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MicroRobotic toolkit to deliver spatiotemporally resolved physicochemical signals and control cell sociology

Periodic Reporting for period 2 - ROBOCHIP (MicroRobotic toolkit to deliver spatiotemporally resolved physicochemical signals and control cell sociology)

Reporting period: 2018-09-01 to 2020-02-29

Much of our understanding of the biological mechanisms that underlie cellular functions has been garnered from studying cells cultured on planar substrates. However, in vivo, cells primarily exist embedded within an information-rich 3D microenvironment that contains multiple extracellular matrix components. The microscale architecture of these fibrous networks constrains spatially where cells can form adhesions and imparts complex mechanical characteristics due to viscoelastic response to loading and fiber alignment. Beyond serving as a mechanical support and communication network for cells, the spatial presentation of diffusible factors are dictated by the structure and porosity of the surrounding ECM. Given these numerous intricacies, making sense of the dynamic communication among cells connected through a structurally complex fiber network in 3D requires a rigorous system identification effort and engineering analysis. To achieve this task, it is essential to develop technologies that can probe the dynamics of multicellular interactions in their social context at the tissue as well as the cellular level.

The distinguishing features of our technological approach are the fine control, extreme dexterity, high-throughput, and multi-dimensionality. We develop microrobotic technologies integrated with state-of-the-art microscopes that allow application of spatiotemporally resolved dynamic stimuli and real-time imaging. From the scientific perspective, our work serves as a test-bed for some of the most intriguing questions in development biology, tissue mechanics and regeneration. We are trying to address the following questions: What are the governing equations that relate the magnitude, timing and direction of force generation to the tissue mechanical properties, such as fibre alignment, viscosity and stiffness? How far, over what time, and in what geometry locally generated forces are transmitted? How cells maintain tissue boundaries, when they decide to move and expand the tissue and how the equilibrium achieved after an injury or change in boundary conditions are other important questions we are trying to address.

The technology we are developing also holds significant potential for translational medicine and targeted therapy. Biological systems are exquisitely sensitive to the location, dose, and timing of physiologic cues and pharmaceuticals. This spatiotemporal sensitivity indicates that diagnostic and therapeutic approaches with minimal off-target effects can be particularly efficacious. The acquired wisdom can aid in developing novel treatments that ensures a microenvironment with a distribution of signals that minimizes disease progression. Our work will also introduce delivery methods that can not only release its contents at the target site in a dose-dependent manner, but also contain and protect the payload during transport. These micromachines will noninvasively collect biological samples from remote pathological sites and make on site measurements for diagnostic purposes or perform intricate tasks like mechanical removal of occlusions.
So far, we developed a number of microrobotic platforms for the tethered and wireless micromanipulation of biological samples. Here, I will highlight three most mature techniques.

The first platform consists of a custom-design six-degrees-of-freedom robotic system that was constructed from piezoelectric stick-slip actuators that have submicron positioning precision. We used microelectromechanical systems technology to form arrays of free-standing 3D fibrous microtissues. We then performed robot-assisted microsurgery, such as local incisions and implantation, to study if and how they recover their original form and structure. In this tissue model, the cells are embedded inside a collagen matrix. The final form of the tissues is defined by the rules of self-assembly as well as boundary conditions. As all the parameters are quantified and under our control, we could recapitulate the experimental conditions with our computational modeling framework. We showed both experimentally via time-lapse microscopy and by using computer simulations that cells at the tissue boundary develop surface stresses and, together with contractile activity of cells residing in the core, drive macroscale deformation and tissue shaping.

The second platform consist of untethered soft robotic microdevices that can perform complex mechanical functions with high dexterity and performance. To this end, we combined the rapid and wireless response of hybrid nanomaterials with state-of-the-art microengineering techniques to develop machinery that can apply physiologically relevant mechanical loading at the cell and tissue scale. Gold nanoparticles efficiently transduce NIR laser light into heat, which in turn drives rapid and powerful collapse of thermoresponsive hydrogels. The microactuators exhibited mechanical properties and performance metrics that were comparable to that of living mammalian cells. NIR illumination provides effective spatiotemporal control over actuation (down to nanoscale spatial resolution at millisecond temporal resolution). The mechanical power generated by the actuators is transformed into desired set of movements with the aid of rationally designed compliant mechanisms. We developed a series of biomimetic models in which micromachines were seamlessly integrated with extracellular matrix or employed to externally load 3D culture models such as cancer spheroids and gastruloids. Preliminary results shed light on the strength and frequency of mechanical signals that lead to symmetry breaking and phenotypic transitions in epithelial constructs.

We developed a wireless microrobotic manipulation platform along with a computational framework to explore dynamical aspects of cellular organization on synthetic fibre networks. Our approach allows application of spatiotemporally defined deformations using magnetically controlled microactuators and mapping of stress using simulations of materials. We record spatial and temporal changes in the distribution and shape of cultured cells along with the topological map of the fibre network using time-lapse confocal microscopy. We constructed an experimentally validated finite element model that can recapitulate these dynamical changes. Application of well-defined forces to the fibres facilitates the characterization of the constitutive properties of the structures. The combined experimental and computational approach has started to reveal the effect of the characteristics of mechanical stimuli along with fibre properties on the tissue architecture.
The microrobotic toolkit that we have developed so far allows us to explore many aspects of mechanobiology that were previously inaccessible. We expect that the automated micromanipulation platforms will become instrumental to handle and operate on engineered tissues, organoids, and embryos. Performing protocols with robotic agents will broaden the number of laboratories that can contribute innovations and increase standardization of procedures across laboratories. We are hoping to reveal a much richer understanding on how mechanical signals are generated, transmitted, and processed by cells interacting through fibrous materials. We are developing cell-mimicking machinery that can be implanted or transported within fibrous matrices, which may revolutionize targeted therapies.
An illustration of untethered soft robotic microdevices mechanically loading microtissues.
An artistic rendering of the microsurgery platform operating on arrays of microtissues.