이 작품은 TJC, KDC, 미국 BDG 홍콩 TRS T13-706 / 11 (KDC), NIH (R01 HL113499)의 연구 보조금위원회에 NIH / NHLBI PEN 계약 HHSN268201000045C에 NIH (1F30HL118923-01A1)에 의해 지원되었다 추가 자금을 RL하는 NIH DRB 5T32GM008553-18에 의해 시스템 및 발달에 NIDCR - 학제 간 교육에 traineeship로 TJC에 제공 한 심장 협회 (12PRE12060254) RJ, 그리고 홍콩 특별 행정구의 연구 그랜트위원회 (/ 11 T13-706가 TBRS) 생물학, 출생 결함 T32HD075735. 저자는 또한 감사 기술 지원을위한 생물 반응기 및 Mamdouh Eldaly 가공과 지원을 뉴욕의 도시 대학은 Zahn 센터에서 아서 Autz을 인정하고 싶습니다. 우리는 또한 아낌없이 인간 중간 엽 줄기 세포를 제공하기 위해 심장 차별화에 대한 조언 박사 케네스 Boheler, 박사 여호수아 토끼 감사합니다.

<ol>
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The authors declare that they have no competing financial interests.

정의 인간 공학 심장 조직 (hECT)의 건축은 인간의 심장 심근 기능의 일관성과 신뢰성있는 모델을 제공 할 수 있습니다. 결정적으로, 시스템의 모든 세포 및 세포 외 성분은 공지되어 있으며, 따라서 분화 과정으로 인한 다른 미지의 세포 유형의 교란의 영향을 제거하고 원하는대로 조작 할 수있다. 빠른 세포 증식과 높은 수율의 균형을 위해서, 이상적으로 분화 사일 도금 후 hESCs는 75 %의 합류점에?...

심장 조직 공학은 최근 두 쥐의 심근 1-6과에서 만든 완전한 기능을, 구타 조직, 인간 줄기 세포 유래 심장 근육 세포 7-12의 결과를 게시하는 여러 그룹으로, 지난 10 년간 크게 발전하고있다. 심근 조직 공학 분야는 두 가지 주요 본질적으로 독립적 인 목적에 의해 구동된다 : 1) 기능을 개선하기 위해 4-6 하트 실패에 이식 할 수 외래 이식을 개발; 2) 생리 및 질병 연구를위한 체외 모델에서 개발, 또는 치료 개발 2,7 선별 도구로합니다. 3 차원 (3-D) 세포 배양 물은 3-D 행렬은 기존의 2-D 단층 세포 배양 물보다 더 자연 심장 미세 환경을 반영하는 바와 같이, 차세대 스크리닝 도구 개발에 필수적인 것으로 간주된다; 실제로 세포 생물학의 일부 측면은 2-D 대 3-D 배양 13,14 근본적으로 다르다 </suP&gt;. 세포 외 기질 및 세포 인구 : 또한, 설계 심장 조직이 완전히 정의 된 구성 요소로 구성되어있다. 세포 외 기질 성분 (일반적 피브린 9 콜라겐 7,8,10)이 엄격하게 통제하면서 전통적인 설계 인간의 심장 조직의 경우, 입력 셀 조성물은 덜의 지시 심장 분화에서 세포의 전체 혼합물로 정의된다 어느 배아 줄기 세포 (ESC 7,9) 또는 유도 만능 줄기 세포 (IPSC 10, 12)는 조직에 추가. 특정 세포주에 사용 된 분화 프로토콜의 효율에 따라, 심근의 생성 비율이 90 % 이상 25 % 미만에서 특정 심근 표현형의 범위 일 수있다 (즉, ventricular-, atrial- 심박 형 또는) 수도도 아닌 심근 분획 (15, 16)을 매우 이질적인하고 차별화 된 심장 m의 성숙을 변경할 수 있습니다 다양17 yocytes. 최근 심근 조직 공학 연구는 심장 리포터 인간 배아 줄기 세포주 8 세포 표면 마커 (18)가 분화 심장 근세포 성분을 분리하는 데 사용되는 하나와, 셀 입력 인구를 제어하려고 하였다. 단지 심장 근육 세포로 구성 초기에 조직이 이상적 일 것 같다 있지만,이 사실이 아닌 경우에; 심장 근육 세포의 1의 비율 : 섬유 아세포가 가장 높은 트의 힘 (8)을 생산 전적으로 심장 근육 세포로 구성 hECTs 일부 그룹 3을 찾는, 기능 조직으로 압축하지 못한다. 생균 정렬 용 표면 마커를 포함하여 다양한 세포의 선택 방법을 이용함으로써, 한정된 세포 집단과 hECTs를 생성 할 수있다. 비 심장 기질 세포의 마커 CD90 19,20 이러한 추정 섬유 아세포 마커로서 당분간 사용할되었지만, 심장 근육 세포 표면 마커는보다 어려웠확인하다. SIRPα 인간 심장 근세포 18 식별 제 심장 표면 마커 사이이고 심장 리니지 고도로 선택적인 것으로 밝혀졌다. 세포 섬유 아세포와 같은 표현형 (Josowitz, 게시되지 않은 관찰)을 나타내는 CD90 + 인구, 거의 순수한 심근 세포를 얻을 수 - 최근에, 우리는 두 번 정렬 SIRPα + 및 CD90에 대한이 있음을 발견했다. 심근 세포와 CD90 + 섬유 아세포 - SIRPα + / CD90의 1 조합 :이 수집 된 결과를 바탕으로, 여기에 우리는 3를 사용하여 hECTs을 만들 설명합니다. 완전히 정의 인간 심장 조직을 설계하는 능력은 강력한 선별 도구를 만드는, 또한 새로운 세포 - 기반 유전자 심장 치료를 조사하기위한 모형을 개발하는 시스템뿐만 아니라 필수적이다. 중간 엽 줄기 세포 (MSC) (21)를 포함한 세포 유형 이용한 심부전 특히, 다수의 세포 치료에서, </SUP&gt;, 심장 줄기 세포 (22) 및 골수 단핵구 23-25은 임상 시험에서 시험되고있다. 초기 결과 많은 21,23,25 유망되었지만, 초기 이득은 종종 26-29 시간에 걸쳐 감소한다. 유사한 경향으로 인해 MSC 보충에 중요한 기능적 이득을 표시 뮤린 설계 심장 조직에서보고되었지만 이득 장기 배양 한 동안 유지되지 않는다. 서브 최적의 성능을 기본 것은 세포 치료를 지배하는 메커니즘의 우리의 제한된 지식이다. 자신의 유익한 영향뿐만 아니라 심근-nonmyocyte 상호 작용의 잠재적 인 부정적 영향을 미치는 방법을 치료 세포의 깊은 이해는 심부전 환자에 대한 최소한의 부작용이 임상 적으로 유의 한 지속적인 혜택을 얻었다 개선 된 치료법의 개발을 활성화합니다. 여기, 우리는 interrog 정의 hECTs의 사용을 설명세포 기반 치료의 메커니즘을 먹었다. 제어 조직 조성물은 심근 성능에 영향을 특정 요소를 식별하는 데 중요하다. 우리 래트 ECTS 1에서 보여준 바와 같이 직접 관심 치료 세포 유형 (예, 중간 엽 줄기 세포)를 hECTs 보충, 심장 근세포의 성능에 미치는 영향을 나타내있다. 다음 다단계 프로토콜 멀티 조직 생물 반응기의 제조이어서, 조직 구조 및 기능 분석에 대한 설명으로 종결 지시 된 심장 줄기 세포의 분화와 함께 시작된다. 우리의 실험은 NIH 승인 H7 인간 배아 줄기 세포 (hESC의) 라인을 사용하여 수행됩니다. 그러나, 다음의 프로토콜들이 또한 추가 hESC의 라인과 유사한 결과 세 유도 다 능성 줄기 세포 (hiPSC) 라인을 사용하여 테스트되었다. 우리는 hECT 제조에서 심근 차별화와 성공의 효율성, 특히 fo를, 세포주 의존 할 수 있다는 것을 발견했다개별 환자에서 파생 된 연구 hiPSC 라인. 이 프로토콜에 따라, 두 개의 6 잘 요리 20 일 동안 차별화 충분히 여섯 정의 된 조직을 만들기 위해, 정렬 후 약 250 만 근육 세포를 얻을 수 1,680,000 hESCs는 (물론 당 140,000 셀), 총으로 도금되어있다.

<table border="0">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td>
		</tr>
		<tr>
			<td>B27 with Insulin</td>
			<td>Life Technologies</td>
			<td>17505055</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Create 6 &#956;M stock, then aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Essential 8 Media</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>H7 Human Embryonic Stem Cells</td>
			<td>WiCell</td>
			<td>WA07</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC Qualified Matrix, Corning Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Thaw on ice at 4 &#176;C overnight then aliquot 150 &#956;l into separate tubes and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td>Create 10 mM stock and aliquot. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum</td>
			<td>Life Technologies</td>
			<td>16010159</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Keep refrigerated</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK Inhibitor)</td>
			<td>Stemgent</td>
			<td>04-0012</td>
			<td>Resuspend to a 10 mM stock concentration, aliquot and store at -20 &#176;C.&#160; Avoid freeze thaw cycles.</td>
		</tr>
		<tr>
			<td>Cell Sorting</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90-FITC</td>
			<td>BioLegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&#160;</td>
			<td>PromoCell</td>
			<td>C-41200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologics</td>
			<td>S11250</td>
			<td></td>
		</tr>
		<tr>
			<td>SIRP&#945;-PE/Cy7</td>
			<td>BioLegend</td>
			<td>323807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Construction</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/0.1% EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-053-CI</td>
			<td>Optional: For collection of supplemental cells of interest</td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M0275-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS Packets</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine Type I</td>
			<td>Life Technologies</td>
			<td>A10644-01</td>
			<td>Keep on ice</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagles Medium (DMEM), High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC0536757</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round-Bottom Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td>With integrated 35 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>50 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat bottom tissue-culture treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, Disposable</td>
			<td>Biologix</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (Teflon)</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ-61</td>
			<td>Or similar, must have a mount for the high speed camera to attach</td>
		</tr>
		<tr>
			<td>Electrical Pacing System</td>
			<td>Astro-Med, Inc</td>
			<td>Grass S88X Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Pixelink</td>
			<td>PL-B741U</td>
			<td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td>
		</tr>
		<tr>
			<td>Plate Temperature Control</td>
			<td></td>
			<td></td>
			<td>Used to maintain media temperature during data acqusition.</td>
		</tr>
		<tr>
			<td>Custom Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LabView Post-tracking Program</td>
			<td></td>
			<td></td>
			<td>available upon request from the authors</td>
		</tr>
	</tbody>
</table>

construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

심장 근육 세포를 얻으려면, Boheler와 리안 분화 방법의 약간 수정 된 버전은 30, 31을 사용합니다. 분화 세포 성장의 로그 단계에서 시작되는지, 또한 출발 인구 (약 75 %가 최적 임) 정렬 후 세포의 가능한 수를 얻기 위해 충분히 융합 인 것이 필수적이다. 일반적 H7 hESCs는 들어 37 ° C로 유지 필수적 8 미디어 및 5 % CO 2 인큐베이터에서 6- 웰 접시의 웰 당 140,000 hESCs는 밀도에서 도금은 ?...

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Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

정의 인간 공학 심장 조직의 건설 심장 세포 치료의 메커니즘을 연구하는

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

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Research

JoVE Journal

Bioengineering

9.4K 조회수.  Icahn School of Medicine at Mount Sinai. This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

비디오: Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

문화를 셀에 충격파 응용 프로그램

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

연구

생체공학

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

소형 조직 배양 시스템에서 마우스 심장 밸브를 배양

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

특별 한 광학 전송 인간의 혈 청에 심장 Biomarkers 척도를 사용 하 여

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

비 계-무료 3 차원 심장 조직 창조의 그물 형 기반 방법

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

인간 조직형 심장 슬라이스의 이중 전압 및 칼슘 광학 매핑에 의한 전임상 심장 전극생리학 평가

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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

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

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

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

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

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

인간 iPSC 유래 심근세포, 심장 섬유아세포 및 내피 세포를 사용하여 3D 심장 미세 조직 배열의 제조

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

인간 심장 병리학을 연구하기 위하여 체외 생체 공학심장 조직에서와 같이 심장 스페로이드

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...