High-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration

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 entrepreneur

The staging of gastritis with the olga system in the italian setting. histological features and gastric cancer risk

BACKGROUND: Recently OLGA (Operative Link on Gastritis Assessment) classification has been proposed to identify high-risk forms of gastritis that can evolve in gastric cancer (stages III and IV). Helicobacter pylori infection and age older than 40 have been considered as independent risk factor for high-risk OLGA stages

Polysaccharide hydrogels for multiscale 3D printing of pullulan scaffolds

Structurally and mechanically similar to the extracellular matrix (ECM), biomimetic hydrogels offer a number of opportunities in medical applications. However, the generation of synthetic microenvironments that simulate the effects of natural tissue niches on cell growth and differentiation requires new methods to control hydrogel feature resolution, biofunctionalization and mechanical properties. Here we show how these goals can be achieved by using a pullulan-based hydrogel, engineered in composition and server as cell-adhesive hydrogel, 3D photo-printable in dimension, ranging from the macro- to the micro-scale dimensions, and of tunable mechanical properties. For this, we used absorbers that limit light penetration, achieving 3D patterning through stereolithography with feature vertical resolution of 200 μm and with overall dimension up to several millimeters. Furthermore, we report the fabrication of 3D pullulan-modified hydrogels by two-photon lithography, with sub-millimetric dimensions and minimum feature sizes down to some microns. These materials open the possibility to produce multiscale printed scaffolds that here we demonstrate to be inert for cell adhesion, but biologically compatible and easily functionalizable with cell adhesive proteins. Under these conditions, successful cell cultures were established in 2D and 3D. Keywords: Hydrogel, Biomaterials, Polysaccharide, Pullulan, 3D printing, Two photon laser lithography, Mesenchymal stromal cell

Postoperative Pancreatic Fistula. Is Minimally Invasive Surgery Better than Open? A Systematic Review and Meta-analysis

Background/Aim: Minimally invasive pancreatico-duodenectomy (PD) is gaining popularity. The aim of this study was to compare the incidence of postoperative pancreatic fistula (POPF) after minimally invasive versus open procedures. Materials and Methods: Following the PRISMA statement, literature research was conducted focusing on papers comparing the incidence of POPF after open pancreaticoduodenectomy (OPD) versus minimally invasive pancreaticoduodenectomy (MIPD). Results: Twenty-one papers were included in this meta -analysis, for a total of 4,448 patients. A total of 2,456 patients (55.2%) underwent OPD, while 1,992 (44.8%) underwent MIPD. Age, ASA score III patients, incidence of pancreatic ductal adenocarcinoma and duct diameter were significantly lower in the MIPD group. No statistically significant differences were found between the OPD and MIPD regarding the incidence of major complications (15.6% vs. 17.0%, respectively, p=0.55), mortality (3.7% vs. 2.4%, p=0.81), and POPF rate (14.3% vs. 12.9%, p=0.25). Conclusion: MIPD and OPD had comparable rates of postoperative complications, postoperative mortality, and POPF

Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)–Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK–Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell’s environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies

High-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration

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 entrepreneursCellule 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 imprenditor

A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling