Bioimaging supported by nanoparticles requires low cost, highly emissive and photostable systems with low cytotoxicity. Carbon dots (C-dots) offer a possible solution, even if controlling their properties is not always straightforward, not to mention their potentially simple synthesis and the fact that they do not exhibit long-term photostability in general. In the present work, we synthesized two C-dots starting from citric acid and tris (hydroxymethyl)-aminomethane (tris) or arginine methyl ester dihydrochloride. Cellular uptake and bioimaging were tested in vitro using murine neuroblastoma and ovine fibroblast cells. The C-dots are highly biocompatible, and after 24 h of incubation with the cells, 100% viability was still observed. Furthermore, the C-dots synthesized using tris have an average dimension of 2 nm, a quantum yield of 37%, high photostability and a zeta potential (ζ) around −12 mV. These properties favor cellular uptake without damaging cells and allow for very effective bioimaging.

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus–oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

Cell spheroids are widely studied for their potential applications in tissue engineering and regenerative medicine. The present work investigated the effects of cryopreservation on spheroids derived from ovine fibroblasts, depending on spheroid size (140 or 220 µm). Specifically, it explored how cryopreservation impacted several biological and physical parameters including cell damage, viability, metabolism, adhesion, proliferation, and spheroid mass density, weight, and diameter at three time points after thawing. A Live/Dead assay provided a visual assessment of cell damage, cell viability and metabolic activity were assessed by an Alamar Blue assay, and a replating assay evaluated cell adhesion and proliferation capabilities. Spheroid mass density, weight, and diameter were quantified by the W8 Biophysical Analyzer, creating accurate biophysical profiles. Real-time PCR (RT-PCR) analysis was employed to uncover gene expression changes following cryopreservation. Our findings indicate that spheroids measuring 140 µm in diameter largely maintained their biophysical features and cell viability post-cryopreservation, whereas those at 220 µm exhibited a decline in both vitality and mass density. The reduced vitality of 220 µm spheroids likely reflects size-related limitations in cryoprotectant diffusion and stress within the core. Overall, this study provides a comprehensive understanding of how cryopreservation affects ovine fibroblast spheroid biophysics and cellular integrity, laying the groundwork for improved preservation techniques for cell spheroids.