RESUMO
Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.
Assuntos
Técnicas de Cultura de Células/métodos , Silício/química , Engenharia Tecidual/métodos , Astrócitos/citologia , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Silício/metabolismoRESUMO
BACKGROUND: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. METHODS: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. FINDINGS: From plasma of 46 healthy donors, we systematically recovered small EV (~250â¯nm of mean diameter; ~3â¯×â¯1010/ml) and large EV (~560â¯nm of mean diameter; ~5â¯×â¯108/ml) lineages ranging from 50 to 700â¯nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).
Assuntos
Biomarcadores Tumorais/sangue , Vesículas Extracelulares , Biópsia Líquida/métodos , Neoplasias/sangue , Neoplasias/diagnóstico , Níquel , Estudos de Casos e Controles , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Citometria de Fluxo , Humanos , Biópsia Líquida/normas , Neoplasias/genética , Neoplasias/metabolismo , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , UltracentrifugaçãoRESUMO
Vibrational properties of inclusion complexes with cyclodextrins are studied by means of Raman spectroscopy and numerical simulation. In particular, Raman spectra of the nonsteroidal, anti-inflammatory drug indomethacin undergo notable changes in the energy range between 1600 and 1700 cm(-1) when inclusion complexes with cyclodextrins are formed. By using both ab initio quantum chemical calculations and molecular dynamics, we studied how to relate such changes to the geometry of the inclusion process, disentangling single-molecule effects, from changes in the solid state structure or dimerization processes.