Neurological disorders are diseases that affect the nervous system and are the leading cause of disability and the second leading cause of death worldwide with an estimated number of 9 million deaths every year. In the last three decades, the number of deaths owing to these disorders increased by 61% (5.5 to 8.8 million) and the number of Disability-Adjusted Life Years (DALYs) by 45% (166 to 241 million). Despite these, no effective treatment options are currently available due to the lack of appropriate research models of the complex human nervous system and the pathological mechanisms of the diseases that affect it. Current approaches to study neurological disorders and neurodevelopment include in vivo animal models and in vitro monolayer cell cultures. These models have limitations when it comes to translating research about disorder pathology, development and treatment to humans. Brain organoids, 3D in vitro models derived from human induced pluripotent stem cells, have the potential to bridge this gap. However, all brain organoids lack vasculature which is essential in brain homeostasis and development. The aim of our project was to obtain a vascularized in vitro 3D model of the brain. To do so the project was divided into three different parts. First, we generated brain organoids from human induced pluripotent stem cells and monitored their growth in culture medium for 30 days following previously published and commercial protocols. The second part of the project focused on the vascularization through laser-based cavitation molding. This is a non-ablative, cell-friendly technique based on femtosecond laser exposure that can generate channels in hydrogels which can later be vascularized by seeding endothelial cells. We tested different hydrogels for their compatibility with cavitation molding and studied the tunability of the optimal one. Lastly, we designed, fabricated and optimized a microfluidic device capable of combining the brain organoid technique and laser cavitation molding of hydrogels to obtain vascularization of brain organoids. The project was conducted at KTH Royal Institute of Technology and Science for Life Laboratory in Professor’s Anna Herland in vitro neural systems lab during an Erasmus project. Together with the KTH team, we successfully generated brain organoids that developed according to protocol. They differentiated properly following the normal development of the in vivo human brain. We then tested Matrigel and collagen and Matrigel combinations of hydrogels for their compatibility with laser cavitation molding. We found that a certain concentration of collagen I is necessary for the phenomenon to occur. We then focused on studying the tunability of the collagen I and Matrigel mixture at a ratio of 1 to 1. We further developed a PDMS microfluidic chip optimized to host the brain organoid embedded in a hydrogel and allow laser-based cavitation molding. We fabricated different chip designs by replica molding and achieved bis-amino silane functionalization of PET membranes then bonded in a sandwich structure between the PDMS slabs. We then developed a protocol for the hydrogel casting, characterized the chip to find the right setting parameters of the laser and lastly patterned channels in the hydrogel. Conclusions. In the end, we managed to develop all the necessary tools that are needed for the vascularization of brain organoids on microfluidic chips. We will further investigate a hydrogel composition that allows both laser cavitation molding and better development of the organoids embedded in it as to cultivate the organoids directly on the chips with molded channels seeded with endothelial cells.
I disturbi neurologici sono malattie che colpiscono il sistema nervoso e sono la principale causa di disabilità e la seconda causa di morte a livello mondiale, con un numero stimato di 9 milioni di decessi ogni anno. Negli ultimi tre decenni, il numero di decessi dovuti a questi disturbi è aumentato del 61% (da 5,5 a 8,8 milioni) e il numero di anni di vita corretti per disabilità (DALYs) del 45% (da 166 a 241 milioni). Nonostante ciò, attualmente non sono disponibili opzioni terapeutiche efficaci a causa della mancanza di modelli di ricerca adeguati del complesso sistema nervoso umano e dei meccanismi patologici delle malattie che lo colpiscono. Gli approcci attuali per studiare i disturbi neurologici e il neurosviluppo includono modelli animali in vivo e colture cellulari monostrato in vitro. Questi modelli hanno dei limiti quando si tratta di tradurre la ricerca sulla patologia, lo sviluppo e il trattamento dei disturbi all'uomo. Gli organoidi cerebrali, modelli 3D in vitro derivati da cellule staminali pluripotenti indotte umane, hanno il potenziale per colmare questa lacuna. Tuttavia, tutti gli organoidi cerebrali mancano di vascolarizzazione, essenziale per l'omeostasi e lo sviluppo del cervello. L'obiettivo del nostro progetto era quello di ottenere un modello in vitro 3D vascolarizzato del cervello. A tal fine, il progetto è stato suddiviso in tre parti diverse. In primo luogo, abbiamo generato organoidi cerebrali da cellule staminali pluripotenti indotte umane e abbiamo monitorato la loro crescita in terreno di coltura per 30 giorni seguendo protocolli commerciali pubblicati precedentemente. La seconda parte del progetto si è concentrata sulla vascolarizzazione mediante laser-based cavitation molding. Si tratta di una tecnica non ablativa e cell-friendly, basata sull'esposizione al laser a femtosecondi, in grado di generare canali negli idrogel che possono poi essere vascolarizzati seminando cellule endoteliali. Abbiamo testato diversi idrogel per verificarne la compatibilità con il cavitation molding e abbiamo studiato la regolabilità di quello ottimale. Infine, abbiamo progettato, fabbricato e ottimizzato un dispositivo microfluidico in grado di combinare la tecnica degli organoidi cerebrali e il laser cavitation molding degli idrogel per ottenere la vascolarizzazione degli organoidi cerebrali. Il progetto è stato condotto presso il KTH Royal Institute of Technology e il Science for Life Laboratory nel laboratorio di sistemi neurali in vitro della Professoressa Anna Herland durante un progetto Erasmus. Insieme al team del KTH abbiamo generato con successo organoidi cerebrali che si sono sviluppati secondo il protocollo. Questi si sono differenziati correttamente seguendo il normale sviluppo del cervello umano in vivo. Abbiamo quindi testato Matrigel e combinazioni di idrogel di collagene e Matrigel per verificarne la compatibilità con il laser cavitation molding. Abbiamo scoperto che è necessaria una certa concentrazione di collagene I perché il fenomeno si verifichi. Ci siamo quindi concentrati sullo studio della regolazione della miscela di collagene I e Matrigel con un rapporto di 1 a 1. Abbiamo poi sviluppato un chip microfluidico in PDMS ottimizzato per ospitare l'organoide cerebrale incorporato in un idrogel e consentire il laser-based cavitation molding. Abbiamo fabbricato diversi modelli di chip mediante stampaggio a replica e abbiamo ottenuto la funzionalizzazione con bis-ammino silano delle membrane PET, poi incollate in una struttura a sandwich tra le parti di PDMS. Abbiamo quindi sviluppato un protocollo per il casting dell'idrogel, caratterizzato il chip per trovare i giusti parametri di impostazione del laser e infine realizzato canali nell'idrogel. Alla fine, siamo riusciti a sviluppare tutti gli strumenti necessari per la vascolarizzazione degli organoidi cerebrali su chip microfluidici. Studieremo ulteriormente una composizione di idrogel che permetta
Vascolarizzazione di organoidi cerebrali su chip microfluidici
GURAU, DOMNICA GABRIELA
2022/2023
Abstract
Neurological disorders are diseases that affect the nervous system and are the leading cause of disability and the second leading cause of death worldwide with an estimated number of 9 million deaths every year. In the last three decades, the number of deaths owing to these disorders increased by 61% (5.5 to 8.8 million) and the number of Disability-Adjusted Life Years (DALYs) by 45% (166 to 241 million). Despite these, no effective treatment options are currently available due to the lack of appropriate research models of the complex human nervous system and the pathological mechanisms of the diseases that affect it. Current approaches to study neurological disorders and neurodevelopment include in vivo animal models and in vitro monolayer cell cultures. These models have limitations when it comes to translating research about disorder pathology, development and treatment to humans. Brain organoids, 3D in vitro models derived from human induced pluripotent stem cells, have the potential to bridge this gap. However, all brain organoids lack vasculature which is essential in brain homeostasis and development. The aim of our project was to obtain a vascularized in vitro 3D model of the brain. To do so the project was divided into three different parts. First, we generated brain organoids from human induced pluripotent stem cells and monitored their growth in culture medium for 30 days following previously published and commercial protocols. The second part of the project focused on the vascularization through laser-based cavitation molding. This is a non-ablative, cell-friendly technique based on femtosecond laser exposure that can generate channels in hydrogels which can later be vascularized by seeding endothelial cells. We tested different hydrogels for their compatibility with cavitation molding and studied the tunability of the optimal one. Lastly, we designed, fabricated and optimized a microfluidic device capable of combining the brain organoid technique and laser cavitation molding of hydrogels to obtain vascularization of brain organoids. The project was conducted at KTH Royal Institute of Technology and Science for Life Laboratory in Professor’s Anna Herland in vitro neural systems lab during an Erasmus project. Together with the KTH team, we successfully generated brain organoids that developed according to protocol. They differentiated properly following the normal development of the in vivo human brain. We then tested Matrigel and collagen and Matrigel combinations of hydrogels for their compatibility with laser cavitation molding. We found that a certain concentration of collagen I is necessary for the phenomenon to occur. We then focused on studying the tunability of the collagen I and Matrigel mixture at a ratio of 1 to 1. We further developed a PDMS microfluidic chip optimized to host the brain organoid embedded in a hydrogel and allow laser-based cavitation molding. We fabricated different chip designs by replica molding and achieved bis-amino silane functionalization of PET membranes then bonded in a sandwich structure between the PDMS slabs. We then developed a protocol for the hydrogel casting, characterized the chip to find the right setting parameters of the laser and lastly patterned channels in the hydrogel. Conclusions. In the end, we managed to develop all the necessary tools that are needed for the vascularization of brain organoids on microfluidic chips. We will further investigate a hydrogel composition that allows both laser cavitation molding and better development of the organoids embedded in it as to cultivate the organoids directly on the chips with molded channels seeded with endothelial cells.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/17425