Biology subjects

Cesare, P.

Publications and source records attributed to Cesare, P..

4 recordsLinked to original sources

bioRxiv2023

A microphysiological system for parallelized morphological and electrophysiological read-out of 3D neuronal cell culture

Three-dimensional in vitro models in microfluidic systems are promising tools for studying cell biology, with complex models using multiple cell types combined with high resolution imaging. Neuronal models demand electrical readout of the activity of networks of single neurons, yet classical planar microelectrode arrays struggle to capture extracellular action potentials when neural soma are suspended distant from the microelectrodes. This study introduces sophisticated microfluidic microelectrode arrays, specifically tailored for electrophysiology of 3D neuronal cultures. Using multilayer photolithography of permanent epoxy photoresists, we developed devices having 12 independent culture modules in a convenient format. Each module has two adjacent compartments for hydrogel-based 3D cell culture, with tunnels allowing projection of neurites between compartments. Microelectrodes integrated in the tunnels record action potentials as they pass between the compartments. Mesh ceilings separate the compartments from overlying wells, allowing for simple cell seeding and later nutrient, gas and waste exchange and application of test substances. Using these devices, we have demonstrated 3D neuronal culture, including electrophysiological recording and live imaging. This microphysiological platform will enable high-throughput investigation of neuronal networks for investigation of neurological disorders, neural pharmacology and basic neuroscience. Further models could include cocultures representing multiple brain regions or innervation models of other organs.

neuroscience↗

bioRxiv2023

Cancer-mediated Axonal Guidance of Sensory Neurons in a Microelectrode-based Innervation MPS

Despite recent advances in the field of microphysiological systems (MPS), availability of models capable of mimicking the interactions between the nervous system and innervated tissues is still limited. This represents a significant challenge in identifying the underlying processes of various pathological conditions, including neuropathic, cardiovascular and metabolic disorders. In this study, we introduce a compartmentalized 3D coculture system that enables physiologically relevant tissue innervation while recording neuronal excitability. By integrating custom microelectrode arrays into tailored glass chips microfabricated via selective laser-etching, we developed an entirely novel class of innervation MPSs (INV-MPS). This INV-MPS allows for manipulation, visualization, and electrophysiological analysis of individual axons innervating complex 3D tissues. Here, we focused on sensory innervation of 3D tumor tissue as a model case study since cancer-induced pain represents a major unmet medical need. The system was compared with existing nociception models and successfully replicated axonal chemoattraction mediated by nerve growth factor (NGF). Remarkably, in the absence of NGF, 3D cancer spheroids cocultured in the adjacent compartment induced sensory neurons to consistently cross the separating barrier and establish fine innervation. Moreover, we observed that crossing sensory fibers could be chemically excited by distal application of known pain-inducing agonists only when cocultured with cancer cells. To our knowledge, this is the first system showcasing morphological and electrophysiological analysis of 3D-innervated tumor tissue in vitro, paving the way for a plethora of studies into innervation-related diseases and improving our understanding of underlying pathophysiology. Significance StatementMPSs integrating innervated tissues are crucial as alternatives to animal models and to enhance the translation of non-clinical research to humans, as nerve fibers play a pivotal role in many organs. To address this need, we developed the INV-MPS, enabling parallelized electro-physiological recording from 3D cocultures of peripheral neurons with complex tissues. We demonstrate the ability of cancer spheroids to direct axonal growth and modulate excitability of nociceptive fibers in a 3D setting. We anticipate that this model will be applicable for closely monitoring innervation in various tissues, including somatic and visceral organs. The INV-MPS, hence, has tremendous potential for human-relevant drug discovery, creating opportunities for more ethical and clinically translatable approaches to study innervation in disease.

neuroscience↗

bioRxiv2021

Comparative transcriptional analysis of the satellite glial cell injury response

Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. The SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNAseq technologies has enabled a new approach to investigate SGCs. Here we publish a dataset from mice subjected to sciatic nerve injury as well as a dataset from dorsal root ganglia cells after 3 days in culture. We use a meta-analysis approach to compare the injury response with that in other published datasets and conclude that SGCs share a common signature following sciatic nerve crush and sciatic ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state, while others start resembling Schwann cell-like precursors. The datasets are available via the Broad Institute Single Cell Portal.

neuroscience↗

bioRxiv2021

A multimodal 3D neuro-microphysiological system with neurite-trapping microelectrodes

Three-dimensional cell technologies as pre-clinical models are emerging tools for mimicking the structural and functional complexity of the nervous system. The accurate exploration of phenotypes in engineered 3D neuronal cultures, however, demands morphological, molecular and especially functional measurements. Particularly crucial is measurement of electrical activity of individual neurons with millisecond resolution. Current techniques rely on customized electrophysiological recording set-ups, characterized by limited throughput and poor integration with other readout modalities. Here we describe a novel approach, using multiwell glass microfluidic microelectrode arrays, allowing non-invasive electrical recording from engineered 3D neural tissues. We demonstrate parallelized studies with reference compounds, calcium imaging and optogenetic stimulation. Additionally, we show how microplate compatibility allows automated handling and high-content analysis of human induced pluripotent stem cell-derived neurons. This microphysiological platform opens up new avenues for high-throughput studies on the functional, morphological and molecular details of neurological diseases and their potential treatment by therapeutic compounds.

neuroscience↗