|Abstract:||The ability of designing, biofabricating and programming engineered tissue constructs, as well as modularly assembling them to achieve the engineering of heterotypic living systems is a key element for the progress of “New Biology”. This perspective consists of utilizing engineering concepts for design of complex systems and combining with the principles and properties of multiple disciplines in cellular biology at various length scales to drive composition of living material that are organized in a way that allows for the emergence of novel functionalities. Advances in this field can constitute an experimental platform that can guide the discovery of factors that allow the emergence of properties that can translate into a richer toolbox with which to address real world problems.
One important example is the development of biological machines, namely consisting of motor neurons as a source of processing and activation and skeletal muscle as the actuator of the system. Towards this goal, advances in the generation of a neuro-muscular driven machine require an expansion of design parameters and arrangement of the assembled parts, guided by parameters established by natural processes. This dissertation presents advances on the expansion of this toolbox for the engineering of multicellular living systems consisting of muscle calls and motor neurons. The dissertation focuses on the modulation of the electrical activity of motor neuronal networks, the biofabrication of neural tissue containing motor neurons, and the redesigning of previous muscle-based walking robots for higher force generation.
First of all, the ability to uncover in-vitro methods to “reprogram” neural networks responsible for guiding the actuation of muscle is critical to engineer the dynamic functionalities needed in these biological machines. In this work, motor neurons were differentiated from mouse embryonic stem cells, which have been transfected to express a GFP reporter under the Hb9 promoter as well as Channelrhodopsin, to enable optogenetic stimulation of these neurons, which was used to design the training regimens. Past efforts have not considered reprogramming in terms of evoking long-term changes in firing patterns of in-vitro networks by training regimens during stages of neural development. Thus, in this dissertation, short and long-term programming of neural networks was explored by using optical stimulation to induce training regimens implemented during neurogenesis and synaptogenesis, and ultimately demonstrating correlation between these and the resulting plastic responses.
Furthermore, to design multicellular engineered living systems subunits that could facilitate the assembly of heterotypic biological machines, a novel biofabrication approach is proposed to form functional in-vitro neural tissue mimics (NTM) using mouse embryonic stem cells, a fibrin matrix, and 3D printed molds. This method can provide a large degree of design flexibility for development of in-vitro functional neural tissue models of varying shapes and forms for applications in the developments of engineered living systems for therapeutic biomedical research, drug discovery and disease modeling, and new engineering and research models. This type of biological manufacturing method is a good representation of the utilization and expansion of biological design principles to guide the creation of self-organized complex functional structures, thus enriching the toolbox for the top-down engineering approach.
Finally, the development of skeletal muscle driven biological machines require optimized approaches for design given the mechanical dynamics between the actuating muscle and the scaffold. This means that advancing these biologically driven soft robots will involve employing different scaffold geometries and cellular constructs to enable a controllable emergence for increased production of force and functionality. Having the flexibility of altering geometry while ensuring tissue viability can enable advancing functional output from these machines through the implementation of new construction concepts and fabrication approaches. Furthermore, a forward engineering approach was developed to design next generation biological machines via direct numerical simulations and subsequent fabrication of high force producing biological machines.
The present thesis aims to propose the development of building blocks and modular technologies surrounding the realization of motor neuronal driven machines, through the implementation of effective training regimens to reprogram neural networks, implementing biofabrication techniques for neural tissues and optimizing design and scaling force produced by the muscle actuators in these biological machines. These stepwise enhancements in the engineering of the subunits are critical for the successful assembly of more complex structures and our understanding of how to build on these designs towards solving real world problems in medicine, environment, and manufacturing.