Development of a "lab on a chip" platform for studying the control of the circadian clock by metabolic cycles

The aim of the thesis is developing an in vitro model that resembles the cyclic dynamic fluctuations that can be correlated to the pertinent biological networks for dissecting the effects the metabolism on circadian clock. The circadian rhythms are an adaptation to the evolutionarily conserved environment, because they allow to coordinate temporally external environmental cycles (eg. light/ dark) with internal biological and physiological processes (eg. sleep/ wake). Forced misalignment of behavioral and circadian cycles in human subjects, have been associated with increased prevalence of obesity, diabetes and cardiovascular disease, in addition to certain cancers and inflammatory disorders. The potential of the microfluidic approach allows dynamic study on circadian behavior of peripheral tissues, opening a new perspective in circadian field

Editorial: 3D-engineered organoids for modelling tissue development and precision medicine

Organoids are small, self-organized three-dimensional (3D) tissue cultures that reproduce organ structure and function, allowing to mimic physiological and pathological conditions and critical functions of organs. These features enable establishing a simplified, scalable, and accessible approach to address the human tissue availability constraints and the gap between animal models and patients. For this reason, organoid research has dramatically increased over the past decade, to create suitable microscale 3D tissue models that can be used for modelling human development, study disease pathophysiology and for personalized medicine

Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures

open10Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability.openScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, Catalin Dacian; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, Gian PaoloScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, CATALIN DACIAN; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, GIAN PAOL

Generation of induced pluripotent stem cells (UCLi024-A) from a patient with argininosuccinate lyase deficiency carrying a homozygous c.437G > A (p.Arg146Gln) mutation

Argininosuccinic aciduria (ASA) is a rare inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. Patients with ASA present with hyperammonaemia due to an impaired urea cycle pathway in the liver, and systemic disease with epileptic encephalopathy, chronic liver disease, and arterial hypertension. A human induced pluripotent stem cell (iPSC) line from the fibroblasts of a patient with ASA with homozygous pathogenic c.437G > A mutation of hASL was generated. Characterization of the cell line demonstrated pluripotency, differentiation potential and normal karyotype. This cell line, called UCLi024-A, can be utilized for in vitro disease modelling of ASA, and design of novel therapeutics

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

Development of a "lab on a chip" platform for studying the control of the circadian clock by metabolic cycles

The aim of the thesis is developing an in vitro model that resembles the cyclic dynamic fluctuations that can be correlated to the pertinent biological networks for dissecting the effects the metabolism on circadian clock. The circadian rhythms are an adaptation to the evolutionarily conserved environment, because they allow to coordinate temporally external environmental cycles (eg. light/ dark) with internal biological and physiological processes (eg. sleep/ wake). Forced misalignment of behavioral and circadian cycles in human subjects, have been associated with increased prevalence of obesity, diabetes and cardiovascular disease, in addition to certain cancers and inflammatory disorders. The potential of the microfluidic approach allows dynamic study on circadian behavior of peripheral tissues, opening a new perspective in circadian field.Lo scopo della tesi è di sviluppare un modello in vitro in grado di mimare fluttuazioni dinamiche e cicliche che possono essere correlate a reti biologiche pertinenti per sezionare gli effetti che il metabolismo ha sull'orologio circadiani. I ritmi circadiani sono un adattamento all'ambiente evolutivamente conservato, in quanto consentono di coordinare cicli ambientali temporalmente esterni (ad es. luce / buio) con i processi biologici e fisiologici interni (ad es. il sonno / veglia). Il disallineamento forzato dei cicli circadiani comportamentali e in soggetti umani, sono stati associati a una maggiore prevalenza di obesità, diabete e malattie cardiovascolari, oltre ad alcuni tipi di cancro e patologie infiammatorie. Le potenzialità dell' approccio microfluidico consente lo studio dinamico sul comportamento circadiano dei tessuti periferici aprendo di una nuova prospettiva nel campo circadian

Fonocardiografia nell'ultrasuono per la valutazione funzionale di dispositivi proteici cardiaci

Il lavoro di tesi prevede lo sviluppo e la validazione di algoritmi di classificazione in grado di individuare in maniera precoce eventuali casi di malfunzionamento dei dispositivi protesici: questo obiettivo risulta importante dal momento che si offre al medico la possibilità di disporre di un nuovo strumento diagnostico, per monitorare la funzionalità delle protesi valvolari cardiache, identificando le possibili cause di malfunzionamento. Le protesi cardiache meccaniche, se da un lato sono indicate nei soggetti con maggiore aspettativa di vita, dall’altro sono soggette al deposito di panno endoteliale e di formazioni trombotiche che ne compromettono la funzionalità rendendo necessaria la sostituzione chirurgica del dispositivo. Pertanto, ci si propone di realizzare una procedura per la determinazione, attraverso metodi non invasivi, della funzionalità delle protesi valvolari cardiache; tale procedura è basata sull’acquisizione del segnale fonocardiografico, sul calcolo del corrispondente spettro di potenza e sulla classificazione degli spettri ottenut

Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices

An integrative approach based on microfluidic design and stem cell biology enables capture of the spatial-temporal environmental evolution underpinning epigenetic remodeling and the morphogenetic process. We examine the body of literature that encompasses microfluidic applications where human induced pluripotent stem cells are derived starting from human somatic cells and where human pluripotent stem cells are differentiated into different cell types. We focus on recent studies where the intrinsic features of microfluidics have been exploited to control the reprogramming and differentiation trajectory at the microscale, including the capability of manipulating the fluid velocity field, mass transport regime, and controllable composition within micro- to nanoliter volumes in space and time. We also discuss studies of emerging microfluidic technologies and applications. Finally, we critically discuss perspectives and challenges in the field and how these could be instrumental for bringing about significant biological advances in the field of stem cell engineering