Integrated Control of Microfluidics – Application in Fluid Routing, Sensor Synchronization, and Real-Time Feedback Control

Microfluidic applications range from combinatorial chemical synthesis to high-throughput screening, with platforms integrating analog perfusion components, digitally controlled microvalves, and a range of sensors that demand a variety of communication protocols. A comprehensive solution for microfluidic control has to support an arbitrary combination of microfluidic components and to meet the demand for easy-to-operate system as it arises from the growing community of unspecialized microfluidics users. It should also be an easy to modify and extendable platform, which offer an adequate computational resources, preferably without a need for a local computer terminal for increased mobility. Here we will describe several implementation of microfluidics control technologies and propose a microprocessor-based unit that unifies them. Integrated control can streamline the generation process of complex perfusion sequences required for sensor-integrated microfluidic platforms that demand iterative operation procedures such as calibration, sensing, data acquisition, and decision making. It also enables the implementation of intricate optimization protocols, which often require significant computational resources. System integration is an imperative developmental milestone for the field of microfluidics, both in terms of the scalability of increasingly complex platforms that still lack standardization, and the incorporation and adoption of emerging technologies in biomedical research. Here we describe a modular integration and synchronization of a complex multicomponent microfluidic platform

Breaking the Third Wall: Implementing 3D-Printing Techniques to Expand the Complexity and Abilities of Multi-Organ-on-a-Chip Devices

The understanding that systemic context and tissue crosstalk are essential keys for bridging the gap between in vitro models and in vivo conditions led to a growing effort in the last decade to develop advanced multi-organ-on-a-chip devices. However, many of the proposed devices have failed to implement the means to allow for conditions tailored to each organ individually, a crucial aspect in cell functionality. Here, we present two 3D-print-based fabrication methods for a generic multi-organ-on-a-chip device: One with a PDMS microfluidic core unit and one based on 3D-printed units. The device was designed for culturing different tissues in separate compartments by integrating individual pairs of inlets and outlets, thus enabling tissue-specific perfusion rates that facilitate the generation of individual tissue-adapted perfusion profiles. The device allowed tissue crosstalk using microchannel configuration and permeable membranes used as barriers between individual cell culture compartments. Computational fluid dynamics (CFD) simulation confirmed the capability to generate significant differences in shear stress between the two individual culture compartments, each with a selective shear force. In addition, we provide preliminary findings that indicate the feasibility for biological compatibility for cell culture and long-term incubation in 3D-printed wells. Finally, we offer a cost-effective, accessible protocol enabling the design and fabrication of advanced multi-organ-on-a-chip devices

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

<div><p><i>Candida albicans</i>, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in <i>C. albicans</i> is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related <i>Candida</i> species, and in an <i>in vivo</i> model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.</p></div

Detection and Quantification through a Lipid Membrane Using the Molecularly Controlled Semiconductor Resistor

The detection of covalent and noncovalent binding events between molecules and biomembranes is a fundamental goal of contemporary biochemistry and analytical chemistry. Currently, such studies are performed routinely using fluorescence methods, surface-plasmon resonance spectroscopy, and electrochemical methods. However, there is still a need for novel sensitive miniaturizable detection methods where the sample does not have to be transferred to the sensor, but the sensor can be brought into contact with the sample studied. We present a novel approach for detection and quantification of processes occurring on the surface of a lipid bilayer membrane, by monitoring the current change through the n-type GaAs-based molecularly controlled semiconductor resistor (MOCSER), on which the membrane is adsorbed. Since GaAs is susceptible to etching in an aqueous environment, a protective thin film of methoxysilane was deposited on the device. The system was found to be sensitive enough to allow monitoring changes in pH and in the concentration of amino acids in aqueous solution on top of the membrane. When biotinylated lipids were incorporated into the membrane, it was possible to monitor the binding of streptavidin or avidin. The device modified with biotin-streptavidin complex was capable of detecting the binding of streptavidin antibodies to immobilized streptavidin with high sensitivity and selectivity. The response depends on the charge on the analyte. These results open the way to facile electrical detection of protein–membrane interactions

Trimera formation is not a general stress response.

<p>Cells exposed to triazoles ketoconazole, voriconazole, and itraconazole formed trimeras at frequencies similar to FLC. Cells that were exposed to caspofungin, an echinocandidn, also produced many trimera-like and multimera-like cells (upper and lower panels). Exposure to toxin 5-FOA as well as heat shock did not result in a significant number of trimeras. No trimeras were detectable following exposure to 2-DOG (unpublished data). Percentages in upper right corner of DAPI image denote frequency of trimera formation in 300–400 cells.</p

Trimeras form <i>in vivo</i>.

<p>Percent of ConA-Texas Red/Eno1-GFP cells showing unbudded (blue), budded (red), trimera-like (purple), and hyphal (green) phenotypes within untreated (left, <i>n</i> = 195) and FLC-treated (right, <i>n</i> = 309) mouse host 48 h after injection. Error bars are 1 standard deviation. Scale bar, 5 µm.</p

Unequal segregation occurs in nuclei with more than one spindle.

<p>(A) Time-lapse microscopy of nuclear segregation patterns in tetraploid/diakaryotic cells. Type I segregation pattern (top two rows, 54% of events), both spindles elongated across the bud neck. Type II (bottom two rows, 46% of events), only one spindle elongated across the bud neck. Numbers denote time (min) of FLC exposure. Scale bars, 5 µm. Total of 13 cells analyzed. (B) Histone H4 (Hhf1)-GFP fluorescence intensity scatter plots. Sister nuclei are plotted relative to each other. Postanaphase cells containing a total of two SPBs (cyan) clustered around 1∶1, indicative of equal segregation (gray region, contains 95% of points from “no drug” cells). Postanaphase cells containing more than two SPBs (magenta) diverged significantly from 1∶1 at 12 h (two-tailed <i>t</i> test, <i>p</i> value <0.05).</p

Cell size correlates with DNA content.

<p>(A) Flow cytometry data plotted as cell size (FSC) versus DNA content (FL1-A) for cultures grown in the absence (top row) or presence (bottom row) of FLC for times indicated. (B) Bar graphs represent percent of cells that fell into colored regions defined in scatterplots above as determined using Gaussian fitting (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001815#s4" target="_blank">Materials and Methods</a>).</p