The authors acknowledge Dr. Timothy Cashman for previous work on this method. This study was supported by funding from the National Institutes of Health (NIH) (R01-HL132226 and K01 HL133424) and the Leducq Foundation International Networks of Excellence Program (CURE-PLaN).

K.D.C. is a co-founder and Chief Scientific Officer of Novoheart and holds equity ownership in the holding company, Medera Biopharmaceutical. Novoheart did not contribute to the funding, planning, or execution of this study; however, the study outcomes could potentially have a financial impact on Novoheart and Medera. The other authors declare that they have no competing interests.

There are numerous linear engineered cardiac tissue models published in the literature, some of which are described in Table 1. Some models involve the direct measurement of the tissue force, but these typically require transferring the construct to a separate muscle bath38. Most models are designed with the tissues permanently anchored at both ends, most commonly to PDMS posts1,2,3,...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64368">Designing a Bioreactor to Improve Data Acquisition and Model the Throughput of Engineered Cardiac Tissues</a>. The title was corrected&#160;from:
Designing a Bioreactor to Improve Data Acquisition and Model the Throughput of Engineered Cardiac Tissues
to:
Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64368">Designing a Bioreactor to Improve Data Acquisition and Model the Throughput of Engineered Cardiac Tissues</a>. The title was corrected.

Erratum: Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Engineered cardiac tissue models come in a diverse array of geometries and configurations for recapitulating various aspects of the native cardiac niche that are difficult to attain with traditional two-dimensional cell culture. One of the most common configurations is the linear tissue strip, with flexible anchors at each end to induce tissue self-assembly and providing the tissue with a defined preload and a readout of the resulting twitch forces1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 
,22,23,24,25,26,27. The force generated can be robustly determined through the optical tracking of the tissue shortening and using elastic beam theory to calculate the force from the measured deflections and the spring constant of the anchors1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 
21,22,25,26,28.
However, cardiac tissue engineering is still an evolving field, and some challenges remain. Specialized equipment, such as custom-made bioreactors and functional assessment devices, are required for each model system10,29,30,31. The size and complexity of the microenvironment of these constructs are often limited by low throughput due to labor-intensive protocols, high numbers of cells, and tissue fragility. To address this, some groups have turned to the fabrication of microtissues containing only hundreds or thousands of cells to facilitate high-throughput assays that are useful for drug discovery. However, this reduced scale complicates the accurate assessment of function12, eliminates key aspects of the native cardiac niche (such as nutrient/oxygen diffusion gradients and complex architecture36), and limits the amount of material available for subsequent molecular and structural analysis (often requiring pooling of the tissues). Table 1 summarizes some of the configurations of linear tissue strip models in the literature1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 
21,22,23,24,25,26,37,38,39,40.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Group</td>
			<td>Cells per tissue</td>
			<td>Tissues per plate</td>
			<td colspan="2">Plate format</td>
			<td colspan="2">Anchoring feature</td>
			<td colspan="2">Functional data acquisition method</td>
			<td>Shared media bath?</td>
			<td>Functional measure- 
			ment in situ?</td>
		</tr>
		<tr>
			<td colspan="2">Yoshida (ECT)38</td>
			<td>4 million</td>
			<td>6</td>
			<td colspan="2">modified 6-well plate*</td>
			<td colspan="2">force transducer</td>
			<td colspan="2">direct force measurement</td>
			<td>no</td>
			<td>no</td>
		</tr>
		<tr>
			<td colspan="2">Chan (hESC-CM-ECTs)26</td>
			<td>310 k</td>
			<td>6</td>
			<td colspan="2">custom 6-well dish&#160;</td>
			<td colspan="2">PDMS posts</td>
			<td colspan="2">direct force measurement</td>
			<td>yes</td>
			<td>no</td>
		</tr>
		<tr>
			<td colspan="2">Feinberg (dyn-EHT)16</td>
			<td>1.5 million</td>
			<td>6</td>
			<td colspan="2">custom 6-well dish&#160;</td>
			<td colspan="2">PDMS wire</td>
			<td colspan="2">tissue shape</td>
			<td>no</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">RADISIC (BioWire)39, 40</td>
			<td>110 k</td>
			<td>8</td>
			<td colspan="2"></td>
			<td colspan="2">polymer wire</td>
			<td colspan="2">wire shape</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Costa (single hECT)1, 2</td>
			<td>1-2million</td>
			<td>4**</td>
			<td colspan="2">10 cm Petri dish**</td>
			<td colspan="2">PDMS posts</td>
			<td colspan="2">optical deflection (edge/object tracking)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Costa (multi-hECT)3&#8211;9</td>
			<td>500 k-1 million</td>
			<td>6</td>
			<td colspan="2">6 cm Petri dish</td>
			<td colspan="2">PDMS posts</td>
			<td colspan="2">optical deflection (edge/object tracking)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Costa (multi-hECT W/ SPoT)</td>
			<td>1 million</td>
			<td>6</td>
			<td colspan="2">6 cm Petri dish</td>
			<td colspan="2">PDMS posts with black caps</td>
			<td colspan="2">optical deflection (object tracking)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Passier (EHT)17</td>
			<td>245 k</td>
			<td>36</td>
			<td colspan="2">12-well plate</td>
			<td colspan="2">PDMS posts with black caps</td>
			<td colspan="2">optical deflection (object tracking)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Vunjak-Novakovic13, 18</td>
			<td>1 million</td>
			<td>12</td>
			<td colspan="2">6 cm Petri dish</td>
			<td colspan="2">PDMS posts with caps</td>
			<td colspan="2">optical deflection (edge detection)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Vunjak-Novakovic (MilliPillar)14</td>
			<td>550 k</td>
			<td>6</td>
			<td colspan="2">custom 6-well dish&#160;</td>
			<td colspan="2">PDMS posts with caps</td>
			<td colspan="2">optical deflection (object tracking); calcium imaging</td>
			<td>no</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Eschenhagen (EHT)10, 19&#8211;21</td>
			<td>1 million</td>
			<td>12</td>
			<td colspan="2">12-well plate</td>
			<td colspan="2">PDMS posts with caps</td>
			<td colspan="2">optical deflection (edge detection of post deflection); calcium imaging</td>
			<td>no</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Zandstra (CaMiRi)22</td>
			<td>25-150 k</td>
			<td>96</td>
			<td colspan="2">96-well plate</td>
			<td colspan="2">PDMS posts with hooks</td>
			<td colspan="2">optical deflection (edge detection)</td>
			<td>no</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Murry23, 24</td>
			<td>900 k</td>
			<td>24</td>
			<td colspan="2">24-well plate</td>
			<td colspan="2">PDMS posts with caps, integrated magnet</td>
			<td colspan="2">magnetic sensor</td>
			<td>no</td>
			<td>yes</td>
		</tr>
		<tr>
			<td colspan="2">Reich (&#181;TUG)11, 12, 25</td>
			<td>undefined</td>
			<td>156</td>
			<td colspan="2">156-well dish</td>
			<td colspan="2">PDMS posts with caps, integrated magnet</td>
			<td colspan="2">optical tracking (fluorescent bead)</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of some linear engineered cardiac tissue models in the literature. Linear engineered cardiac tissue models vary in size, throughput, anchoring feature designs, and the facilitation of shared medium baths, as well as the requirements for a separate muscle bath system for functional characterization. * The researchers used a commercially available engineered tissue system based on the dimensions of a standard 6-well plate.&#160;** A modular system in which single-tissue bioreactors are anchored to any plastic culture dish in the desired number and location.
This paper describes the latest protocol for fabricating our established model of linear human engineered cardiac tissue (hECT)1,2,3,4,5,6,7,8,9,15,27 and methods for assessing hECT contractile function. Each multi-tissue bioreactor accommodates up to six hECTs in a shared medium bath and is composed two &#34;rack&#34; pieces made of the silicone elastomer polydimethylsiloxane (PDMS) mounted on a rigid polysulfone frame. Each PDMS rack contains six&#160;flexible&#160;integrated force-sensing posts that are 0.5 mm in diameter and 3.25 mm long, and together, two racks provide six pairs of posts, each of which holds one hECT. Inversion of the bioreactor helps overcome any hindrance to the visualization of the hECTs from below due to water condensation from the culture medium or distortions from the meniscus of the air-liquid interface. Each contraction of an hECT causes deflection of the integrated end-posts, and the optical measurement of the deflection signal is processed into a force versus time tracing representing the contractile function of the hECT1,2,3,4,5,6,7,8,9,15,27. Compared to the single-tissue bioreactors typically used for tissues of this size, the multi-tissue design improves the experimental throughput and enables the study of paracrine signaling between adjacent tissues of potentially different cellular composition. This system has been validated in published studies describing applications in disease modeling4,8, paracrine signaling6,7, heterocellular culture5,9, and therapeutic screening7,9.
In this system, the hECTs are designed to be approximately 6 mm long and 0.5 mm in diameter to facilitate robust optical tracking of force measurements with low noise. Furthermore, aspects of tissue complexity such as diffusion gradients and cellular organization are balanced with a manageable requirement of 1 million cells per tissue. With standard CCD camera technology, forces as weak as 1 &#181;N (representing less than 5 &#181;m post deflection) generate a clear signal, ensuring that even extremely weak contractile function, as observed with some hECT disease models, can be accurately measured. This also facilitates the detailed analysis of the twitch force curve, thus enabling the high-content analysis of up to 16 contractility metrics41, including developed force, rates of contraction (+dF/dt) and relaxation (&#8722;dF/dt), and beat rate variability.
This protocol begins with instructions for fabricating the bioreactor components. Special attention is paid to the steps to maximize the hECT yield, reduce technical variability in the tissue function, and optimize the quality and depth of the tissue assessment. Most cardiac tissue engineering studies do not report rates of tissue loss during fabrication and long-term testing, although it is a well-known challenge in the field and reduces the throughput and efficiency of the studies27. The tissue engineering methods described here have been refined over the years to ensure retention of all hECTs in most of the bioreactors (regardless of how the PDMS racks are fabricated). However, even a 5%-20% loss of tissues can significantly affect the statistical power, particularly in smaller experiments limited by the number of cardiomyocytes available (e.g., due to differentiation challenges with some diseased cell lines4 or due to the high cost of commercially purchased cardiomyocytes), or by the treatment condition (e.g., limited availability or high cost of various treatment compounds).
This protocol describes the fabrication of stable post trackers (SPoTs), a new feature of the PDMS racks, which function as caps at the ends of the force-sensing posts that hold the hECTs27. It is demonstrated how the cap geometry significantly reduces the hECT loss from falling or pulling off the posts, thus opening new opportunities for culturing hECTs with a greater variety of stiffnesses and tensions, which are challenging to culture on uncapped posts. Additionally, the SPoTs provide a high-contrast object to improve the optical tracking of the hECT contraction through a consistent and well-defined shape27. This is followed by a description of culturing human induced pluripotent stem cells (iPSCs) and cardiomyocyte differentiation based on prior published protocols3,42,43 and an explanation of hECT fabrication, culture, and functional measurements.
This article also addresses the need to measure tissue function at physiological temperature. Human myocardium (fetal as well as adult healthy and diseased tissue), as well as heart tissue from a wide range of animal species (including rats, cats, mice, ferrets, and rabbits)44,45, displays a marked increase in the frequency-matched twitch force at temperatures of 28 &#176;C-32 &#176;C compared to physiological temperature-a phenomenon known as hypothermic inotropy45,46. However, the effects of temperature on engineered myocardial tissue function remain understudied. Many recent engineered cardiac tissue models in the literature are designed to be functionally assessed at 37 &#176;C to approximate physiological conditions13,14,37. However, to our knowledge, the temperature-dependent effects on the force generated by engineered cardiac tissues have not been systematically investigated. This protocol describes a pacing electrode design that minimizes heat loss during testing, as well as allowing for the incorporation of an insulated heating element into the setup for functional measurements, which can maintain the hECTs at physiological temperature without compromising sterility27. We then report some of the observed effects of temperature on hECT function, including on the developed force, spontaneous beating frequency, +dF/dt, and &#8722;dF/dt. Altogether, this paper provides the details required to manufacture this multi-tissue force-sensing bioreactor system to fabricate human engineered cardiac tissues and to assess their contractile function, and a set of data is presented that provides a basis for comparison for measurements at room temperature and at 37 &#176;C27.

<table><tbody><tr><td>0.25 mm diamete 304 Stainless Steel Wire</td><td>McMaster Carr</td><td>6517K61&amp;nbsp;</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>1.7 mL Microtubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10 cm dishes (20 mm tall)</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>Drummond</td><td>6-000-010</td><td></td></tr><tr><td>10 N NaOH</td><td>Fisher Scientific</td><td>SS225-1</td><td>dilute 1:10 in sterile distilled water</td></tr><tr><td>10X Modified Eagle Medium</td><td>Sigma Aldrich</td><td>M0275</td><td></td></tr><tr><td>20 - 200 &amp;mu;L Micropipette</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>200 &amp;mu;L MicroPipette Tips</td><td>VWR</td><td>76322-150</td><td></td></tr><tr><td>5 mL Serological Pipette</td><td>Drummond</td><td>6-000-005</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>6 cm Petri Dish</td><td>Corning</td><td>353002</td><td></td></tr><tr><td>6 Watt LED Dual Gooseneck Illuminator</td><td>AmScope</td><td>&amp;nbsp;LED-6W&amp;nbsp;</td><td></td></tr><tr><td>6-Well Plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>90 degree angle mirror</td><td>Edmund Optics</td><td>45-594</td><td></td></tr><tr><td>Acrylic bonding glue</td><td>SCIGRIP</td><td>#4</td><td></td></tr><tr><td>Adjustable 10 cm x 10 cm jack</td><td>Fisher Scientific</td><td>14-673-50</td><td></td></tr><tr><td>Aluminum 6061</td><td>McMaster Carr</td><td>9008K82</td><td></td></tr><tr><td>A-Plan 10X Objective Lens</td><td>ZEISS</td><td>1020-863</td><td></td></tr><tr><td>Autoclave Bags</td><td>Propper</td><td>21002</td><td></td></tr><tr><td>B-27 supplement</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>B-27 supplement (without insulin)</td><td>ThermoFisher</td><td>A1895601</td><td></td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Black ABS</td><td>Ultimaker</td><td></td><td>2.85 mm wide</td></tr><tr><td>Bovine Collagen I</td><td>Gibco</td><td>A1064401</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>Class II Biosafety Cabinet</td><td>Labconco</td><td>3430009</td><td></td></tr><tr><td>Clear Acrylic Sheeting</td><td>estreetplastics</td><td>1002502436</td><td>6.25 mm thick</td></tr><tr><td>CNC Vertical Mill</td><td>Haas</td><td>VF-1</td><td></td></tr><tr><td>Conductive Graphite Bars</td><td>McMaster Carr</td><td>1763T33</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Ham&#039;s F-12 Nutrient Mix</td><td>ThermoFisher</td><td>11330032</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>A4094</td><td>Dilute to 70% in water</td></tr><tr><td>EVE Automated Cell counter</td><td>NanoEntek</td><td>E1000</td><td></td></tr><tr><td>EVE Cell Counting Slide</td><td>NanoEntek</td><td>EVS-050</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10438026</td><td></td></tr><tr><td>Fine Curved Forceps</td><td>Fine Science Tools</td><td>11253-25</td><td></td></tr><tr><td>Forma Series II Water Jacketed CO2 Incubator</td><td>Thermo Electron Corporation</td><td>3110</td><td>AKA &quot;incubator&quot;. With HEPA class 100 filter</td></tr><tr><td>Fusion360 software</td><td>Autodesk</td><td></td><td>AKA &quot;CAD software&quot;</td></tr><tr><td>Glass Hemocytometer</td><td>Reichert</td><td>1475</td><td>0.1 mm deep</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3784</td><td></td></tr><tr><td>hESC qualified matrigel</td><td>Corning</td><td>354277</td><td>AKA &quot;basement membrane matrix&quot;. Store in frozen aliquots</td></tr><tr><td>High Speed CCD Camera</td><td>PixelLINK</td><td>P7410</td><td></td></tr><tr><td>Inverted Microscope</td><td>Carl Zeiss Werk</td><td>Axiovert 40 CFL</td><td>10X phase contrast objective</td></tr><tr><td>IWR-1</td><td>Selleck Chem</td><td>S7086</td><td></td></tr><tr><td>LabView Software</td><td>National Instruments</td><td>2016</td><td></td></tr><tr><td>Laminar flow clean bench</td><td>NuAire</td><td>NU-201-330</td><td>necessary for hECT functional analysis</td></tr><tr><td>Laptop</td><td>AsusTek</td><td>Strix</td><td>Intel Core i&amp;amp; processor ,CPU 2.8GHz, 16GB RAM</td></tr><tr><td>Laser Cutting Machine</td><td>Epilog</td><td>Helix 24</td><td></td></tr><tr><td>Magnification headset</td><td>ExcelBlades</td><td>70020</td><td>Recommended for steps requiring fine manipulations</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>Version 2019b or later</td><td>AKA &quot;data analysis software&quot;</td></tr><tr><td>Micro Vannas Scissors, 3 mm blade</td><td>WPI Instruments</td><td>501839</td><td></td></tr><tr><td>Microscope Boom Stand</td><td>Olympus</td><td>SZ2-STU1</td><td></td></tr><tr><td>Penicillin-Streptomycin stock solution</td><td>ThermoFisher</td><td>15140122</td><td>10,000 IU/ml penicillin; 10,000 &amp;mu;g/ml streptomycin</td></tr><tr><td>Phosphate-buffered saline without divalent cations</td><td>Sigma Aldrich</td><td>P3813</td><td>Diluted in distilled water to 1X and 10X concentrations</td></tr><tr><td>Pipette Controller</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>PixelLINK Capture OEM</td><td>PixelLINK</td><td>10.2.1.6</td><td>AKA &quot;Camera Software&quot;</td></tr><tr><td>Polysulfone</td><td>McMaster Carr</td><td>86735K73</td><td>translucent amber color</td></tr><tr><td>Polytetrafluoroethylene (PTFE)</td><td>McMaster Carr</td><td>8545K176&amp;nbsp;</td><td>Black, molded</td></tr><tr><td>ReLeSR</td><td>Stem Cell Technologies</td><td>5872</td><td>AKA &quot;iPSC dissociation media&quot;</td></tr><tr><td>Rosewell Park Memorial Institute 1640 Media</td><td>ThermoFisher</td><td>11875135</td><td></td></tr><tr><td>Silicone Sheeting</td><td>SMI manufacturing</td><td></td><td>glossy, 0.02 in thickness, durometer 40</td></tr><tr><td>Size 10/0 Blue, Green, Red, and Yellow Glass Seed Beads</td><td>Michael&#039;s</td><td></td><td>color should withstand autoclaving</td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>14-373</td><td>used for mixing PDMS</td></tr><tr><td>Square Pulse Stimulator&amp;nbsp;</td><td>Astro-Med / Grass Technologies</td><td>S88X</td><td></td></tr><tr><td>Stainless Steel Razoblades</td><td>GEM</td><td>62-0179-CTN</td><td>preferred over non-stainless steel due to lower hardness</td></tr><tr><td>Stemflex</td><td>ThermoFisher</td><td>A3349401</td><td>AKA &quot;iPSC culture media&quot;</td></tr><tr><td>Sterile distilled water</td><td>ThermoFisher</td><td>5230</td><td></td></tr><tr><td>Sylgard 170 -&amp;nbsp; Silicone Elastomer Encapsulant Black 0.9 kg Kit</td><td>Dow</td><td>DOWSIL 170 2LB KIT</td><td>AKA black Polydimethylsiloxane (black PDMS)</td></tr><tr><td>Sylgard 184 - Silicone Elastomer Clear 1 lb Kit</td><td>Dow</td><td>DC 184 SYLGARD 0.5KG 1.1LB KIT</td><td>AKA Polydimethylsiloxane (PDMS)</td></tr><tr><td>Temperature-controlled heated stage</td><td>Okolab</td><td>H401-HG-SMU</td><td>Set height to 10 cm</td></tr><tr><td>Thermoplastic 3D printer</td><td>Ultimaker</td><td>Ultimaker 3</td><td></td></tr><tr><td>Thiazovivin</td><td>Selleck Chem</td><td>S1459</td><td></td></tr><tr><td>Trypan Blue</td><td>NanoEntek</td><td>EBT-001</td><td></td></tr><tr><td>Vacuum Chamber</td><td>Bel-Art Parts</td><td>F42027-0000</td><td></td></tr><tr><td>Variable Speed Mini Band Saw</td><td>Micro-Mark</td><td>82203</td><td></td></tr><tr><td>Variable Speed Miniature Drill Press</td><td>Micro-Mark</td><td>82959</td><td></td></tr><tr><td>Vibration Isolation Table</td><td>Labconco</td><td>3618000</td><td></td></tr><tr><td>Weighing Boats</td><td>VWR</td><td>10803-140</td><td></td></tr><tr><td>Talon Cylinder Bench Clamp</td><td>VWR</td><td>97035-528</td><td>AKA screw clamp</td></tr></tbody></table>

designing a bioreactor to improve data acquisition and model throughput of engineered cardiac tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Following the above protocol, cardiomyocytes were generated from a healthy iPSC line used previously by our group9,15 and fabricated into hECTs after 8-61 days in culture. Figure 9A shows representative images of hECTs as viewed from the bottom, which were created without (top) and with (bottom) SPoTs. Functional measurements were taken at room temperature (23 &#176;C) and at physiological temperature (36 &#176;C) between 37 days and...

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Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

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JoVE Journal

Bioengineering

2.3K Views.  Icahn School of Medicine at Mount Sinai. Ons in-vitromodel van driedimensionale stamcelafgeleide menselijke hartspier verbetert de biotrouw van traditionele tweedimensionale celkweek en elimineert de verschillen tussen soorten die gepaard gaan met proefdierproeven. Ons multi-weefsel bioreactorontwerp met stabiele posttrackers maximaliseert het weefselsucces en verbetert de nauwkeurigheid van diepgaande karakterisering van de gemanipuleerde hartweefselfunctie. Aangezien hartfalen wereldwijd nog steeds de belangrijkste doodsoorzaak is...

Video: Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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.

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

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.

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

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

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.

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

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

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

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

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.

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

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

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