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Giulitti, Stefano (2014) High-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration. [Tesi di dottorato]

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Abstract (inglese)

Human cells and tissues are key systems to study human biology and physiology, and to develop new strategies and targeting drugs for human diseases. Since the study and testing on human beings may not be acceptable due to exposure to risks and practical and ethical concerns, in vitro strategies are of paramount importance to rely on human organism and avoid non-fully predictive animal models. The demand of research in clinical and industrial fields for effective, representative and affordable strategies is undoubtedly increasing.
Conventional cell culture systems and drug discovery are normally performed in vessels with a characteristic dimension in the order of centimeters. Nutrients are delivered to cells through liquid media containing balanced saline buffers and oligo-elements. A reasonable amount of medium is necessary to homogeneously cover a cell layer and must exchanged with fresh media to maintain a proper amount of available nutrients and remove released waste products. Many studies and applications require expensive reagents and are subjected to limited data throughput. The discovery of reprogramming process by 2012 Nobel Prize Yamanaka opened breakthrough new perspective on research and clinical applications. Basically, from a patient’s skin biopsy it is now possible to derive induced pluripotent stem cells (iPSC) and to obtain new tissues for an ad hoc self-repair. So far, human iPSC (hiPSC) have not been applied to clinics due to some unexplored aspects on their derivation, non clinical-grade methods and the significative cost of hiPSC derivation per patient. The down-scale of reprogramming process could provide an unique opportunity to derive cost-effective hiPSC and obtain valuable human in vitro tissues.
The aim of this thesis is the development of a comprehensive platform for the reprogramming of human cells at the microscale. To this end, we focused on the development of cell microenvironment which is composed by both soluble and solid components.
During this thesis, synthetic and biodegradable hydrogels were developed. The large-scale production of mechanically-tunable poly-acrylamide-based substrates were fundamental to reveal the interaction occurring between substrate stiffness and cell behavior and fate. Engineering of biodegradable hydrogels has revealed the potential to develop in vitro functional tissues and to integrate them at a later stage in patients. Chemical modifications were transferred to topological substrate control and in turn in microfluidic platforms.
Microfluidic chip environment and management was designed in order to allow long-term adhesion, culture and biologically relevant cell behaviors. Adhesion proteins fundamental for cell attachment and growth were modified and integrated with the micronized substrates. Since medium for microfluidic cell culture relies on perfusion, continuous or periodic flow could be applied. Thus, we studied the management of media delivery in order to determine the best strategy for long-term cell cultures.
The achievements obtained with both substrate and microfluidic cell culture development was applied to the generation of a new platform for hiPSC derivation, differentiation and testing at the microscale. For the first time, it is possible to obtain human iPSC clones in microfluidics with a remarked reduction of minimum requirements (materials, reagents, overall expenses).
The production of cost effective hiPSC can lead to a mass production of characterized and functional tissues that can be either integrated in 3D developed constructs and serve as valuable tissue source derivation for drug development. Our platform opens new perspectives in studying and treating both abundant and rare diseases involving both scientists and entrepreneurs

Abstract (italiano)

Cellule e tessuti umani sono sistemi essenziali per lo studio della biologia e fisiologia del corpo umano e per lo sviluppo di nuove strategie e farmaci per la cura di varie patologie. Il coinvolgimento di persone in casi studio di ricerca e testing farmacologici espone i soggetti ad elevato rischio e introduce problematiche tecniche ed etiche non facilmente risolvibili. Lo sviluppo di nuove strategie in vitro è di fondamentale importanza per ricavare informazioni sull’organismo umano e limitare l’uso di sistemi animali non pienamente predittivi. La richiesta di sistemi efficaci, rappresentativi e a basso costo in campo clinico ed industriale è indubbiamente in aumento.
I sistemi convenzionali per colture cellulari sono normalmente costituiti da recipienti con dimensioni caratteristiche dell’ordine dei centimetri. I nutrienti sono veicolati alle cellule tramite mezzi di coltura liquidi che contengono buffer salini e oligoelementi. Un quantitativo di medium minimo è necessario per garantire un battente omogeneo al di sopra della coltura cellulare e deve essere sostituito periodicamente per apportare nuovi nutrienti e rimuovere i prodotti di scarto. Molti studi e applicazioni richiedono reagenti costosi e sono soggetti a una ridotta capacità di ricavare dati. La scoperta del processo di riprogrammazione cellulare da parte del Premio Nobel 2012 Yamanaka hanno aperto nuove esaltanti prospettive in ambito di ricerca e applicazioni cliniche. In tale processo, da una biopsia cutanea di un paziente è possibile ricavare cellule staminali pluripotenti indotte (iPSC) e derivare nuovi tessuti per una riparazione autologa ad hoc dei tessuti.
Ad oggi, le iPSC umane (hiPSC) non sono ancora state utilizzate in ambito clinico a causa di aspetti sulla loro derivazione non ancora pienamente caratterizzati, di metodologie non a livello clinico e del costo significativo della derivazione di hiPSC per singolo paziente. La micronizzazione del processo di riprogrammazione può dare un’opportunità notevole per la derivazione di hiPSC a basso costo e per ottenere tessuti umani in vitro.
Scopo di questa tesi è lo sviluppo di una piattaforma per la riprogrammazione di cellule umane in microscala. Per la sua realizzazione, abbiamo focalizzato la ricerca sullo sviluppo di un microambiente cellulare che tenga conto sia dell’ambiente solubile che dei componenti solidi per l’adesione cellulare.
Durante questo dottorato, sono stati sviluppati degli idrogel sintetici e biodegradabili. La produzione su larga scala di substrati a rigidità variabile a base di poliacrilammide è stata fondamentale per rivelare le interazioni tra la rigidità del substrato e il comportamento e destino cellulare. L’ingegnerizzazione di idrogel biodegradabili ha rivelato il potenziale nello sviluppare tessuti in vitro funzionali e la loro integrazione nel paziente. Il know-how acquisito sulle modifiche chimiche è stato trasferito al controllo della topologia del substrato e all’interno dell’ambiente microfluidico.
L’ambiente microfluidico e la sua amministrazione sono stati ottimizzati per garantire l’adesione e la crescita cellulare a lungo-termine e registrare importanti fenomeni biologici. Le proteine di adesione fondamentali per la crescita delle cellule sono state modificate e integrate in un ambiente in microscala. In microfluidica, poiché il medium necessario alle colture viene perfuso all’interno del ciruito, un flusso continuo o periodico possono essere applicati. Abbiamo così studiato l’amministrazione della distribuzione del medium per determinare le migliori strategie per colture a lungo termine in microfluidica.
I risultati ottenuti nello sviluppo dei substrati e ambienti microfluidici per colture cellulari sono stati applicati alla generazione di una nuova piattaforma per la derivazione delle hiPSC, differenziamento e validazione in microscala. Per la prima volta in letteratura, è possibile ottenere cloni hiPSC in microfluidica con una riduzione sostanziale dei requisiti minimi (materiali, reagenti, spese globali).
La produzione di hiPSC a basso costo può portare a una produzione di massa di tessuti caratterizzati e funzionali che possono in seguito essere integrati in supporti 3D e servire come valida fonte di derivazione per lo sviluppo di nuovi farmaci. La nostra piattaforma apre nuove prospettive nello studio e trattamento di malattie diffuse e rare coinvolgendo scienziati e imprenditori

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Tipo di EPrint:Tesi di dottorato
Relatore:Elvassore, Nicola
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > INGEGNERIA INDUSTRIALE > INGEGNERIA CHIMICA, DEI MATERIALI E DELLA PRODUZIONE
Data di deposito della tesi:30 Gennaio 2014
Anno di Pubblicazione:30 Gennaio 2014
Parole chiave (italiano / inglese):human reprogramming, on-a-chip, microscale, efficient, one-step, assay, differentiation, clinical
Settori scientifico-disciplinari MIUR:Area 09 - Ingegneria industriale e dell'informazione > ING-IND/34 Bioingegneria industriale
Area 05 - Scienze biologiche > BIO/10 Biochimica
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/06 Fluidodinamica
Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/22 Scienza e tecnologia dei materiali
Struttura di riferimento:Dipartimenti > Dipartimento di Ingegneria Industriale
Codice ID:6799
Depositato il:14 Nov 2014 09:49
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