A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells

Abstract

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped “cell traps”, each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion / repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.

A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection

“…[39][40][41][42][43] Our results indicate that when chemical stimulation is to be used it is signicant to optimise the ow conditions in order to avoid mechanical stimulation, however, this aspect is not commonly addressed in publications.…”

“…36 The development of electrochemical methods and analysis platforms for population based neurotransmitter detection, specically for DA, is a new emerging eld 25,[35][36][37][38] with scarce applications in microuidic environment. 29,[39][40][41][42][43] We measured DA release from PC12 cells induced by both chemical and mechanical (increased ow rate) stimulation in the microuidic environment aer EIS monitoring of cell adhesion and growth.…”

Profiles of secreted neuropeptides and catecholamines illustrate similarities and differences in response to stimulation by distinct secretagogues

“…These neurotransmitters are secreted from chromaffin cells in a regulated manner from secretory vesicles upon stimulation by sympathetic activation. Secretion of these catecholamine and neuropeptide transmitters (ie., enkephalin, NPY, galanin, and others) have been studied individually from chromaffin cells (Zhang et al, 2006; Hook et al, 2008; Smith and Eiden, 2012; Ges et al, 2013) and other neuronal cell types (Eiden, 2013). But characterization of the secreted profiles of co-released neurotransmitters in response to stimulation by different secretagogues has not yet been fully defined.…”

Vesicular exocytosis and microdevices – microelectrode arrays

“…14 The electrochemical recording has been readily achieved, thus extracted data contributed to the issues dealing with the release dynamics at the top and bottom of the cell. It has to be emphasized that an ingenious strategy for trapping the cells has been recently published by Baudenbacher et al 17 By considering MEAs with interdigitated platinum band electrodes (50 µm width) and PDMS channels, U-shaped cell traps were added to the microsystem in order to stop and position small groups of chromaffin cells at the UME surfaces. 15 In both cases, it cannot be excluded that the mechanical or hydrodynamic stress applied to cells may alter the cell response.…”