Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions

Centaur: A Mobile Dexterous Humanoid for Surface Operations

Future human and robotic planetary expeditions could benefit greatly from expanded Extra-Vehicular Activity (EVA) capabilities supporting a broad range of multiple, concurrent surface operations. Risky, expensive and complex, conventional EVAs are restricted in both duration and scope by consumables and available manpower, creating a resource management problem. A mobile, highly dexterous Extra-Vehicular Robotic (EVR) system called Centaur is proposed to cost-effectively augment human astronauts on surface excursions. The Centaur design combines a highly capable wheeled mobility platform with an anthropomorphic upper body mounted on a three degree-of-freedom waist. Able to use many ordinary handheld tools, the robot could conserve EVA hours by relieving humans of many routine inspection and maintenance chores and assisting them in more complex tasks, such as repairing other robots. As an astronaut surrogate, Centaur could take risks unacceptable to humans, respond more quickly to EVA emergencies and work much longer shifts. Though originally conceived as a system for planetary surface exploration, the Centaur concept could easily be adapted for terrestrial military applications such as de-Gig, surveillance and other hazardous duties

Spatiotemporal NF-κB dynamics encodes the position, amplitude, and duration of local immune inputs

Infected cells communicate through secreted signaling molecules like cytokines, which carry information about pathogens. How differences in cytokine secretion affect inflammatory signaling over space and how responding cells decode information from propagating cytokines are not understood. By computationally and experimentally studying NF-κB dynamics in cocultures of signal-sending cells (macrophages) and signal-receiving cells (fibroblasts), we find that cytokine signals are transmitted by wave-like propagation of NF-κB activity and create well-defined activation zones in responding cells. NF-κB dynamics in responding cells can simultaneously encode information about cytokine dose, duration, and distance to the cytokine source. Spatially resolved transcriptional analysis reveals that responding cells transmit local cytokine information to distance-specific proinflammatory gene expression patterns, creating "gene expression zones."Despite single-cell variability, the size and duration of the signaling zone are tightly controlled by the macrophage secretion profile. Our results highlight how macrophages tune cytokine secretion to control signal transmission distance and how inflammatory signaling interprets these signals in space and time

SBC2008-193021 COMPUTATIONAL SIMULATION OF A MEMS-BASED MICROACTUATOR FOR TISSUE ENGINEERING APPLICATIONS

INTRODUCTION The relationship between the 3D microstructure of tissueengineered constructs (TECs) and their resulting mechanical and biological function is critical in providing TECs with clinically meaningful mechanical properties in reasonable incubation times. We hypothesize that the next generation of TECs must incorporate a controllable and optimized microstructure (and resulting mechanical properties) if they are to mechanically and biologically mimic tissue function. While the development of a robustly engineered tissue replacement will undoubtedly require simultaneous biochemical and biomechanical stimulation, this paper will focus on the development of a device to impose localized micro-mechanical stimulation. In this paper a MEMS-based device is introduced that can differentially stimulate the mechanical microenvironment of TECs using a noninvasive magnetic actuation mechanism. The device consists of a bed of micro-flaps (MFs) that are doped with a para/ferromagnetic material. Since the fabrication of such a device can be costly, the purpose of the current work is to develop a computational tool that can aid in device design. Finite element models (FEMs) are introduced to determine the relationship between MF/magnet size/properties and horizontal MF deflection. Towards this end, a 2D magneto-structural model was created to guide the development of a microdevice with a desired MF deflection

A Mechanostimulation System for Revealing Intercellular Calcium Communication in HUVEC Networks

Abstract -This paper reports a mechanostimulation system for studying mechanically induced intercellular calcium signaling in networks of human umbilical vein endothelial cells (HUVECs). By incorporating a capacitive (comb drive) force probe and plasma lithography cell patterning, the roles of biophysical factors, including force, duration, and network architecture, in calcium intercellular communication can be investigated systematically. Particularly, we observed cancellation of calcium waves in linear networks and bi-directional splitting in cross junctions. The effects of key biophysical factors on intercellular calcium wave propagation were studied. These results demonstrate the applicability of the mechanostimulation system in studying intercellular calcium signaling and reveal the robustness of calcium signaling in HUVEC networks, which mimics the vasculature

MICROENVIRONMENTS FOR STUDY OF MYOGENESIS SPATIAL ORGANIZATION AND ENDOTHELIAL CELL SMALL MESSENGER SIGNALING

In complex organisms, the combined actions of multiple cells enable higher order functions while using only the building blocks of individual cells. Understanding the regulation of groups of cells is thus critical in order to uncover how single cells combine to produce higher levels of functionality. One principle input which regulates cell behavior is the surrounding cellular environment, many aspects of which are inherently nano- or microscale in nature. A system has therefore been developed which can replicate important micro- and nanoscale aspects of the cellular environment by providing inputs to groups of cells which mimic those found physiologically. This has been accomplished by developing a cell sensitive plasma surface patterning technique termed plasma lithography that produces area selective functional group modification to provide cell attachment guidance at sizes ranging from 100 nm to millimeters. This surface patterning system has further been coupled with additional inputs such as chemical and mechanical stimulation in order to investigate several areas where higher order functionality is observed based upon interactions of single cells. These investigations include study of an autocatalytic feedback mechanism which is involved in muscle formation and the behavior of small messenger signaling in networks of vascular cells

High-Content Quantification of Single-Cell Immune Dynamics

Cells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-κB, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages