Name: construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy
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Description: Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

Detta arbete stöddes av NIH (1F30HL118923-01A1) till TJC, NIH / NHLBI PEN kontrakt HHSN268201000045C till KDC, forskningsanslaget råd Hongkong TRS T13-706 / 11 (KDC), NIH (R01 HL113499) till BDG, den amerikanska Heart Association (12PRE12060254) till RJ, och forskningsbidrag rådet Hongkong (TBRS, T13-706 / 11) RL Ytterligare finansiering lämnades till TJC av NIH DRB 5T32GM008553-18 och som en praktikplats på NIDCR-tvärvetenskaplig utbildning inom Systems and Develop biologi och fosterskador T32HD075735. Författarna vill också tacksamt erkänna Arthur Autz på The Zahn centrum av staden College of New York för att få hjälp med att bearbeta bioreaktorn och Mamdouh Eldaly för tekniskt stöd. Vi vill också tacka Dr Kenneth Boheler för råd om hjärt differentiering, och Dr Joshua Hare för generöst ger humana mesenkymala stamceller.

Obs: Utför alla cell manipulationer i aseptiska betingelser med användning av ett HEPA-filtrerad klass II biologiskt säkerhetsskåp och sterilisera alla lösningar genom att filtrera dem genom ett 0,2 pm filter. Utföra vävnads uppbyggnad och funktion testning i antingen samma aseptiska förhållanden eller ett dragskåp med laminärt flöde. 1. Sådd av H7 hESCs i Förberedelse för hjärt differentiering <ol><li> (Dag 1) Förberedelse av Basement Membrane Matrix <ol><li> Tina 150 pl ...

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Konstruktion av definierade mänskliga tekniska hjärtvävnad (Hect) kan ge en mer konsekvent och tillförlitlig modell av human hjärt myocyte funktion. Kritiskt, är alla cellulära och extracellulära komponenter i systemet är kända och kan manipuleras enligt önskemål, på så sätt avlägsna den confounding påverkan av andra okända celltyper till följd av differentieringsprocessen. Att balansera snabb celltillväxt och hög avkastning, är det att föredra att differentieringen börjar vid 75% sammanflytning ...

Hjärtvävnad engineering har avancerat kraftigt under det senaste decenniet, med flera grupper publicera resultaten av fullt fungerande, slå vävnader tillverkad av både murina cardiomyocytes 1-6 och, på senare tid, mänskliga stamceller härledda hjärtmyocyter 7-12. Hjärtvävnadsteknik fält drivs av två primära och väsentligen oberoende mål: 1) att utveckla exogena transplantat som kan transplanteras in inte hjärtan för att förbättra funktionen 4-6; och 2) att utveckla in vitro-modeller för att studera fysiologi och sjukdom, eller som screeningverktyg för terapeutisk utveckling 2,7. Tredimensionell (3-D) cellodling anses nödvändig för att utveckla nästa generations analysverktyg, som 3-D matris återspeglar en mer naturlig hjärtmikro än traditionella 2-D monolager cellodling; faktiskt vissa aspekter av cellbiologi är fundamentalt olika i 2-D vs 3-D kulturer 13,14 </sup&gt;. Dessutom är manipulerade hjärtvävnader tillverkade av helt definierade komponenter: en extracellulär matris, och en cellpopulation. För traditionella manipulerade humana hjärtvävnader, medan den extracellulära matriskompositionen (vanligen fibrin 9 eller kollagen 7,8,10) är strikt kontrollerad, ingångscellkomposition är mindre väl definierad, med hela blandningen av celler från en riktad hjärt differentiering av antingen embryonala stamceller (ESC 7,9) eller inducerade pluripotenta stamceller (IPSC 10,12) läggs till vävnaderna. Beroende på den specifika cellinjen och effektiviteten i differentieringsprotokoll som används, kan den resulterande andel av kardiomyocyter variera från mindre än 25% till över 90%, den specifika cardiomyocyte fenotyp (dvs ventricular-, atrial- eller pacemaker-liknande) kan också variera, även icke-cardiomyocyte fraktion kan vara mycket heterogen 15,16 och ändra löptiden för den differentierade hjärt myocytes 17. Senaste hjärtvävnad ingenjörsarbete har försökt att styra ingångs population av celler, med antingen en hjärt reporter human embryonal stamcellslinje 8 eller cellytmarkörer 18 används för att isolera hjärt myocyte komponenten av differentiering. Även initialt en vävnad som består av endast hjärtmyocyter verkar vara den idealiska, är detta i själva verket inte fallet; hECTs som består endast av hjärtmyocyter misslyckas med att kompaktera i funktionella vävnader, med vissa grupper att hitta en 3: 1 förhållande av hjärtmyocyter: fibroblaster som producerar den högsta rycka kraft 8. Genom att använda olika urvals cell metoder, inklusive ytmarkörer för levande cellsortering, är det möjligt att skapa hECTs med definierade cellpopulationer. Medan markörer för icke-hjärt stromaceller har funnits under en längre tid, såsom den förmodade fibroblast markören CD90 19,20, har ytmarkörer av hjärtmuskelceller varit svårareatt identifiera. SIRPα var bland de första hjärt ytmarkörer identifierats för mänskliga hjärtmuskelceller 18 och har visat sig vara mycket selektiva för hjärt härstamning. Nyligen har vi funnit att dubbelsortering för SIRPα + och CD90 - celler ger nästan rena kardiomyocyter, med CD90 + populationen som uppvisar en fibroblast-liknande fenotyp (Josowitz, opublicerade observationer). Baserat på dessa insamlade fynd, här beskriver vi skapar hECTs med hjälp av en 3: 1 blandning av SIRPα + / CD90 - hjärtmuskelceller och CD90 + fibroblaster. Förmågan att konstruera en helt definierad human hjärtvävnad är viktigt inte bara för att skapa robusta screeningverktyg, men även för att utveckla modellsystem för att undersöka nya cell- och genbaserade hjärt terapier. I synnerhet många cellterapi för hjärtsvikt, utnyttjar celltyper inklusive mesenkymala stamceller (MSC) 21 </sup&gt;, har hjärt stamceller 22 och benmärgsmononukleära celler 23-25, testats i kliniska prövningar. Även om många av de första resultaten har varit lovande 21,23,25, minskar den initiala fördel ofta över tiden 26-29. En liknande trend har rapporterats i murina konstruerad hjärtvävnader, som visar en betydande funktionell fördel på grund av MSC tillskott, men fördelen är inte upprätt under långtidsodling en. Bakom sub-optimal prestanda är vår begränsade kunskap om de mekanismer som styr cellterapier. En djupare förståelse av hur terapeutiska celler utöva sin välgörande inflytande, liksom eventuella negativa konsekvenser av myocyt-nonmyocyte interaktioner, skulle göra det möjligt att utveckla förbättrade behandlingar ger kliniskt signifikanta och varaktiga fördelar, med minimala biverkningar för patienter med hjärtsvikt. Här beskriver vi användningen av definierade hECTs att interrogåt mekanismerna för cellbaserad terapi. Den kontrollerade vävnadssammansättning är viktigt att identifiera specifika faktorer som påverkar cardiomyocyte prestanda. Direkt komplettera hECTs med den terapeutiska celltypen av intresse (t.ex. MSC) kan avslöja effekterna på hjärt myocyte prestanda, som vi har visat i rått ECT 1. Följande flerstegsprotokoll börjar med riktad hjärtstamcellsdifferentiering, följt av tillverkning av flervävnads bioreaktor, och sluta med en beskrivning av vävnads konstruktion och funktionell analys. Våra experiment utförs med hjälp av NIH-godkända H7 mänskliga embryonala stamceller (hESC) linje. Emellertid har följande protokoll också testats med användning av en ytterligare hESC linje och tre inducerade pluripotenta stamceller (hiPSC) linjer med liknande resultat. Vi har funnit att effektiviteten i cardiomyocyte differentiering och framgång i Hect tillverkning kan vara cellinje beroende, särskilt for hiPSC linjer som härrör från enskilda patienter. Genom att följa detta protokoll, två 6-brunnar pläterad med totalt 1,68 miljoner hESCs (140.000 celler per brunn), vilket ger cirka 2,5 miljoner myocyter efter differentiering i 20 dagar och sortering, tillräckligt för att göra sex definierade vävnader.

<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.

För att få hjärtmuskelceller, är en något modifierad version av Boheler och Lian differentieringsmetoder som används 30,31. Det är absolut nödvändigt att differentieringen startar under log-fasen av celltillväxt, men också att utgångspopulationen är tillräckligt konfluent för att erhålla en användbar antal celler efter sortering (ca 75% är optimalt). Typiskt för H7 hESCs, plätering vid en densitet av 140.000 hESCs per brunn i en 6-brunnsskål i väsentlig 8 medier och 5% CO2 ink...

Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

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.

Konstruktion av definierade mänskliga Engineered hjärtvävnader att studera mekanismer för Cardiac Cellterapi

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

Research

JoVE Journal

Bioengineering

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 ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Odling Mouse hjärtklaffar i miniatyr 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 ...

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

Automatiserad Contraction analys av Human Engineered hjärtvävnad för Cardiac Drug Safety 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

Använda extra optisk överföring att kvantifiera hjärt biomarkörer i humant Serum

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

Protokoll för mikroRNA överföring till benmärgen-härrör hematopoetiska stamceller att aktivera Cell teknik kombinerat med magnetiska inriktning

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

En netto mögel-baserade metod byggnadsställning-fri tredimensionella hjärtvävnad skapelsens

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

Preklinisk hjärtelektrofysiologisk bedömning av dubbel spänning och kalciumoptisk kartläggning av mänskliga organotypiska hjärtskivor

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

Beredning av Mesh-formade Engineered Hjärt vävnader som härrör från mänskliga iPS-celler för In Vivo hjärtinfarkt Reparation

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

Tillverkning av 3D Cardiac Microtissue Arrays med mänskliga iPSC-härledda kardiomyocyter, hjärtfibblaster och endotelceller

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

Hjärt sfäroider som in vitro Bioengineered hjärtvävnader att studera människans hjärta Patofysiologi

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