Site-Specific Immobilization of Living Cells in 2 and 3 Dimensions on Surfaces and in Fluidic Devices

There is a substantial need for new chemical tools with the capability to arrange multiple cell types in 2- and 3-dimensions on surfaces in a rapid and precise manner. Such tools would provide scientists with models for the study of mono and cocultures of cells leading to a variety of applications including the development and screening of drugs, cellular synthetic biology, and the assembly and mechanistic study of in vitro tissue and biofilm mimics. Current live-cell patterning tools do not have the ability to produce assemblies of multiple cell types rapidly and repeatedly with good control over dimensional cell localization. However, by exploiting DNA complementarity, I have developed a patterning method that overcomes the previous limitations. In my approach, surfaces are patterned with single strands of DNA enabling hybridization-directed deposition of cells displaying complementary DNA sequences on their surface. Successive rounds of photolithography and etching provide a means to develop a glass or silicon surface selectively. Following this, the exposed areas are covalently modified with unique single-stranded DNA (ssDNA) capture probes. This method has enabled cell patterning of multiple cell types in a site-specific manner, with good resolution and feature sizes of less than 10 µm (the size of a single mammalian cell)./n/nIn previous work, the DNA-patterning method was limited to mammalian cells or to cells with chemically accessible amines. Herein, a protocol is developed to modify oxidized cell surface diols with hydrazide-ssDNA; this new chemical biology technology expands DNA-directed cell patterning to include Gram positive and negative bacteria, eukaryotes with cell walls, and protists like algae. My method is the first for directly and site-specifically patterning multiple microorganisms on one substrate using ssDNA. Together with the previous mammalian cell modification technique, DNA-directed cell patterning now accommodates all kingdoms of life, and can be performed on a variety of materials and in microfluidic devices./nCompared to previous photolithographic-based ssDNA patterning methods, my direct photopatterning method for covalently modifying a substrate surface reduces the process complexity as well as the amount of time and reagent needed to pattern multiple DNA sequences on a surface. This is the first demonstration of a direct, chemoselective photopatterning method capable of incorporating multiple unique chemical species on one surface. Using an oxidative coupling reaction between aniline and an azidophenol, which acts as a photocaged iminoquinone, UV initiated chemical reactivity is demonstrated for functionalization of surfaces with ssDNA. By irradiating through a photomask, the mask features are transferred to a surface of interest for creation of complex patterns directly and covalently on an aniline substrate. This procedure can be expanded to encompass the patterning of any molecule type that displays an azidophenol moiety, allowing for the prospective use of this technique for the creation of complex patterns of molecules ranging from small molecules to protein assemblies./nIn summary, I developed a new method to derivitize the surface of living cells with cell walls with ssDNA for site-specific patterning. To reduce the time and complexity of multi-sequence DNA patterning, a new photopatterning method was also developed to allow for the direct and covalent modification of surfaces with multiple ssDNA sequences. Finally, these methods were used to arrange cells in complex designs on glass surfaces and in fluidic channels. Together, these procedures have enabled the assembly of on-chip cocultures for optical and impedance monitoring of tissue models consisting of endothelial cells, vascular smooth muscle cells, T-cells, and/or basophils for the real-time study of allergy response or vasculature assembly and growth. Overall, these advancements in ssDNA-directed patterning methods provide heretofore-unprecedented tools for rapid and site-specific assembly of any desirable combination of cell type and molecule and for the functional study of these mono or coculture on surfaces and in microfluidic devices.

Advisor: Francis, Matthew B
Mathies, Richard A