Supervisor: Stefano Vassanelli
Co-supervisor: Dr. Giacomo Cappellini e Dr.ssa Elisa Tentori
Co-supervisor Department/Company: Dipartimento di Scienze Biomediche
Creation Date: 06/10/2026 10:36
The proposed thesis is part of the project Development of an ex vivo innervated human skin model on High-Density Microelectrode Array (HD-MEA) platforms, which aims to develop and characterize an ex vivo model of reinnervated human skin. The model is based on human skin biopsies obtained from plastic and reconstructive surgery procedures and subsequently reinnervated using sensory neurons derived from the Dorsal Root Ganglia (DRG) of neonatal rats. The structural and functional properties of sensory neurons in vitro will be investigated through a multimodal approach integrating structural analyses, patch-clamp recordings, and HD-MEA electrophysiology. This characterization represents a fundamental and necessary step prior to their integration into the subsequent co-culture experiments.
The thesis will primarily focus on the acquisition and management of electrophysiological recordings using the MaxOne platform (MaxWell Biosystems), together with the subsequent quantitative analysis of the acquired data. HD-MEA experiments will be performed through the MaxLab Live Scope, the software environment controlling the MaxOne system, which provides dedicated acquisition workflows for complementary levels of electrophysiological characterization, including Activity Scan, Network recordings, and Axon Tracking. The Activity Scan assay will be used to sequentially screen the entire HD-MEA sensing area and map the spatial distribution of spontaneous neuronal activity. Electrode-wise firing rate and extracellular spike amplitude will be used to identify active regions of the culture and to guide the selection of recording electrodes for subsequent high-resolution and network-level recordings. The Axon Tracking analysis will further allow the study of action potential propagation along axonal structures by reconstructing their trajectories, branching patterns, direction of propagation, latency, and axonal conduction velocity. The integration of these features will provide a comprehensive functional characterization of sensory neurons in vitro at both the cellular and subcellular levels, establishing the baseline characterization required for their subsequent integration into co-culture experiments.
An additional component of the thesis will involve the characterization of pharmacological responses. Different compounds will be added to the culture medium present on the chip, and changes in electrophysiological activity will be monitored through pre- and post-treatment recordings. The effects of treatment will be assessed by evaluating changes in firing rate, extracellular action potential characteristics, electrical footprints, and, where detectable, axonal propagation. Potential dose-response relationships and the reversibility of drug-induced effects through washout experiments will also be investigated.
As a last step, the focus will be extended from the characterization of sensory neurons in vitro to the functional characterization of the reinnervated skin, with particular attention to the electrophysiological response of the skin following its interaction with the sensory neurons. Furthermore, additional compounds will be evaluated through topical administration directly onto the reinnervated skin, with the aim of assessing their effects on the electrophysiological parameters described above. This experimental approach will make it possible to investigate the functional response of the reinnervated tissue to compounds applied through a route that more closely reflects their interaction with the skin in physiological and translational contexts.
Dataset type: Data to be acquired
Dataset description: The dataset will mainly consist of electrophysiological recordings acquired from both sensory neuronal cultures and reinnervated skin preparations acquired using the MaxOne HD-MEA platform (MaxWell Biosystems). Recordings will include spontaneous spiking activity recording, using the MaxLab Live Scope workflows: Activity Scan, network recordings, and Axon Tracking acquisitions. Data will be collected across different Days in Vitro (DIV), during electrical stimulation protocols and following pharmacological or cosmetic compound application. In neuronal cultures, compounds will be administered through the culture medium and their effects on electrophysiological activity will be assessed before, during, and after treatment, including a washout phase where appropriate to evaluate response reversibility and potential dose-response relationships. Following the establishment of cutaneous reinnervation, compounds will instead be applied topically to the skin, and the electrophysiological response of the reinnervated tissue will be assessed under acute and longitudinal conditions.
List of Methods: Sensory neurons will be isolated from the DRG of neonatal rats (P1-P3) using enzymatic digestion followed by mechanical dissociation, pre-plating, and antimitotic treatment to obtain an enriched neuronal population. Following isolation, neurons will be maintained under controlled culture conditions (37 °C, 5% CO₂, approximately 97% relative humidity) and seeded either on Petri dishes for patch-clamp experiments or directly onto MaxOne chips (MaxWell Biosystems) for HD-MEA recordings. At DIV9, an approximately 5-mm ex vivo human skin biopsy obtained from abdominoplasty procedures will be gently positioned onto the neuronal culture to establish the co-culture model. The tissue will be maintained in co-culture for a further 6-7 days using an Air-Liquid Interface (ALI) system, with daily replacement of the culture medium. Following the co-culture period, the establishment of cutaneous reinnervation will be assessed by immunofluorescence, using PGP9.5 and βIII-tubulin as neuronal markers. At DIV15–18, electrophysiological recordings will be acquired from the neuronal cultures using both patch-clamp and HD-MEA approaches. Analysis. Spontaneous neuronal activity will be characterized both temporally and spatially, with particular emphasis on firing rate and its distribution in time and across the electrode array. Changes in neuronal activity will be quantified across DIV and following pharmacological administration. Responses to electrical stimulation will also be analyzed to determine how stimulation protocols modify the temporal and spatial organization of network activity. Axon Tracking data will be used to characterize action potential propagation along axonal processes. Analyses will include axonal trajectories and branching patterns, propagation direction and latency, and conduction velocity. Particular attention will be given to changes in propagation dynamics across DIV and following compound administration, including possible alterations in conduction latency and velocity.
• Biomarkers, Precision Medicine and Drug Development • Elaborazione di segnali biologici • Metodi statistici per la bioingegneria • Neurophysiology, Neural Computation and Neurotechnologies • Modeling Methodology for Physiology and Medicine