Less Is More: Oligomer Extraction and Hydrothermal Annealing Increase PDMS Adhesion Forces for Materials Studies and for Biology-Focused Microfluidic Applications.

Cues in the micro-environment are key determinants in the emergence of complex cellular morphologies and functions. Primary among these is the presence of neighboring cells that form networks. For high-resolution analysis, it is crucial to develop micro-environments that permit exquisite control of network formation. This is especially true in cell science, tissue engineering, and clinical biology. We introduce a new approach for assembling polydimethylsiloxane (PDMS)-based microfluidic environments that enhances cell network formation and analyses. We report that the combined processes of PDMS solvent-extraction and hydrothermal annealing create unique conditions that produce high-strength bonds between solvent-extracted PDMS (E-PDMS) and glass-properties not associated with conventional PDMS. Extraction followed by hydrothermal annealing removes unbound oligomers, promotes polymer cross-linking, facilitates covalent bond formation with glass, and retains the highest biocompatibility. Herein, our extraction protocol accelerates oligomer removal from 5 to 2 days. Resulting microfluidic platforms are uniquely suited for cell-network studies owing to high adhesion forces, effectively corralling cellular extensions and eliminating harmful oligomers. We demonstrate the simple, simultaneous actuation of multiple microfluidic domains for invoking ATP- and glutamate-induced Ca2+ signaling in glial-cell networks. These E-PDMS modifications and flow manipulations further enable microfluidic technologies for cell-signaling and network studies as well as novel applications.

Identifying Candidate Biomarkers of Ionizing Radiation in Human Pulmonary Microvascular Lumens Using Microfluidics-A Pilot Study.

The microvasculature system is critical for the delivery and removal of key nutrients and waste products and is significantly damaged by ionizing radiation. Single-cell capillaries and microvasculature structures are the primary cause of circulatory dysfunction, one that results in morbidities leading to progressive tissue and organ failure and premature death. Identifying tissue-specific biomarkers that are predictive of the extent of tissue and organ damage will aid in developing medical countermeasures for treating individuals exposed to ionizing radiation. In this pilot study, we developed and tested a 17 µL human-derived microvascular microfluidic lumen for identifying candidate biomarkers of ionizing radiation exposure. Through mass-spectrometry-based proteomics, we detected 35 proteins that may be candidate early biomarkers of ionizing radiation exposure. This pilot study demonstrates the feasibility of using humanized microfluidic and organ-on-a-chip systems for biomarker discovery studies. A more elaborate study of sufficient statistical power is needed to identify candidate biomarkers and test medical countermeasures of ionizing radiation.

Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay.

Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.

Microfluidics and Metabolomics Reveal Symbiotic Bacterial-Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

Label-free time- and space-resolved exometabolite sampling of growing plant roots through nanoporous interfaces.

Spatial and temporal profiling of metabolites within and between living systems is vital to understanding how chemical signaling shapes the composition and function of these complex systems. Measurement of metabolites is challenging because they are often not amenable to extrinsic tags, are diverse in nature, and are present with a broad range of concentrations. Moreover, direct imaging by chemically informative tools can significantly compromise viability of the system of interest or lack adequate resolution. Here, we present a nano-enabled and label-free imaging technology using a microfluidic sampling network to track production and distribution of chemical information in the microenvironment of a living organism. We describe the integration of a polyester track-etched (PETE) nanofluidic interface to physically confine the biological sample within the model environment, while allowing fluidic access via an underlying microfluidic network. The nanoporous interface enables sampling of the microenvironment above in a time-dependent and spatially-resolved manner. For demonstration, the diffusional flux through the PETE membrane was characterized to understand membrane performance, and exometabolites from a growing plant root were successfully profiled in a space- and time-resolved manner. This method and device provide a frame-by-frame description of the chemical environment that maps to the physical and biological characteristics of the sample.

Genetic Selection for Small Molecule Production in Competitive Microfluidic Droplets.

Biosensors can be used to screen or select for small molecule production in engineered microbes. However, mutations to the biosensor that interfere with accurate signal transduction are common, producing an excess of false positives. Strategies have been developed to avoid this limitation by physically separating the production pathway and biosensor, but these approaches have only been applied to screens, not selections. We have developed a novel biosensor-mediated selection strategy using competition between cocultured bacteria. When applied to the biosynthesis of cis,cis-muconate, we show that this strategy yields a selective advantage to producer strains that outweighs the costs of production. By encapsulating the competitive cocultures into microfluidic droplets, we successfully enriched the muconate-producing strains from a large population of control nonproducers. Facile selections for small molecule production will increase testing throughput for engineered microbes and allow for the rapid optimization of novel metabolic pathways.

Increasing access to microfluidics for studying fungi and other branched biological structures.

BACKGROUND: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics." RESULTS: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi. CONCLUSIONS: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Accessing microfluidics through feature-based design software for 3D printing.

Additive manufacturing has been a cornerstone of the product development pipeline for decades, playing an essential role in the creation of both functional and cosmetic prototypes. In recent years, the prospects for distributed and open source manufacturing have grown tremendously. This growth has been enabled by an expanding library of printable materials, low-cost printers, and communities dedicated to platform development. The microfluidics community has embraced this opportunity to integrate 3D printing into the suite of manufacturing strategies used to create novel fluidic architectures. The rapid turnaround time and low cost to implement these strategies in the lab makes 3D printing an attractive alternative to conventional micro- and nanofabrication techniques. In this work, the production of multiple microfluidic architectures using a hybrid 3D printing-soft lithography approach is demonstrated and shown to enable rapid device fabrication with channel dimensions that take advantage of laminar flow characteristics. The fabrication process outlined here is underpinned by the implementation of custom design software with an integrated slicer program that replaces less intuitive computer aided design and slicer software tools. Devices are designed in the program by assembling parameterized microfluidic building blocks. The fabrication process and flow control within 3D printed devices were demonstrated with a gradient generator and two droplet generator designs. Precise control over the printing process allowed 3D microfluidics to be printed in a single step by extruding bridge structures to 'jump-over' channels in the same plane. This strategy was shown to integrate with conventional nanofabrication strategies to simplify the operation of a platform that incorporates both nanoscale features and 3D printed microfluidics.

Bacterial-fungal interactions: ecology, mechanisms and challenges.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.

Phase correlation imaging of unlabeled cell dynamics.

We present phase correlation imaging (PCI) as a novel approach to study cell dynamics in a spatially-resolved manner. PCI relies on quantitative phase imaging time-lapse data and, as such, functions in label-free mode, without the limitations associated with exogenous markers. The correlation time map outputted in PCI informs on the dynamics of the intracellular mass transport. Specifically, we show that PCI can extract quantitatively the diffusion coefficient map associated with live cells, as well as standard Brownian particles. Due to its high sensitivity to mass transport, PCI can be applied to studying the integrity of actin polymerization dynamics. Our results indicate that the cyto-D treatment blocking the actin polymerization has a dominant effect at the large spatial scales, in the region surrounding the cell. We found that PCI can distinguish between senescent and quiescent cells, which is extremely difficult without using specific markers currently. We anticipate that PCI will be used alongside established, fluorescence-based techniques to enable valuable new studies of cell function.

Material-mediated proangiogenic factor release pattern modulates quality of regenerated blood vessels.

Hydrogels designed to sustainably release bioactive molecules are extensively used to enhance tissue repair and regenerative therapies. Along this line, numerous efforts are made to control the molecular release rate and amount. In contrast, few efforts are made to control the molecular release pattern, and, subsequently, modulate the spatial organization of newly forming tissues, including blood vessels. Therefore, using a hydrogel printed to release vascular endothelial growth factor (VEGF) into a pre-defined pattern, this study demonstrates that spatial distribution of VEGF is important in guiding growth direction of new blood vessels, and also in retaining the structural integrity of pre-existing vasculature. Guided by a computational model, we fabricated a patch composed of micro-sized VEGF-releasing poly(ethylene glycol) diacrylate (PEGDA) hydrogel cylinders using an ink-jet printer. Interestingly, hydrogel printed with computationally optimized spacing created anisotropically aligned vasculature exclusively when the printed gel pattern was placed parallel to pre-existing blood vessels. In contrast, vascular sprouting from placing the printed gel pattern perpendicular to pre-existing vessels resulted in deformation and structural disintegration of the original vasculature. We envision that this study will be useful to better understand angiogenesis-modulated neovascularization and further improve the treatment quality for various wounds and tissue defects.

Measuring physical properties of neuronal and glial cells with resonant microsensors.

Microelectromechanical systems (MEMS) resonant sensors provide a high degree of accuracy for measuring the physical properties of chemical and biological samples. These sensors enable the investigation of cellular mass and growth, though previous sensor designs have been limited to the study of homogeneous cell populations. Population heterogeneity, as is generally encountered in primary cultures, reduces measurement yield and limits the efficacy of sensor mass measurements. This paper presents a MEMS resonant pedestal sensor array fabricated over through-wafer pores compatible with vertical flow fields to increase measurement versatility (e.g., fluidic manipulation and throughput) and allow for the measurement of heterogeneous cell populations. Overall, the improved sensor increases capture by 100% at a flow rate of 2 µL/min, as characterized through microbead experiments, while maintaining measurement accuracy. Cell mass measurements of primary mouse hippocampal neurons in vitro, in the range of 0.1-0.9 ng, demonstrate the ability to investigate neuronal mass and changes in mass over time. Using an independent measurement of cell volume, we find cell density to be approximately 1.15 g/mL.

Micro-patterning of mammalian cells on suspended MEMS resonant sensors for long-term growth measurements.

MEMS resonant mass sensors can measure the mass of individual cells, though long-term growth measurements are limited by the movement of cells off the sensor area. Micro-patterning techniques are a powerful approach to control the placement of individual cells in an arrayed format. In this work we present a method for micro-patterning cells on fully suspended resonant sensors through select functionalization and passivation of the chip surface. This method combines high-resolution photolithography with a blanket transfer technique for applying photoresist to avoid damaging the sensors. Cells are constrained to the patterned collagen area on the sensor by pluronic acting as a cell adhesion blocker. This micro-patterning method enables long-term growth measurements, which is demonstrated by a measurement of the change in mass of a human breast cancer cell over 18 h.

Ultra-localized single cell electroporation using silicon nanowires.

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Micromechanical properties of hydrogels measured with MEMS resonant sensors.

Hydrogels have gained wide usage in a range of biomedical applications because of their biocompatibility and the ability to finely tune their properties, including viscoelasticity. The use of hydrogels on the microscale is increasingly important for the development of drug delivery techniques and cellular microenvironments, though the ability to accurately characterize their micromechanical properties is limited. Here we demonstrate the use of microelectromechanical systems (MEMS) resonant sensors to estimate the properties of poly(ethylene glycol) diacrylate (PEGDA) microstructures over a range of concentrations. These microstructures are integrated on the sensors by deposition using electrohydrodynamic jet printing. Estimated properties agree well with independent measurements made using indentation with atomic force microscopy.

Over a century of neuron culture: from the hanging drop to microfluidic devices.

The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) - that neurons can be cultured outside the body - to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chemotemporal control) within sub-cellular environments of three-dimensional fluidic spaces (microfluidic devices) and materials (e.g., hydrogels). They will enable new insights into the complexities of neuronal development and pathology.

New perspectives on neuronal development via microfluidic environments.

Understanding the signals that guide neuronal development and direct formation of axons, dendrites, and synapses during wiring of the brain is a fundamental challenge in developmental neuroscience. Discovery of how local signals shape developing neurons has been impeded by the inability of conventional culture methods to interrogate microenvironments of complex neuronal cytoarchitectures, where different subdomains encounter distinct chemical, physical, and fluidic features. Microfabrication techniques are facilitating the creation of microenvironments tailored to neuronal structures and subdomains with unprecedented access and control. The design, fabrication, and properties of microfluidic devices offer significant advantages for addressing unresolved issues of neuronal development. These high-resolution approaches are poised to contribute new insights into mechanisms for restoring neuronal function and connectivity compromised by injury, stress, and neurodegeneration.

Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes.

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca2+ increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca2+-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC-MS/MS and CE-MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca2+-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.

Characterization of mass and swelling of hydrogel microstructures using MEMS resonant mass sensor arrays.

The use of hydrogels for biomedical engineering, and for the development of biologically inspired cellular systems at the microscale, is advancing at a rapid pace. Microelectromechanical system (MEMS) resonant mass sensors enable the mass measurement of a range of materials. The integration of hydrogels onto MEMS resonant mass sensors is demonstrated, and these sensors are used to characterize the hydrogel mass and swelling characteristics. The mass values obtained from resonant frequency measurements of poly(ethylene glycol)diacrylate (PEGDA) microstructures match well with the values independently verified through volume measurements. The sensors are also used to measure the influence of fluids of similar and greater density on the mass measurements of microstructures. The data show a size-dependent increase in gel mass when fluid density is increased. Lastly, volume comparisons of bulk hydrogels with a range polymer concentration (5% to 100% (v/v)) show a non-linear swelling trend.

Pattern analysis and spatial distribution of neurons in culture.

The nervous system is a complex, highly-ordered, integrated network of cells. Dispersed cultures of neurons enable investigations into intrinsic cellular functions without the complexities inherent in the intact nervous system. This culture process generates a homogeneously dispersed population that is assumed to be spatially random. Despite the vast number of studies utilizing dispersed neurons, few studies address the spatial distribution of large populations of neurons, in vitro. We used ink-jet printing and surface chemistry to define patterned areas of poly-lysine adhesion (∼50 µm spots) juxtaposed against a fluorinated-silane background. We quantitatively analysed populations of patterned neurons on printed protein spots, and unpatterned neurons. Using a microarray scanner, we acquired large images (72 mm × 22 mm) of patterns, and neurons with and without patterns. Fast Fourier transformation (FFT) image analysis was used to determine global alignment of neurons to patterns. Through point pattern analysis, we described the spatial organization of dispersed neurons with, or without, patterned substrates. Patterned neurons show spatial organization characteristics reminiscent of printed patterns, with spatial distributions representative of unpatterned neurons. Most notably, both patterned and unpatterned neurons show departure from null models of complete spatial randomness (CSR; a homogeneous Poisson process) at shorter distances with conformity to CSR occurring at longer distances. Cellular morphometrics show that when compared to their unpatterned counterparts, spot-patterned neurons exhibit a significant increase (p < 0.0001) in the mean dendritic circularity and an increase in the number of more circular neurons. Through neurite tracing, we show that dendritic processes are also highly confined to patterned areas, and that they are on average 58% shorter than dendrites of neurons without patterns. Our findings show that patterned areas change the spatial organization of the somata and dendrites of cultured neurons, and that traditional neuronal cultures deviate from CSR.