Name: cardiac muscle cell-based actuator and self-stabilizing biorobot - part 2
Uploaded: 2017-05-09T15:00:00
Description: Watch this Scientific Journal Video about Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 at JoVE.com

M. T. Holley is supported by the Graduate Fellows program of the Louisiana Board of Regents, and C. Danielson is supported by the Howard Hughes Medical Institute Professors Program. This study is supported by NSF Grant No: 1530884.

All procedures described here have been carried out using an approved protocol and in accordance with the regulations of the Institutional Animal Care and Use Committee of the University of Notre Dame.
1. Cell Seeding and Culture
<ol>
	<li>Before starting, prepare the required items: a small funnel, pipettes, and warm Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin antibiotic (DMEM complete).</li>
	<li>Take T-25 flasks along with t...

<ol>
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NeuroEng Rehabil. 1, 6 (2004).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototactic+guidance+of+a+tissue-engineered+soft-robotic+ray.">Phototactic guidance of a tissue-engineered soft-robotic ray.</a> Science. 353 (6295), 158-162 (2016).</li><li>Cvetkovic, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensionally+printed+biological+machines+powered+by+skeletal+muscle.">Three-dimensionally printed biological machines powered by skeletal muscle.</a> Proc. Natl. Acad. Sci. 111, 10125-10130 (2014).</li><li>Williams, B. J., Anand, S. V., Rajagopalan, J., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-propelled+biohybrid+swimmer+at+low+Reynolds+number.">A self-propelled biohybrid swimmer at low Reynolds number.</a> Nat. Commun. 5, (2014).</li><li>Holley, M. T., Nagarajan, N., Danielson, C., Zorlutuna, P., Park, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+muscle-based+actuators+for+self-stabilizing+swimming+biorobots.">Development and characterization of muscle-based actuators for self-stabilizing swimming biorobots.</a> Lab. Chip. 16, 3473-3484 (2016).</li><li>Hopkins, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+physiology.">Skeletal muscle physiology.</a> Contin Educ Anaesth Crit Care Pain. 6, 1-6 (2006).</li><li>Nawroth, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-engineered+jellyfish+with+biomimetic+propulsion.">A tissue-engineered jellyfish with biomimetic propulsion.</a> Nat Biotechnol. 30 (8), 729-797 (2012).</li><li>Ehler, E., Moore-Morris, T., Lange, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Neonatal+Mouse+Cardiomyocytes.">Isolation and Culture of Neonatal Mouse Cardiomyocytes.</a> J. Vis. Exp. JoVE. (79), e50154 (2013).</li><li>Bers, D. 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The procedure outlined here describes a successful seeding method for PDMS-based actuators and biorobots, which facilitates the attachment of cardiomyocytes. Furthermore, the process of image acquisition and the subsequent analysis that characterizes the behavior of the cells and the performance of the devices was described.
We observed spontaneous contraction of cells on the cantilever arms after 24 h; the intensity of contractions continued to increase steadily over time and reached a maximu...

Biorobots are devices based on living cells that are incorporated within a mechanical backbone that is usually composed of soft, elastic materials, such as PDMS or hydrogels1. The cells undergo rhythmic contractions, either spontaneously or in response to stimuli, and thus function as an actuator. The power generated from cell contraction drives various biorobots. Mammalian heart cells (cardiomyocytes) and skeletal muscle cells are often used for biorobot actuation due to their contractile properties. Aside from cardiomyocyte and skeletal muscle cells, other cell types, such as insect muscle tissues2 and explanted muscle tissues3, have been used. Insect muscle tissues enable the operation of biological actuators at room temperature.
The function and performance of a biorobot are chiefly determined by the strength and consistency of the biological actuator (i.e.&#160;muscle cells), while the mechanical backbone structure primarily determines the mechanisms of locomotion, stability, and power. Since these devices are solely driven by forces generated by cells, there are no chemical pollutants or operating noises. Therefore, they form an energy-efficient alternative to other conventional robots. Various literature sources have discussed the different methods to integrate living cells and tissues into biorobots1,4,5. Advances in microfabrication and tissue engineering techniques have enabled the development of biorobots that can walk, grip, swim, or pump5,6. In general, cells are cultured directly onto the mechanical (polymeric) backbone as a confluent cell sheet or they are molded into 3-dimensional actuating structures within scaffolds such as rings and strips. Most often, biorobots are fabricated using cardiomyocyte sheets6,7, as these cells have an innate ability to exhibit spontaneous contraction without external stimuli. On the other hand, reports on skeletal muscle cell sheets are limited due to their need for stimuli to initiate contractions in vitro in order to initiate membrane depolarization8.
This protocol first describes how to seed cardiomyocytes on a functionalized biological actuator made of a thin PDMS cantilever. It then describes in detail the seeding and analysis of the swimming profiles. The cantilever is functionalized with a cell adhesive protein such as fibronectin and is seeded confluently with cardiomyocytes. As the cells seeded on the device contract, they cause the cantilever to bend and thus to act as an actuator. Over time, as the cells mature, we trace the changes in surface stress on the device by analyzing videos of the cantilever bending. The biological actuator developed here can be used to determine the contractile properties of any cell type, such as the fibroblasts or induced pluripotent stems cells, as they undergo differentiation.
Much of the earlier research on biorobots has been focused on developing biological actuators, while optimization of the biorobot architecture and functional capabilities were largely neglected. Recently, a few studies have demonstrated the implementation of swimming modes in biorobots that are inspired by nature. For example, swimming biorobots with flagella-based motion6, jellyfish propulsion9, and bio-hybrid rays4 have been engineered. Unlike other works in literature, here we focus on varying the properties of the mechanical backbone to create a self-stabilizing structure. The biorobot developed in this study is capable of maintaining a constant pitch, roll, and immersion depth as it swims. These parameters can be modified by varying the thickness of each base composite. The fabrication steps involved in developing the PDMS actuator, the submergible biorobot, and the functionalization of the device are described in detail in Part 1 of this two-part article, as well as in our recent work7.The technique developed here can pave the way for the development of novel, highly efficient biorobots for various applications, such as cargo delivery.
The isolation process followed in this study is similar to the process described in an earlier work10, as well as in recently published work7. The microfabrication methods used for fabricating the PDMS actuators and biorobot devices are described in detail in Part 1 of this two-part manuscript. The protocol section of this manuscript describes the steps involved in seeding cardiomyocytes onto the fabricated PDMS actuator and the biorobot following their functionalization with cell adhesive proteins.

<table>
	<tbody>
		<tr>
			<td>Chemicals and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiomyocytes (primary cardiac cells)</td>
			<td>Charles River</td>
			<td>NA</td>
			<td>Isolated from 2-day old neonatal Sprague Dawley rats</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle&#8217;s media (DMEM)</td>
			<td>Hyclone Laboratories</td>
			<td>16750-074</td>
			<td>with 4500 mg/L glucose, 4.0 mM L-glutamine, and 110 mg/L sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetalclone III serum</td>
			<td>Hyclone industries, GE</td>
			<td>16777-240</td>
			<td>Fetal bovin serum (FBS)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffer (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole dihydrocholride powder (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Solution (1 mg/ml)</td>
		</tr>
		<tr>
			<td>Calcein-AM and ethidium homodimer-1 kit (Live/Dead Assay)</td>
			<td>Molecular Probes</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Fluo-4, AM</td>
			<td>Molecular Probes</td>
			<td>F14217</td>
			<td>calcium indicator dye</td>
		</tr>
		<tr>
			<td>Tyrodes salt solution</td>
			<td>Sigma-Aldrich</td>
			<td>T2397</td>
			<td>buffer solution</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Molecular Probes</td>
			<td>P3000MP</td>
			<td>nonionic surfactant-20 % solution in Dimethylsiloxane (DMSO)</td>
		</tr>
		<tr>
			<td>16% Parafomaldehyde</td>
			<td>Electron microscopy</td>
			<td>15710</td>
			<td>Caution: Irritant and combustible</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100 100 mL</td>
			<td>cell lyses detergent, (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether)</td>
		</tr>
		<tr>
			<td>ProLong gold antifade reagent</td>
			<td>Molecular Probes</td>
			<td>P10144</td>
			<td>Mounting agent</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Phalloidin</td>
			<td>Molecular Probes</td>
			<td>A12381</td>
			<td>Actin filament marker</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 594 conjugate</td>
			<td>Molecular Probes</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>pha</td>
			<td>Molecular Probes</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-connexin 43 antibody</td>
			<td>Abcam</td>
			<td>ab11370</td>
			<td>Gap junction marker</td>
		</tr>
		<tr>
			<td>Anti-cardiac troponin I antibody</td>
			<td>Abcam</td>
			<td>ab10231</td>
			<td>Contractile protein</td>
		</tr>
		<tr>
			<td>16% EM grade paraformaldehyde solution</td>
			<td>Electron microscopy</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Elsevier</td>
			<td></td>
			<td>Sylgard 184</td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Thor Labs</td>
			<td>DCC1545M</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light strip</td>
			<td>NA</td>
			<td>NA</td>
			<td>Any white LED without spectrum emission</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikkon C2</td>
			<td>NA</td>
			<td>Confocal microscope with three filter set.</td>
		</tr>
		<tr>
			<td>Zooming lens</td>
			<td>Infinity</td>
			<td>Model# 252120</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>NA</td>
			<td>Used in Section 4) for biological actuator analysis.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institute of Health</td>
			<td>NA</td>
			<td>Java-based image processing software. Used in Section 5) for biorobot analysis. 
			Free Image Processing and Analysis software in java. (https://imagej.nih.gov/ij/)</td>
		</tr>
		<tr>
			<td>Thor Cam</td>
			<td>Thor Labs</td>
			<td>NA</td>
			<td>Camera operating software</td>
		</tr>
	</tbody>
</table>

cardiac muscle cell-based actuator and self-stabilizing biorobot - part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

The biological actuator made of a thin PDMS cantilever (25 &#181;m in thickness) and cardiomyocytes constitutes the core of the swimming biorobot, as shown in the schematic and screenshot of the devices in Figure 1. The cells start to exhibit contractions after 24 h in culture, and bending of the cantilever arms was observed by day 2. The side profile of the device was recorded every day, and the surface stress was quantified from the bending of the cantilever arms using ...

Watch this Scientific Journal Video about Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 at JoVE.com

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

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