Micropatterning of 3D Microenvironments for Living Biosensor Applications.

Micro-scale printing and patterning of living cells has multiple applications including tissue engineering, cell signaling assays, and the fabrication of cell-based biosensors. In this work, a molecular printing instrument, the Bioforce Nano eNabler, was modified to enable micron-scale -quill-pen based printing of mammalian cells in a 3D hyaluronan/gelatin based hydrogel. Specifically, photo-initiated -thiol-ene click chemistry was used to couple the thiol groups of thiolated hyaluronan/thiolated gelatin to the alkene groups of 4-arm polyethylene glycol (PEG)-norbornene molecules. Rapid photopolymerization enabled direct printing and controlled curing of living cells within the hydrogel matrix. The resulting hydrogels were biocompatible with human adipose-derived stem cells, NIH-3T3 cells, and mouse embryonic stem cells. The utility of this printing approach was also explored for cell-based biosensors. Micro-printed cells expressing a redox sensitive variant of the green fluorescent protein (roGFP-R12) showed a measurable fluorescent response to addition of oxidizing and then reducing agents. This work represents a novel approach to micron-scale cell patterning, and its potential for living, cell-based biosensors.

Characterization of Thiol-Ene Crosslinked PEG Hydrogels.

The properties of synthetic hydrogels can be tuned to address the needs of many tissue-culture applications. This work characterizes the swelling and mechanical properties of thiol-ene crosslinked PEG hydrogels made with varying prepolymer formulations, demonstrating that hydrogels with a compressive modulus exceeding 600 kPa can be formed. The amount of peptide incorporated into the hydrogel is shown to be proportional to the amount of peptide in the prepolymer solution. Cell attachment and spreading on the surface of the peptide-functionalized hydrogels is demonstrated. Additionally, a method for bonding distinct layers of cured hydrogels is used to create a microfluidic channel.

Regulating specific growth factor signaling using immobilized branched ligands.

VEGF-binding peptide ligands are incorporated into hydrogel microspheres and reduce the amount of growth factor in solution. VEGF binding affinity is enhanced by creating ligands with a dimer structure. The spheres are able to knock down VEGF-mediated HUVEC growth and reduce calcium signaling. The binding interaction is reversible, allowing the spheres to be used as a VEGF delivery vehicle.

Specific VEGF sequestering and release using peptide-functionalized hydrogel microspheres.

Growth factor signaling plays an essential role in regulating processes such as tissue development, maintenance, and repair. Gene expression levels, diffusion, degradation, and sequestration by extracellular matrix components all play a role in regulating the concentration of growth factors within the cellular microenvironment. Herein, we describe the synthesis and characterization of hydrogel microspheres that mimic the ability of the native extracellular matrix to reversibly bind vascular endothelial growth factor (VEGF) out of solution. A peptide ligand derived from the VEGF receptor 2 (VEGFR2) was covalently incorporated into the hydrogel microspheres in order to achieve binding affinity and specificity. In addition to being able to both bind and release VEGF in a controllable manner, the microspheres were also shown to affect human umbilical vein endothelial cell (HUVEC) proliferation. The resulting microspheres may enable new strategies to specifically upregulate or downregulate growth factor signaling in the cellular microenvironment.

Facile formation of dynamic hydrogel microspheres for triggered growth factor delivery.

Dynamic hydrogels have emerged as an important class of biomaterials for temporal control over growth factor delivery. In this study we formed dynamic hydrogel microspheres from protein-polymer conjugates using an aqueous two-phase suspension polymerization process. This polymerization process enabled rapid microsphere formation without the use of an organic phase, surfactants, mechanical strain or toxic radical initiators. The microspheres' size distribution was modulated by varying the protein-polymer conformation in the pre-polymer solution. Notably, the protein's ligand-induced, nanometer-scale conformational change translated to maximum hydrogel volume changes of 76±10%. The magnitude of the microspheres' volume change was tuned by varying the crosslinking time and ligand identity. After characterizing the microspheres' dynamic properties, we encapsulated two important therapeutic proteins, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2), in the hydrogel microspheres and characterized how the microspheres' dynamic properties controlled their release. Significantly, the aqueous two-phase suspension polymerization process enabled high encapsulation efficiencies (65.8±4.8% and 79.5±3.0% for VEGF and BMP-2, respectively). Also, the microspheres' ligand-induced volume change triggered VEGF and BMP-2 release at specific, predetermined times. There are hundreds of proteins that undergo well-characterized conformational changes that could be processed into hydrogel microspheres via aqueous two-phase suspension polymerizations. Therefore, this approach could be used to form dynamic, growth-factor-releasing hydrogel microspheres that respond to a broad range of specific biochemical ligands.

A general route for the synthesis of functional, protein-based hydrogel microspheres using tailored protein charge.

pH-induced protein aggregation facilitated the formation of nucleation centers for the chain growth polymerization of hydrogel microspheres.

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment.

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.

Microfluidic logic gates and timers.

We use surface tension-based passive pumping and fluidic resistance to create a number of microfluidic analogs to electronic circuit components. Three classes of components are demonstrated: (1) OR/AND, NOR/NAND, and XNOR digital microfluidic logic gates; (2) programmable, autonomous timers; and (3) slow, perfusive flow rheostats. The components can be implemented with standard pipettes and provide a means of non-electronic and autonomous preprogrammed control with potential utility in cell studies and high throughput screening applications.

Microfluidic flow-flash: method for investigating protein dynamics.

We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or O2 binding to myoglobin after the laser-induced photolysis of CO from myoglobin by UV/visible absorbance spectral imaging.

PDMS absorption of small molecules and consequences in microfluidic applications.

Microfluidic devices made out of polydimethylsiloxane (PDMS) have many physical properties that are useful for cell culture applications, such as transparency and gas permeability. Another distinct characteristic of PDMS is its ability to absorb hydrophobic small molecules. Partitioning of molecules into PDMS can significantly change solution concentrations and could potentially alter experimental outcomes. Herein we discuss PDMS absorption and its potential impact on microfluidic experiments.

Multilevel microfluidics via single-exposure photolithography.

This communication reports a new method to form multilevel features in a single layer of SU-8 photoresist to facilitate the generation of 3D microfluidic chips. The method utilizes the spatial dependence of diffracted light intensity to selectively overexpose masked regions of photoresist and requires only a UV light source and a single transparency mask. 3D structures are formed within microfluidic channels using this selective overexposure method, with feature sizes being determined by the exposure dose and mask feature sizes. The dimensions of the internal features and the microfluidic channels can be varied independently according to these parameters, and any number of different heights can be obtained in a single exposure step. The method provides a simple means of forming 3D microfluidic structures with integrated features, including mixing structures, flow stabilization ridges, and separation weirs to increase the capabilities of microfluidic chips in a variety of microchemical applications.