Muscle-driven Bioelectronic Robots (eBiobots) with Remote Control

Introduction:: Bioengineering approaches that combine living cellular components with three-dimensional scaffolds to generate motion can be used to develop a new generation of miniature robots. Integrating on-board electronics and remote control in these biological machines will enable various applications across engineering, biology, and medicine. Here, we present hybrid bioelectronic robots equipped with battery-free and microinorganic light emitting diodes for wireless control and real-time communication. Centimeter-scale walking robots were computationally designed and optimized to host on-board optoelectronics with independent stimulation of multiple optogenetic skeletal muscles, achieving remote command of walking, turning, plowing, and transport functions both at individual and collective levels. This work paves the way toward a class of biohybrid machines able to combine biological actuation and sensing with on-board computing.


Materials and Methods:: Optogenetic C2C12 myoblasts transfected with blue light–sensitive ion channel channelrhodopsin-2 tagged with tdTomato, namely, ChR2 (H134R), were cultured in the growth medium composed of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) of fetal bovine serum, 1% (v/v) of ʟ-glutamine, and 1% (v/v) of penicillin-streptomycin (Lonza). Five million cells/ml of ChR2 C2C12s were seeded in a 3D-printed mold after mixing with 30% (v/v) of Matrigel, fibrinogen (4 mg/ml), and thrombin with fibrinogen (0.5 U/mg of fibrinogen). To fabricate eBiobots, the cell/gel solution was seeded on 3D-printed mold. The growth medium was added to the seeded tissues with 3D-printed structures after 90 min of seeding, followed by 3 days of incubation for the compaction of cell/gel mixture with the same medium in 37°C. Three days after seeding, the medium was switched into the differentiation medium, DMEM containing 10% (v/v) of horse serum, 1% (v/v) of penicillin/streptomycin, 1% (v/v) of ʟ-glutamine, aminocaproic acid (1 mg/ml), and insulin-like growth factor-1 (1 ng/ml; Sigma-Aldrich) to differentiate myoblasts to myotubes in muscle actuator. The centimeter-scale (1.4 cm by 0.7cm) scaffold was designed to hold the optogenetic device (5 mm in diameter) in the center. The eBiobot was then deployed in the antenna arena to test its mobility and controllability. To systematically improve the eBiobot’s performance, we characterized the variation of muscle forces in each batch of the experiment, feeding that information into the next design iteration.


Results, Conclusions, and Discussions:: The battery-free, wireless optogenetic devices exploited resonant magnetic induction between paired transmission and a receiving antenna system operating at radio frequency (RF; 13.56 MHz) to power microinorganic light-emitting diodes to actuate the optogenetic muscles. Constituent layers for the wireless electronic module included a thin double-sided flexible circuit board composed of rolled annealed copper layers (18 μm) separated by a polyimide film (75 μm) and a conformal encapsulation layer of parylene (about 14 μm) to isolate the device from the cell culture medium. This encapsulation strategy has been proven to protect devices for over 80 days in saline solution and for over 60 days after in vivo implantation in rodents. In a step toward enhanced maneuverability, a second eBiobot inspired by bipedal architecture and biological motor units was designed to have two muscle actuators in combination with a central microcontroller (μC), which extends laterally to bring two circular plates that hold five μ-ILEDs in intimate proximity with the muscle actuators. To further highlight the eBiobot's ability to maneuver, we challenged it to traverse an obstacle course.

In this work, we present biohybrid electronic robots powered by optogenetic skeletal muscles and controlled by wireless optoelectronics. The robot architecture and locomotion performance were computationally optimized through a staged iterative process, incorporating fabrication and experimental testing for robustness. However, future use of cells from other organisms such as insects could potentially enable room temperature operation. Although the molecular weight of the hydrogels can be designed to produce a specific mechanical modulus and the hydrogel can be 3D-printed to produce a desired shape, the hydrogel scaffolds required hydration to maintain their printed form and the mechanical properties to produce the desired functional response upon the muscle actuation.

In conclusion, the building blocks demonstrated here could be used to design higher-order structures and systems that could combine the advantages of living tissues, 3D-printed additive manufacturing, and optoelectronics and pave the way for biohybrid miniature robots with integrated electronics and multicellular engineered living systems for a wide range of potential applications.


Acknowledgements (Optional): : This work was funded by NSF Emerging Frontiers in Research and Innovation C3 SoRo Grant 1830881; NSF Science and Technology Center Emergent Behavior of Integrated Cellular Systems Grant CBET0939511 (to R.B.); the Querrey Simpson Institute for Bioelectronics, Northwestern University.

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