Name: cardiac muscle-cell based actuator and self-stabilizing biorobot - part 1
Uploaded: 2017-07-11T13:00:00
Description: Watch this Scientific Journal Video about Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1 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 Howard Hughes Medical Institute Professors Program. This study is supported by NSF Grant No: 1530884. The authors would like to thank the support of the cleanroom at the Center for Advanced Microstructures and Devices (CAMD).

1. Calculate Mass of PDMS and Additives
<ol>
	<li>Use the following equation to find the mass of PDMS needed for specific heights in the following procedures, 
	M = &#1009;*V = &#1009; * Height*Area&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (1), 
	where &#39;Height&#39; is the height of the layer, &#39;Area&#39; is the area of a container that the PDMS will be cured in, &#39;&#1009;&#39; is the density of the mixture and &#39;V&#39; is the volume. 
	NOTE: Densities for height calculations are...

<ol>
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Various locomotion mechanisms can be found among aquatic swimmers16. The locomotion mechanism of the biorobot in this study uses fin-based locomotion, specifically ostraciiform locomotion. Ostraciiform swimmers propel themselves by wagging a tail (cantilever) and having a rigid body (layered base)16. Fish such as the boxfish and cowfish use this type of locomotion. Ostraciiform swimmers are typically slow and have inefficient body dimensions. Although ostraciiform swimming ...

Biological actuators and biorobots are being actively studied to provide an alternative to conventional robotics for numerous applications. Biorobots that walk5,6,7,8, swim1,2,3,4, pump9,10, or grip11,12,13 have already been developed. Similarly, muscle cells can be incorporated into a 3D rolled PDMS structure14. Often, the biorobot backbones are fabricated using soft lithography techniques with materials such as hydrogels and PDMS (polydimethylsiloxane). These are attractive choices because of their flexibility, biocompatibility, and easily tunable stiffness. Living muscle cells are usually incorporated with these materials to provide force generation through contraction. Mammalian heart muscle cells (cardiomyocytes) and skeletal muscle cells have dominantly been used for actuation. Besides these two, insect muscle tissues have been used to operate biorobots at room temperature3. In this two-part study, cardiomyocytes were chosen because of their spontaneous contraction6.
Much of earlier research on biorobots was focused on developing the biological actuators while optimization of the biorobot architecture and the development of essential functionalities for the biorobots were largely neglected. Recently, a few reports demonstrated the implementation of different swimming modes which were inspired by propulsion modes found in nature. These methods incorporate PDMS films and muscle cells to mimic various natural propulsion methods. For instance, flagella-based propulsion1, biomimetic jellyfish propulsion2, bio-hybrid ray4, and thin film PDMS swimming devices13 have been reported.
In this paper, we present the fabrication process of self-stabilizing swimming biorobots which can maintain immersion depth as well as pitch and roll. The biorobot has a solid base or body, which is propelled by a single cantilever with cardiomyocytes attached to its surface. The cardiomyocytes cause the cantilever to bend in a longitudinal direction when they contract. This form of swimming is classified as ostraciiform swimming. The ability to add additional functionalities on the base is a unique advantage of ostraciiform swimming. For instance, the base can be utilized to provide excess buoyancy to carry additional cargos or control circuitry for cardiomyocyte contraction.
Stability of the biorobot was often overlooked in previous studies of biorobots. In this study, we implemented self-stabilization by designing the base with different composite PDMS materials of varying mass densities. The biorobot thus exhibits resistance to external disturbances and maintains its submersion depth, pitch and roll, unaided. The first layer is microballoon PDMS (MB-PDMS), i.e PDMS mixed with microballoons, which lowers the density of the biorobot, enabling it to float in media. The second layer is the PDMS cantilever, and its thickness is tailored such that force generated by the cardiomyocytes can dramatically bend the cantilever from 45&#176; to 90&#176;. The bottom layer is nickel-PDMS (Ni-PDMS), i.e. PDMS mixed with nickel powder. This layer performs multiple functions. It is magnetic, and therefore allows the biorobot to be anchored at the bottom of the medium, during cell seeding, with a magnet. The nickel mixture is of higher density than the MB-PDMS and medium, and ensure an upright position of the biorobot while floating. The weight of this layer generates a restoring torque on the biorobot at any pitch and roll. Also, the volume ratio between the Ni-PDMS and the MB-PDMS maintains the submersion depth. The presented protocols would be highly useful to researchers interested in characterizing the beating force of muscle cells and tissues, as well as those who wish to build swimming biorobots.
The seeding of the functionalized biological actuator and biorobot devices, the mechanical and biochemical characterization of the cells, and the quantitative analysis of the device function are described in detail in Part 2 of this two-part article as well as in the recent work15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>184 sil elast kit&#160; 0.5kg</td>
			<td>Sylgard 184</td>
		</tr>
		<tr>
			<td>Nickel Powder</td>
			<td>Sigma-Aldrich</td>
			<td>266981-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenolic microballoons</td>
			<td>US Composites</td>
			<td>BJO-0930</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td></td>
			<td></td>
			<td>4 inch diameter</td>
		</tr>
		<tr>
			<td>PWM101 light-duty spinner</td>
			<td></td>
			<td></td>
			<td>Spin- coater</td>
		</tr>
		<tr>
			<td>Positive photoresist (S1808)</td>
			<td>Dow Corning</td>
			<td>DEM-10018197</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M206 mechanical convection oven</td>
			<td></td>
			<td></td>
			<td>Convection oven</td>
		</tr>
		<tr>
			<td>Laser engraver</td>
			<td>Universal Laser System</td>
			<td>VLS2.30</td>
			<td>Utilizes a 10W, 10.6 &#181;m wavelength, CO2 Laser</td>
		</tr>
		<tr>
			<td>Universal Laser Systems Application</td>
			<td>Universal Laser System</td>
			<td></td>
			<td>Application for running the VLS 2.30</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>Numerical analysis program</td>
		</tr>
		<tr>
			<td>Scotch Tape</td>
			<td>Scotch Brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solid-glass beads</td>
			<td>Sigma-Aldrich</td>
			<td>Z265926-1EA</td>
			<td>Soda-lime glass, diameter 3 mm</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td>EL303</td>
			<td></td>
		</tr>
		<tr>
			<td>BD-20AC Laboratory Corona Treater</td>
			<td>Electrotechnic Products</td>
			<td>12051A</td>
			<td>Corona Discharger</td>
		</tr>
		<tr>
			<td>Ultrasonic Bath 1.9L</td>
			<td>Fisher Scientific</td>
			<td>15-337-402</td>
			<td>40 kHz industrial transducer</td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffer (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (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 bovine serum</td>
		</tr>
		<tr>
			<td>Penicillin-G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
	</tbody>
</table>

cardiac muscle-cell based actuator and self-stabilizing biorobot - part 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

The biological actuator and biorobot have very similar fabrication processes, as the biorobot is a natural extension of the biological actuator (Figure 1). The biological actuator was developed first to establish techniques required for the biorobot, to analyze the force generated by the cells, and to characterize the cell maturation mechanically and biochemically, both of which are described in detail in Part 2 of this two-part article as well as in our recently publishe...

Watch this Scientific Journal Video about Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1 at JoVE.com

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

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