Microfluidic device using a gold pillar array and integrated electrodes for on-chip endothelial cell immobilization, direct RBC contact, and amperometric detection of nitric oxide.

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC-derived ATP. These studies incorporate on-chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC-EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum-black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8 ± 0.2 nM NO per endothelial cell (average ± SEM, n = 8). Pretreatment of RBCs with glybenclamide inhibited treprostinil-induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92 ± 0.1 nM NO per endothelial cell, n = 7). In the future, this device can be used to study interactions between many other cell types (both adherent and non-adherent cell lines) and incorporate other detection schemes.

Insert-based microfluidics for 3D cell culture with analysis.

We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.

Use of 3D Printing and Modular Microfluidics to Integrate Cell Culture, Injections and Electrochemical Analysis.

Fabrication of microchip-based devices using 3-D printing technology offers a unique platform to create separate modules that can be put together when desired for analysis. A 3-D printed module approach offers various advantages such as file sharing and the ability to easily replace, customize, and modify the individual modules. Here, we describe the use of a modular approach to electrochemically detect the ATP-mediated release of nitric oxide (NO) from endothelial cells. Nitric oxide plays a significant role in the vasodilation process; however, detection of NO is challenging due to its short half-life. To enable this analysis, we use three distinct 3-D printed modules: cell culture, sample injection and detection modules. The detection module follows a pillar-based Wall-Jet Electrode design, where the analyte impinges normal to the electrode surface, offering enhanced sensitivity for the analyte. To further enhance the sensitivity and selectivity for NO detection the working electrode (100 µm gold) is modified by the addition of a 27 µm gold pillar and platinum-black coated with Nafion. The use of the pillar electrode leads to three-dimensional structure protruding into the channel enhancing the sensitivity by 12.4 times in comparison to the flat electrode (resulting LOD for NO = 210 nM). The next module, the 3-D printed sample injection module, follows a simple 4-Port injection rotor design made of two separate components that when assembled can introduce a specific volume of analyte. This module not only serves as a cheaper alternative to the commercially available 4-Port injection valves, but also demonstrates the ability of volume customization and reduced dead-volume issues with the use of capillary-free connections. Comparison between the 3-D printed and a commercial 4-Port injection valve showed similar sensitivities and reproducibility for NO analysis. Lastly, the cell culture module contains electrospun polystyrene fibers with immobilized endothelial cells, resulting in 3-D scaffold for cell culture. With the incorporation of all 3 modules, we can make reproducible ATP injections (via the 3-D printed sample injection module) that can stimulate NO release from endothelial cells cultured on a fibrous insert in the cell culture module which can then be quantitated by the pillar WJE module (0.19 ± 0.03 nM/cell, n = 27, 3 inserts analyzed each day, on 9 different days). The modular approach demonstrates the facile creation of custom and modifiable fluidic components that can be assembled as needed.

Microchip-based 3D-Cell Culture Using Polymer Nanofibers Generated by Solution Blow Spinning.

Polymer nano/micro fibers have found many applications including 3D cell culture and the creation of wound dressings. The fibers can be produced by a variety of techniques that include electrospinning, the primary disadvantage of which include the requirement for a high voltage supply (which may cause issues such as polymer denaturation) and lack of portability. More recently, solution blow spinning, where a high velocity sheath gas is used instead of high voltage, has been used to generate polymer fibers. In this work, we used blow spinning to create nano/microfibers for microchip-based 3D cell culture. First, we thoroughly investigated fiber generation from a 3D printed gas sheath device using two polymers that are amenable to cell culture (polycaprolactone, PCL and polystyrene, PS) as well as the parameters that can affect PCL and PS fiber quality. Using the 3D printed sheath device, it was found that the pressure of the sheath N2 and the concentration of polymer solutions determine if fibers can be produced as well as the resulting fiber morphology. In addition, we showed how these fibers can be used for 3D cell culture by directly depositing PCL fibers in petri dishes and well plates. It is shown the fibers have good compatibility with RAW 264.7 macrophages and the PCL fiber scaffold can be as thick as 178 ± 14 µm. PCL fibers created from solution blow spinning (with the 3D printed sheath device) were then integrated with a microfluidic device for the first time to fabricate a 3D cell culture scaffold with a flow component. After culturing and stimulating macrophages on the fluidic device, it was found that the integrated 3D fibrous scaffold is a better mimic of the extracellular matrix (as opposed to a flat, 2D substrate), with enhanced nitrite accumulation (product of nitric oxide release) from macrophages stimulated with lipopolysaccharide. PS fibers were also made and integrated in a microfluidic device for 3D culture of endothelial cells, which stayed viable for at least 72 hours (48 hours under the flowing conditions). This approach will be useful for future studies involving more realistic microchip-based culture models for studying cell-to-cell communication.

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.

Simultaneous analysis of vascular norepinephrine and ATP release using an integrated microfluidic system.

BACKGROUND: Sympathetic nerves are known to release three neurotransmitters: norepinephrine, ATP, and neuropeptide Y that play a role in controlling vascular tone. This paper focuses on the co-release of norepinephrine and ATP from the mesenteric arterial sympathetic nerves of the rat. NEW METHOD: In this paper, a quantification technique is described that allows simultaneous detection of norepinephrine and ATP in a near-real-time fashion from the isolated perfused mesenteric arterial bed of the rat. Simultaneous detection is enabled with 3-D printing technology, which is shown to help integrate the perfusate with different detection methods (norepinephrine by microchip-based amperometery and ATP by on-line chemiluminescence). RESULTS: Stimulated levels relative to basal levels of norepinephrine and ATP were found to be 363nM and 125nM, respectively (n=6). The limit of detection for norepinephrine is 80nM using microchip-based amperometric detection. The LOD for on-line ATP detection using chemiluminescence is 35nM. COMPARISON WITH EXISTING METHOD: In previous studies, the co-transmitters have been separated and detected with HPLC techniques. With HPLC, the samples from biological preparations have to be derivatized for ATP detection and require collection time before analysis. Thus real-time measurements are not made and the delay in analysis by HPLC can cause degradation. CONCLUSIONS: In conclusion, the method described in the paper can be used to successfully detect norepinephrine and ATP simultaneously and in a near-real-time fashion.