Bioinspired Soft Robot with Incorporated Microelectrodes

Summary

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.

Abstract

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.

Introduction

Modern state-of-the-art soft robots can mimic the hierarchical structures and muscle dynamics of many living organisms, such as the jellyfish^1,2, sting ray², octopus³, bacteria⁴, and sperm⁵. Mimicking the dynamics and architecture of natural systems offers higher performances in terms of both energetic and structural efficiency⁶. This is intrinsically related to the soft nature of natural tissue (e.g., skin or muscle tissue with a Young's modulus between 10⁴−10⁹ Pa) which allows for higher degrees of freedom and superior deformation and adaptability when compared with standard engineered actuators (e.g., a Young's modulus usually between 10⁹−10¹² Pa)⁶. Cardiac muscle-based soft-actuators, especially, show superior energy efficiency due to their self-actuation as well as their potential for autorepair and regeneration when compared to a mechanically based robotic system⁷. However, the fabrication of soft robots is challenging due to the necessity of integrating different components with different physical, biological, and mechanical properties into the one system. For example, engineered synthetic systems need to be integrated with living biological systems, not only providing them with structural support but also influencing and modulating their actuation behavior. In addition, many microfabrication methods require harsh/cytotoxic processes and chemicals that decrease the viability and function of any living components. Therefore, new approaches are necessary to enhance the functionality of the soft robots and to control and modulate their behavior.

To successfully integrate living components with good viability, a hydrogel-based scaffold is an excellent material to create the body of a soft robot. A hydrogel's physical and mechanical properties can easily be tuned to create microenvironments for living components such as muscle tissues^8,9. Also, it can easily adopt various microfabrication techniques, resulting in the creation of hierarchical structures with high fidelity^1,2,10. Flexible electronic devices can be incorporated into the soft robot to control its behavior with electrical stimulation. For example, optogenetic techniques to engineer electrogenic cells (e.g., cardiomyocytes), which show a light-dependent electrophysiological activation, have been used to develop a polydimethylsiloxane (PDMS)-based soft robotic sting ray guided by light that was able to recreate the undulatory movement of the fish in vitro². Although optogenetic techniques have shown excellent controllability, the work presented uses electrical stimulation, a conventional and traditional simulation method. This is because electrical stimulation via flexible microelectrodes is easy and simple compared to optogenetic techniques, which require extensive development processes¹¹. The use of flexible electronic devices can allow for long-term stimulation and standard/simple fabrication processes as well as tunable biocompatibility and physical and mechanical properties^12,13.

Here, we present an innovative method to fabricate a bioinspired soft robot, actuated by the beating of engineered cardiac muscle tissue and controlled by electrical stimulation through embedded flexible Au microelectrodes. The soft robot is designed to mimic the muscle and cartilage structure of the sting ray. The sting ray is an organism with a relatively easy to mimic structure and movement compared to other swimming species. The muscles are recreated in vitro by seeding cardiomyocytes on an electrically conductive hydrogel micropattern. As previously reported, incorporating electrically conductive nanoparticles such as CNT in the GelMA hydrogel not only improves the electrical coupling of the cardiac tissue, but also induces an excellent in vitro tissue architecture and arrangement^8,9. The cartilage joints are then mimicked using a mechanically robust PEGDA hydrogel pattern that acts as the mechanically robust substrate of the whole system. Flexible Au microelectrodes with a serpentine pattern are embedded in the PEGDA pattern to locally and electrically stimulate the cardiac tissue.

Protocol

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the institutional Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital.

1. GelMA synthesis

1. Dissolve 10 g of gelatin in 100 mL of Dulbecco's phosphate-buffered saline (DPBS) using a magnetic stirrer at 50 °C.
2. Add 8 mL of methacrylic anhydride slowly while stirring the gelatin prepolymer solution at 50 °C for 2 h. Dilute the reacted gelatin solution with preheated DPBS at 50 °C.
3. Transfer the diluted solution into dialysis membranes (molecular weight cutoff = 12–14 kDa) and place them into deionized (DI) water. Perform dialysis at 40 °C for about 1 week.
4. Filter the dialyzed GelMA prepolymer solution using a sterile filter (pore size = 0.22 µm) and transfer 25 or 30 mL of the solution into 50 mL tubes and store at -80 °C for 2 days.
5. Freeze-dry the frozen GelMA prepolymer solution using a freeze dryer for 5 days.

2. Preparation of poly(ethylene glycol) diacrylate (PEGDA) prepolymer solution

1. Dissolve 200 mg (20% of total solution) of PEGDA (MW = 1,000) with 5 mg (0.5% of total solution) of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (photo-initiator, PI) in 1 mL of DPBS.
2. Incubate the prepolymer solution at 80 °C for 5 min.

3. Preparation of GelMA-coated CNT dispersed stock solution

1. Dissolve 80 mg of GelMA (used as a biosurfactant) in 4 mL of DPBS and then add 20 mg of COOH functionalized multiwalled carbon nanotubes (MWCNTs) into the GelMA prepolymer solution.
2. Sonicate the MWCNT-laden GelMA prepolymer solution for 1 h (0.66Hz, 100 Watt).
   NOTE: During the sonication process, the solution must be immersed in a water bath at ~15 °C to prevent evaporation of solvent due to the rise in temperature.

4. Preparation of 1 mg/mL CNT containing 5% GelMA prepolymer solution

1. Dissolve 50 mg of GelMA and 5 mg (0.5% of total solution) of PI in 0.8 mL of DPBS at 80 °C for 10 min.
2. Add 0.2 mL of the prepared CNT stock solution (step 3). Vortex and incubate the solution at 80 °C for 10 min.

5. Preparation of a 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) coated glass slide

1. Wash the glass slides (thickness = 1 mm, size = 5.08 cm x 7.62 cm) with pure ethanol.
2. Stack the cleaned slides vertically in a 250 mL beaker and spread 3 mL of TMSPMA on top of them using a syringe. Cover the beaker with aluminum foil to prevent evaporation of TMSPMA.
3. Incubate the slides in an 80 ˚C oven for 1 day.
4. Wash the coated glass slides by dipping them into pure ethanol, then dry.
5. Store the coated glass slides wrapped in aluminum foil at room temperature (RT).
   NOTE: Try to minimize touching the surfaces of the TMSPMA-coated glass slides.

6. Fabrication of the flexible Au microelectrodes

1. Design a shadow mask using computer-aided design (Supplementary File 3).
2. Fabricate and purchase a shadow mask.
3. Wash the glass slide (thickness = 1 mm, size = 3 cm x 4 cm) with acetone and dry with a compressed air gun.
4. Attach the shadow mask to the glass substrates using double sided tape, then put them in an E-beam evaporator and wait until the chamber pressure reaches at least 10^-6 Torr.
   NOTE: The two pieces of tape were placed manually on the support at a distance short enough to host the glass and large enough to fit the entire pattern. This step takes around 45–60 min.
5. Deposit a 200 nm thick Au layer by E-beam evaporator (e.g., with Denton EE-4, vacuum = 10^-6 Torr, power = 2.6%, rate = 2 Å/s) and cut the fabricated microelectrodes using a dicing saw machine (electrodes size = 7.38 mm x 8.9 mm x 200 nm).

7. Fabrication of an Au microelectrode-integrated micropatterned multilayered hydrogel scaffold

NOTE: The result of this procedure is a membrane where a micropatterned PEGDA hydrogel is in the bottom layer, a micropatterned CNT-GelMA hydrogel is on top, and the Au microelectrodes are between the two layers. This configuration ensures a better flexibility to the electrode and limits the risk of breaking.

1. Design and fabricate two photomasks to create the micropatterned PEGDA (1^st photomask) and the CNT-GelMA hydrogel (2^nd photomask) layers. See Supplementary File 2–3. The design can be done by using CAD software.
   NOTE: See Figure 2B, E.
2. Place 50 μm spacers made by stacking one layer of commercial invisible tape (Thickness: 50 μm) on a TMSPMA coated glass. Pour 15 μL of 20% PEGDA prepolymer solution on top of the TMSPMA coated glass, then cover with gold microelectrode. Place the 1st photomask for the glass slide (micropatterned PEGDA) on top of the gold microelectrode and expose the whole construct to UV light (200 W mercury vapor short arc lamp with 320–390 nm filter) at 800 mW of intensity and 8 cm distance for 110 s.
   NOTE: See Figure 1A.
3. Add DPBS to surround the glass slide and detach the micropatterned PEGDA hydrogel together with the Au microelectrodes from the uncoated glass substrate carefully after 5–10 min to obtain the glass slide that has the micropatterned PEGDA hydrogel with the Au microelectrodes.
   NOTE: See Figure 1B. Due to the TMSPMA coating, the construct is transferred from the uncoated glass substrate to the TMSPMA-coated one. Detach carefully because the Au microelectrodes can break easily during this step (Figure 3).
4. Place 100 μm spacers made by stacking two layers of commercial transparent tape (thickness = 50 µm) on the bottom of a Petri dish. Deposit a drop of 20 μL CNT-GelMA prepolymer solution between the spacers and then flip the glass slide obtained in 7.3 and fix it onto the dish with adhesive tape.
5. Rotate the device upside down and place the 2^nd photomask on top of the glass slide. Expose under UV light at 800 mW of intensity and 8 cm distance for 200 s.
   NOTE: See Figure 1C. Alignment of the 2nd mask is important.
6. Wash the obtained scaffold with DPBS and with cell culture medium that includes 10% fetal bovine serum (FBS).
7. Leave them overnight in the 37 °C incubator before seeding the cells.

8. Neonatal rat cardiomyocytes isolation and culture

1. Isolate hearts from 2-day-old Sprague-Dawley rats following protocols approved by the Institute's Committee on Animal Care⁸.
2. Put the heart pieces on the shaker overnight (around 16 h) in 0.05% trypsin without EDTA in HBSS in a cold room.
3. Collect the heart pieces with a pipette gun and minimize the amount of trypsin, then put them in a 50 mL tube with 10 mL of warm cardiac media (10% FBS, 1% P/S, 1% L-glutamine).
4. Swirl slowly (~60 rpm) in a 37 °C water bath for 7 min. Remove the media carefully from the tube with a 10 mL pipette and leave the heart pieces in the tube.
5. Add 7 mL of 0.1% collagenase type 2 in HBSS and swirl in a 37 °C water bath for 10 min.
6. Mix with a 10 mL pipette 10x gently to disrupt the heart pieces. Remove the media from the tube with a 1 mL pipette.
7. Add 10 mL of 0.1% collagenase type 2 in HBSS and swirl quickly (~120–180 rpm) in a 37 °C water bath for 10 min, then check if the heart pieces are dissolving.
8. Mix with a 10 mL pipette, then repeat with a 1 mL pipette to break the last heart pieces.
9. Once the solution looks homogeneous, place a 70 µm cell strainer on a new 50 mL tube and pipette the solution 1 mL at a time on strainer.
10. Centrifuge the heart cell solution at 180 x g for 5 min at 37 °C.
    NOTE: If there are still some heart pieces or mucus which did not dissolve, repeat steps 8.7–8.9 again.
11. Carefully remove all the liquid above the cell pellet and resuspended the cells in 2 mL of cardiac media.
12. Add 2 mL of cardiac media from the tube wall carefully to resuspend the cells and avoid breaking them.
13. Add the suspended cells into a T175 flask with warm cardiac media drop by drop. Put the flask in a 37 °C incubator for 1 h to allow cardiac fibroblasts to attach to the bottom.
    NOTE: At this preplating step, the cardiac fibroblasts will attach to the flask while the cardiomyocytes will remain in the suspension medium.
14. Collect the media from the flask that contains the cardiomyocytes and put it into a 50 mL tube.
15. Count the cells, then centrifuge at 260 x g for 5 min at 37 °C.
16. Resuspend and seed the cells on top of the fabricated soft robot in step 7. Pour specific volume of cardiac media with the cardiomyocytes at a concentration of 1.95 × 10⁶ cell/mL drop by drop onto the entire surface of the device.
17. Incubate the samples at 37 °C and change the media with 5 mL cell culture media with 2% FBS and 1% L-glutamine on the first and the second days after seeding. Change the media every time the color of the media shifts.

9. Cell staining for alignment analysis

1. Remove the media and wash with DPBS for 5 min at RT.
2. Fix the cells using 4% paraformaldehyde (PFA) for 20 min at RT. Then wash with DPBS for 5 min at RT.
3. Incubate the cells with 0.1% triton in DPBS at RT for 1 h. Wash 3x with PBS for 5 min at RT.
4. Incubate the cells with 10% goat serum in DPBS at RT for 1 h.
5. Incubate the cells with a primary antibody (sarcomeric α-actinin and connexin-43) in 10% goat serum in DPBS at 4 °C for ~14–16 h.
6. Wash 3x with DPBS for 5 min at RT. Incubate the cells with the secondary antibody in 10% goat serum in DPBS at RT for 1 h.
7. Wash 3x with DPBS for 5 min at RT, then counterstain cells with 4',6-diamidino-2-phenylindole (DAPI) in DI water (1:1,000) for 10 min at RT. Wash 3x with DPBS for 5 min at RT.
8. Take fluorescence images using an inverted laser scanning confocal microscope.

10. Actuator testing and behavior evaluation

1. Spontaneous beating of the cardiomyocytes on the soft robot
   1. Incubate bioinspired actuators at 37 °C for 5 days and refresh the media on day 1 and 2 and when necessary (i.e., when the media turns yellow). Use an inverted optical microscope to take images daily (5x and/or 10x). Record cell movements using video capture software on the microscope's live window for 30 s at 20 frames per second (5x and/or 10x) when the contractile activity starts (generally around day 3).
   2. At day 5, detach the membranes by gently lifting from the edge with a cover slide.
      NOTE: If the cells show a strong beating behavior, the membranes will detach by themselves due to the mechanical action of the contractions.
2. Bulk electrical signal stimulation
   1. Using a 3 cm spaced PDMS as a holder, affix two carbon rod electrodes with platinum (Pt) wire in a 6 cm Petri dish filled with cardiac media. Then carefully transfer the soft robot into the Petri dish.
   2. Apply a square waveform with 50 ms pulse width, DC offset value 0 V, and peak voltage amplitude between 0.5 and 6 V. The frequency varies between 0.5, 1.0, and 2.0 Hz with a duty cycle between 2.5%, 5%, and 10%, respectively. Record macroscale contractions using a commercially available camera.
3. Electrical stimulation with the Au microelectrodes
   1. After fabrication of Au microelectrode-integrated multilayered hydrogel scaffold, attach two copper wires to the Au electrodes though an external square port using silver paste.
   2. Cover the silver paste with a thin layer of PDMS precured at 80 °C for 5 min. Then put the samples on a hot plate at 45 °C for 5 h to fully crosslink the PDMS.
   3. After seeding cardiomyocyte, apply a square wave electrical stimulus on the copper wires with DC offset value 1 V, peak voltage amplitude between 1.5 and 5 V, and frequencies of 0.5, 1.0, and 2.0 Hz respectively.

Results

Flow diagram of the steps for developing the Au microelectrode-incorporated bioinspired soft robot
The aim of the soft robot design was to build a membrane capable of actuating a swimming movement with minimal complexity. The structure must be able to sustain strong flexions repeatedly over time (about 1 Hz) and be able to keep its shape while achieving a strong beating. By selectively photo crosslinking the polymer using photomasks, we fabricated a hierarchically structured scaffold comprised of a...

Discussion

Using this method, we were able to successfully fabricate a batoid fish-like bioinspired soft robot with an integrated self-actuating cardiac tissue on a multilayer structured scaffold that is controlled by embedded Au microelectrodes. Due to two distinct micropatterned hydrogel layers made of PEGDA and CNT–GelMA hydrogels, the bioinspired scaffold showed good mechanical stability and ideal cell alignment and maturation. The PEGDA pattern layer, which serves as a cartilage joint of the skeletal architecture in a st...

Materials

Name	Company	Catalog Number	Comments
250 mL Beaker	PYREX	1000-250CNEa
2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone	Sigma-Aldrich	410896
3-(Trimethoxysilyl)propyl methacrylate	Milipore	M6514
37° Water bath	VWR	W6M
4',6-diamidino-2-phenylindole (DAPI)	Sigma-Aldrich	D9542
50mL Conical Centrifuge Tubes	Falcon	14-959-49A
70 µm Cell Strainer	Falcon	352350
80° incubator	VWR	1370GM
Alexa Fluor 488 goat anti-mouse IgG (H+L)	Invitrogen	A11029
Alexa Fluor 594 goat anti-rabbit IgG (H+L)	Invitrogen	A11037
Alexa Fluor 488 Phalloidin	Invitrogen	A12379
Antibiotic/Antimycotic solution	ThermoFisher Scientific	15240062
Anti-Connexin 43/GJAI antibody	Abcam	ab11370	Rabbit polyclonal
Anti-Sarcomeric α-actinin	Abcam	ab9465	Mouse monoclonal
Benchtop Freeze Dryers	Labconco	77500-00 K
Biosafety cabinet	Sterilgard	A/B3
Carbon rod electrodes	SGL Carbon Group	6971105
Centrifuge	Eppendorf	5804
CO2 incubator	Forma Scientific	3110
Collagenase, Type II, Powder	Gibco	17-101-015
Confocal Microscope	Zeiss	LSM 880
COOH Functionalized Carbon Nanotubes	NanoLab	PD30L5-20-COOH
Dicing saw machine	Giorgio Technology	DAD-321
DMEM, High Glucose	Gibco	11-965-118
DPBS without Calcium and Magnesium	Gibco	14-190-144
E-beam evaporator	CHA	57367
Fetal Bovine Serum	Gibco	10-437-028
Gelatin	Sigma-Aldrich	G9391	Type B, 300 bloom from porcine skin
Glass slide	VWR	48382-180
HBSS without Calcium, Magnesium or Phenol Red	Gibco	14-175-079
Inverted optical microscope	Olympus	CK40
Magnetic hotplate	Corning	PC-420
methacrylic anhydride	Sigma-Aldrich	276695	Contains 2,000ppm topanol A as inhibitor
Nunc EasYFlask 175cm2	ThermoFisher Scientific	159910
Olicscope	Siglent	SDS1052DL+
Paraformaldehyde Aqueous Solution -16%	Electron Microscopy Sciences	15710
PDMS SYLGARD 184	Sigma-Aldrich	761036
Photomask	Mini micro stencil inc
Platinum wire	Alfa Aesar	AA43014BU
Polyethylene glycol dimethcrylate	Polysciences Inc.	15178-100
Regenerated Cellulose Dialysis Tubing	Fisherbrand	21-152-14
Silver Epoxy Adhesive	MG Chemicals	8330S
Stericup Quick Release-GP Sterile Vacuum Filtration System	Millipore	S2GPU02RE
Ultra sonicator	Qsonica	Q500
UV Curing System	OmniCure	S2000
Vortex mixer	Scientific Industry	SI-0246A
Waveform generator	Agilent	33500B
Wrap Aluminium foil	Reynolds	N/A