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.

Figure 1

A.) CAD design of the 3D printed culture device. The device is printed together as an assembly of 2 parts consisting of the bottom portion with slots for the inserts tapered to a funnel and the top portion with threads to accommodate commercial fittings. B.) Picture showing the printed device. A pin is epoxied to the end of the device for simple integration with analysis devices. C.) The inserts made up of PS fibers electrospun onto PS sheets. The sheets are then laser cut into individual inserts securing the fibers to the sheet. D.) Cross-sectional view of the slots for the inserts to fit in. E.) Cross-sectional view with the inserts inside the housing.

Figure 2

A.) Micrograph showing an insert with PC12 cells immobilized onto the fibers (cells stained with acridine orange); B-C.) Confocal images taken of a section of the collage-coated PS inserts. B.) Micrograph showing clumped cells on top with cells in fibers below. C.) Same view of the cells with the clumped cells on top removed. D.-F) Scanning electron microscopy images showing PC12 fixed onto PS fibers. D.) Well split groups of cells permeate the fibers throughout. E.) Clumps of cells branching out to hold the substrate. F.) Cells bunching together on the surface of a thin sheet of fibers.

Figure 3

A.) Schematic of the straight channel setup for the flow based electrochemical detection of PC12 catecholamine release. B.) Picture showing the integration of the flow device containing cells with the automated syringe pump and 4-port valve system and the analysis device.

Figure 4

A and B.) Bar graphs comparing the detected release of catecholamines from both PC12 and PC12 cells pre-loaded with dopamine. The concentration of detected catecholamine release was 4 ± 1 µM and 11 ± 2 µM or PC12 and preloaded PC12 cells respectively (n=5). Reserpine inhibition resulted in catecholamine levels released below the lowest detectable concentration (n=3). C.) Shows a comparison of the concentration released between both sets of cells and per cell respectively.

Figure 5

A.) Picture of the electrophoresis setup. The cell device can be seen connected with a capillary to the automated syringe and valve system. The device is connected to the bilayer valving chip. B.) Picture showing the integration of the cell device with the electrophoresis chip. The steel pin can be seen integrated with the 1-µL droplet. C-E.) Electropherograms of C.) 100 µM norepinephrine and dopamine D.) Detected release of norepinephrine and dopamine from pre-loaded PC12 cells E.) The release of norepinephrine and dopamine from cells pre-loaded with 1 mM of both for 1 hr prior to the experiments. E.) A bar graph showing a comparison of the norepinephrine and dopamine detected from the pre-loaded PC12 cells (n=4).