir&#243;, 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=para-Nitroblebbistatin,+the+non-cytotoxic+and+photostable+Myosin+inhibitor.">para-Nitroblebbistatin, the non-cytotoxic and photostable Myosin inhibitor.</a> Angewandte Chemie International Edition. 53 (31), 8211-8215 (2014).</li></ol>

케언 리서치(Cairn Research Ltd)는 비디오 파일의 제작 비용을 충당하여 이 출판물을 지원했습니다.

여기서 우리는 전기 생리학적 모델링 및 심장 약물 스크리닝에 적합한 격리된 iPSC-CM에서 상세한 AP 프로파일을 쉽게 획득하기 위한 기본 프로토콜을 설명합니다. 우리는 표시기 기능과 방법론 적 충실도를 모두 시사하는 희소하게 시드 된 iPSC-CM에서 정기적이고 견고한 AP를 감지합니다.
iPSC 재프로그래밍을 위한 광범위한 상용 방법론과 심장 분화 프로토콜에 대한 표준화 부족...

심근세포의 전기생리학적 모델링과 심장 약물 스크리닝을 위한 효율적인 플랫폼 구축은 다양한 부정맥 장애에 대한 치료 전략의 개발에 필수적이다. 유도 된 다능성 줄기 세포 (iPSC) 기술의 급속한 확장은 고립 된 환자 파생 심근 세포 (iPSC-CM)를 사용하여 인간 질병 모델링 및 약리학적 조사에 유망한 진입을 일으켰습니다. 패치 클램프(current-clamp)를 통해 이들 세포의 전기생리학적 특성화를 위한 "골드 스탠다드" 기술은 작용 전위(AP) 형태및 지속시간을 정량화할 수 있지만, 이 방법은 매우 복잡하고 느리며 높은 처리량 데이터 수집1에적합하지 않다. iPSC-CM은 성인 토착 심근세포2에비해 확장막 전위 및 누출 전류가 증가하는 것으로 정기적으로 보고된다. iPSC-CM에서 관찰된 더 작은 세포 크기 및 감소된 막 정전용량이 현재 클램프 기술을 사용할 때 일부 체계적인 오류를 생성할 수 있으며, 아마도 이러한 편차를 설명할 수 있는 것으로나타났다3. iPSC-CM 플랫폼의 유용성을 극대화하기 위해 iPSC-CM의 단일 셀 수준에서 막 전압 변화를 특성화할 때 처리량을 늘리고 데이터 정확도를 보장하는 추가 방법이 중요합니다.
전압 민감염료(VSD)는 기존의 기술4와비교한 심장 AP 역학의 더 빠르고 비침습적이며 동등한 분석을 제공하는 제안된 방법이다. 최근 연구는 정확하게 심장 AP5를정량화하는 비율 전압 민감한 프로브 광합성의 적합성을 입증했다. 더욱이, 광학 광합성 접근법을 용이하게 확장하는 능력은 치료 약물 개발(예를 들어 CiPA)에서 중요한 대규모 심폐성 스크린에 이 기술을 빌려준다. 마이크로 전극 어레이 및 전압 감지 광학 기술을 이용한 시각 장애인 다중 현장 연구에서 표준화된 심폐성 프로토콜의 개발은 이 접근법6의핵심 가치를 입증했다.
많은 전능성 염료는 시판되고, 새로운 프로브의 지속적인 합성 개발은 다양한 심장 및 신경 구조물에 걸쳐 그들의 효과를 간소화하기위한 흥미로운 잠재력을 보여줍니다. 이상적인 VSD는 증가 된 운동과 감도를 가지며 정전 용량 부하, 광 표백 및 세포 독성을 줄입니다. 최근 합성 된 VF2.1Cl (FluoVolt)은 새로운 전압 플루어 (VF) 제품군7의다른 구성원이 공유하는 새로운 와이어 기반 분자 구조로 인해 이러한 유익한 특성의 대부분을 표현한다. 간단한 프로브가 플라즈마 멤브레인에 분자및 전기적으로 컨쥬게이트되는 일반적인 전기 색소와 는 달리,이 염료는 변형 된 형광성 불소 호레 (FITC)와 전자가 풍부한 기증자를 페어링 하는 수동적으로 삽입된 멤브레인 스패닝 합성 와이어로 구성됩니다. 기계학적 세부 사항은 그림 1에서제공됩니다. 이 염료는 멤브레인 전압 변동에 대한 우수한 감도를 나타내며, 비교 가능한 속도7에서다른 일반적인 프로브에서 볼 수 있는 ~10%와 달리 100mV당 배출 강도의 27% 변화를 나타낸다. 또한, 와이어 기반 PeT 시스템은 셀룰러 전기장과 직접 상호 작용하지 않아 최소한의 전기 간섭과 셀룰러 정전 용량 부하의 무시할 수 있는 변화를 일으킵니다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg"> 그림 1: VF2.1Cl 염료의 화학, 스펙트럼 및 기계론 파라미터. (A)VF2.1Cl. 분자 피처의 화학적 구조는 플라즈마 멤브레인내로 삽입을 용이하게 하는 페닐렌 비닐렌 분자 와이어 내의 다중 알킬 군을 포함한다. FITC 프로브에 접합된 음전하 황포산 단은 세포외 표면의 불소 안정화를 보장하고 지질 이중층의 전기장에 비해 수직 삽입 부근에 보조를 지원한다. (b)표적 세포의 혈장 막에 수직 VF2.1Cl을 포함시키는 단순화된 회로도. (C)VF2.1Cl 염료의 흡수 및 방출 스펙트럼. 스펙트럼은 표준 FITC 및 GFP 프로브와 동일합니다. (D)VF2.1Cl의 기계주의 적 행동 모드 묘사. 휴식 조건 (과극성)에서, 음의 세포 내 전압은 로스트랄 불소로 전환기쪽으로 무료 전자를 구동한다. 전자 풍부는 광 유발 전자 전달(PeT)이 광 흥분 상태에서 나오는 통로로서 선호되고, 효과적으로 형광을 담금질할 수 있도록 보장한다. 대조적으로, 탈극막 전위는 광학 여기에 형광을 선호하는 하향 전자 운동에 영향을 미칩니다. 결과 형광 반응은 멤브레인 전압과 선형적으로 관련되어 있으며 세포 전기 생리학적 운동학에 대한 상세한 측두정보를 수집하기 위해 정확하게 활용할 수 있습니다. (E)대표적인 브라이트필드(upper) 및 470nm(하부)의 형광은 VF2.1Cl로 로드된 leporine 심근세포의이미지(이하)단일 하중심근세포의 Z 스택이다. 화살표는 세포 막에 VF2.1Cl의 명확한 국소화영역을 나타낸다. 이미지는 X-lightv3 회전 디스크 공초점 헤드로 구성된 회전 디스크 공초점 시스템으로 50 μm 핀홀 패턴을 획득했습니다. LDI-7 조명기; Prime95B 카메라와 PlanApo Lambda 100x 목표. 스케일 바: 20 μm. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
VF2.1Cl에 컨쥬게이트된 FITC 프로브는 표준 및 GFP 필터 구성에 따라 효과적으로 사용할 수 있도록 보장하며, 형광 이미징 플랫폼의 공통 특징인 단일 채널 수집 시스템만 필요합니다. 이 염료를 가진 조밀한 인간 iPSC-CM 단층의 분석은 최근에보고되었습니다 8,9,10,11. 우리의 프로토콜은 조밀한 싱크로나단층의 전기 및 파라크리안 영향에 흔들리지 않는 단일 격리 된 iPSC-CM에 대한 조사와 복잡한 공초점 또는 넓은 현장 이미징 준비와는 달리 저렴하고 사용자 정의 가능한 광측정 시스템의 사용으로 인해 이러한 연구와 다릅니다.
여기서는 고립된 인간 iPSC 유래 심근세포및 원심근세포로부터 견고한 광학 AP를 빠르게 획득하고 분석하기 위한 프로토콜을 설명합니다(보충 파일참조). 우리는 단일 셀 광측정 측정을위한 아트 플랫폼의 사용자 정의 상태와 함께 VF2.1Cl을 사용합니다. 이러한 실험 프로토콜은 대학 의료 센터 괴팅겐의 윤리위원회에 의해 승인되었습니다 (번호 10/9/15).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>HN77.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamanin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y27632</td>
			<td>Stemolecule</td>
			<td>04-0012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslips, Thickness 0</td>
			<td>Thermo Scientific</td>
			<td>CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual OptoLED Power Supply&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET535/50m</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etched Neubauer Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cubes&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T565lpxr Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T660lpxr Dichroic Beamsplitter</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC-20 Dual Channel Temperature Controller&#160;</td>
			<td>npi Electronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

기존의 세포내 미세 전기 전극 기술은 심근세포 전기 생리학을 정량화하는 매우 복잡하고 노동 집약적이며 일반적으로 낮은 처리량에서 수행됩니다. 유도 된 다능성 줄기 세포 (iPSC) 기술의 신속하고 지속적인 확장은 심장 혈관 연구에서 새로운 표준을 제시하고 대체 방법은 이제 단일 세포 수준에서 전기 생리 학적 데이터의 처리량을 증가하는 데 필요한. VF2.1Cl은 최근 유래된 전압 민감 성 염료로, 멤브레인 전위변동에 대한 신속한 단일 채널, 높은 크기 응답을 제공한다. 다른 기존 전압 지표보다 우수한 운동학을 보유하고 있으며 기존의 미세 전극 기술과 동등한 기능성 데이터를 제공합니다. 여기서는 모듈식및 매우 저렴한 광합성 시스템을 사용하여 외부적으로 진행되는 인간 iPSC 파생 심근세포에서 단순화되고 비침습적인 작용 잠재적 특성화를 입증합니다.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg"> 도 3: 분리된 토착 심근세포 및 인간 유도만능 줄기세포의 광학 작용 전위(AP) 프로파일유래 심근세포(iPSC-CM). (A)APD 50 및 APD90(n= 7, 오른쪽)의 평균 ± SEM을 가진 단일 모린 심근세포(중앙)의 대표적인 광학 AP. (B)APD 50 및 APD90(n= 48, 오른쪽)의 평균 ± SEM...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

여기서는 고속 모듈식 광혈약 시스템을 사용하여 유도된 다능줄기 세포 유래 심근세포로부터의 작용 잠재력의 광학 획득 및 특성화를 설명합니다.

인간 유도 만능 줄기 세포 유래 심근 세포에서 단세포 광학 작용 잠재적 측정

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

single-cell-optical-action-potential-measurement-human-induced

Research

JoVE Journal

Bioengineering

4.0K 조회수.  University Medical Center Goettingen, Goettingen, Germany. 여기서는 고속 모듈식 광혈약 시스템을 사용하여 유도된 다능줄기 세포 유래 심근세포로부터의 작용 잠재력의 광학 획득 및 특성화를 설명합니다.

비디오: Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

마우스 중심에 액션 후보와 칼슘 과도의 광학 매핑

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

연구

생체공학

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

개발, 확대,<em&gt; 생체 내</em&gt; 인간 배아 줄기 세포 (hESCs에)에서 인간 NK 세포의 모니터링 및 및 유도 된 pluripotent 줄기 세포 (유도 만능)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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 ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

재발 동작 전위 기록을 위한 멀티웰 마이크로 전극 어레이의 인간 iPSC 유래 심근구 네트워크

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

화학적으로 정의된 조건하에서 인간 유도된 다능성 줄기 세포에서 성숙한 신장 분피의 유도된 분화

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

인간 다능성 줄기 세포 구상체를 운반하는 코어-쉘 마이크로캡슐의 미세유체 제조

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

인간 유도 만능 줄기 세포로 설계된 Isogenic 신장 사구체 칩

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

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 ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

만능줄기세포(Pluripotent Stem Cells)에서 인간 혈관 오가노이드(Human Blood Vessel Organoids)의 생성

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

인간 유도 만능 줄기 세포 유래 심장 스페로이드의 생성, 고처리량 스크리닝 및 바이오뱅킹

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...