Cell type-specific changes in transcriptomic profiles of endothelial cells, iPSC-derived neurons and astrocytes cultured on microfluidic chips.

In vitro neuronal models are essential for studying neurological physiology, disease mechanisms and potential treatments. Most in vitro models lack controlled vasculature, despite its necessity in brain physiology and disease. Organ-on-chip models offer microfluidic culture systems with dedicated micro-compartments for neurons and vascular cells. Such multi-cell type organs-on-chips can emulate neurovascular unit (NVU) physiology, however there is a lack of systematic data on how individual cell types are affected by culturing on microfluidic systems versus conventional culture plates. This information can provide perspective on initial findings of studies using organs-on-chip models, and further optimizes these models in terms of cellular maturity and neurovascular physiology. Here, we analysed the transcriptomic profiles of co-cultures of human induced pluripotent stem cell (hiPSC)-derived neurons and rat astrocytes, as well as one-day monocultures of human endothelial cells, cultured on microfluidic chips. For each cell type, large gene expression changes were observed when cultured on microfluidic chips compared to conventional culture plates. Endothelial cells showed decreased cell division, neurons and astrocytes exhibited increased cell adhesion, and neurons showed increased maturity when cultured on a microfluidic chip. Our results demonstrate that culturing NVU cell types on microfluidic chips changes their gene expression profiles, presumably due to distinct surface-to-volume ratios and substrate materials. These findings inform further NVU organ-on-chip model optimization and support their future application in disease studies and drug testing.

Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings.

Annual prevalence of the use of common illicit drugs and new psychoactive substances (NPS) is high, despite the often limited knowledge on the health risks of these substances. Recently, cortical cultures grown on multi-well microelectrode arrays (mwMEAs) have been used for neurotoxicity screening of chemicals, pharmaceuticals, and toxins with a high sensitivity and specificity. However, the use of mwMEAs to investigate the effects of illicit drugs on neuronal activity is largely unexplored. We therefore first characterised the cortical cultures using immunocytochemistry and show the presence of astrocytes, glutamatergic and GABAergic neurons. Neuronal activity is concentration-dependently affected following exposure to six neurotransmitters (glutamate, GABA, serotonin, dopamine, acetylcholine and nicotine). Most neurotransmitters inhibit neuronal activity, although glutamate and acetylcholine transiently increase activity at specific concentrations. These transient effects are not detected when activity is determined during the entire 30min exposure window, potentially resulting in false-negative results. As expected, exposure to the GABAA-receptor antagonist bicuculline increases neuronal activity. Exposure to a positive allosteric modulator of the GABAA-receptor (diazepam) or to glutamate receptor antagonists (CNQX and MK-801) reduces neuronal activity. Further, we demonstrate that exposure to common drugs (3,4-methylenedioxymethamphetamine (MDMA) and amphetamine) and NPS (1-(3-chlorophenyl)piperazine (mCPP), 4-fluoroamphetamine (4-FA) and methoxetamine (MXE)) decreases neuronal activity. MXE most potently inhibits neuronal activity with an IC50 of 0.5µM, whereas 4-FA is least potent with an IC50 of 113µM. Our data demonstrate the importance of analysing neuronal activity within different time windows during exposure to prevent false-negative results. We also show that cortical cultures grown on mwMEAs can successfully be applied to investigate the effects of different (illicit) drugs on neuronal activity. Compared to investigating multiple single endpoints for neurotoxicity or neuromodulation, such as receptor activation or calcium channel function, mwMEAs can provide information on integrated aspects of drug-induced neurotoxicity more rapidly. Therefore, this approach could contribute to a faster insight in possible health risks and shorten the regulation process.