Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.

Aging is a complex, multifaceted degenerative process characterized by a progressive accumulation of macroscopic and microscopic modifications that cause a gradual decline of physiological functions. During the last few years, strategies to ease and counteract senescence or even rejuvenate cells and tissues were proposed. Here we investigate whether young cell secretome-derived extracellular vesicles (EVs) ameliorate the cellular and physiological hallmarks of aging in senescent cells. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli, directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after exposure to young cell secretome. The results obtained demonstrate that young cells release EVs endowed with the ability to counteract aging. In addition, comparison between young and aged cell secretomes shows a significantly higher miR-200 content in EVs produced using fibroblasts isolated from young donors. The effect exerted by young cell secretome-derived EVs is transient, but can be stabilized using a young ECM microenvironment. This finding indicates a synergistic interaction occurring among molecular effectors and ECM-derived stimuli that cooperate to control a unique program, driving the cell clock. The model described in this paper may represent a useful tool to finely dissect the complex regulations and multiple biochemical and biomechanical cues driving cellular biological age.

The weaning period is a stressful period for the gastrointestinal tract (GIT) of piglets. This work aims to evaluate the effects of the commercial polyphenol-based product GreenFIS^® on: (1) GIT health and performance of 60 weaned piglets; (2) digestibility in 18 growing pigs. Three diets were tested: a control diet (C), C plus 2.5 g of GreenFIS^®/kg C (T1), and C plus 5 g of GreenFIS^®/kg C (T2). After the post-weaning trial three piglets per treatment were sacrificed for the GIT histological analysis. No differences between diets were recorded in terms of growing performance or clinical and biochemical blood parameters. The GIT histological analysis did not show any indicators of inflammation for any of the groups. The feces of the two extreme treatments (C and T2) were analyzed for microbiota, revealing a greater presence of the Ruminococcus bromii group, positively associated with starch degradation, in T2. In the second experiment six pigs per treatment were randomly chosen for the digestibility study. The inclusion of GreenFIS^® at both levels led to a higher fecal digestibility of gross energy (86.2%, 89.1%, and 89.5%, for C, T1, and T2, respectively) and crude protein (87.0%, 90.2%, and 90.0%). In conclusion, the additive did not improve, in the excellent experimental hygienic conditions, the gut health, but it did increase nutrient digestibility.

In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200′s ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor’s cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms.

Astaxanthin (AST) is a natural compound derived from shellfish, microorganisms, and algae, with several healthy properties. For this reason, it is widely used in the diet of humans and animals, such as pigs, broilers, and fish, where its addition is related to its pigmenting properties. Moreover, AST’s ability to reduce free radicals and protect cells from oxidative damage finds application during the weaning period, when piglets are exposed to several stressors. To better elucidate the mechanisms involved, here we generate ad hoc pig and rainbow trout in vitro platforms able to mimic the intestinal mucosa. The morphology is validated through histological and molecular analysis, while functional properties of the newly generated intestinal barriers, both in porcine and rainbow trout models, are demonstrated by measuring trans-epithelial electrical resistance and analyzing permeability with fluorescein isothiocyanate–dextran. Exposure to AST induced a significant upregulation of antioxidative stress markers and a reduction in the transcription of inflammation-related interleukins. Altogether, the present findings demonstrate AST’s ability to interact with the molecular pathways controlling oxidative stress and inflammation both in the porcine and rainbow trout species and suggest AST’s positive role in prevention and health.

Milk is an essential component of the diet. Among its diverse molecular constituents, it contains nanoscale entities, known as extracellular vesicles (EVs), which play a pivotal role in intercellular communication. In particular, milk-derived EVs (MEVs) influence intestinal homeostasis by mitigating inflammatory responses, modulating gut microbiota composition, and contributing to epithelial integrity preservation and restoration. Currently, there are no information regarding their impact on intestinal connective tissue. Here, we investigate bovine MEV effects on the porcine gut stromal compartment, exposing intestinal decellularized bio-scaffolds repopulated with primary intestinal stromal fibroblasts, to different MEV concentrations (10⁶, 10⁸, and 10¹⁰ particles/mL). We observed a dose-dependent effect of MEVs on stromal fibroblast proliferation rate at concentrations higher than 10⁶ particles/mL. In addition, when MEVs were used to pre-condition the decellularized intestinal bio-scaffolds prior to cell repopulation, fibroblast growth was further boosted. Overall, these findings suggest that MEVs may play a significant role in promoting tissue remodeling and repair. This activity appears particularly relevant for enhancing intestinal homeostasis and resilience, as stromal fibroblasts contribute to the maintenance of gut integrity, barrier function, and immune balance. Moreover, the data here presented suggests the possibility of using MEVs to develop serum-free, chemically defined culture media for the generation of advanced three-dimensional (3D) models and intestinal artificial organs.