Microscale technologies to decode EV-mediated cell behavior

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A penetration efficiency model for the optimization of solid conical microneedles’ geometry

Microneedles (MNs) are promising alternatives to pills and traditional needles as drug delivery systems due to their fast, localized, and relatively less painful administration. Filling a knowledge gap, this study investigated and optimized the most influential geometrical factors determining the penetration efficiency of MNs. The effects of height, base diameter, and tip diameter were analyzed using the finite element method, with results showing that the most influencing factor was base diameter, followed by height. Moreover, the taper angle, which is dependent on all the geometrical factors, was found to directly affect the penetration efficiency at a fixed height. An additional model was developed to relate the height and taper angle to penetration efficiency, and the results were experimentally validated by compression testing of MN array prototypes printed using two-photon photolithography. The numerical model closely predicted the experimental results, with a root mean square error of 9.35. The results of our study have the potential to aid the design of high-penetration efficiency MNs for better functionality and applicability

Microfluidic Device Generating Stable Concentration Gradients for Long-Term Cell Culture: Application to Wnt3a Regulation of B-catenin signaling

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of B-catenin signaling was chosen as a case study. The highly conserved Wnt-activated B-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of B-catenin pathway, leading to translocation of B-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/B-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the B-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the B-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of B-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells

Microfluidic Bioreactor for Dynamic Regulation of Early Mesodermal Commitment in Human Pluripotent Stem Cells

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization and assembly that are orchestrated by cascades of regulatory signals. The complex and dynamic in vivo milieu cannot be replicated using standard in vitro techniques. Microscale technologies now offer potential for conducting highly controllable and sophisticated experiments at biologically relevant scales, with real-time insights into cellular responses. We developed a microbioreactor providing time sequences of space-resolved gradients of multiple molecular factors in three-dimensional (3D) cell culture settings, along with a versatile, high-throughput operation and imaging compatibility. A single microbioreactor yields up to 120 data points, corresponding to 15 replicates of a gradient with 8 concentration levels. Embryoid bodies (EBs) obtained from human embryonic and induced pluripotent stem cells (hESC, hiPSC) were exposed to concentration gradients of Wnt3a, Activin A, BMP4 and their inhibitors, to get new insights into the early-stage fate specification and mesodermal lineage commitment. We were able to evaluate the initiation of mesodermal induction by measuring and correlating the gene expression profiles to the concentration gradients of mesoderm-inducing morphogens. We propose that the microbioreactor systems combining spatial and temporal gradients of molecular and physical factors to hESC and hiPSC cultures can form a basis for predictable in vitro models of development and disease

Hybrid Piezoresistive 2D MoS2/PEGDA/PANI Covalent Hydrogels for the Sensing of Low‐to‐Medium Pressure

Wearable technologies are attracting increasing attention in the materials science field, prompting a quest for active components with beneficial functional attributes whilst ensuring human and environmental safety. Hydrogels are highly biocompatible platforms with interesting mechanical properties, which can be exploited for the construction of strain sensors. In order to improve the directionality of their strain response and combine it with electrical properties to fabricate piezoresistive devices, it is possible to incorporate various types of nanofillers within the polymeric network of the hydrogels. 2D materials are ideal nanofillers thanks to their intrinsic two-dimensional anisotropy and unique electronic properties. Herein, the covalent functionalization of 2D 1T-MoS2 is exploited to build robust hybrid cross-linked networks with a polyethylene glycol diacrylate gel (PEGDA). The conductivity of this nanocomposite is also further improved by inducing the interfacial polymerization of aniline. The resulting free-standing samples demonstrate a linear and highly reversible piezoresistive response in a pressure range compatible with that of peripheral blood, while also featuring good compatibility with human skin cells, thereby making them interesting options for incorporation into wearable strain sensors

Design and development of microscale technologies and microfluidic platforms for the in vitro culture of stem cell

The impelling needs related to the processes of drug and therapy development for the cure of diseases such as Duchenne Muscular Dystrophy or myocardial infarction, led to an increasing demand for the development of innovative methods and strategies. It is also clear how stem cells could represent a fundamental source for the production of artificial human tissues to be employed in such processes. This perspective, would require both sophisticated tools for the control of stem cells differentiation and their integration within procedures apt to satisfy the fundamental requirements for obtaining tissue-model on which perform pharmacological or therapeutic studies. Among the fundamental requisites are: micronization, versatility, low-cost and highthroughput. The main aims of this thesis have been the design, development and fabrication of microscale technologies capable of both reproducing a biomimetic stimulation inspired to the in vivo cell microenvironment and responding to the above mentioned technological requirements. We performed a semi-quantitative analysis of the characteristic times of microscale phenomena that lead to the generation of operative diagrams that would prove useful in the design and development phases of experimental strategies. We developed microfluidic microbioreactors that have been used for biological studies involving cell cultures. In particular, we investigated the role and effect of concentration gradients on the fundamental Wnt signaling pathway. We developed techniques for obtaining a topological control at the microscale of cell cultures on hydrogels with tunable mechanical properties. These techniques were successfully applied and interfaced with relevant biological systems such as primary human myoblasts from dystrophic patients and human embryonic stem cells-derived cardiomyocytes. We finally proposed a prototype of an integrated microfluidic platform capable of coupling the topological stimulations to the control of the soluble microenvironment on cell cultures. The obtained results open new and interesting perspectives for both de efficient development of drugs or therapies for defined diseases and for gaining deeper insights into the complexity of biological systems.Le impellenti necessità legate allo sviluppo di nuovi farmaci e di terapie innovative per la cura di malattie dell’apparato muscolare, quali ad esempio la Distrofia Muscolare di Duchenne o l’infarto miocardico, hanno portato alla crescente domanda di nuovi metodi e tecnologie. E’ chiaro inoltre come le cellule staminali possano costituire una risorsa fondamentale per la generazione di tessuti umani artificiali da impiegare in tali processi. Tale prospettiva, richiederebbe da un lato strumenti sofisticati per il controllo e il differenziamento delle cellule staminali e dell’altro, l’integrazione di questi all’interno di procedure in grado di soddisfare i requisiti fondamentali dei modelli sui quali operare per lo sviluppo di nuovi farmaci o strategie terapeutiche. Tra i requisiti fondamentali da rispettare si citano quindi: micronizzazione, versatilità, basso costo e highthroughput. Obiettivo fondamentale di questa tesi sono stati la progettazione, sviluppo e fabbricazione di tecnologie su scala micrometrica in grado di riprodurre una stimolazione biomimetica ispirata al microambiente cellulare in vivo e, nello stesso tempo, di rispondere ai requisiti tecnologici descritti sopra. E’ stata effettuata un’analisi semi quantitativa basata sull’analisi dei tempi caratteristi dei fenomeni su microscala, che ha portato alla produzione di diagrammi operativi da impiegarsi nelle fasi di progettazione e sviluppo di tali strategie sperimentali. Sono stati sviluppati microbioreattori all’interno di piattaforme microfluidiche applicate poi allo studio di sistemi cellulari. In particolare, sono stati effettuati studi biologi sull’effetto di gradienti di concentrazione all’interno dell’importante signaling cellulare del Wnt. E’ stata realizzata una tecnica per l’organizzazione topologica su microscala di colture cellulari su substrati in hydrogel dalle proprietà meccaniche definite. Le tecnologie sviluppate sono state impiegate per casi studio dall’elevato valore scientifico e sono state interfacciate con colture di particolare interesse quali mioblasti umani distrofici e cellule cardiache derivate da staminali embrionali umane. Infine, è stato proposto un prototipo di piattaforma microfluidica in grado di accoppiare le stimolazioni di tipo topologico al controllo dell’ambiente solubile su colture cellulari. I risultati ottenuti aprono nuove ed interessanti prospettive sia per lo sviluppo di nuovi farmaci che di strategie terapeutiche volti alla cura di patologie ed allo studio approfondito della complessità dei sistemi biologici

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing

Image analysis data for publicatio

Microfluidic approaches for producing lipid-based nanoparticles for drug delivery applications

Review article on Microfluidic approaches for producing lipid-based nanoparticles for drug delivery application

EVs from hypoxic NB cells stimulate migration and invasion of target cells via miR-210-3p

Tumor hypoxia stimulates release of extracellular vesicles (EVs) that facilitate short- and long-range intercellular communication and metastatization. Albeit hypoxia and EVs release are known features of Neuroblastoma (NB), a metastasis-prone childhood malignancy of the sympathetic nervous system, whether hypoxic EVs can facilitate NB dissemination is unclear. Here we show that EVs from hypoxic NB cells promote pro-metastasis features in vitro and in an in vivo model. EVs from NB cells cultured at different oxygen tensions did not differ for type and abundance of surface markers and for biophysical properties. However, EVs derived from hypoxic NB cells were more potent than their normoxic counterpart in inducing NB cells migration and colony formation. We characterized and compared the microRNA (miRNA) cargo of EVs from NB cells cultured at different oxygen tensions and found that miR-210-3p was the most upregulated. Mechanistically, we identified that the pro-metastatic features of EVs from hypoxic NB cells derived from a miR-210-3p enrichment in their cargo. Indeed, overexpression of miR-210-3p in normoxic EVs conferred them pro-metastatic features, whereas miR-210-3p silencing suppressed the metastatic ability of hypoxic EVs both in vitro and in in vivo zebrafish model. Our data identify a role for hypoxic EVs in the cellular and microenvironmental changes favoring NB dissemination

Comparative Study of Spheroids (3D) and Monolayer Cultures (2D) for the In Vitro Assessment of Cytotoxicity Induced by the Mycotoxins Sterigmatocystin, Ochratoxin A and Patulin

Research data and manuscript for a Comparative Study of Spheroids (3D) and Monolayer Cultures (2D) for the In Vitro Assessment of Cytotoxicity Induced by the Mycotoxins Sterigmatocystin, Ochratoxin A and Patuli