Name: designing a bioreactor to improve data acquisition and model throughput of engineered cardiac tissues
Uploaded: 2023-06-02T12:50:00
Description: Watch this Scientific Journal Video about Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues at JoVE.com

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).

This protocol used a de-identified iPSC line, SkiPS 31.3 (originally reprogrammed using dermal fibroblasts from a healthy 45 year old male)47, and was, thus, exempt from specific Institutional Review Board approval, in concordance with the institution&#39;s human research ethics committee guidelines. Perform all the cell and hECT manipulation in aseptic conditions in a HEPA-filtered class II biological safety cabinet or laminar flow work bench. Sterilize all the non-sterile solutions by filtration...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.25 mm diamete 304 Stainless Steel Wire</td>
			<td >McMaster Carr</td>
			<td >6517K61&#160;</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 &#956;L Micropipette</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td></td>
		</tr>
		<tr >
			<td >200 &#956;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>&#160;LED-6W&#160;</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&#39;s Modified Eagle Medium/Ham&#39;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 &#34;incubator&#34;. With HEPA class 100 filter</td>
		</tr>
		<tr >
			<td >Fusion360 software</td>
			<td>Autodesk</td>
			<td></td>
			<td>AKA &#34;CAD software&#34;</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 &#34;basement membrane matrix&#34;. 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&#38; 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 &#34;data analysis software&#34;</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 &#956;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 &#34;Camera Software&#34;</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&#160;</td>
			<td>Black, molded</td>
		</tr>
		<tr >
			<td >ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td>AKA &#34;iPSC dissociation media&#34;</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&#39;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&#160;</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 &#34;iPSC culture media&#34;</td>
		</tr>
		<tr >
			<td >Sterile distilled water</td>
			<td>ThermoFisher</td>
			<td>5230</td>
			<td></td>
		</tr>
		<tr >
			<td >Sylgard 170 -&#160; 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...

Watch this Scientific Journal Video about Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues at JoVE.com

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 ...

Our invitro model of three-dimensional stem cell derived human heart muscle improves the biofidelity of traditional two-dimensional cell culture while eliminating the interspecies differences associated with laboratory animal testing. Our multi-tissue bioreactor design with stable post trackers maximizes tissue success and improves the accuracy of in-depth characterization of engineered cardiac tissue function. As heart failure continues to be the leading cause of death worldwide, our engineered cardiac tissues enable researchers to model human cardiac diseases, as well as screen potential therapies.To begin, position four aluminum negative master casts in the cast holder so that the post holes align with the dead space opposite the triangle shelves. Place the apparatus between two parallel brackets and clamp the sides together using a rectangular piece of 0.5 millimeter thick silicone sheeting as a gasket to prevent leakage of the liquid PDMS. In a shallow container, mix 0.5 milliliters of PDMS curing agent with five milliliters of PDMS elastomer base at a one to 10 ratio and stir vigorously for five minutes.Degas the PDMS mixture in a vacuum chamber under a strong vacuum until the bubbles disappear. Next, pour the PDMS mixture onto the casting apparatus, overfilling to ensure complete coverage of each slot. Add small colored glass beads to the body of the PDMS racks opposite to the side with the posts for the unique identification of each rack.Place the casting apparatus leveled horizontally into the vacuum chamber under a strong vacuum for at least 12 hours. Allow the PDMS to cure at room temperature away from dust for 48 hours to enable complete curing and maximum strength of the delicate posts. Remove the clamp, brackets, and silicone sheeting from the casting apparatus.Using a stainless steel razor blade, trim away the PDMS film on the top of the casting apparatus and frame supports. Use fingers to separate the PDMS racks from the sides of the cast holder. Insert a blunted stainless steel razor blade into the dead space between the cast and the cast holder and pry them apart, ensuring that the PDMS filling the dead space remains with the cast holder.Then, using a sharp stainless steel blade, cut away the remaining PDMS films and the dead space PDMS from the tips of the posts. Using the fingers, slowly separate the PDMS rack from the cast on the side opposite the posts. Continue to alternate sides until the posts are free of the master casts.Free all the PDMS racks and the posts as previously demonstrated, and use a sharp razor blade to trim away any remaining excess PDMS from the racks. Using a thermoplastic fused deposition modeling 3D printer, print the components of the stable post tracker, or SPoT, casting apparatus. Ensure a secure press fit between the 3D printed pieces and between the PDMS racks and the three-pronged jig.Also confirm that the PDMS racks fit snugly with the posts just reaching the bottom of the wells without being bent. Mix 0.5 milliliters of black PDMS part A to 0.5 milliliters of part B in a small weighing boat, or a shallow container until the solution is uniform in color. Degas the mixed black PDMS in a vacuum chamber under a strong vacuum for 20 minutes.Pour the degassed black PDMS onto the 3D printed base to fill the holes and tap to ensure no bubbles remain. Scrape as much excess PDMS off the base as possible. Snap the three-pronged piece onto the base and place the PDMS racks in the grooves on the three-pronged jig, ensuring that the ends of the posts dip into the black PDMS in the circular wells.Allow the black PDMS to cure at room temperature away from dust for 48 hours. Slide out the three-pronged piece, minimizing tension on the posts. Using small forceps, scrape away the thin black PDMS film surrounding each SPoT, then insert fine tipped, bent forceps into the SPoT well to free it from the 3D printed base.Inspect the SPoTs and trim any remaining black PDMS film from the casting process using fine vannas scissors. Ensure that the finished posts are of the correct length by fitting the PDMS racks onto the polys cell phone frame, and then sliding this onto the black base plate. After autoclaving the bioreactor and preparing the cell mixture, proceed to fabricate the hECTs.Wearing sterile gloves, remove the black base plate from the autoclaved bag containing the bioreactor parts and place it in a 60 milliliter dish with the wells facing up. Pipette 44 microliters of the cell mixture into each well slowly to avoid introducing bubbles. Wearing a fresh pair of sterile gloves, remove the polysulfone frame with the PDMS racks from the autoclave bag.Lower the frame onto the base plate such that the ends of the frame fit into the grooves at the end of the base plate. Ensuring that the posts are all straight and the frame is not tilted, place the bioreactor into a 60 millimeter dish. Add one milliliter of 10%FBS in cardiomyocyte maintenance medium to the 60 milliliter dish to increase the humidity as the hECTs solidify.Place the dish without a lid into a high profile 100-millimeter dish and cover it with a 100-millimeter dish lid, then return the bioreactor to a 37 degrees Celsius, 5%carbon dioxide incubator to allow the collagen to form a gel with the cells in suspension. After two hours, remove the dish from the incubator. Add 13 milliliters of 10%FBS in the cardiomyocyte maintenance medium, tilting the dish to allow the medium to flow between the PTFE base plate and the PDMS racks.Inspect the bioreactor from the side for no air bubbles and return the dish to the incubator. If air is trapped, tilt the bioreactor out of the medium to let the bubble break and slowly lower it again, or use a micro pipette with a gel loading tip to siphon the air without disturbing the posts. Inspect the hECT compaction through the window in the frame.Over 24 to 96 hours, the hECTs compact and become opaquer. Remove the base plate when the hECTs are compacted by at least 30%compared to the original diameter. Fill the 60 millimeter dish containing the bioreactor with cardiomyocyte maintenance medium until the liquid flushes with the lip of the dish and add 14 milliliters to a new 60-millimeter dish.While wearing sterile gloves, flip the bioreactor over in its dish so that the base plate is on top. After inspecting for trapped air bubbles, slowly lift the base plate, keeping it level. Ensure the post tips are in focus.Turn on the thresholding switch and adjust the slider until the SPoTs are nicely demarcated and do not change shape as the hECT contracts. Use the rectangle tool to draw a rectangle around one of the SPoTs, and click on the set one button inside the post boundaries box to set the rectangle position around the SPoT, ensuring that the SPoT always remains within the boundary of the rectangle. Repeat the process to the other post and record it under set two.Adjust the object size settings to prevent the program from tracking smaller objects, and ensure that the number of objects tracked in each rectangle remains constant. The interface shows the measured distance between the tracked objects in real time. Use this graph to monitor the noise.Under the pacing frequency hertz header, indicate the range of desired frequencies and the desired interval for stepping from min to max. In the boxes to the right, choose the desired setting time to allow the hECT to adjust to the new pacing frequency, then specify the recording time and pacing voltage. Begin the program by clicking on the start program button.After data from one frequency has been recorded, increase the stimulator frequency by the desired interval for a new recording until the max frequency has been reached. Representative images of hECTs are shown here as viewed from the bottom, created without SPoTs and with SPoTs. The SPoTs provided a single defined shape to track during the data acquisition.In some extreme instances, the tracking object was even obscured. A more reliable shape for optical tracking and noise reduction enabled the measurement of weak tissues with a developed force as low as one micro Newton. The SPoTs provided a cap geometry that prevented the hECT loss.The measured hECT function was stable over extended culture time in the current pacing setup with the heated stage. Compared to measurements at physiological temperature, hECTs showed altered contraction dynamics at room temperature, with slower rates of contraction and relaxation. At higher frequencies, the hECTs tended to be weaker at room temperature.At lower frequencies, the hECTs tended to be stronger at room temperature. When paced at 36 degrees Celsius, the hECTs had a higher spontaneous beat rate and a more expansive range of capturing frequencies. The temperature controlled environment ensures the physiologic relevance of the measured hECTs function.Additionally, the shared media bath of the multi-tissue bioreactor facilitates studies of paracrine signaling between hECTs of different cellular compositions. The addition of SPoTs to our multi tissue bioreactor allows researchers to efficiently study diseased myocardium with abnormally high or low tension, which would otherwise slip off the ends of uncapped posts.

Research

JoVE Journal

Bioengineering

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

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 ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

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 ...

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

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

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

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

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

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 ...