Use of electrospinning and dynamic air focusing to create three-dimensional cell culture scaffolds in microfluidic devices.

Organs-on-a-chip has emerged as a powerful tool for pharmacological and physiological studies. A key part in the construction of such a model is the ability to pattern or culture cells in a biomimetic fashion. Most of the reported cells-on-a-chip models integrate cells on a flat surface, which does not accurately represent the extracellular matrix that they experience in vivo. Electrospinning, a technique used to generate sub-micron diameter polymer fibers, has been used as an in vitro cell culture substrate and for tissue engineering applications. Electrospinning of fibers directly into a fully sealed fluidic channel using a conventional setup has not been possible due to issues of confining the fibers into a discrete network. In this work, a dynamic focusing method was developed, with this approach enabling direct deposition of electrospun fibers into a fully sealed fluidic channel, to act as a matrix for cell culture and subsequent studies under continuous flowing conditions. Scanning electron microscopy of electrospun polycaprolactone fibers shows that this method enables the formation of fibrous layers on the inner wall of a 3D-printed fluidic device (mean fiber size = 1.6 ± 0.6 µm and average pore size = 113 ± 19 µm(2)). Cells were able to be cultured in this 3D scaffold without the addition of adhesion proteins. Media was pumped through the channel at high flow rates (up to 400 µL min(-1)) during a dynamic cell culture process and both the fibers and the cells were found to be strongly adherent. A PDMS fluidic device was also prepared (from a 3D printed mold) and coated with polycaprolactone fibers. The PDMS device enables optical detection and confocal imaging of cultured cells on the fibers. Finally, macrophages were cultured in the devices to study how the fibrous scaffold can affect cell behavior. It was found that under lipopolysaccharide stimulation, macrophages cultured on PCL fibers inside of a channel secreted significantly more cytokines than those cultured on a thin layer of PCL in a channel or directly on the inner channel wall. Overall, this study represents a new approach for in vitro cell studies, where electrospinning can be used to easily and quickly create 3D scaffolds that can improve the culture conditions in microfluidic devices.

Integrating 3D Cell Culture of PC12 Cells with Microchip-Based Electrochemical Detection.

Developing in vitro cell culture models that accurately mimic in vivo processes in a manner that also enables near real-time analysis of neurotransmitters is an important research area. New technologies being developed such as 3D scaffolds for cell culture and 3D printed microfluidics provide an opportunity for such advancements. In this work, PC12 cells were used as a model system and they were immobilized onto a 3D scaffold of polystyrene (PS) fibers. These fibers were created by electrospinning onto PS sheets, which were laser cut and, after cell seeding, inserted into a 3D printed microfluidic device. The 3D printed device was designed with threads for connecting commercial fittings (to integrate automated pumps and a 4-port injection system) and a steel pin for simple coupling with PDMS/polystyrene analytical devices. A straight PDMS channel was used for simple (and continuous) flow-based detection by sealing onto a PS base containing an embedded gold array working electrode and a platinum pseudo-reference. Electrochemical detection of stimulated catecholamine release was demonstrated. The insert-based system was then integrated with a bilayer valving PDMS device (for microchip electrophoresis) sealed onto a PS base (with electrodes for electrochemical detection). This base was embedded with a Pd decoupler (for grounding the separation voltage and adsorbing hydrogen) and a 33 µm carbon fiber working electrode for in-channel detection. PC12 cells were stimulated in the 3D cell culture device, and the valving/electrophoresis microchip was able to separate and detect dopamine and norepinephrine release. This work demonstrates the ability to integrate 3D cell scaffolds with microchip-based analysis for detection of multiple analytes released from cells.

Benzobis(imidazolium) salts as templates for the self-assembly of starburst [24]pseudorotaxanes and their corresponding carbene-based classic [22]pseudorotaxanes.

Benzobis(imidazolium) salts ([BBI-H2-R4]2+, R = alkyl, aryl) interact with crown ethers through a combination of hydrogen bonds, ion-dipole, and π-π stacking interactions to form starburst [24]pseudo-rotaxanes. This new recognition motif allows the extension of four side-arms directly from the cavity of the crown ether, thus positioning the wheel component in a straddled orientation onto the axle, while their carbene-based derivatives show the classical shape of regular [22]pseudorotaxanes.

Enhanced Microchip Electrophoresis Separations Combined with Electrochemical Detection Utilizing a Capillary Embedded in Polystyrene.

The ability to use microchip-based electrophoresis for fast, high-throughput separations provides researchers with a tool for close-to real time analysis of biological systems. While PDMS-based electrophoresis devices are popular, the separation efficiency is often an issue due to the hydrophobic nature of PDMS. In this study, a hybrid microfluidic capillary device was fabricated to utilize the positive features of PDMS along with the electrophoretic performance of fused silica. A capillary loop was embedded in a polystyrene base that can be coupled with PDMS microchannels at minimal dead volume interconnects. A method for cleaning out the capillaries after a wet-polishing step was devised through the use of 3D printed syringe attachment. By comparing the separation efficiency of fluorescein and CBI-glycine with both a PDMS-based serpentine device and the embedded capillary loop device, it was shown that the embedded capillary loop device maintained higher theoretical plates for both analytes. A Pd decoupler with a carbon or Pt detection electrode were embedded along with the loop allowing integration of the electrophoretic separation with electrochemical detection. A series of catecholamines were separated to show the ability to resolve similar analytes and detect redox active species. The release of dopamine and norepinephrine from PC 12 cells was also analyzed showing the compatibility of these improved microchip separations with high ionic cell buffers associated with cell culture.

3D-printed Microfluidic Devices: Fabrication, Advantages and Limitations-a Mini Review.

A mini-review with 79 references. In this review, the most recent trends in 3D-printed microfluidic devices are discussed. In addition, a focus is given to the fabrication aspects of these devices, with the supplemental information containing detailed instructions for designing a variety of structures including: a microfluidic channel, threads to accommodate commercial fluidic fittings, a flow splitter; a well plate, a mold for PDMS channel casting; and how to combine multiple designs into a single device. The advantages and limitations of 3D-printed microfluidic devices are thoroughly discussed, as are some future directions for the field.

Integrated hybrid polystyrene-polydimethylsiloxane device for monitoring cellular release with microchip electrophoresis and electrochemical detection.

In this work, a polystyrene (PS)-polydimethylsiloxane (PDMS) hybrid device was developed to enable the integration of cell culture with analysis by microchip electrophoresis and electrochemical detection. It is shown that this approach combines the fundamental advantages of PDMS devices (the ability to integrate pumps and valves) and PS devices (the ability to permanently embed fluidic tubing and electrodes). The embedded fused-silica capillary enables high temporal resolution measurements from off-chip cell culture dishes and the embedded electrodes provide close to real-time analysis of small molecule neurotransmitters. A novel surface treatment for improved (reversible) adhesion between PS and PDMS is described using a chlorotrimethylsilane stamping method. It is demonstrated that a Pd decoupler is efficient at handling the high current (and cathodic hydrogen production) resulting from use of high ionic strength buffers needed for cellular analysis; thus allowing an electrophoretic separation and in-channel detection. The separation of norepinephrine (NE) and dopamine (DA) in highly conductive biological buffers was optimized using a mixed surfactant system. This PS-PDMS hybrid device integrates multiple processes including continuous sampling from a cell culture dish, on-chip pump and valving technologies, microchip electrophoresis, and electrochemical detection to monitor neurotransmitter release from PC 12 cells.