The widespread adoption of microfluidic devices among the neuroscience and neurobiology communities has enabled addressing a broad range of questions at the molecular, cellular, circuit, and system levels. Here, we review biomedical engineering approaches that harness the power of microfluidics for bottom-up generation of neuronal cell types and for the assembly and analysis of neural circuits. Microfluidics-based approaches are instrumental to generate the knowledge necessary for the derivation of diverse neuronal cell types from human pluripotent stem cells, as they enable the isolation and subsequent examination of individual neurons of interest. Moreover, microfluidic devices allow to engineer neural circuits with specific orientations and directionality by providing control over neuronal cell polarity and permitting the isolation of axons in individual microchannels. Similarly, the use of microfluidic chips enables the construction not only of 2D but also of 3D brain, retinal, and peripheral nervous system model circuits. Such brain-on-a-chip and organoid-on-a-chip technologies are promising platforms for studying these organs as they closely recapitulate some aspects of in vivo biological processes. Microfluidic 3D neuronal models, together with 2D in vitro systems, are widely used in many applications ranging from drug development and toxicology studies to neurological disease modeling and personalized medicine. Altogether, microfluidics provide researchers with powerful systems that complement and partially replace animal models.

Neuronal networks derived from human induced pluripotent stem cells have been exploited widely for modeling neuronal circuits, neurological diseases, and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features, we improved a glia–neuron co-culture protocol within multi-electrode arrays, facilitating continuous assessment of electrical features in weekly intervals. By full-field optogenetic stimulation, we detected an earlier onset of neuronal firing and burst activity compared with spontaneous activity. Full-field stimulation enhanced the number of active neurons and their firing rates. Compared with full-field stimulation, which evoked synchronized activity across all neurons, holographic stimulation of individual neurons resulted in local activity. Single-cell holographic stimulation facilitated to trace propagating evoked activities of 400 individually stimulated neurons per multi-electrode array. Thereby, we revealed precise functional neuronal connectivity motifs. Holographic stimulation data over time showed increasing connection numbers and strength with culture age. This holographic stimulation setup has the potential to establish a profound functional testbed for in-depth analysis of human-induced pluripotent stem cell-derived neuronal networks.

Comprehensive electrophysiological characterizations of human induced pluripotent stem cell (hiPSC)-derived neuronal networks are essential to determine to what extent these in vitro models recapitulate the functional features of in vivo neuronal circuits. High-density micro-electrode arrays (HD-MEAs) offer non-invasive recording with the best spatial and temporal resolution possible to date. For 3 months, we tracked the morphology and activity features of developing networks derived from a transgenic hiPSC line in which neurogenesis is inducible by neurogenic transcription factor overexpression. Our morphological data revealed large-scale structural changes from homogeneously distributed neurons in the first month to the formation of neuronal clusters over time. This led to a constant shift in position of neuronal cells and clusters on HD-MEAs and corresponding changes in spatial distribution of the network activity maps. Network activity appeared as scarce action potentials (APs), evolved as local bursts with longer duration and changed to network-wide synchronized bursts with higher frequencies but shorter duration over time, resembling the emerging burst features found in the developing human brain. Instantaneous firing rate data indicated that the fraction of fast spiking neurons (150–600 Hz) increases sharply after 63 days post induction (dpi). Inhibition of glutamatergic synapses erased burst features from network activity profiles and confirmed the presence of mature excitatory neurotransmission. The application of GABAergic receptor antagonists profoundly changed the bursting profile of the network at 120 dpi. This indicated a GABAergic switch from excitatory to inhibitory neurotransmission during circuit development and maturation. Our results suggested that an emerging GABAergic system at older culture ages is involved in regulating spontaneous network bursts. In conclusion, our data showed that long-term and continuous microscopy and electrophysiology readouts are crucial for a meaningful characterization of morphological and functional maturation in stem cell-derived human networks. Most importantly, assessing the level and duration of functional maturation is key to subject these human neuronal circuits on HD-MEAs for basic and biomedical applications.