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 PDMS = 0.965 g/mL, Ni-PDMS = 1.639 g/mL, MB-PDMS = 0.648 g/mL.</li>
	<li>Use equation (1) to estimate the mass of PDMS needed, for a given container, to obtain a specific height (5 mm) for the base of the biological actuator. The resulting density of PDMS is 0.965 g/mL. 
	NOTE: The ratio is 10:1 base to curing agent by weight. 
	Mbase = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq1.jpg" />)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (2) 
	Mcuring agent = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq2.jpg" />)</li>
	<li>Use equation (1) to find the mass of Ni-PDMS needed, for a given container, to obtain a specific height (1.5 mm) of the bottom base of the biorobot. 
	NOTE: The ratios are 1:1.88 (Nickel Powder to PDMS by weight) and 1:1.71:0.171 (Nickel Powder to PDMS Base to PDMS curing agent by weight). The resulting density of Ni-PDMS will be 1.639 g/mL. 
	MNickel = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq3.jpg" />)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (3) 
	Mbase = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq4.jpg" />) 
	Mcuring agent = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq5.jpg" />)</li>
	<li>Similarly, use equation (1) to find the mass of MB-PDMS needed, for a given container, to obtain a specific height (3.5 mm) of the top base of the biorobot. 
	NOTE: The ratios are 1:5 (microballoons to PDMS by weight) and 1:4.54:0.454 (microballoons to PDMS base to PDMS curing agent by weight). The resulting density of MB-PDMS will be 0.648 g/mL. 
	MMicroballoon = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq6.jpg" />)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (4) 
	Mbase = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq7.jpg" />) 
	Mcuring agent = &#1009;*V = &#1009;*V*(<img alt="Equation" src="/files/ftp_upload/55642/55642eq8.jpg" />)</li>
	<li>Check the dynamic stability of the biorobot with the desired dimension and geometry using the analysis scripts; see the supplementary information, &#39;Biorobot_dynamic_stability.m&#39; and &#39;CG_CB_calculation.m&#39;.</li>
</ol>
2. Fabrication of Biological Actuators on a Stationary Base
NOTE: See Figure 1a.
<ol>
	<li>Spin-coat a thin film of PDMS (see Figure 1a-1 and a2). The thickness of the resulting PDMS film will be 25 &#181;m.
	<ol>
		<li>Place a silicon wafer on a photoresist spinner and flip the pump switch on to produce suction. 
		NOTE: The silicon wafer has a 4-inch diameter and 500 &#181;m thickness.</li>
		<li>Pour positive photoresist (e.g. S1808) onto the silicon wafer until the wafer is completely covered. Program the spinner to spin at 2,000 rpm for 20 s. Then, engage the spinner by pressing on the foot pedal. Turn off the suction after spinning.</li>
		<li>Heat a hot plate up to 120 &#176;C. Use wafer tweezers to pick up the silicon wafer from the spinner and place the silicon wafer directly on the hotplate. Cover the wafer with a shallow petri dish and bake for 10 min. 
		NOTE: An oven can be used to bake the wafer using the same temperature and duration. Figure 1a-1 depicts this process.</li>
		<li>Place a plastic container on a weighing scale and zero it out. Pour 6 g of PDMS base into the container and add 0.6 g of PDMS curing agent. Mix the PDMS thoroughly for 5 min. 
		NOTE: After mixing, the mixture should be confluent with bubbles.</li>
		<li>Place the container of mixed PDMS into a vacuum chamber. Reduce the pressure of the vacuum chamber to 100 mbar and leave the container in the chamber for 30 min. Break the vacuum, and remove the container. Keep the container covered until use.</li>
		<li>Place the silicon wafer with the baked photoresist layer on the spinner. Slowly pour the entire degassed PDMS mixture on the wafer. 
		NOTE: Pour slowly so that no new bubbles are introduced into the mixture.</li>
		<li>Set the spinner to 1,200 rpm for 5 min. Turn on the spinner suction and engage the spinner. Turn off the suction after spinning. 
		NOTE: These settings result in a 25 &#181;m thick layer of PDMS.</li>
		<li>Heat an oven to 40 &#176;C. Use wafer tweezers to pick up the silicon wafer from the spinner, then place it in the oven. Bake the wafer overnight and then cool the wafer at room temperature. 
		NOTE: Figure 1a-2 depicts this process.</li>
	</ol>
	</li>
	<li>Laser engraving of the thin-film PDMS layer.
	<ol>
		<li>Turn on the power switch of the laser engraver and its exhaust. Turn on the computer connected to the laser engraver. Open the laser engraver software.</li>
		<li>Under the &#34;File&#34; option, open the biological actuator design file shown in Figure 2e.
		<ol>
			<li>Press the &#34;Settings&#34; button. Click on &#34;Blue&#34; and change the power setting to 3% and speed to 4%. Click &#8220;Set&#8221;. Click on &#34;Black&#34; and change the &#34;Mode&#34; to skip. Then click &#8220;Set&#8221;. Do the same for &#34;Red&#34;. Press the &#34;Apply&#34; button to finish the settings.&#8221;</li>
			<li>Push the &#34;Activate the Engraver&#34; button at the top right.</li>
		</ol>
		</li>
		<li>Press the &#34;Relocate&#34; button to move the design into the center of the software&#39;s screen.</li>
		<li>Press the &#34;Focus View&#34; button in the program and click on the edge of the biorobot on the screen. This will move the guiding laser dot of the laser engraver to the corresponding point.</li>
		<li>Move the wafer manually with tweezers, so that the point on the wafer corresponding to the point clicked in 2.2.4 is directly under the guiding laser dot.</li>
		<li>Press the &#34;Start engraving the prior job&#34; button to start the engraving process. Remove the wafer after the engraving is completed. Turn off all equipment. 
		NOTE: The &#34;Start engraving the prior job&#34; button is the large green triangle. Do not look directly at the engraving process as the laser can damage the eyes. Figure 1a-3 depicts this process.</li>
	</ol>
	</li>
	<li>Preparation and fabrication of the biological actuator base.
	<ol>
		<li>Pour glass beads (3 mm diameter) into a 15 mL tube. Immerse the beads with 70% ethanol in DI water for 24 h. Remove the ethanol and fill the tube with DI water for 24 h. Pour out the DI water and place the tube on a hotplate at 50 &#176;C to facilitate drying of the glass beads.</li>
		<li>Add 3 g to the amount of PDMS found in equation (1) to account for the PDMS that will stick to container sides during pouring. Use equation (2) to find PDMS base and curing agent amounts.</li>
		<li>Place a plastic container on a weighing scale and zero it out. Pour the amount of PDMS base found in step 2.3.2 into the container and zero it out. Then pour the amount of PDMS curing agent found in step 2.3.2 into the container.</li>
		<li>Mix the PDMS thoroughly for 5 min. 
		NOTE: PDMS is used at a ratio of 10:1 base to curing agent. The mixture should have many bubbles.</li>
		<li>Place a container to be used for baking on a scale and zero out. Carefully pour out the correct amount of PDMS found in step 2.3.2 (and mixed in step 2.3.4) into the container. Drop cleaned glass beads throughout the PDMS mixture at regular intervals. Leave a minimum of 5 mm of space surrounding each bead for the biological actuator base.</li>
		<li>Place the container into a vacuum chamber. Reduce the vacuum pressure to 100 mbar and turn off the vacuum pump. After 30 min, break the vacuum and remove the container. Keep covered until use. 
		NOTE: The pressure in the chamber may rise slowly over time as the mixture degasses and the vacuum chamber leaks. If the pressure increases substantially over 100 mbar, turn on the vacuum pump to restore the pressure to 100 mbar.</li>
		<li>Heat a hotplate to 40 &#176;C. Carefully place the container of PDMS and the glass beads on the hot plate. Cover the container and bake overnight.</li>
	</ol>
	</li>
	<li>Biological actuator assembly. 
	NOTE: The following procedure can be done with the naked eye.
	<ol>
		<li>Cut cubes (5 mm x 5 mm x 5 mm) out of the bulk PDMS made in part 2.3 using a razor blade. 
		NOTE: One bead should be in the center of each cube.</li>
		<li>Clean all sides of each biological actuator base, to remove any contaminates on the base surfaces, by pressing the base into the tape and remove. Repeat for each side.</li>
		<li>Redo steps 2.3.2 to 2.3.6 to make a small amount of liquid PDMS. Dip the tip of a needle into the liquid PDMS. Place a drop of the liquid PDMS on the engraved base area of the wafer patterned in step 2.2. Smear the droplet of PDMS so that it completely covers the 5 mm x 5 mm base area. 
		NOTE: The base area is the middle square section in Figure 2a.</li>
		<li>Use tweezers to place the cleaned cube from step 2.4.2 on the base area that is covered with liquid PDMS.</li>
		<li>Repeat step 2.4.3 from &#34;Place a drop of liquid PDMS&#34; to the end and step 2.4.4 for each device that will be made.</li>
		<li>Heat a hotplate to 40 &#176;C. Carefully place the silicon wafer with the assemblies on the hot plate. Cover the wafer and bake overnight. 
		NOTE: Keep the assemblies attached until use. Figure 1a-4 depicts the final device.</li>
	</ol>
	</li>
</ol>
3. Fabrication of Biorobots (Figure 1b)
<ol>
	<li>Spin-coating and laser-engraving a thin PDMS film
	<ol>
		<li>Repeat all steps in 2.1 and 2.2 using a new silicon wafer. This will result in a silicon wafer with a thin film of PDMS and a thin film of the photoresist, which is engraved with a biorobot design. 
		NOTE: While repeating step 2.2, use the biorobot design for laser engraving instead of the biological actuator design previously used. Figures 1b-1 and b-3 depicts these processes.</li>
	</ol>
	</li>
	<li>Preparation and fabrication of PDMS composites. 
	NOTE: The following procedure can be done with the naked eye.
	<ol>
		<li>Pour phenolic microballoons into a 50 mL tube until full. Fill the tube with 70% ethanol in DI-water and let it sit for 24 h. Pour out the ethanol, add DI water, and let it sit for 24 h. Pour out the DI water, and then place the tube on a hotplate at 50 &#176;C to facilitate the drying of the microballoons before use.</li>
		<li>Use equation (1) with the MB-PDMS density and 3.5 mm height to find the volume of PDMS required. Add 3 g to the total amount, to account for the material that will remain in the container after pouring. Use equation (3) to find the PDMS base and curing agent amounts. Measure out the appropriate amount of PDMS base, curing agent, and microballoons using the scale.</li>
		<li>Use equation (1) with Ni-PDMS density and 1.5-mm height to find the volume of PDMS needed. Add 3 g to the total amount as in step 3.2.2. Use equation (2) to find the PDMS base and curing agent amounts. Measure out the appropriate amount of PDMS base, curing agent, and nickel powder using the scale.</li>
		<li>Mix each mixture of MB-PDMS and Ni-PDMS for 5 min. Carefully pour the correct amount of MB-PDMS and Ni-PDMS calculated in 3.2.2 and 3.2.3 into separate containers using a scale. 
		NOTE: The mixtures should be thoroughly mixed by a metal or glass rod without scratching the bottom surface of the mixing container. The mixture will be confluent with bubbles.</li>
		<li>Place both containers into a vacuum chamber. Reduce its pressure to 100 mbar for 30 min. Break the vacuum and remove the containers. Keep covered until use.</li>
		<li>Heat a hotplate to 40 &#176;C. Place containers with MB-PDMS and Ni-PDMS on the hot plate. Cover each container and bake overnight. 
		NOTE: Store with a lid until use.</li>
	</ol>
	</li>
	<li>Biorobot assembly.
	<ol>
		<li>Cut biorobot bases of dimensions respective to each biorobot size from Ni-PDMS and MB-PDMS using a razor blade. See Figure 2b-2d for base designs. 
		NOTE: The thicknesses of Ni-PDMS is 1.5 mm and that of MB-PDMS is 3.5 mm.</li>
		<li>Clean all sides of the biorobot bases to remove any contaminants on the surfaces, by pressing the base into the tape and removing. Repeat for each side.</li>
		<li>Turn on a corona discharger. Bring the tip of the corona discharger 1 cm above the Ni-PDMS base, which is placed on a metal plate with a cleanroom tissue in between. Move the tip around the base and continue for 15 s to treat the surface. 
		NOTE: A discharge should occur between the corona discharger and the wafer. If it does not, bring the tip closer until a discharge occurs.</li>
		<li>Repeat step 3.3.3 to treat the surface of the base of a biorobot engraved in step 3.1 for the same duration. Use tweezers to place the Ni-PDMS treated side onto the treated side of the film. Let the device sit for 5 min. 
		NOTE: This will strongly bond the two parts. See Figure 1b4.</li>
		<li>Use sharp tweezers to peel the biorobot cantilever from the wafer and place it on the bottom of the Ni-PDMS base. Use tweezers to remove the entire assembly from the wafer. 
		NOTE: The cantilever will be attached to the Ni-PDMS base. Figure 1b-5 and b-6 depicts this.</li>
		<li>Place a small drop of uncured PDMS (10:1 base to curing agent) on the top of the MB-PDMS base. Use tweezers to place the side of the Ni-PDMS with the thin film PDMS on the MB-PDMS with the uncured PDMS. Place the assembly in a plastic petri dish, and then place this on a hotplate at 40 &#176;C to cure overnight. 
		NOTE: Figure 1b-7 depicts the final device.</li>
	</ol>
	</li>
</ol>
4. Functionalization of the Devices
NOTE: Below, we describe the process of preparing the devices for cell seeding.
<ol>
	<li>Prepare the required materials: Fibronectin solution (50 &#181;g/mL), Phosphate Buffer Saline solution (PBS), Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin antibiotic (DMEM complete).</li>
	<li>Place 100 &#181;L of fibronectin solution into the center of a T-25 culture flask (bottom surface when the flask is sitting upright). Maintain separate flasks for each device.</li>
	<li>Place the biorobot or the biological actuator facing down over the droplet of fibronectin solution. Ensure that the cantilever is unfolded and immersed within the droplet. Incubate at 37 &#8451; for 30 min.</li>
	<li>After the incubation, remove the fibronectin solution and wash with PBS twice.</li>
	<li>Remove the PBS and fill the flask with 10 mL of DMEM. Incubate at 37 &#8451; for 1 h to facilitate degassing of the PDMS. To submerge the biorobots in 10 mL of media, use a magnet to hold the device at the bottom of the flask. Place the flask with the samples in an ultrasonication bath for 5 min to remove the bubbles. 
	NOTE: During the incubation period, air bubbles form on the PDMS surface, which is referred to as degassing here. The Ni-PDMS used in the biorobot assembly is magnetic. The biological actuator does not need a magnet because it will remain at the bottom of the flask due to the weight of the glass bead. The biorobot or the biological actuator assembly is now ready for seeding, which is explained in detail in part 2.</li>
</ol>

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The authors have nothing to disclose

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><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>184 sil elast kit&amp;nbsp; 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 10 W, 10.6 &amp;micro;m wavelength, CO&lt;sub&gt;2&lt;/sub&gt; 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 1.9 L</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&amp;rsquo;s Phosphate Buffer (PBS)</td><td>Sigma-Aldrich</td><td>D1408-100ML</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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 ...

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

Research

JoVE Journal

Bioengineering

8.1K Views.  Louisiana State University. 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.

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

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

Neuroscience

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Fabrication of Carbon-Based Ionic Electromechanically Active Soft Actuators

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, compliant and biomimetic nature of this deformation, actuators made of the laminate have received increasing interest in soft robotics and (bio)medical applications. However, methods to easily fabricate the active material in large (even industrial) quantities and with a high batch-to-batch and within-batch repeatability are needed to transfer the knowledge from laboratory to industry. This protocol describes a simple, industrially scalable and reproducible method for the fabrication of ionic carbon-based electromechanically active capacitive laminates and the preparation of actuators made thereof. The inclusion of a passive and chemically inert (insoluble) middle layer (e.g., a textile-reinforced polymer network or microporous Teflon) distinguishes the method from others. The protocol is divided into five steps: membrane preparation, electrode preparation, current collector attachment, cutting and shaping, and actuation. Following the protocol results in an active material that can, for example, compliantly grasp and hold a randomly shaped object as demonstrated in the article.

This article describes a fast and simple manufacturing process of ionic electromechanically active composite materials for actuators in biomedical, biomimetic and soft robotics applications. The key fabrication steps, their importance for the actuators&#39; final properties, and some of the main characterization techniques are described in detail.

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, ...

Engineering

Manufacturing, Control, and Performance Evaluation of a Gecko-Inspired Soft Robot

This protocol presents a method for manufacturing, control, and evaluation of the performance of a soft robot that can climb inclined flat surfaces with slopes of up to 84&#176;. The manufacturing method is valid for the fast pneunet bending actuators in general and might, therefore, be interesting for newcomers to the field of actuator manufacturing. The control of the robot is achieved by means of a pneumatic control box that can provide arbitrary pressures and can be built by only using purchased components, a laser cutter, and a soldering iron. For the walking performance of the robot, the pressure-angle calibration plays a crucial role. Therefore, a semi-automated method for the pressure-angle calibration is presented. At high inclines (&#62; 70&#176;), the robot can no longer reliably fix itself to the walking plane. Therefore, the gait pattern is modified to ensure that the feet can be fixed on the walking plane.

This protocol provides a detailed list of steps to be performed for the manufacturing, control and evaluation of the climbing performance of a gecko-inspired soft robot.

This protocol presents a method for manufacturing, control, and evaluation of the performance of a soft robot that can climb inclined flat surfaces with ...

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.

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. ...

Revolutionizing Stroke Rehabilitation with Enhanced Grasping and Finger Recovery

A Flexible Wearable Supernumerary Robotic Limb for Chronic Stroke Patients

In this study, we present a flexible wearable supernumerary robotic limb that helps chronic stroke patients with finger rehabilitation and grasping movements. The design of this innovative limb draws inspiration from bending pneumatic muscles and the unique characteristics of an elephant's trunk tip. It places a strong emphasis on crucial factors such as lightweight construction, safety, compliance, waterproofing, and achieving a high output-to-weight/pressure ratio. The proposed structure enables the robotic limb to perform both envelope and fingertip grasping. Human-robot interaction is facilitated through a flexible bending sensor, detecting the wearer's finger movements and connecting them to motion control via a threshold segmentation method. Additionally, the system is portable for versatile daily use. To validate the effectiveness of this innovation, real-world experiments involving six chronic stroke patients and three healthy volunteers were conducted. The feedback received through questionnaires indicates that the designed mechanism holds immense promise in assisting chronic stroke patients with their daily grasping activities, potentially improving their quality of life and rehabilitation outcomes.

Author Spotlight: Enhancing Grasping Abilities for Hemiplegic Patients with Flexible Robotic Limbs 

This protocol introduces a flexible wearable supernumerary robotic limb tailored to assist in finger rehabilitation for stroke patients. The design incorporates a bending sensor to facilitate seamless human-robot interaction. Validation through experiments involving both healthy volunteers and stroke patients underscores the efficacy and dependability of the proposed study.

In this study, we present a flexible wearable supernumerary robotic limb that helps chronic stroke patients with finger rehabilitation and grasping ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...