A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

BACKGROUND: Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required. METHODS: Primary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared. RESULTS: The tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1ß, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture. CONCLUSIONS: The tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.

Rat primary cortical cell tri-culture to study effects of amyloid-beta on microglia function.

Introduction: The etiology and progression of sporadic Alzheimer's Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (Aß) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in Aß clearance, and alterations in microglial function due to aging or disease may result in Aß accumulation 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 microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and Aß clearance) and proteomic response to exogenous Aß. Methods: We established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled Aß (FITC-Aß) particles for varying durations. Images were analyzed to determine the number of FITC-Aß particles after specific lengths of exposure. A group of cells were stained for ßIII-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). Results and discussion: FITC-Aß particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aß particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-Aß significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-Aß addition revealed a significant increase in proinflammatory cytokines (TNF-α, IL-1α, IL-1ß, IL-6) in tri-culture, but not co-culture. In addition, Aß addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and Aß clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.

Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures.

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.

Cultured Vagal Afferent Neurons as Sensors for Intestinal Effector Molecules.

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.

Experiential Learning in a Biomedical Device Engineering Course: Proposal Development and Raw Research Data-Based Assignments.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s43683-022-00094-z.

Primary Cortical Cell Tri-Culture-Based Screening of Neuroinflammatory Response in Toll-like Receptor Activation.

The activation of toll-like receptors (TLRs) in the central nervous system (CNS) can lead to neuroinflammation and contribute to many neurological disorders, including autoimmune diseases. Cell culture models are powerful tools for studying specific molecular and cellular mechanisms that contribute to these disease states and identifying potential therapeutics. However, most cell culture models have limitations in capturing biologically relevant phenomena, due in part to the non-inclusion of necessary cell types. Neurons, astrocytes, and microglia (critical cell types that play a role in neuroinflammation) all express at least a subset of TLRs. However, the response of each of these cell types to various TLR activation, along with their relative contribution to neuroinflammatory processes, is far from clear. In this study, we demonstrate the screening capabilities of a primary cortical cell tri-culture of neuron, astrocyte, and microglia from neonatal rats. Specifically, we compare the neuroinflammatory response of tri-cultures to that of primary neuron-astrocyte co-cultures to a suite of known TLR agonists. We demonstrate that microglia are required for observation of neurotoxic neuroinflammatory responses, such as increased cell death and apoptosis, in response to TLR2, 3, 4, and 7/8 activation. Additionally, we show that following TLR3 agonist treatment, microglia and astrocytes play opposing roles in the neuroinflammatory response, and that the observed response is dictated by the degree of TLR3 activation. Overall, we demonstrate that microglia play a significant role in the neuroinflammatory response to TLR activation in vitro and, hence, the tri-culture has the potential to serve as a screening platform that better replicates the in vivo responses.

Influence of microchannel geometry on device performance and electrophysiological recording fidelity during long-term studies of connected neural populations.

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.

Chemically-Gated and Sustained Molecular Transport through Nanoporous Gold Thin Films in Biofouling Conditions.

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).

Multilayer poly(3,4-ethylenedioxythiophene)-dexamethasone and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate-carbon nanotubes coatings on glassy carbon microelectrode arrays for controlled drug release.

The authors present an electrochemically controlled, drug releasing neural interface composed of a glassy carbon (GC) microelectrode array combined with a multilayer poly(3,4-ethylenedioxythiophene) (PEDOT) coating. The system integrates the high stability of the GC electrode substrate, ideal for electrical stimulation and electrochemical detection of neurotransmitters, with the on-demand drug-releasing capabilities of PEDOT-dexamethasone compound, through a mechanically stable interlayer of PEDOT-polystyrene sulfonate (PSS)-carbon nanotubes (CNT). The authors demonstrate that such interlayer improves both the mechanical and electrochemical properties of the neural interface, when compared with a single PEDOT-dexamethasone coating. Moreover, the multilayer coating is able to withstand 10 × 106 biphasic pulses and delamination test with negligible change to the impedance spectra. Cross-section scanning electron microscopy images support that the PEDOT-PSS-CNT interlayer significantly improves the adhesion between the GC substrate and PEDOT-dexamethasone coating, showing no discontinuities between the three well-interconnected layers. Furthermore, the multilayer coating has superior electrochemical properties, in terms of impedance and charge transfer capabilities as compared to a single layer of either PEDOT coating or the GC substrate alone. The authors verified the drug releasing capabilities of the PEDOT-dexamethasone layer when integrated into the multilayer interface through repeated stimulation protocols in vitro, and found a pharmacologically relevant release of dexamethasone.

Highly Stable Glassy Carbon Interfaces for Long-Term Neural Stimulation and Low-Noise Recording of Brain Activity.

We report on the superior electrochemical properties, in-vivo performance and long term stability under electrical stimulation of a new electrode material fabricated from lithographically patterned glassy carbon. For a direct comparison with conventional metal electrodes, similar ultra-flexible, micro-electrocorticography (µ-ECoG) arrays with platinum (Pt) or glassy carbon (GC) electrodes were manufactured. The GC microelectrodes have more than 70% wider electrochemical window and 70% higher CTC (charge transfer capacity) than Pt microelectrodes of similar geometry. Moreover, we demonstrate that the GC microelectrodes can withstand at least 5 million pulses at 0.45 mC/cm2 charge density with less than 7.5% impedance change, while the Pt microelectrodes delaminated after 1 million pulses. Additionally, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) was selectively electrodeposited on both sets of devices to specifically reduce their impedances for smaller diameters (<60 µm). We observed that PEDOT-PSS adhered significantly better to GC than Pt, and allowed drastic reduction of electrode size while maintaining same amount of delivered current. The electrode arrays biocompatibility was demonstrated through in-vitro cell viability experiments, while acute in vivo characterization was performed in rats and showed that GC microelectrode arrays recorded somatosensory evoked potentials (SEP) with an almost twice SNR (signal-to-noise ratio) when compared to the Pt ones.

Investigation Into the Effects of Nucleotide Content on the Electrical Characteristics of DNA Plasmid Molecular Wires.

In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between [Formula: see text] in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100 Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nanoscale DNA molecules (<50bp), we were able to demonstrate that nucleotide content can affect the conductivity of micrometer length DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nanoscale DNA molecules may still be applicable for DNA wires at the micro scale.