Tiziana A. L. Brevini

Genotype-to-phenotype (G2P) research in farmed animals has entered a phase in which deep genome annotation, multi-omics and AI-enabled prediction can nominate variants, regulatory mechanisms and cellular pathways at scale, but causal validation remains a major bottleneck. In vitro cellular systems, from tractable primary cells to organoids and other advanced models, provide experimentally controlled contexts in which massively parallel reporter assays and CRISPR-based perturbations can test variant effects and define cellular phenotypes. Here, we examine how in vitro approaches can be deployed across host–pathogen interactions, genotype-by-environment responses, nutrition, adaptation and One Health research, and outline priorities for integrating in vivo, in vitro and in silico data into mechanistic G2P workflows.

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.

Several early developmental events remain unclear due to ethical and technical limitations related to the use of natural embryos. To overcome this problem, over the last decade, different approaches aimed at the generation of in vitro blastocyst-like models have been developed.Here, we describe a protocol that combines miR-200-mediated cell reprogramming and mechanical stimuli to create 3D spheroids phenotypically similar to 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 differentiated into trophoblast (TR)-like cells, using an ad hoc induction protocol, or encapsulated into polytetrafluoroethylene (PTFE) micro-bioreactors to generate inner cell mass (ICM)-like spheroids. Subsequently, TR-like cells and ICM-like spheroids are encapsulated and co-cultured in the same micro-bioreactor and then transferred to microwells to allow further differentiation and boost blastoid development.The generated 3D artificial blastoids, phenotypically similar to natural embryos, could find a useful application in the field of assisted reproduction technologies to further characterize the events taking place during early embryogenesis as well as to study embryo disorders.

Purpose To investigate the effects of microplastics (MPs) on the human endometrium in vitro. Methods A predictive 3D endometrial in vitro model was generated using highly porous scaffolds where human endometrial stromal (hESC) and epithelial (hEEC) cells were co-cultured for 35 days. The newly generated endometrial barrier was then exposed to different MP concentrations (from 0.25 to 50 mg/ml) for 24 h and 48 h, respectively. Histological staining and functional analyses were performed to assess the endometrial barrier integrity. Molecular studies and collagen deposition were evaluated to investigate the possible activation of pro-apoptotic and pro-fibrotic related pathways. Results MP exposure for 24 h does not affect endometrial barrier integrity nor collagen synthesis and deposition. Similar responses are detected when concentrations between 0.25 and 1 mg/ml are used for 48 h. In contrast, 48-h incubations with higher doses (10–50 mg/ml MPs) induce epithelial barrier alterations, reduce TEER values and decrease ZO1 and CDH1 gene transcription. This is accompanied by the activation of pro-fibrotic signalling pathways resulting in collagen increment, which often accompanies endometriosis-related alterations. Conclusion The data obtained suggest MP ability to exert deleterious effects in vitro on human endometrium, with a possible negative impact on its functionality and receptivity.

Extremely low-frequency electromagnetic field (ELF-EMF) radiation alters the steroidogenic activity of porcine conceptuses during the peri-implantation period. This study investigated whether short exposure (2 h) to ELF-EMF radiation may induce changes in the transcriptomic profile of conceptuses and their DNA methylation levels, with a focus on the promoter regions of differentially expressed genes of interest. Porcine conceptuses were collected on days 15–16 of pregnancy, preincubated, and then exposed in vitro to ELF-EMF at a frequency of 50 Hz. ELF-EMF treatment affected the expression of 21 protein-coding transcripts, including solute carrier family 34 member 1 (SLC34A1), hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2), apolipoprotein M (APOM), regucalcin (RGN), heat shock protein family A (Hsp70) member 6 (HSPA6), fibrinogen beta chain, and fibrinogen gamma chain (FGG). There were evaluated 21 GO annotations for biological process terms, nine GO annotations for cellular component terms and two KEGG pathways. The ELF-EMF exposure increased approximately 16 times genomic DNA methylation in conceptuses. ELF-EMF-induced changes in methylation were observed in the promoter regions of APOM, HSD17B2, FGG, and SLC34A1. The results determined 116 predicted single nucleotide variant substitutions within RNA editing sites. In conclusion, ELF-EMF radiation appears to interact with DNA methylation levels and to affect the expression of genes involved mainly in cellular homeostasis, conceptuses development, and attachment, suggesting the need for further investigation to elucidate better the impact of ELF-EMF exposure on the physiology of early conceptuses.

Microplastics (MPs) in fish can cross the intestinal barrier and are often bioaccumulated in several tissues, causing adverse effects. While the impacts of MPs on fish are well documented, the mechanisms of their cellular internalization remain unclear. A rainbow-trout (Oncorhynchus mykiss) intestinal platform, comprising proximal and distal intestinal epithelial cells cultured on an Alvetex scaffold, was exposed to 50 mg/L of MPs (size 1–5 µm) for 2, 4, and 6 h. MP uptake was faster in RTpi-MI compared to RTdi-MI. Exposure to microplastics compromised the cellular barrier integrity by disrupting the tight-junction protein zonula occludens-1, inducing significant decreases in the transepithelial-electrical-resistance (TEER) values. Consequently, MPs were internalized by cultured epithelial cells and fibroblasts. The expression of genes related to endocytosis (cltca, cav1), macropinocytosis (rac1), and tight junctions’ formation (oclna, cldn3a, ZO-1) was analyzed. No significant differences were observed in cltca, oclna, and cldn3a expression, while an upregulation of cav1, rac1, and ZO-1 genes was detected, suggesting macropinocytosis as the route of internalization, since also cav1 and ZO-1 are indirectly related to this mechanism. The obtained results are consistent with data previously reported in vivo, confirming its validity for identifying MP internalization pathways. This could help to develop strategies to mitigate MP absorption through ingestion.

In recent years, great interest has been focused on the development of highly reproducible 3D in vitro models that are able to mimic the physiological architecture and functionality of native tissues. To date, a wide range of techniques have been proposed to recreate an intestinal barrier in vitro, including synthetic scaffolds and hydrogels, as well as complex on-a-chip systems and organoids. Here, we describe a novel protocol for the generation of an artificial intestine based on the creation of decellularized bio-scaffolds and their repopulation with intestinal stromal and epithelial cells. Organs collected at the local slaughterhouse are subjected to a decellularization protocol that includes a freezing/thawing step, followed by sequential incubation in 1% SDS for 12 h, 1% Triton X-100 for 12 h, and 2% deoxycholate for 12 h. At the end of the procedure, the generated bio-scaffolds are repopulated with intestinal fibroblasts and then with epithelial cells. The protocol described here represents a promising and novel strategy to generate an in vitro bioengineered intestine platform able to mimic some of the complex functions of the intestinal barrier, thus constituting a promising 3D strategy for nutritional, pharmaceutical, and toxicological studies.

During mammalian implantation, complex and well-orchestrated interactions between the trophectoderm of implanting blastocysts and the maternal endometrium lead to a successful pregnancy. On the other hand, alteration in endometrium-blastocyst crosstalk often causes implantation failure, pregnancy loss, and complications that result in overall infertility. In domestic animals, this represents one of the major causes of economic losses and the understanding of the processes taking place during the early phases of implantation, in both healthy and pathological conditions, is of great importance, to enhance livestock system efficiency. Here we develop highly predictive and reproducible functional tridimensional (3D) in vitro models able to mimic the two main actors that play a key role at this developmental stage: the blastocyst and the endometrium. In particular, we generate a 3D endometrial model by co-culturing primary epithelial and stromal cells, isolated from sow uteri, onto highly porous polystyrene scaffolds. In parallel, we chemically reprogram porcine adult dermal fibroblasts and encapsulate them into micro-bioreactors to create trophoblast (TR) spheroids. Finally, we combine the generated artificial endometrium with the TR spheroids to model mammalian implantation in vitro and mimic the embryo-maternal interactions. The protocols here described allow the generation of reproducible and functional 3D models of both the maternal compartment as well as the implanting embryo, able to recreate in vitro the architecture and physiology of the two tissues in vivo. We suggest that these models can find useful applications to further elucidate early implantation mechanisms and to study the complex interactions between the maternal tissue and the developing embryos.

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.