Michael Junkin

Probing cell migration in confined environments by plasma lithography.

Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell-substrate interactions, and cell-cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell-microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory behavior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.

Cellular self-organization by autocatalytic alignment feedback

Myoblasts aggregate, differentiate and fuse to form skeletal muscle during both embryogenesis and tissue regeneration. For proper muscle function, long-range self-organization of myoblasts is required to create organized muscle architecture globally aligned to neighboring tissue. However, how the cells process geometric information over distances considerably longer than individual cells to self-organize into well-ordered, aligned and multinucleated myofibers remains a central question in developmental biology and regenerative medicine. Using plasma lithography micropatterning to create spatial cues for cell guidance, we show a physical mechanism by which orientation information can propagate for a long distance from a geometric boundary to guide development of muscle tissue. This long-range alignment occurs only in differentiating myoblasts, but not in non-fusing myoblasts perturbed by microfluidic disturbances or other non-fusing cell types. Computational cellular automata analysis of the spatiotemporal evolution of the self-organization process reveals that myogenic fusion in conjunction with rotational inertia functions in a self-reinforcing manner to enhance long-range propagation of alignment information. With this autocatalytic alignment feedback, well-ordered alignment of muscle could reinforce existing orientations and help promote proper arrangement with neighboring tissue and overall organization. Such physical self-enhancement might represent a fundamental mechanism for long-range pattern formation during tissue morphogenesis.

Evaporation-induced assembly of biomimetic polypeptides

We report an evaporation assisted plasma lithography (EAPL) process for guided self-assembly of a biomimetic silk-elastinlike protein (SELP). We demonstrate the formation of SELP structures from millimeter to submicrometer range on plasma-treatment surface templates during an evaporation-induced self-assembly process. The self-assembly processes at different humidities and droplet volumes were investigated. The process occurs efficiently in a window of optimized operating conditions found to be at 70% relative humidity and 8μl volume of SELP solution. The EAPL approach provides a useful technique for the realization of functional devices and systems using these biomimetic materials.

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. This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level. This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

Mechanically Induced Intercellular Calcium Communication in Confined Endothelial Structures

Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures.

Calcium Wave Propagation in Networks of Endothelial Cells: Model-based Theoretical and Experimental Study

In this paper, we present a combined theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. We consider multicellular structures composed of a single chain of cells as well as a chain of cells with a side branch, namely a “T” structure. In the experiments, we investigate the result of applying mechano-stimulation to induce signaling in the form of calcium waves along the chain and the effect of single and dual stimulation of the multicellular structure. The experimental results provide evidence of an effect of architecture on the propagation of calcium waves. Simulations based on a model of calcium-induced calcium release and cell-to-cell diffusion through gap junctions shows that the propagation of calcium waves is dependent upon the competition between intracellular calcium regulation and architecture-dependent intercellular diffusion.

Adjustable nanomanufacturing using template-guided self-assembly

Template-guided self-assembly is one of the most promising approaches for creating functional nanosystems and effective methods are needed in order to generate micro and nanoscale templates for a diverse type of materials necessary for different applications. In this paper a plasma lithography technique is demonstrated which can directly pattern a wide variety of substrates including polystyrene, PDMS and glass for self-assembly of nanomaterials. The technique employs a deformable mold made using PDMS replication molding. The mold protects selective areas of the substrate from plasma surface treatment, which will spatially alter the surface properties of the substrate. Nanomaterials will then selectively self-assemble onto the pattern. Three-dimensional control is achieved by means of mold geometry and self-assembly conditions. Nanomaterials including nanoparticles, proteins, and salts have been patterned on configurations from arrays of lines and dots to arbitrary shapes in sizes ranging from millimeters to hundreds of nanometers.

A mechanostimulation system for revealing intercellular calcium communication in HUVEC networks

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.

Plasma lithography for probing collective cell behaviors

Collective cell motions play essential roles in the regulation of various complex biological processes such as embryogenesis, tissue regeneration, and carcinogenesis. In this study, we present a novel plasma lithography technique for creating surface functionalized confinements toward the investigation of collective cell migration. The ability to confine cells one-dimensionally using plasma lithography is demonstrated to facilitate quantification of collective cell migratory behaviors including the overall speed and the probability of velocity change upon cell-cell contact. This quantitative information in cell-cell interactions will allow us to understand the dynamics of collective cell migration from a systems biology perspective.

An integrated mechanostimulation system for probing architecture based calcium signaling in HUVEC cells

Dynamic signal conduction in endothelial networks plays an important role in endothelial function, and characteristics of the network architecture itself are theorized to play a role in this function. We have therefore developed an integrated mechanostimulation system to create spatiotemporal stimuli including geometric cues, fluidic shear, mechanical deformation, and tunable surface stiffness for probing intercellular communication in artificial networks of human umbilical vein endothelial (HUVEC) cells. The system enables detection of architecture dependent (e.g. linear, grid, and branching patterns), spatiotemporal calcium propagation characteristics such as speed, contact length, and repeated stimulation dependence due to mechanostimulation at the single cell level.