The intestine, considered the visceral brain, plays a fundamental role in the absorption of nutrients. Intestinal disorders can result from genetic, pathological and / or environmental causes and can lead to mild disorders, or more serious conditions, such as ulcers and cancer. Rapid diagnosis and therapy are essential to restore proper functioning, therefore, to date, several animal models and 2D in vitro models have been used as methods for studying intestinal pathologies. However, the former is limited by interspecies mismatch and ethical criticalities while the latter cannot faithfully recreate the complexity of the physiological environment. These limitations have led to the need for new culture systems able to better control the generation of functional tissues. Technological progress in the field of microfabrication has led to the development of microfluidic devices to be used in the field of disease modelling, diagnostics, and tissue engineering. This work focuses on Organ-on-a-chip (OoC) technology; OoC are micro-engineered platforms that mimic the functional units of organs. Such devices can finely regulate the culture environment and provide for physical stimulation that modulates the cellular response by promoting differentiation, proliferation, phenotype control and migration. Among the OoCs capable of exerting mechanical cues, BiomimX® uBeat® Stretch, which uses pneumatic actuation to generate stimulation, is worth being mentioned for their versatility to help in mimicking different tissue types such as cardiac and intestinal ones. Such pneumatic actuation is controlled by uBox®, an electronic unit designed by the same company The aim of this work was to improve this electronic control system which generates pressure waves characterized by pulsed waveforms. In particular, the possibility of generating customised pressure waves has been introduced in order to better simulate physiological stimulation such as intestinal peristaltic movements. The system consists of an Arduino board that generate a digital voltage system that is transformed and amplified to control a voltage-driven pressure regulator. Once the system was developed, an accurate mechanical characterization was performed to verify its correct functioning and the correct actuation when it is connected to the uBeat® Stretch platforms. As a further step, a biological experiment aimed at reproducing the intestinal epithelium was performed to validate the designed control system and to compare its stimulation pattern with the uBox® one. To do so Caco-2 and HT-29 MTX cells were cocultured on-chip and cultured for 9 days with and without stimulation. Brightfield and fluorescent microscopy methods were used to analyse culture outcome and samples were stained for proliferation marker Ki-67, marker ZO-1 for epithelial cohesion and specific tissue proteins (Villin, Mucin). The system has proven to be able to provide adequate stimulation, being able to generate any sine wave between 0 and 1 bar, with a maximum frequency of 2 Hz. Furthermore, during the biological experiment it has been shown that the uBeat® Stretch devices are able to provide an ideal environment for cell survival and proliferation. Finally, the results showed that mechanically stimulated devices promoted the generation of three-dimensional structures with respect to the non-stimulated condition. Further developments of this work could involve the implementation of screen graphics of the designed control unit and biological experiments to evaluate drug uptake and intestinal epithelium absorption as compared to standard culture techniques such as transwell method. Once the epithelial barrier on a chip is completely characterised and optimised it will represent a powerful tool to speed up drug development process.
L'intestino, considerato il cervello viscerale, svolge un ruolo fondamentale nell'assorbimento dei nutrienti. I disturbi intestinali possono derivare da cause genetiche, patologiche e/o ambientali e possono portare a disturbi lievi, come nausea e diarrea, o disturbi più gravi, come ulcera e cancro. Diagnosi e terapia rapide sono essenziali per ripristinare il corretto funzionamento, pertanto, ad oggi, sono stati utilizzati diversi modelli animali e modelli 2D in vitro come metodi per lo studio delle patologie intestinali. Tuttavia, i primi sono limitati dalle differenze interspecie e i secondi non sono in grado ricreare fedelmente la complessità dell'ambiente fisiologico. Tali limitazioni hanno portato alla necessità di sviluppare nuovi sistemi di coltura in grado di controllare meglio la generazione di un tessuti funzionale. Il progresso tecnologico nel campo della microfabbricazione ha portato allo sviluppo di dispositivi microfluidici da utilizzare nel campo della modellazione delle malattie, della diagnostica e dell'ingegneria dei tessuti. Questo lavoro si concentra sui dispositivi Organ-on-a-chip (OoC), piattaforme di coltura micro-ingegnerizzate che permettono di ricreare in-vitro le unità funzionali degli organi. Tali dispositivi sono in grado di regolare finemente l'ambiente di coltura e fornire stimolazioni fisiche che modulano la risposta cellulare promuovendo differenziazione, proliferazione, controllo del fenotipo e migrazione. Tra gli OoC in grado di esercitare stimoli meccanici merita di essere citata la piattaforma uBeat® Stretch dell’azienda BiomimX®, che impiega un meccanismo di attuazione pneumatica per generare la stimolazione. Tale stimolazione è guidata da un sistemo di controllo elettronico, uBox®, sviluppato dalla stessa azienda. Lo scopo di questo lavoro è stato il miglioramento di tale sistema di controllo che, ad oggi, genera onde di pressione caratterizzate da forme d'onda a impulsi. In particolare, è stata introdotta la possibilità di generare onde di pressione personalizzate al fine di simulare al meglio stimoli fisiologici come i movimenti peristaltici intestinali. Il sistema è costituito da una scheda Arduino che genera un sistema di tensione digitale che viene trasformato e amplificato per controllare un regolatore di pressione pilotato in tensione. Una volta sviluppato il sistema, è stata eseguita un'accurata caratterizzazione meccanica per verificarne il corretto funzionamento. Come ulteriore passo, è stato eseguito un esperimento biologico volto a riprodurre l'epitelio intestinale per validare il sistema di controllo progettato e per confrontare il suo pattern di stimolazione con quello uBox®. Con questo scopo, due popolazioni cellulari, Caco-2 e HT-29 MTX, sono state coltivate su chip per nove giorni e stimolate con i due sistemi di controllo differenti. Sono stati adottati metodi di microscopia a campo chiaro e microscopia a fluorescenza per analizzare l'esito della coltura e i campioni sono stati processati per evidenziare con tecniche di immunofluorescenza il marcatore di proliferazione Ki-67, il marcatore ZO-1 per valutare l’integrità dell'epitelio e le proteine tessuto-specifiche (Villina, Mucina). Il sistema si è dimostrato in grado di fornire una stimolazione adeguata, potendo generare qualsiasi onda sinusoidale di ampiezza compresa tra 0 e 1 bar, con una frequenza massima di 2 Hz. Inoltre, durante l’esperimento biologico si è dimostrato che i dispositivi uBeat® Stretch, sono in grado di fornire un ambiente ideale per la sopravvivenza e la proliferazione cellulare delle linee scelte. Infine, i risultati hanno evidenziato che i dispositivi stimolati meccanicamente hanno promosso una maggiore generazione di strutture tridimensionali rispetto a quelli statici. Ulteriori sviluppi di questo lavoro potrebbero comportare l'implementazione della grafica dello schermo dell'unità di controllo progettata e esperimenti biologici per valutare l'assorbimento di farmaci e l'assorbimento dell'epitelio intestinale rispetto alle tecniche di coltura standard come il metodo transwell. Una volta che la barriera epiteliale su un chip sarà completamente caratterizzata e ottimizzata, rappresenterà un potente strumento per accelerare il processo di sviluppo di farmaci.
Re-design of a controller for the mechanical stimulation of Gut-on-a-chip devices
Pigazzini, Andrea
2020/2021
Abstract
The intestine, considered the visceral brain, plays a fundamental role in the absorption of nutrients. Intestinal disorders can result from genetic, pathological and / or environmental causes and can lead to mild disorders, or more serious conditions, such as ulcers and cancer. Rapid diagnosis and therapy are essential to restore proper functioning, therefore, to date, several animal models and 2D in vitro models have been used as methods for studying intestinal pathologies. However, the former is limited by interspecies mismatch and ethical criticalities while the latter cannot faithfully recreate the complexity of the physiological environment. These limitations have led to the need for new culture systems able to better control the generation of functional tissues. Technological progress in the field of microfabrication has led to the development of microfluidic devices to be used in the field of disease modelling, diagnostics, and tissue engineering. This work focuses on Organ-on-a-chip (OoC) technology; OoC are micro-engineered platforms that mimic the functional units of organs. Such devices can finely regulate the culture environment and provide for physical stimulation that modulates the cellular response by promoting differentiation, proliferation, phenotype control and migration. Among the OoCs capable of exerting mechanical cues, BiomimX® uBeat® Stretch, which uses pneumatic actuation to generate stimulation, is worth being mentioned for their versatility to help in mimicking different tissue types such as cardiac and intestinal ones. Such pneumatic actuation is controlled by uBox®, an electronic unit designed by the same company The aim of this work was to improve this electronic control system which generates pressure waves characterized by pulsed waveforms. In particular, the possibility of generating customised pressure waves has been introduced in order to better simulate physiological stimulation such as intestinal peristaltic movements. The system consists of an Arduino board that generate a digital voltage system that is transformed and amplified to control a voltage-driven pressure regulator. Once the system was developed, an accurate mechanical characterization was performed to verify its correct functioning and the correct actuation when it is connected to the uBeat® Stretch platforms. As a further step, a biological experiment aimed at reproducing the intestinal epithelium was performed to validate the designed control system and to compare its stimulation pattern with the uBox® one. To do so Caco-2 and HT-29 MTX cells were cocultured on-chip and cultured for 9 days with and without stimulation. Brightfield and fluorescent microscopy methods were used to analyse culture outcome and samples were stained for proliferation marker Ki-67, marker ZO-1 for epithelial cohesion and specific tissue proteins (Villin, Mucin). The system has proven to be able to provide adequate stimulation, being able to generate any sine wave between 0 and 1 bar, with a maximum frequency of 2 Hz. Furthermore, during the biological experiment it has been shown that the uBeat® Stretch devices are able to provide an ideal environment for cell survival and proliferation. Finally, the results showed that mechanically stimulated devices promoted the generation of three-dimensional structures with respect to the non-stimulated condition. Further developments of this work could involve the implementation of screen graphics of the designed control unit and biological experiments to evaluate drug uptake and intestinal epithelium absorption as compared to standard culture techniques such as transwell method. Once the epithelial barrier on a chip is completely characterised and optimised it will represent a powerful tool to speed up drug development process.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/188361