Die Autoren danken Alessandra Moretti und Dennis Schade für ihre Art Beitrag des Materials. Wir erkennen die große Unterstützung der iPS und EHT-Arbeitsgruppe am Institut für Experimentelle Pharmakologie und Toxikologie des UKE. Die Arbeit der Autoren wird durch Zuschüsse aus dem DZHK (Deutsches Zentrum für Herz-Kreislaufforschung) und dem Bundesministerium für Bildung und Forschung (BMBF), der Deutschen Forschungsgemeinschaft (DFG Es 88 / 12-1, HA 3423 unterstützt / 5-1 ), British National Center für das Ersatz-Refinement &amp; Reduktion von Tieren in der Forschung (NC3Rs CRACK-IT 35.911-259.146 gewähren), die British Heart Foundation RM / 13/30157, der Europäischen Forschungsrat (Advanced Grant IndivuHeart), die deutsche Herzstiftung und die Freie und Hansestadt Hamburg.

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IM, TE und AH sind Mitbegründer der EHT Technologies GmbH, Deutschland.

Ausgeführt Herzgewebe bieten eine wertvolle Option für den Werkzeugkasten der kardiovaskulären Forschung. EHTs in dem 24-Well - Format haben für Krankheit Modellierung 8, 14, Arzneimittelsicherheit Screening 7, 8, 10, 11, 15, oder basisches Cardiovascular Research 16,<...

Herznebenwirkungen wie die Arzneimittel-induzierte Long-QT-Syndrom haben Rücknahme aus dem Markt in den letzten Jahren geführt. Statistiken zeigen , dass etwa 45% aller Abhebungen fällig sind , um unerwünschte Auswirkungen auf das Herz - Kreislauf - System 1. Dieses Medikament Versagen nach dem teueren Entwicklungsprozess und die Genehmigung ist das Worst-Case-Szenario für Pharmaunternehmen. Forschungs- und Entwicklungsabteilungen konzentrieren sich daher auf dem Nachweis von solchen unerwünschten kardiovaskulären Wirkungen frühzeitig zu erkennen. Aus wirtschaftlichen und ethischen Bedenken, die Bemühungen um Tierversuche zu reduzieren und ersetzen sie durch neue in - vitro - Screening - Tests sind im Gange. Eine Reihe von etablierten Assays sind in den Vereinigten Staaten Food and Drug Administration (FDA) und der Europäischen Arzneimittel - Agentur (EMA) Richtlinien für die präklinische Bewertung von proarrhythmischen Arzneimittelwirkungen 2 enthalten. Die Technologie von somatischen Zellen durch Differenzierung gefolgt Umprogrammierung vonmenschlichen induzierten pluripotenten Stammzellen (hiPSC) verstärkt dieses Forschungsgebiet 3. Es bietet nun die Möglichkeit , neue Medikamentenkandidaten auf dem menschlichen Kardiomyozyten in vitro zu screenen und vermeidet Probleme mit Wechselwirkungen zwischen den Arten Unterschieden. Aktuelle Herzdifferenzierungsprotokolle 4, 5 bieten unbegrenzte Versorgung von Kardiomyozyten ohne ethische Bedenken. Die Messung der Kontraktionskraft ist die wichtigste und am besten charakterisierte jedoch in vivo Parameter von Kardiomyozyten, nicht gut etabliert. Dies hängt mit der relativen Unreife 6 des menschlichen induzierten pluripotenten Stammzellen gewonnenen Herzmuskelzellen (hiPSC-CM) im Vergleich zu dem erwachsenen Kardiomyozyten. Eine mögliche Weiterentwicklung ist 3-dimensionale Herzgewebe aus einzelnen Zellen zu konstruieren , 7 (engineered Herzgewebe, EHT). Das EHT - Protokoll basiert auf einzelnen murinen oder humanen Kardiomyozyten Einbettung 8 </sup&gt;, 9, 10 in Fibrinhydrogel zwischen zwei flexiblen Polydimethylsiloxan (PDMS) Pfosten 11 in 24-Well - Format. Innerhalb von wenigen Tagen beginnen die Kardiomyozyten spontan als einzelne Zellen zu kontrahieren und starten Mobilfunknetze zu bilden. Nach 7-10 Tagen sind makroskopische Kontraktionen des gesamten Gewebes sichtbar. Während dieses Prozesses wird die extrazelluläre Matrix umgestaltet, was zu einer Abnahme des Durchmessers und der Länge führt. Die Verkürzung der EHT Ergebnisse in der PDMS Biege Post auch während der Ruhe, Kardiomyozyten in der Entwicklung von EHT zu Dauerbelastung ausgesetzt wird. EHTs weiterhin auxotonisches Muskelkontraktionen über mehrere Wochen durchzuführen. Human EHTs zeigen Reaktionen auf physiologische und pharmakologische Stimulation anzeigt , deren Eignung für Wirkstoff - Screening und Krankheit 7 modellieren. In diesem Manuskript präsentieren wir ein robustes und einfaches Protokoll für die Generatiauf der menschlichen EHT, und die automatisierten Analyse von Kontraktilität konzentrationsabhängigen Veränderungen des Kontraktionsmusters in Gegenwart von hERG Kanalinhibitoren.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EHT analysis intrument</td>
			<td>EHT Technologies GmbH</td>
			<td>A0001</td>
			<td>Software is included</td>
		</tr>
		<tr>
			<td>EHT PDMS rack</td>
			<td>EHT Technologies GmbH</td>
			<td>C0001</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT PTFE spacer</td>
			<td>EHT Technologies GmbH</td>
			<td>C0002</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT electrode</td>
			<td>EHT Technologies GmbH</td>
			<td>P0001</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT pacing adapter/cable</td>
			<td>EHT Technologies GmbH</td>
			<td>P0002</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well-plate</td>
			<td>Nunc</td>
			<td>144530</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well-cell culture plate</td>
			<td>Nunc</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube, graduated&#160;</td>
			<td>Sarstedt</td>
			<td>62,554,502</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sarstedt</td>
			<td>831,830</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flask</td>
			<td>Integra</td>
			<td>182 101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirrer Variomag/ Cimarec Biosystem Direct&#160;</td>
			<td>Thermo scientific</td>
			<td>70101</td>
			<td>Adjust rotor speed to 40 rpm</td>
		</tr>
		<tr>
			<td>T175 cell culture flask&#160;</td>
			<td>Sarstedt&#160;</td>
			<td>831,812,002</td>
			<td></td>
		</tr>
		<tr>
			<td>V-shaped sedimentation rack&#160;</td>
			<td>Custom made at UKE Hamburg</td>
			<td>na</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#215; DMEM</td>
			<td>Gibco</td>
			<td>52100</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Phospho-L-ascorbic acid trisodium salt</td>
			<td>Sigma Aldrich</td>
			<td>49752</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin-A&#160;</td>
			<td>R&#38;D systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>15510-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma Aldrich</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia</td>
			<td>Baxter GmbH</td>
			<td>1428</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 PLUS insulin&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>R&#38;D systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II&#160;</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom</td>
			<td>F0415</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase II, type V (from bovine spleen)</td>
			<td>Sigma&#160;</td>
			<td>D8764</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin&#160;</td>
			<td>abcam</td>
			<td>ab120843</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-921</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen (bovine)</td>
			<td>Sigma Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex&#160;</td>
			<td>Gibco</td>
			<td>A1413302</td>
			<td>For coating: 1:200 dilution</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+/Mg2+&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Life technologies</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum albumin&#160;</td>
			<td>Biological Industries</td>
			<td>05-720-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>Sigma Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipidmix&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L5146</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>For EHT reconsitutionmix.</td>
		</tr>
		<tr>
			<td>N-Benzyl-p-Toluenesulfonamide</td>
			<td>TCI</td>
			<td>B3082-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o MgCl2/CaCl2</td>
			<td>Biochrom</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco</td>
			<td>21875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#223;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma Aldrich</td>
			<td>T7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8158</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biorbyt</td>
			<td>orb6014</td>
			<td></td>
		</tr>
		<tr>
			<td>hiPSC</td>
			<td>Custom made at UKE hamburg</td>
			<td>na</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell cardiomyocytes kit</td>
			<td>Cellular Dynamics International</td>
			<td>CMC-100-010-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluricyte cardiomyocyte kit</td>
			<td>Pluriomics</td>
			<td>PCK-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cor.4U - HiPSC cardiomyocytes kit</td>
			<td>Axiogenesis AG</td>
			<td>Ax-C-HC02-FR3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellartis cardiomyocytes</td>
			<td>Takara Bio USA, Inc.</td>
			<td>Y10075</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated contraction analysis of human engineered heart tissue for cardiac drug safety screening

Cardiac Tissue Engineering beschreibt Techniken dreidimensionale Krafterzeugungs engineered Gewebe zu bilden. Für die Umsetzung dieser Verfahren in der Grundlagenforschung und die präklinische Entwicklung von Medikamenten, ist es wichtig, Protokolle zur automatisierten Generierung und Analyse unter standardisierten Bedingungen zu entwickeln. Hier präsentieren wir eine Technik Engineered Herzgewebe (EHT) von Kardiomyozyten verschiedener Spezies zu erzeugen (Ratte, Maus, Mensch). Die Technik beruht auf der Anordnung eines Fibrin-Gel, das dissoziiert Kardiomyozyten zwischen elastischer Polydimethylsiloxan (PDMS) Einträgen in einem 24-Well-Format. Dreidimensionale, krafterzeugende EHTs bilden innerhalb von zwei Wochen nach dem Gießen. Dieses Verfahren ermöglicht die Erzeugung von mehreren hundert EHTs pro Woche und ist technisch nur durch die Verfügbarkeit von Kardiomyozyten (0,4-1,0 × 10 6 / EHT) begrenzt. Bewertung der auxotonisches Muskelkontraktionen in einem modifizierten Inkubationskammer mit einem mechan durchgeführtische Verriegelung für 24-Well-Platten und eine Kamera auf der Oberseite dieser Kammer angeordnet. Eine Software steuert eine Kamera auf einem XYZ-Achsensystem an jede EHT bewegt. EHT Kontraktionen werden durch eine automatisierte Figur Erkennungsalgorithmus erkannt wird, und wird eine Kraft berechnet, basierend auf Verkürzung der EHT und die elastische Neigung und die Geometrie der PDMS Beiträge. Dieses Verfahren ermöglicht die automatisierte Analyse von hohen Zahl von EHT unter standardisierten und sterilen Bedingungen. Die zuverlässige Erkennung von Arzneimittelwirkungen auf Kardiomyozyten Kontraktion ist von entscheidender Bedeutung für die Herzarzneimittelentwicklung und Sicherheitspharmakologie. Wir zeigen, mit dem Beispiel der hERG Kanalinhibitor E-4031, dass das menschliche EHT System Arzneimittelantworten auf der Kontraktion Kinetik des menschlichen Herzens nachbildet, angibt, dass es ein vielversprechendes Werkzeug für die kardialen Arzneimittelsicherheit Screening sein.

Herzdifferenzierung und Herstellung von EHT HiPSC wurde auf reduziertem Wachstumsfaktor Basalmembranmatrix erweitert, dissoziiert mit EDTA und embryoid bodies (EBs) über Nacht in Spinnerflaschen gebildet. Nach mesodermalen Induktion für drei Tage wurde kardiale Differenzierung mit dem Wnt-Inhibitor ausgelöst. Nach ~ 17 Tagen Differenzierung Protokoll wurde schlagen EBs in einzelne Zellen dissoziiert mit Kollagenase Typ II <str...

Watch this Scientific Journal Video about Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening at JoVE.com

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

Hier zeigen wir die Herstellung von humanen konstruierte Herzgewebe aus induzierten pluripotenten Stammzellen (hiPSC) abgeleitetes Kardiomyozyten. Wir stellen eine Methode Kontraktionskraft und beispielhafte Veränderung der Kontraktionsmuster durch den hERG-Kanal-Hemmer E-4031 zu analysieren. Dieses Verfahren zeigt hohe Robustheit und die Eignung für das Herz Wirkstoff-Screening.

Automatische Kontraktions Analyse menschlicher Engineered Herzgewebe für Herz Drug Safety Screening

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

automated-contraction-analysis-human-engineered-heart-tissue-for

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Bioengineering

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

12.5K Aufrufe.  University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research).  Hier zeigen wir die Herstellung von humanen konstruierte Herzgewebe aus induzierten pluripotenten Stammzellen (hiPSC) abgeleitetes Kardiomyozyten. Wir stellen eine Methode Kontraktionskraft und beispielhafte Veränderung der Kontraktionsmuster durch den hERG-Kanal-Hemmer E-4031 zu analysieren. Dieses Verfahren zeigt hohe Robustheit und die Eignung für das Herz Wirkstoff-Screening. 

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

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Biolumineszenz-Bildgebung für die Beurteilung von Immunantworten nach der Implantation von Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Forschung

Biotechnik

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Selbstberichts Gerüste für 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Analyse von Membranrohrnetze in Herzmuskelzellen von Vorhöfe und Kammern

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Hohlfaser-Bioreaktoren für<em&gt; In Vivo</em&gt; -ähnlichen Mammalian Gewebekultur

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

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.

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.

Bau von Defined Menschen Engineered Herzgewebe zur Untersuchung Mechanismen der Herzzelltherapie

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

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Ein kombiniertes 3D-Gewebezüchtungen<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Lungentumormodell für Hintergründe in bestimmten Mutations Drug Wirksamkeit vorhersagen

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Einsatz von Hochdurchsatz-automatisierte Microbioreactor-System für die Produktion des Modells IgG1 in CHO-Zellen

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

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.

Vorbereitung von Mesh-Shaped Engineered Cardiac Tissues derived from Human iPS Cells for In Vivo Myokard repair

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

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.

Herzsphäroide als in vitro bioengineered Herzgewebe zur Untersuchung der menschlichen Herzpathophysiologie

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

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Entwicklung von 3D-organisiertem menschlichem Herzgewebe innerhalb einer mikrofluidischen Plattform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...