Die Autoren erkennen die folgende Finanzierungsquelle: Magie, dass Angelegenheiten Fonds für Herz-Kreislauf-Forschung.

1. Vorbereitung von Kardiomyozyten
<ol><li>6-Well-Platten mit Basalmembran Matrix und Kultur Mensch-induzierte pluripotente Stammzellen (HiPSCs) wie zuvor beschrieben17zu beschichten.</li>
	<li>HiPSCs in HiPSC-CMs mit der zuvor beschriebenen Methoden18zu unterscheiden.</li>
	<li>16-18 d Post-Differenzierung aussetzen der Herzmuskelzellen durch Spülen jedes gut mit 2 mL 1 x Phosphat-gepufferte Kochsalzlösung (PBS) ohne Kalzium und Magnesium, gefolgt von Inkubation mit 1 mL/auch von Trypsin oder Zelle Dissoziation Reagenz (siehe Tabelle des Materialien) für 5 min bei Raumtemperatur.</li>
	<li>Das Trypsin oder Zelle Dissoziation Reagenz zu neutralisieren (siehe Tabelle der Materialien) über ein gleiches Volumen von Roswell Park Memorial Institute (RPMI) Zelle Medien mit B-27 (RPMI/B-27 Zelle Medien) ergänzt. Pipette rauf und runter um alle eingehalten Zellen zu lockern.</li>
	<li>Sammeln Sie die abgehängten Herzzellen mit einer serologischen 10-mL-Pipette und übertragen Sie sie auf eine 50 mL konische Rohr.</li>
	<li>Zentrifugieren der Zellsuspension bei 250 X g für 5 min bei Raumtemperatur einen Zelle Pellet zu erhalten.</li>
	<li>Das Pellet in 10 mL RPMI/B-27 Zelle Medien aufzuwirbeln.</li>
	<li>Kombinieren Sie 20 µL Zellsuspension mit einer gleichen Menge von 0,4 % Trypan blau Lösung und mischen Sie vorsichtig.</li>
	<li>Verwenden Sie eine manuelle Hemocytometer zu zählen und erhalten die Konzentration und Zelle Tragfähigkeit der Zellsuspension.</li>
</ol>2. Vorbereitung der Fibroblasten
<ol><li>Initiieren einer Kultur einer menschlichen kardialen Fibroblasten (HCF) (Erwachsene ventrikuläre Typ)-Zelllinie wie zuvor16beschrieben.</li>
	<li>Aussetzen der TREIBGASEN durch Inkubation mit einer entsprechenden Menge an Trypsin oder Zelle Dissoziation Reagenz (siehe Tabelle der Materialien) für 5 min bei Raumtemperatur16. Für ein T175 Fläschchen wurde 10 mL Trypsin oder Zelle Dissoziation-Reagenz verwendet.</li>
	<li>Das Trypsin oder Zelle Dissoziation Reagenz zu neutralisieren (siehe Tabelle der Materialien) ein gleiches Volumen des Mediums mit, dann übertragen Sie die Probe auf eine 50 mL konische Rohr und Zentrifugieren die Zellsuspension bei 250 X g für 5 min bei Raumtemperatur zu erhalten eine Pellet.</li>
	<li>Das Pellet in 10 mL von Fibroblasten Wachstumsmedium Aufschwemmen (siehe Tabelle der Materialien).</li>
	<li>20 µL der HCF Zellsuspension mit einer gleichen Menge von 0,4 % Trypan blau Lösung kombinieren und vorsichtig mischen.</li>
	<li>Verwenden Sie eine automatisierte Zelle Zähler oder manuelle Hemocytometer zu zählen und erhalten die Konzentration und Zelle Tragfähigkeit des neuen Zellsuspension.</li>
</ol>3. Vorbereitung der Endothelzellen
<ol><li>Initiieren Sie eine Kultur der menschlichen nabelader Endothelzellen (HUVECS) Zeile wie oben beschrieben17. Aussetzen der Mediumwechsel durch Inkubation mit einer entsprechenden Menge an Trypsin oder Zelle Dissoziation Reagenz (siehe Tabelle der Materialien) für 3 min bei Zimmertemperatur17. Das Trypsin oder Zelle Dissoziation Reagenz zu neutralisieren (siehe Tabelle der Materialien) ein gleiches Volumen des Mediums verwenden, übertragen Sie dann auf eine 50 mL konische Rohr und Zentrifugieren die Zellsuspension bei 250 X g für 5 min bei Raumtemperatur ein Pellet zu erhalten. Hinweis: Für eine Flasche T175 war 10 mL Trypsin oder Zelle Dissoziation Reagenz verwendet.</li>
	<li>Das Pellet in 10 mL von Endothelzellen Wachstumsmedium Aufschwemmen (siehe Tabelle der Materialien).</li>
	<li>Kombinieren Sie 20 µL der HUVECS Suspension zu und Färben Sie es mit einer gleichen Menge von 0,4 % Trypan blau Lösung und mischen Sie vorsichtig.</li>
	<li>Verwenden Sie eine automatisierte Zelle Zähler oder eine manuelle Hemocytometer zu zählen und erhalten die Konzentration und Zelle Tragfähigkeit der Zellsuspension.</li>
</ol>4. Schaffung von hängenden Tropfen Sphäroide
<ol><li>Legen Sie die hängenden Tropfen Gerät enthält 850 Mikroporen (jeweils mit einem Durchmesser von 350 µm) in sterilen 6-Well-Platten.</li>
	<li>Die drei Arten von Zellen zu isolieren, wie oben beschrieben: HiPSC-CMs und TREIBGASEN Mediumwechsel. Kombinieren sie in einem Verhältnis von 70 % iPSC-CMs, 15 % TREIBGASEN und 15 % Mediumwechsel in einem 50 mL konische Röhrchen mit RPMI/B-27 Zelle Medien in einer Konzentration von 2.475.000 Zellen pro mL.</li>
	<li>4 mL Zellsuspension (2.475.000 Zellen pro mL) in jede Vertiefung einer ultra-niedrigen Anlage hängen Drop-System (mit Zusammenarbeit von 350 µm Durchmesser) sitzt in einem 6-Well-Platte zu verzichten.</li>
	<li>Sphäroide bilden sich spontan innerhalb von 12 h der Verzicht auf der Zellsuspension in den hängenden Tropfen Gerät. Kultur für eine Gesamtmenge von 72 h (3 d) bei 37 ° C, 5 % CO2und 95 % Luftfeuchtigkeit für die Reifung der Sphäroide weiter.</li>
	<li>Nach 72 h Kultur, Ernte der Sphäroide durch die Poren an der Unterseite der hängenden System indem das Gerät in eine Schale mit Medium und wirbelnden es sanft um die Sphäroide aus der hängenden freizugeben fallen Tropfen Gerät.</li>
</ol>5. 3-d-Patch-Erstellung unter Verwendung neuartige Net Form
<ol><li>Die Basis, quadratische Bodenplatte Bodennetz und 10 geschichteten Seite Netze montiert sind, um die neuartige Form mit Hilfe von ein paar sterilen Pinzette zu erstellen. Zuerst, stapeln Sie und richten Sie die Base, quadratische Bodenplatte, und unten Sie Netz mit den Eckpfosten. Dann Stapeln und Schicht 10 Seite Netze in wechselnden Richtungen um ein Netz von feinen Edelstahl-Zinken zu erstellen.</li>
	<li>Spülen Sie die Füllung mit PBS oder Medium zu reduzieren die Oberflächenspannung und übertragen Sie anschließend die zusammengebaute Form auf die Füllung-Basis.</li>
	<li>Befeuchten Sie die net Kavität mit PBS oder Medium in der gleichen Methode. Füllen Sie langsam die Sphäroide in vorausgesetzten Hohlraum der gewünschten Größe (2 x 2 x 1 mm, 4 x 4 x 1 mm oder 6 x 6 x 1 mm). Hinweis: Stellen Sie sicher, dass 120 % des erwarteten Volumens der Sphäroide in den Hohlraum zugeführt wird, so dass die Zelle Aggregate in engen Zusammenhang stehen und statisch bleiben.</li>
	<li>Montieren Sie die obere Netz und quadratische Deckplatte auf die Eckpfosten und sichern Sie den Stopper und Holding Röhren eine Auswaschung der Sphäroide zu verhindern.</li>
	<li>Übertragen Sie gefüllte net Schimmel-System auf eine 6-Well-Platte mit 6 mL RPMI/B-27 Zelle Medien oder in einen sterilen Behälter mit genügend Mittel zur Deckung der gesamten zusammengesetzten Form.</li>
	<li>7-10 Tage bei 3.000-4.000 X ginkubieren Sie 6-Well-Platte mit dem gefüllten Schimmel-System auf einen schwingenden Shaker. Hinweis: Die Dauer der Inkubation wird durch die Größe der kardialen Patches entschieden. Mit größeren Patches müssen die Inkubationszeit für kardiale Patch Reifung erhöht werden.</li>
</ol>6. Entfernen des Patches aus der neuartigen Net Form
<ol><li>Entfernen Sie das Schimmel-System aus den Medien und legen Sie es auf die sterilisierten Umgang mit Matte in eine sterile 10-cm-Schüssel.</li>
	<li>Entfernen Sie das halteröhrchen, Stopper, quadratische Deckplatte und im besten Netz. Schieben Sie vorsichtig mit einer sterilen Pinzette heraus die geschichteten Seite Netze eins nach dem anderen. Nach Decannulation wird ein intakter Herz Patch oben auf das Bodennetz erhalten.</li>
	<li>Das Bodennetz mit dem Patch mit sterilen Pinzette Abholung und transfer das Bodennetz in eine 35-mm-Schale mit 5 mL RPMI/B27 Medien. Lösen Sie vorsichtig den Patch von net unten durch das Netz langsam umschwenken, in der Schale oder mit einem sterilen Zelle Schaber. Nach der Loslösung von der Bodennetz kann das Herzgewebe mit RPMI/B27 Medien kultiviert werden.</li>
	<li>Weiter zu den frei schwebenden 3-d-kardiale Patch in der 35-mm-Schale zu inkubieren. Funktionale synchron schlagen kann bereits 24 Stunden nach der Entnahme aus der Form beobachtet werden.</li>
	<li>Nach dem Entfernen des Patchs aus dem net Werkzeug, zu beobachten, dass seine Form mit Integrität und der Intensität der synchron schlagen steigt mit der Zeit erhalten bleibt. Hinweis: Die 3-d-net Schimmel-basierte kardiale Patches ausgestellt elektrische Integration der Komponente Cardiospheres nach Decannulation. Wir beobachteten eine Tracht Prügel-Frequenz von 60 bis 80 Schlägen pro Minute, die länger als 60 Tage beibehalten.</li>
</ol>

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Die Autoren haben nichts preisgeben.

Die Bedeutung dieser Methode liegt in ihrer Reproduzierbarkeit und die Wirksamkeit der daraus resultierenden vielschichtigen Herzgewebe. Im Bereich der kardialen Gewebetechnik ist eines der aktuellen Ziele zu identifizieren, eine Methode um schlagen, vielschichtig und funktionale 3-d-kardiale Patches zu konstruieren. Wir berichten über eine effiziente und reproduzierbare Methode vielschichtigen kardialen Gewebe durch manuelle Direktsaat der Sphäroide bestehend aus Herzzellen, Endothelzellen und Fibroblasten in eine neu...

Das Ziel des aktuellen Herzgewebe Engineering ist, eine Therapie zu ersetzen oder zu reparieren, die Struktur und Funktion des verletzten Herzmuskelgewebe1zu entwickeln. Methoden zum Erstellen von 3-d-Herzgewebe Modelle präsentiert die wichtigeren kontraktilen und elektrophysiologischen Eigenschaften des nativen Herzgewebe haben2,3rasch ausgedehnt. Eine Vielzahl von Strategien haben erforscht und in Studien4,5verwendet worden. Diese Methoden reichen von den Einsatz von speziellen synthetischen und natürlichen bioaktiven Hydrogele, wie Gelatine, Kollagen, Fibrin und Peptide6, auf Bio-Tinte Ablagerung Technologien2 und Bioprinting Technologien7.
Es hat sich gezeigt, dass Gerüst-freie Methoden als Biomaterial-basierte Methoden, ohne die Nachteile der Einbeziehung ausländischer Gerüst Material8vergleichbare Gewebe produzieren können. Oren Caspi Et Al. zeigten, dass die Einbeziehung der verschiedenen Arten von Zellen die Generation stark vaskularisierte menschlichen entwickelt Herzgewebe9ermöglicht. Kinn Et Al. entwickelt eine 3-d-Druckverfahren für kardiale Patch Kreation aus Sphäroide. Daraus resultierende Patches bestehen aus Herzzellen, Fibroblasten und Endothelzellen in einem 70:15:15 Verhältnis10. Sphäroide nachweislich wirksame "Bausteine" der Gerüst-freie Herzgewebe Schöpfung, wie sie sind resistent gegen Hypoxie und verfügen über ausreichende mechanische Integrität für Implantation11,12. Frühere Studien haben gezeigt, verschiedene Herstellungsmethoden für Sphäroid Schöpfung, einschließlich der Verwendungdes der hängenden drop-Methode, Spinner Fläschchen13, mikrofluidische Systeme14und nicht anhaftende Kultur Oberflächen unbeschichtet oder beschichtet mit Agarose Mikro-Gußformen15. In diesem Protokoll verwenden wir die hängenden Tropfen Gerät, enthält Mikroporen für Hunderte von Sphäroide gleichzeitig kondensiert.
Diese Studie stellt eine neuartige und effiziente Gerüst-freie Methode für Herzgewebe Erstellung, manuell die Sphäroide in einen quadratischen Formhohlraum Aussaat und Inkubation des Gewebes auf einen Shaker für Reifung. Unter üblichen statischen Kulturbedingungen ist Sauerstoffdiffusion beschränkt sich auf die äußeren Aspekte der Gewebe-Konstrukt, was zentrale Nekrose. Mit der net Form Tauchen alle Sphäroide ausgesät in die Form jedoch in Medien mit einer Konstanten fluidische Bewegung, so dass für die verstärkte Verbreitung von Nährstoffen und Sauerstoff. Darüber hinaus diese Form-basierte Methode ermöglicht die gleichzeitige Erstellung von unterschiedlich großen Gewebe Patches mit minimalem manuellen Aufwand und das resultierende Gewebe kann leicht aus der Form entfernt werden. Diese neuartige Methode ermöglicht die effiziente und reproduzierbare Erstellung von Gerüst-frei, vielschichtigen kardiale Patches.

<table><tbody><tr><td>Human Cardiac fibroblasts (HCF)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>FM-2 Consists of Basal Medium</td><td>Sciencell</td><td>2331</td><td>HCF culture medium</td></tr><tr><td>Human umbilical vein endothelial cells (HUVEC)</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>EGM+Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC5035</td><td>HUVEC culture medium</td></tr><tr><td>E8 media&amp;nbsp;</td><td>Invitrogen</td><td>A1517001</td><td>HiPSC culture medium</td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Invitrogen</td><td>A1413202</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X)</td><td>Thermo Fisher</td><td>12604013</td><td>Trypsin and Cell dissociation reagent</td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11875093</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>B-27 supplement (50x)</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Novel net mold&amp;nbsp;</td><td>TissueByNet Co.,Ltd</td><td>NM25-1</td><td></td></tr><tr><td>Hanging drop plate</td><td>Kuraray Co.,Ltd</td><td>MPc350</td><td></td></tr><tr><td>6 well plates&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS-3516</td><td></td></tr></tbody></table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

Dieses Protokoll beschreibt eine neuartige und einfache net Schimmel-basierte Methode, dreidimensionale (3D) kardialen Gewebe ohne zusätzliche Gerüst-Material. Human-induzierte pluripotente Stammzellen-Zelle abgeleitet Kardiomyozyten (iPSC-CMs), menschliche kardialen Fibroblasten (TREIBGASEN) und menschliche nabelader Endothelzellen (Mediumwechsel) zu isolieren und verwendet, um eine Zellsuspension mit 70 % iPSC-CMs, 15 % TREIBGASEN und 15 % Mediumwechsel generieren. Sie sind in eine extrem niedrige Befestigungssystem "hängenden Tropfen", die Mikroporen für Hunderte von Sphäroide gleichzeitig kondensiert enthält Co kultiviert. Die Zellen aggregieren und spontan schlagende Sphäroide nach 3 Tagen der kokultur bilden. Die Sphäroide werden geerntet, in einen Roman Formhohlraum entkernt und auf einen Shaker im Brutschrank kultiviert. Die Sphäroide werden Reife Funktionsgewebe ca. 7 Tage nach der Aussaat. Die daraus resultierende mehrschichtige Gewebe bestehen aus verschmolzenen Sphäroide mit zufriedenstellenden Strukturintegrität und synchron schlagen Verhalten. Diese neue Methode hat viel versprechende Potenzial als eine reproduzierbare und kostengünstige Methode, um technische Gewebe für die Behandlung der Herzinsuffizienz in der Zukunft zu schaffen.

In unseren Experimenten haben wir eine Zellsuspension 70 % iPSC-CMs, 15 % TREIBGASEN und 15 % Mediumwechsel in RPMI/B-27 Zelle Medien in einer Konzentration von 2.475.000 Zellen pro mL verwendet. Nach dem Erstellen der Zellsuspension, verzichtet wir 4 mL Zellsuspension in jede Vertiefung der Dateianhang Ultra low Drop Aufhängesystem, wie unter Punkt 4.3 des Protokolls beschrieben. Die Verwendung des Behangs fundsystem führte die spontane Bildung von Hunderten Sphäroide nach 3 Tagen der...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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

Eine Net Schimmel basierende Methode der dreidimensionalen Gerüst-freie Herzgewebe Schöpfung

Dieses Protokoll beschreibt eine net Schimmel basierende Methode um dreidimensionale Gerüst-freie kardialen Gewebe mit zufriedenstellenden Strukturintegrität und synchron schlagen Verhalten zu erstellen.

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

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Research

JoVE Journal

Bioengineering

8.8K Aufrufe.  The First Hospital of Jilin University. Dieses Protokoll beschreibt eine net Schimmel basierende Methode um dreidimensionale Gerüst-freie kardialen Gewebe mit zufriedenstellenden Strukturintegrität und synchron schlagen Verhalten zu erstellen.

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

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kapillar-Force-Lithographie für Herz-Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Forschung

Biotechnik

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Generation Ausgerichtet Functional Myokardgewebe Durch Mikrokontaktdrucken

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Tissue Engineering: Aufbau eines Multizelluläre 3D Gerüst für die Lieferung von Layered Zellschichten

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal Cardiac Scaffolds: Neuartige Matrices für Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Schaffung von Herzgewebe, die mechanische Integration von Sphäroiden mit 3D-Bioprinting ermöglicht

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

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.

Herstellung von 3D-Cardiac Microtissue Arrays unter Verwendung von humanen iPSC-abgeleiteten Kardiomyozyten, kardialen Fibroblasten und Endothelzellen

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

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

Schrittweise Zellaussaat auf tessellierten Gerüsten zur Untersuchung keimender Blutgefäße

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

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 ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

Erstellung patientenspezifischer Silikon-Herzmodelle mit Anwendungen in präoperativen Plänen und Hands-on-Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...