Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Mehl BT

PROVIDER: S-EPMC6594695 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Mehl Benjamin T BT Martin R Scott RS

Analytical methods : advancing methods and applications 20190129 8

Developing <i>in vitro</i> cell culture models that accurately mimic <i>in vivo</i> 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 ...[more]

PMID: 31244918

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Project description: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.

Project description: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.

Dataset Information

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

Publications

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

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