Biological building blocks for 3D printed cellular systems

Advancements in the fields of tissue engineering, biomaterials, additive manufacturing, synthetic and systems biology, data acquisition, and nanotechnology have provided 21st-century biomedical engineers with an extensive toolbox of techniques, materials, and resources. These “building blocks” could include biological materials (such as cells, tissues, and proteins), biomaterials (bio-inert, -instructive, -compatible, or -degradable), soluble factors (growth factors or small molecules), and external signals (electrical, chemical, or mechanical). “Forward engineering” attempts to integrate these building blocks in different ways to yield novel systems and machines that, by promoting new relationships and interactions among their individual components, are greater than the sum of their parts. Drawing from an extensive reserve of parts and specifications, these bio-integrated forward-engineered cellular machines and systems could acquire the ability to sense, process signals, and produce force, and could also contain a countless array of applications in drug screening and delivery, programmable tissue engineering, and biomimetic machine design. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. In contrast to traditional machines that use external energy to produce an output, muscle cells can be fueled by glucose and other biomolecules. While cardiac cell driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel “bio-bots” powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot upon applied electrical stimulation. The muscle strips were composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. The hierarchical organization, modularity, and scalable nature of mature skeletal muscle fibers (which can be combined in parallel to increase force production, for example), lends itself to “building with biology.” Few systems have shown net movement from an autonomous, freestanding biological machine composed of skeletal muscle, and even fewer have attempted to incorporate multiple cell types for greater functionality. Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking. We also present a modular heterotypic cellular system, made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons embedded in an extracellular matrix. Site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allowed for muscle contraction via chemical stimulation of motor neurons with glutamate, a major excitatory mammalian neurotransmitter, with the frequency of contraction increasing with glutamate concentration. The addition of the nicotinic receptor antagonist tubocurarine chloride halted the contractions, indicating that muscle contraction was motor neuron-induced. We also present a thorough characterization and optimization of a co-culture system that harnesses the potential of engineered skeletal muscle tissue as the actuating component in a biological machine through the incorporation of motor neurons, and creates an environment that is amenable to both cell types and prime for functional neuromuscular formation. With a bio-fabricated system permitting controllable mechanical and geometric attributes on a range of length scales, our novel engineered cellular system can be utilized for easier integration of other modular “building blocks” in living cellular and biological machines. We are poised to design the next generation of complex biological machines with controllable function, specific life expectancy, and greater consistency. In the future, we envision that this system can be used for applications beyond bio-robotics and muscular actuators; as a functioning heterotypic co-culture, the muscle- neuron arrangement is also a highly relevant machine for the study of neuromuscular diseases and related drug toxicity studies. These results could prove useful for the study of disease-specific models, treatments of myopathies such as muscular dystrophy, and tissue engineering applications

A 3D Muscle-ECM Construct to Power a Biological Actuator

Our research focuses on the use of skeletal muscle as a power source for soft robotic biological machines. A supportive extracellular matrix (ECM) environment is important for the maturation and maintenance of many cells and tissues, including muscle. Here, we embed skeletal muscle myoblasts (precursor cells) in a solution of ECM proteins such as collagen and fibrin, allowing them to grow and differentiate in a physiologically relevant 3D environment, to create a biological machine that actuates upon muscle contraction and responds to various external stimuli. Immuno-fluorescent staining offers a simple and visually appealing method to verify the presence of certain cellular components or proteins in a biological sample. The top-left image demonstrates a region of the muscle, which shows sarcomeric myosin (a motor protein involved in muscle contraction) in green, channelrhodopsin-2 (a membraneion channel that opens and allows for cell contraction in response to blue light) in red, and cell nuclei in blue. The colors of the remaining images have been inverted or altered for aesthetic appeal, with the relevant sub-cellular localization remaining visible. The result is an art form that is appealing to scholars of all backgrounds while still conveying important biological information

The development of a skeletal muscle bio-actuator using 3-D stereolithography

Over the past decade, a new scientific discipline has emerged, integrating mechanics with biology to create complex engineered living systems. The building blocks – different cell types in an instructive environment – can be assembled in various ways to promote the emergence (or natural evolution and interaction) of the cells in a system with well-defined functionality. These functions could include sensing, information processing, protein expression, and actuation, among countless others. This Thesis presents a novel cellular system capable of actuation and fabricated using cells and hydrogels. A stereolithographic 3D printing technique (SLA) was used to create a hydrogel backbone made of a beam connecting two pillars that supports a muscle strip created from skeletal muscle cells in a fibrin-based matrix. The entire device is termed a “bio-bot,” or biological robot. Contraction of the cells within the muscle strip produced enough force to move the pillars and displace the bio-bot on a surface in a liquid medium. This Thesis is focused on the development and characterization (mechanical and biological) of the skeletal muscle-based biological actuator. The use of the SLA allowed for easy modifications of the polymerized part’s geometry and material properties. Increasing the energy dose of polymerization produced a stiffer beam that restricted bending and increased the passive tension in the muscle strip. 15-19 days after cell seeding, the bio-bots displayed spontaneous contraction that resulted in a net displacement of up to ~6 mm in 10 minutes. During this time span, a maximum velocity of over 1890 μm/min was achieved. Future plans are focused on controlling the activity of the bio-bot using optogenetics

Biodegradable Monocrystalline Silicon Photovoltaic Microcells As Power Supplies For Transient Biomedical Implants

Bioresorbable electronic materials serve as foundations for implantable devices that provide active diagnostic or therapeutic function over a timeframe matched to a biological process, and then disappear within the body to avoid secondary surgical extraction. Approaches to power supply in these physically transient systems are critically important. This paper describes a fully biodegradable, monocrystalline silicon photovoltaic (PV) platform based on microscale cells (microcells) designed to operate at wavelengths with long penetration depths in biological tissues (red and near infrared wavelengths), such that external illumination can provide realistic levels of power. Systematic characterization and theoretical simulations of operation under porcine skin and fat establish a foundational understanding of these systems and their scalability. In vivo studies of a representative platform capable of generating ≈60 µW of electrical power under 4 mm of porcine skin and fat illustrate an ability to operate blue light-emitting diodes (LEDs) as subdermal implants in rats for 3 d. Here, the PV system fully resorbs after 4 months. Histological analysis reveals that the degradation process introduces no inflammatory responses in the surrounding tissues. The results suggest the potential for using silicon photovoltaic microcells as bioresorbable power supplies for various transient biomedical implants

Optogenetic skeletal muscle-powered adaptive biological machines

Complex biological systems sense, process, and respond to their surroundings in real time. The ability of such systems to adapt their behavioral response to suit a range of dynamic environmental signals motivates the use of biological materials for other engineering applications. As a step toward forward engineering biological machines (bio-bots) capable of nonnatural functional behaviors, we created a modular light-controlled skeletal muscle-powered bioactuator that can generate up to 300 µN (0.56 kPa) of active tension force in response to a noninvasive optical stimulus. When coupled to a 3D printed flexible bio-bot skeleton, these actuators drive directional locomotion (310 µm/s or 1.3 body lengths/min) and 2D rotational steering (2°/s) in a precisely targeted and controllable manner. The muscle actuators dynamically adapt to their surroundings by adjusting performance in response to “exercise” training stimuli. This demonstration sets the stage for developing multicellular bio-integrated machines and systems for a range of applicationsNational Science Foundation (U.S.) (Science and Technology Center Emergent Behavior of Integrated Cellular Systems (EBICS) Grant CBET-0939511)National Science Foundation (U.S.) (Graduate Research Fellowship, Grant DGE-1144245)National Science Foundation (U.S.) (NSF Cellular and Molecular Mechanics and Bionanotechnology (CMMB) Integrative Graduate Education and Research Traineeship (IGERT) at UIUC (Grant 0965918)

Mapping Astrocyte Transcriptional Signatures in Response to Neuroactive Compounds

Astrocytes play central roles in normal brain function and are critical components of synaptic networks that oversee behavioral outputs. Despite their close affiliation with neurons, how neuronal-derived signals influence astrocyte function at the gene expression level remains poorly characterized, largely due to difficulties associated with dissecting neuron- versus astrocyte-specific effects. Here, we use an in vitro system of stem cell-derived astrocytes to identify gene expression profiles in astrocytes that are influenced by neurons and regulate astrocyte development. Furthermore, we show that neurotransmitters and neuromodulators induce distinct transcriptomic and chromatin accessibility changes in astrocytes that are unique to each of these neuroactive compounds. These findings are highlighted by the observation that noradrenaline has a more profound effect on transcriptional profiles of astrocytes compared to glutamate, gamma-aminobutyric acid (GABA), acetylcholine, and serotonin. This is demonstrated through enhanced noradrenaline-induced transcriptomic and chromatin accessibility changes in vitro and through enhanced calcium signaling in vivo. Taken together, our study reveals distinct transcriptomic and chromatin architecture signatures in astrocytes in response to neuronal-derived neuroactive compounds. Since astrocyte function is affected in all neurological disorders, this study provides a new entry point for exploring genetic mechanisms of astrocyte–neuron communication that may be dysregulated in disease

Three-Dimensionally Printed Biological Machines Powered by Skeletal Muscle

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel “bio-bots” with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼156 μm s−1, which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design