Cardiac fibrosis plays a central role in the progression of heart failure, resulting from the excessive accumulation of extracellular matrix (ECM) proteins that compromise myocardial function. Despite its clinical relevance, studying this pathology in vitro remains challenging due to the lack of physiologically relevant models, both 2D and 3D. In particular, in vitro evaluation of cardiac fibrosis is often limited to end-point analyses, which are typically invasive and destructive. As such, these methods do not enable continuous monitoring of disease progression over time but rather provide information of its state at the end of the experimental timeline. In the last few years, Organs-on-Chip (OoC) platforms have been offering a valid solution by better replicating the complexity of human tissues, including both their physiological and pathological state. Moreover, these microfluidic platforms can be integrated with sensors for direct or indirect monitoring of cellular constructs by evaluating parameters such as the viability of the cells, metabolic activity and ECM deposition. This thesis aims to develop and integrate an impedance-based sensing system into a 3D OoC platform for real-time monitoring of cardiac fibrosis onset and progression, by correlating impedance measurements with extracellular matrix deposition associated with the fibrotic process. The impedance measuring setup was designed and optimized throughout the project, supported by both technical and biological validation. First, the measuring setup was optimized by comparing different electrode configurations and evaluating the impact of the width of the channel containing the cellular constructs. To study the relationship between cardiac fibrosis and impedance, experiments using human ventricular cardiac fibroblasts (hCFs) embedded in fibrin gel were conducted. These cells were chosen because they play a relevant role not only in the physiological remodeling of the heart, but also in the pathological remodeling that drives fibrotic responses. Fibrosis was successfully induced by treating hCFs with TGF-β1, a growth factor that triggers a signaling pathway known to promote fibroblasts-to-myofibroblast transition and ECM proteins production. During these experiments, impedance values were monitored and compared between the cells stimulated with TGF-β1 and control devices, to assess the potential correlation between impedance variation and the fibrotic behavior. To enhance ECM deposition and highlight differences between experimental conditions, hCFs were subjected to combined chemical and mechanical stimulation, following the protocol described by Visone et al. (2023). As anticipated, the dual stimulation led to strong cell activation and substantial ECM deposition, which was reflected in increased impedance measurement. Furthermore, to strengthen this analysis, different cell types known for their intrinsic capacity to produce ECM, such as chondrocytes and mesenchymal cells, were tested using the proposed setup with encouraging results. Following the optimization and characterization of the on-chip model of cardiac fibrosis, this project focused on evaluating the therapeutical effects of extracellular vesicles (EVs) to prevent disease progression. Cardiac fibroblasts were pre-treated with EVs at varying doses prior to either chemical or mechanical stimulation. Notably, the EVs – in particular at low concentration – effectively inhibited fibroblast activation under both conditions, confirming their anti-fibrotic potential in a 3D model.
La fibrosi cardiaca gioca un ruolo fondamentale nella progressione dell’insufficienza del cuore, che deriva dall’accumulo eccessivo di proteine della matrice extracellulare (ECM) che compromette la funzionalità del miocardio. Nonostante la sua rilevanza clinica, lo studio in vitro di questa patologia risulta ancora complesso a causa della mancanza di modelli fisiologicamente rilevanti, sia bidimensionali che tridimensionali. In particolare, la valutazione in vitro della fibrosi cardiaca è spesso limitata ad analisi end-point, che sono generalmente invasive e distruttive. Di conseguenza, questi metodi non permettono un monitoraggio continuo nel tempo della progressione della malattia, ma forniscono in- formazioni solo sul suo stato solo a conclusione dell’esperimento. Negli ultimi anni, le piattaforme di Organs-on-Chip (OoC) si sono affermate come una valida soluzione, gra- zie alla loro capacità di replicare più fedelmente la complessità dei tessuti umani, sia in condizioni fisiologiche che patologiche. Inoltre, queste piattaforme microfluidiche possono essere integrate con sensori per il monitoraggio diretto o indiretto dei costrutti cellulari, attraverso la valutazione di parametri come la vitalità cellulare, l’attività metabolica e la deposizione di matrice extracellulare. Questa tesi ha l’obbiettivo di sviluppare e integrare un sistema di misura basato sull’impedenza all’interno di una piattaforma 3D OoC, per il monitoraggio in tempo reale dell’insorgenza e della progressione della fibrosi cardiaca, correlando le misure di impedenza alla deposizione di matrice extracellulare associata al processo fibrotico. Il sistema di misura d’impedenza è stato progettato e ottimizzato nel corso del progetto, supportato sia da validazione tecnica che biologica. In una prima fase, il setup di misura è stato ottimizzato confrontando diverse configurazioni di elettrodi e valutando l’impatto della larghezza del canale contenente i costrutti cellulari. Per studiare la relazione tra fibrosi cardiaca e impedenza, sono stati condotti esperimenti utilizzando fibroblasti cardiaci umani ventricolari (hCFs) incapsulati in un gel di fibrina. Queste cellule sono state scelte perché svolgono un ruolo rilevate non solo nel rimodellamento fisiologico del cuore, ma anche nel rimodellamento patologico che guida la risposta fibrot- ica. La fibrosi è stata indotta con successo trattando gli hCFs con TGF-β1, un fattore di crescita in grado di attivare una via di segnalazione nota per promuovere la transizione dei fibroblasti in miofibroblasti e la produzione di proteine della ECM. Durante gli esperimenti, i valori di impedenza sono stati monitorati e confrontati tra le cellule trattate con TGF-β1 e i controlli, al fine di valutare la potenziale correlazione tra la variazione di impedenza e il comportamento fibrotico. Per potenziare la deposizione di ECM e amplificare le differenze tra condizioni sperimentali, gli hCFs sono stati trattati combinando la stimolazione chimica e la stimolazione meccanica, seguendo il protocollo descritto da Visone et al. (2023). Come previsto, la doppia stimolazione ha indotto una forte attivazione cellulare e una consistente deposizione di matrice extracellulare, evidenziata in un aumento delle misurazioni di impedenza. Per rafforzare ulteriormente questa analisi, altri tipi cellulari noti per la loro capacità intrinseca di produrre ECM, come i condrociti e le cellule mesenchimali, sono stati testati con il sistema proposto con risultati promettenti. In seguito all’ottimizzazione e caratterizzazione del modello di fibrosi cardiaca su chip, il progetto si è focalizzato sulla valutazione degli effetti ter- apeutici delle vescicole extracellulari (EVs) nel prevenire la progressione della malattia. I fibroblasti cardiaci sono stati pre-trattati con EVs a dosaggi variabili, prima di essere sot- toposti a stimolazione chimica o meccanica. In particolare, le EVs – soprattutto a basse concentrazioni – hanno dimostrato di inibire efficacemente l’attivazione dei fibroblasti in entrambe le condizioni, confermando il loro potenziale anti-fibrotico in un modello 3D.
Assessing micro-tissue remodeling via electrical impedance in an Organ on Chip platform
DONATONI, GIORGIA
2024/2025
Abstract
Cardiac fibrosis plays a central role in the progression of heart failure, resulting from the excessive accumulation of extracellular matrix (ECM) proteins that compromise myocardial function. Despite its clinical relevance, studying this pathology in vitro remains challenging due to the lack of physiologically relevant models, both 2D and 3D. In particular, in vitro evaluation of cardiac fibrosis is often limited to end-point analyses, which are typically invasive and destructive. As such, these methods do not enable continuous monitoring of disease progression over time but rather provide information of its state at the end of the experimental timeline. In the last few years, Organs-on-Chip (OoC) platforms have been offering a valid solution by better replicating the complexity of human tissues, including both their physiological and pathological state. Moreover, these microfluidic platforms can be integrated with sensors for direct or indirect monitoring of cellular constructs by evaluating parameters such as the viability of the cells, metabolic activity and ECM deposition. This thesis aims to develop and integrate an impedance-based sensing system into a 3D OoC platform for real-time monitoring of cardiac fibrosis onset and progression, by correlating impedance measurements with extracellular matrix deposition associated with the fibrotic process. The impedance measuring setup was designed and optimized throughout the project, supported by both technical and biological validation. First, the measuring setup was optimized by comparing different electrode configurations and evaluating the impact of the width of the channel containing the cellular constructs. To study the relationship between cardiac fibrosis and impedance, experiments using human ventricular cardiac fibroblasts (hCFs) embedded in fibrin gel were conducted. These cells were chosen because they play a relevant role not only in the physiological remodeling of the heart, but also in the pathological remodeling that drives fibrotic responses. Fibrosis was successfully induced by treating hCFs with TGF-β1, a growth factor that triggers a signaling pathway known to promote fibroblasts-to-myofibroblast transition and ECM proteins production. During these experiments, impedance values were monitored and compared between the cells stimulated with TGF-β1 and control devices, to assess the potential correlation between impedance variation and the fibrotic behavior. To enhance ECM deposition and highlight differences between experimental conditions, hCFs were subjected to combined chemical and mechanical stimulation, following the protocol described by Visone et al. (2023). As anticipated, the dual stimulation led to strong cell activation and substantial ECM deposition, which was reflected in increased impedance measurement. Furthermore, to strengthen this analysis, different cell types known for their intrinsic capacity to produce ECM, such as chondrocytes and mesenchymal cells, were tested using the proposed setup with encouraging results. Following the optimization and characterization of the on-chip model of cardiac fibrosis, this project focused on evaluating the therapeutical effects of extracellular vesicles (EVs) to prevent disease progression. Cardiac fibroblasts were pre-treated with EVs at varying doses prior to either chemical or mechanical stimulation. Notably, the EVs – in particular at low concentration – effectively inhibited fibroblast activation under both conditions, confirming their anti-fibrotic potential in a 3D model.| File | Dimensione | Formato | |
|---|---|---|---|
|
2025_07_Donatoni_Tesi_01.pdf
non accessibile
Descrizione: testo tesi
Dimensione
15.62 MB
Formato
Adobe PDF
|
15.62 MB | Adobe PDF | Visualizza/Apri |
|
2025_07_Donatoni_Executive Summary_02.pdf
non accessibile
Descrizione: testo executive summary
Dimensione
1.81 MB
Formato
Adobe PDF
|
1.81 MB | Adobe PDF | Visualizza/Apri |
I documenti in POLITesi sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/10589/240380