By Prof. Jan H. Hoh, Johns Hopkins School of Medicine

In multicellular organisms cells have highly specialized functions that depend on signals from their local microenvironment.  Understanding the relationship between these signals and the cellular responses requires the ability to quantify them.  In many simple cases there are direct ways for doing so, such as measuring the concentration of a ligand or the steepness of a gradient. However, important signals are often compositionally and spatially complex. For example, in a developing nervous system axonal growth is directed by the composition and distribution of molecules in the extracellular environment.  Likewise, the migration of cancer cells involves responses to the organization of specific molecules in the surroundings.  One powerful approach to understanding how cells process spatial signals is based on using micropatterned substrates to control the distribution of signaling molecules.  Here we propose that the Shannon information theory formalism provides a robust and useful way to quantify the organization of proteins in micropatterned systems.  To demonstrate the use of informational entropy as a metric we microfluidically patterned of lines of fibronectin with varying information content.  Fibroblasts grown on these patterns were sensitive to very small changes in informational entropy (6.6 bits), and the responses depended on the scale of the pattern.

By Dr. Jim Johnson, Ph.D., Senior Scientist, BioForce Nanosciences, Inc.

Cell biology studies involving the manipulation, perturbation or detection of material in and near small clusters or even single eukaryotic cells have importance in cell science.  The means to place, affix and manipulate cells became available with advances in MEMs technology and microcontact printing, surface chemistries and developments in the biology of extracellular matrix proteins and cell ligands.

New techniques enabling the deposition of femtoliter to attoliter amounts of proteins in solutions have created the ability to produce single and multicomponent protein patterns in virtually an infinite variety thus facilitating study of many cell biology problems. Some of the more salient problems and solutions to the application of ECM proteins to substrata, immobilization protocols with emphasis on the retention of protein functionality, the microenvironment needed for attachment of cells, and the responses of cells to attachment on surfaces will be discussed in this webinar.

Introduction:

-Single cells in isolation-differentiation morphogenesis
-Single cell biosensors
-Cell interactions – distance, signal transduction, communication
-Agent transposition through cells
-Agent transfer between cells
-Interaction and interference with substrates
-Mixed cell populations- XY formats
-Multidimensional tissue constructs

Salient Factors and Deliberations:

-Control of spot size, pitch, repetitions (array of arrays), Change on the fly
-Amount of substance in spot, substance density/area
-Flexibility of pattern design and spacing
-Multicomponent patterns, (interlacing dot maps, line maps)
-Single spot  –  Multicomponent gradient patterns
-Others

Prof. Nathaniel C. Cady
College of Nanoscale Science & Engineering
University at Albany

Spatial control of biomolecules is critically important for the development of biosensors and for studying cellular interactions with surfaces.  Precision patterning of biomolecules onto sensor surfaces requires detailed knowledge of both surface properties and the interactions between the printed molecules and the substrate material.  With the goal of producing ultra-sensitive electronic biosensors, our laboratory has developed strategies for printing nucleic acids and proteins onto advanced semiconductor materials.  Our results demonstrate that printed biomolecules can be directly adsorbed onto these materials with high spatial control and retention of functionality.  We have also developed bacterial arrays to study cell signaling and quorum sensing within defined microenvironments.  These studies highlight the use of precision printing with the BioForce Nano eNabler system and key parameters involved in surface preparation and choice of surface adhesion / adsorption strategies.

Dr. Ying Mei
Department of Chemical Engineering
Massachusetts Institute of Technology

Recent developments in micro- and nanoscale technology have enabled the generation of extracellular-matrix (ECM) protein microarrays with well-defined geometries. These patterned surfaces have shown utility for the study and control of a variety of cellular behaviors. In particular, the patterning of proteins with feature sizes smaller than a single cell has demonstrated potential application for use as tools to control cellular activity. To date, most research has been limited to studies with single protein factors due to the technical limitations of existing printing methods. Herein, we describe the development of a microscale direct writing (MDW) technology for the generation of complex ECM protein arrays at subcellular feature size with multiple components. Automated printing techniques based on atomic force microscopy (AFM) were developed to allow programmable generation of cell-compatible surfaces, with multiple ECM proteins, at a subcellular feature size of 6–9 micrometers. Cell-compatible, two-component ECM protein arrays were systematically generated with varying spacing and composition. These arrays were then studied for their effects on the cellular attachment and spreading of a model cell line, mouse myofibroblasts. Interestingly, the precise tuning of spacing and placement of two components at subcellular resolution can lead to an increase in cellular alignment. Given the complexity of the in vivo cellular microenvironment, we believe the MDW methods described here could prove generally applicable for the study and optimization of biomaterial surfaces.