Search for: All records

The gut–brain axis embodies the bi-directional communication between the gastrointestinal tract and the central nervous system (CNS), where vagal afferent neurons (VANs) serve as sensors for a variety of gut-derived signals. The gut is colonized by a large and diverse population of microorganisms that communicate via small (effector) molecules, which also act on the VAN terminals situated in the gut viscera and consequently influence many CNS processes. However, the convoluted in vivo environment makes it difficult to study the causative impact of the effector molecules on VAN activation or desensitization. Here, we report on a VAN culture and its proof-of-principle demonstration as a cell-based sensor to monitor the influence of gastrointestinal effector molecules on neuronal behavior. We initially compared the effect of surface coatings (poly-L-lysine vs. Matrigel) and culture media composition (serum vs. growth factor supplement) on neurite growth as a surrogate of VAN regeneration following tissue harvesting, where the Matrigel coating, but not the media composition, played a significant role in the increased neurite growth. We then used both live-cell calcium imaging and extracellular electrophysiological recordings to show that the VANs responded to classical effector molecules of endogenous and exogenous origin (cholecystokinin serotonin and capsaicin) in a complex fashion. We expect this study to enable platforms for screening various effector molecules and their influence on VAN activity, assessed by their information-rich electrophysiological fingerprints. 

Neuroinflammation plays a central role in many neurological disorders, ranging from traumatic brain injuries to neurodegeneration. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. In order to study neuroinflammation and its electrophysiological fingerprints, there is a need for in vitro models that accurately capture the in vivo phenomena. In this study, we employed a new tri-culture of primary rat neurons, astrocytes, and microglia in combination with extracellular electrophysiological recording techniques using multiple electrode arrays (MEAs) to determine the effect of microglia on neural function and the response to neuroinflammatory stimuli. Specifically, we established the tri-culture and its corresponding neuron-astrocyte co-culture (lacking microglia) counterpart on custom MEAs and monitored their electrophysiological activity for 21 days to assess culture maturation and network formation. As a complementary assessment, we quantified synaptic puncta and averaged spike waveforms to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons. The results demonstrate that the microglia in the tri-culture do not disrupt neural network formation and stability and may be a better representation of the in vivo rat cortex due to its more similar E/I ratio as compared to more traditional isolated neuron and neuron-astrocyte co-cultures. In addition, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following pro-inflammatory lipopolysaccharide exposure, highlighting the critical role of microglia in capturing electrophysiological manifestations of a representative neuroinflammatory insult. We expect the demonstrated technology to assist in studying various brain disease mechanisms. 

Abstract There is a need for novel teaching approaches to train biomedical engineers that are conversant across disciplines and have the technical skills to address interdisciplinary scientific and technological challenges. Here, we describe a graduate-level miniaturized biomedical device engineering course that has been taught over the last decade in in-person, remote, and hybrid formats. The course employs experiential learning components, including a proposal development and review that mimic the National Institutes of Health process and technical assignments that use raw research data to simulate a research experience. The effectiveness of the course was measured via pre-/post-course concept inventory surveys as well as course evaluations with targeted questions on the learning instruments. Statistical comparison of pre-/post-course survey scores suggests that the course was effective in students achieving the learning objectives, and comparison of relative increase in pre-/post-course survey scores across different instruction formats (i.e., in-person, remote, hybrid) showed minimal difference, suggesting that the teaching elements are readily transferrable to remote instruction. 

Compartmentalized microfluidic neural cell culture platforms, which physically separate axons from the neural soma using a series of microchannels, have been used for studying a wide range of pathological conditions and basic neuroscience questions. While each study has different experimental needs, the fundamental design of these devices has largely remained unchanged and a systematic study to establish long-term neural cultures in this format is lacking. Here, we investigate the influence of microchannel geometry and cell seeding density on device performance particularly in the context of long-term studies of synaptically-connected, yet fluidically-isolated neural populations of neurons and glia. Of the different experimental parameters, the microchannel height was the principal determinant of device performance, where the other parameters offer additional degrees of freedom in customizing such devices for specific applications. We condense the effects of these parameters into design rules and demonstrate their utility in engineering a microfluidic neural culture platform with integrated microelectrode arrays. The engineered device successfully recorded from primary rat cortical cells for 59 days in vitro with more than on order of magnitude enhancement in signal-to-noise ratio in the microchannels. 

BackgroundMicroglia play a critical role in neurodegenerative disorders, such as Alzheimer's disease, where alterations in microglial function may result in pathogenic amyloid-β (Aβ) accumulation, chronic neuroinflammation, and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of critical cell types in the same culture. ObjectiveWe employed a rat primary tri-culture (neurons, astrocytes, and microglia) model and compared it to co-culture (neurons and astrocytes) and mono-culture (microglia) to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ. MethodsThe cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Epifluorescence microscopy images were analyzed to quantify the number of FITC-Aβ particles and assess cytomorphological features. Cytokine profiles from conditioned media were obtained. Live-cell imaging was employed to extract microglia motility parameters. ResultsFITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. FITC-Aβ treatment significantly increased microglia size, but had no significant effect on neuronal surface coverage or astrocyte size. Upon FITC-Aβ treatment, there was a significant increase in proinflammatory cytokines in tri-culture, but not in co-culture. Aβ treatment altered microglia motility evident as a swarming-like motion. ConclusionsThe results suggest that neuron-astrocyte-microglia interactions influence microglia function and highlight the utility of the tri-culture model for studies of neuroinflammation, neurodegeneration, and cell-cell communication. 

Sustained release and replenishment of the drug depot are essential for the long-term functionality of implantable drug-delivery devices. This study demonstrates the use nanoporous gold (np-Au) thin films for in-plane transport of fluorescein (a small-molecule drug surrogate) over large (mm-scale) distances from a distal reservoir to the site of delivery, thereby establishing a constant flux of molecular release. In the absence of halides, the fluorescein transport is negligible due to a strong non-specific interaction of fluorescein with the pore walls. However, in the presence of physiologically relevant concentration of ions, halides preferentially adsorb onto the gold surface, minimizing the fluorescein–gold interactions and thus enabling in-plane fluorescein transport. In addition, the nanoporous film serves as an intrinsic size-exclusion matrix and allows for sustained release in biofouling conditions (dilute serum). The molecular release is reproducibly controlled by gating it in response to the presence of halides at the reservoir (source) and the release site (sink) without external triggers (e.g., electrical and mechanical).