MicroBioRobots for Single Cell Manipulation

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable micro actuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a bio-integrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 μm coated with a monolayer of the swarming Serratia marcescens. The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments, which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment

Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues

Cellular contractility, migration, and extracellular matrix (ECM) mechanics are critical for a wide range of biological processes including embryonic development, wound healing, tissue morphogenesis, and regeneration. Even though the distinct response of cells near the tissue periphery has been previously observed in cell-laden microtissues, including faster kinetics and more prominent cell-ECM interactions, there are currently no models that can fully combine coupled surface and bulk mechanics and kinetics to recapitulate the morphogenic response of these constructs. Mailand \textit{et al.} (2019) had shown the importance of active elastocapillarity in cell-laden microtissues, but modeling the distinct mechanosensitive migration of cells on the periphery and the interior of highly deforming tissues has not been possible thus fur, especially in the presence of active elastocapillary effects. This paper presents a framework for understanding the interplay between cellular contractility, migration, and ECM mechanics in dynamically morphing soft tissues accounting for distinct cellular responses in the bulk and the surface of tissues. The major novelty of this approach is that it enables modeling the distinct migratory and contractile response of cells residing on the tissue surface and the bulk, where concurrently the morphing soft tissues undergoes large deformations driven by cell contractility. Additionally, the proposed model is validated through simulation results that capture the changes in shape and cell concentration for wounded and intact microtissues, enabling the interpretation of experimental data.Comment: 20 pages, 13 figure

Surface and bulk stresses drive morphological changes in fibrous microtissues

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.Published versio

Mechanical Characterization and Shape Optimization of Fascicle-Like 3D Skeletal Muscle Tissues Contracted with Electrical and Optical Stimuli

Here we present a quantitative approach to constructing effective 3D muscle tissues through shape optimization and load impedance matching with electrical and optical stimulation. We have constructed long, thin, fascicle-like skeletal muscle tissue and optimized their form factor through mechanical characterization. A new apparatus was designed and built which allowed us to measure force-displacement characteristics with diverse load stiffnesses. We have found that a) there is an optimal form factor that maximizes the muscle stress, b) the energy transmitted to the load can be maximized with matched load stiffness, and c) optical stimulation using channelrhodopsin2 in the muscle tissue can generate twitch force as large as its electrical counterpart for well developed muscle tissue. Using our tissue construct method we found an optimal initial diameter of 500 microns outperformed tissues using 250 microns by more than 60% and tissues using 760 microns by 105%. Using an optimal load stiffness, our tissues have generated 12 pJ of energy at a peak generated stress of 1.28 kPa. Additionally, the difference in optically stimulated twitch performance vs. electrically stimulated is a function of how well the overall tissue performs, with average or better performing strips having less than 10% difference. The unique mechanical characterization method used is generalizable to diverse load conditions and will be used to match load impedance to muscle tissue impedance for a wide variety of applications.National Science Foundation (U.S.) (Grant No. CBET-0939511)Singapore-MIT Alliance for Research and Technology (BioSyM IRG)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular System

Adaptive locomotion of artificial microswimmers

Bacteria can exploit mechanics to display remarkable plasticity in response to locally changing physical and chemical conditions. Compliant structures play a striking role in their taxis behavior, specifically for navigation inside complex and structured environments. Bioinspired mechanisms with rationally designed architectures capable of large, nonlinear deformation present opportunities for introducing autonomy into engineered small-scale devices. This work analyzes the effect of hydrodynamic forces and rheology of local surroundings on swimming at low Reynolds number, identifies the challenges and benefits of utilizing elastohydrodynamic coupling in locomotion, and further develops a suite of machinery for building untethered microrobots with self-regulated mobility. We demonstrate that coupling the structural and magnetic properties of artificial microswimmers with the dynamic properties of the fluid leads to adaptive locomotion in the absence of on-board sensors

Adaptive locomotion of artificial microswimmers.

Bacteria can exploit mechanics to display remarkable plasticity in response to locally changing physical and chemical conditions. Compliant structures play a notable role in their taxis behavior, specifically for navigation inside complex and structured environments. Bioinspired mechanisms with rationally designed architectures capable of large, nonlinear deformation present opportunities for introducing autonomy into engineered small-scale devices. This work analyzes the effect of hydrodynamic forces and rheology of local surroundings on swimming at low Reynolds number, identifies the challenges and benefits of using elastohydrodynamic coupling in locomotion, and further develops a suite of machinery for building untethered microrobots with self-regulated mobility. We demonstrate that coupling the structural and magnetic properties of artificial microswimmers with the dynamic properties of the fluid leads to adaptive locomotion in the absence of on-board sensors.ER

Single Cell Manipulation using Ferromagnetic Composite Microtransporters

For biomedical applications, such as single cell manipulation, it is important to fabricate microstructures that can be powered and controlled wirelessly in fluidic environments. In this letter, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic microtransporters driven by external magnetic fields. Microtransporters were fabricated using a simple, single step fabrication method and can be produced in large numbers. We demonstrate that they can be navigated to manipulate single cells with micron-size precision without disturbing the local environment