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

Summary

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.

Abstract

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.

Introduction

Biological actuators and biorobots are being actively studied to provide an alternative to conventional robotics for numerous applications. Biorobots that walk^5,6,7,8, swim^1,2,3,4, pump^9,10, or grip^11,12,13 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° to 90°. 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.

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Protocol

1. Calculate Mass of PDMS and Additives

1. Use the following equation to find the mass of PDMS needed for specific heights in the following procedures,
   M = ϱ*V = ϱ * Height*Area          (1),
   where 'Height' is the height of the layer, 'Area' is the area of a container that the PDMS will be cured in, 'ϱ' is the density of the mixture and 'V' 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.
2. 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.
   M_base = ϱ*V = ϱ*V*()          (2)
   M_{curing agent} = ϱ*V = ϱ*V*()
3. 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.
   M_Nickel = ϱ*V = ϱ*V*()          (3)
   M_base = ϱ*V = ϱ*V*()
   M_{curing agent} = ϱ*V = ϱ*V*()
4. 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.
   M_Microballoon = ϱ*V = ϱ*V*()          (4)
   M_base = ϱ*V = ϱ*V*()
   M_{curing agent} = ϱ*V = ϱ*V*()
5. Check the dynamic stability of the biorobot with the desired dimension and geometry using the analysis scripts; see the supplementary information, 'Biorobot_dynamic_stability.m' and 'CG_CB_calculation.m'.

2. Fabrication of Biological Actuators on a Stationary Base

NOTE: See Figure 1a.

1. Spin-coat a thin film of PDMS (see Figure 1a-1 and a2). The thickness of the resulting PDMS film will be 25 µm.
   1. 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 µm thickness.
   2. 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.
   3. Heat a hot plate up to 120 °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.
   4. 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.
   5. 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.
   6. 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.
   7. 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 µm thick layer of PDMS.
   8. Heat an oven to 40 °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.
2. Laser engraving of the thin-film PDMS layer.
   1. 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.
   2. Under the "File" option, open the biological actuator design file shown in Figure 2e.
      1. Press the "Settings" button. Click on "Blue" and change the power setting to 3% and speed to 4%. Click “Set”. Click on "Black" and change the "Mode" to skip. Then click “Set”. Do the same for "Red". Press the "Apply" button to finish the settings.”
      2. Push the "Activate the Engraver" button at the top right.
   3. Press the "Relocate" button to move the design into the center of the software's screen.
   4. Press the "Focus View" 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.
   5. 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.
   6. Press the "Start engraving the prior job" button to start the engraving process. Remove the wafer after the engraving is completed. Turn off all equipment.
      NOTE: The "Start engraving the prior job" 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.
3. Preparation and fabrication of the biological actuator base.
   1. 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 °C to facilitate drying of the glass beads.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Heat a hotplate to 40 °C. Carefully place the container of PDMS and the glass beads on the hot plate. Cover the container and bake overnight.
4. Biological actuator assembly.
   NOTE: The following procedure can be done with the naked eye.
   1. 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.
   2. 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.
   3. 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.
   4. Use tweezers to place the cleaned cube from step 2.4.2 on the base area that is covered with liquid PDMS.
   5. Repeat step 2.4.3 from "Place a drop of liquid PDMS" to the end and step 2.4.4 for each device that will be made.
   6. Heat a hotplate to 40 °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.

3. Fabrication of Biorobots (Figure 1b)

1. Spin-coating and laser-engraving a thin PDMS film
   1. 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.
2. Preparation and fabrication of PDMS composites.
   NOTE: The following procedure can be done with the naked eye.
   1. 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 °C to facilitate the drying of the microballoons before use.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. Heat a hotplate to 40 °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.
3. Biorobot assembly.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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 °C to cure overnight.
      NOTE: Figure 1b-7 depicts the final device.

4. Functionalization of the Devices

NOTE: Below, we describe the process of preparing the devices for cell seeding.

1. Prepare the required materials: Fibronectin solution (50 µg/mL), Phosphate Buffer Saline solution (PBS), Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin antibiotic (DMEM complete).
2. Place 100 µL 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.
3. 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 ℃ for 30 min.
4. After the incubation, remove the fibronectin solution and wash with PBS twice.
5. Remove the PBS and fill the flask with 10 mL of DMEM. Incubate at 37 ℃ 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.

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Results

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...

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Discussion

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 ...

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Materials

Name	Company	Catalog Number	Comments
Polydimethylsiloxane (PDMS)	Dow Corning	184 sil elast kit  0.5kg	Sylgard 184
Nickel Powder	Sigma-Aldrich	266981-100G
Phenolic microballoons	US Composites	BJO-0930
Silicon wafers			4 inch diameter
PWM101 light-duty spinner			Spin- coater
Positive photoresist (S1808)	Dow Corning	DEM-10018197
Hotplate
Vacuum chamber
M206 mechanical convection oven			Convection oven
Laser engraver	Universal Laser System	VLS2.30	Utilizes a 10 W, 10.6 µm wavelength, CO2 Laser
Universal Laser Systems Application	Universal Laser System		Application for running the VLS 2.30
Matlab	MathWorks		Numerical analysis program
Scotch Tape	Scotch Brand
Solid-glass beads	Sigma-Aldrich	Z265926-1EA	Soda-lime glass, diameter 3 mm
Scale	Mettler Toledo	EL303
BD-20AC Laboratory Corona Treater	Electrotechnic Products	12051A	Corona Discharger
Ultrasonic Bath 1.9 L	Fisher Scientific	15-337-402	40 kHz industrial transducer
Fibronectin from bovine plasma	Sigma-Aldrich	F1141
Dulbecco’s Phosphate Buffer (PBS)	Sigma-Aldrich	D1408-100ML
Dulbecco’s Modified Eagle Medium (DMEM)	Hyclone Laboratories	16750-074	With 4500 mg/L glucose, 4.0 mM L-glutamine, and 110 mg/L sodium pyruvate.
Fetalclone III serum	Hyclone Industries, GE	16777-240	Fetal bovine serum
Penicillin-G sodium salt	Sigma-Aldrich	P3032