Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases.

The release of extracellular vesicles (EVs) is a common language, used by living organisms from different kingdoms as a means of communication between them. Extracellular vesicles are lipoproteic particles that contain many biomolecules, such as proteins, nucleic acids, and lipids. The primary role of EVs is to convey information to the recipient cells, affecting their function. Plant-derived extracellular vesicles (PDEVs) can be isolated from several plant species, and the study of their biological properties is becoming an essential starting point to study cross-kingdom communication, especially between plants and mammalians. Furthermore, the presence of microRNAs (miRNAs) in PDEVs represents an interesting aspect for understanding how PDEVs can target the mammalian genes involved in pathological conditions such as cancer, inflammation, and oxidative stress. In particular, this review focuses on the history of PDEVs, from their discovery, to purification from various matrices, and on the functional role of PDEV-RNAs in cross-kingdom interactions. It is worth noting that miRNAs packaged in PDEVs can be key modulators of human gene expression, representing potential therapeutic agents.

The cross-talk between plant-derived nanovesicles (PDNVs) and mammalian cells has been explored by several investigations, underlining the capability of these natural nanovesicles to regulate several molecular pathways. Additionally, PDNVs possess biological proprieties that make them applicable against pathological conditions, such as hepatic diseases. In this study we explored the antioxidant properties of lemon-derived nanovesicles, isolated at laboratory (LNVs) and industrial scale (iLNVs) in human healthy hepatocytes (THLE-2) and in metabolic syndrome induced by a high-fat diet (HFD) in the rat. Our findings demonstrate that in THLE-2 cells, LNVs and iLNVs decrease ROS production and upregulate the expression of antioxidant mediators, Nrf2 and HO-1. Furthermore, the in vivo assessment reveals that the oral administration of iLNVs improves glucose tolerance and lipid dysmetabolism, ameliorates biometric parameters and systemic redox homeostasis, and upregulates Nrf2/HO-1 signaling in HFD rat liver. Consequently, we believe LNVs/iLNVs might be a promising approach for managing hepatic and dysmetabolic disorders.

Lemon essential oil (LEO) is known for its aromatic and healthy properties; however, less consideration is given to the biological properties of the fractions obtained from LEO. This study aims to evaluate the ability of a citral-enriched fraction obtained from LEO (Cfr-LEO) to counteract lipopolysaccharide (LPS)-mediated inflammation, oxidative stress, and epithelial–mesenchymal transition (EMT) in healthy human hepatocytes. Human immortalized hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS at various time points. We report that the pretreatment with Cfr-LEO counteracts LPS-mediated effects by inhibiting inflammation, oxidative stress, and epithelial–mesenchymal transition in THLE-2. In particular, we found that pretreatment with Cfr-LEO reduced NF-κB activation and the subsequent proinflammatory cytokines release, ROS production, and NRF2 and p53 expression. Furthermore, the pretreatment with Cfr-LEO showed its beneficial effect in counteracting LPS-induced EMT. Taken together, these results support Cfr-LEO application in the nutraceutical research field not only for its organoleptic properties, conferred by citral enrichment, but also for its biological activity. Our study could lay the basis for the development of foods/drinks enriched with Cfr-LEO, aimed at preventing or alleviating chronic conditions associated with liver dysfunction.

Hepatic ischemia–reperfusion injury (IRI) is a major clinical challenge in liver surgery and transplantation, contributing to postoperative complications and graft dysfunction. The pathogenesis of hepatic IRI is complex and multifactorial, involving ischemia-induced metabolic consequences, oxidative stress, inflammatory responses, endothelial dysfunction, and the activation of immune pathways upon reperfusion. Despite extensive research efforts, the translation of preclinical findings into effective clinical interventions remains limited. This review provides a critical overview of the principal models used to investigate hepatic IRI. Conventional two-dimensional in vitro systems, including monoculture and co-culture models, offer controlled environments for mechanistic studies and high-throughput screening, but fail to fully reproduce the structural and cellular complexity of the liver microenvironment. Animal models, particularly those based on mice, rats, and pigs, remain essential for studying the systemic and multicellular aspects of hepatic IRI. Nevertheless, species-specific physiological differences, ethical concerns, high costs, and limited translational predictability represent significant limitations. In this context, three-dimensional liver models have emerged as promising alternatives capable of bridging the gap between in vitro systems and animal experimentation. By more accurately recapitulating tissue architecture, cell–cell interactions, and functional heterogeneity, 3D platforms offer enhanced physiological relevance and translational potential. We discuss the strengths and limitations of each experimental approach and highlight the role of advanced 3D models as complementary tools that may enable more accurate investigations of hepatic IRI and accelerate the development of effective therapeutic strategies.

Hepatic ischemia-reperfusion injury (IRI) is one of the main clinical challenges in liver surgery and transplantation, contributing to postoperative complications and graft dys-function. The pathogenesis of hepatic IRI is complex and multifactorial, involving meta-bolic consequences induced by ischemia, oxidative stress, inflammatory responses, endo-thelial dysfunction, and the activation of immune pathways upon reperfusion. Despite extensive research efforts, the translation of preclinical findings into effective clinical in-terventions has been limited. This review provides a critical overview of the most im-portant models employed to investigate hepatic IRI. Conventional two-dimensional (2D) in vitro systems, including monocultures and co-culture models, offer controlled envi-ronments for mechanistic studies and high-throughput screening but fail to fully repro-duce the structural and cellular complexity of the liver microenvironment. Animal mod-els, particularly those based on mice, rats, and pigs, remain essential for studying the sys-temic and multicellular aspects of hepatic IRI. However, species-specific physiological differences, ethical concerns, high costs, and limited translational predictability represent significant limitations. In this context, three-dimensional (3D) liver models have emerged as promising alternatives able to bridge the gap between in vitro systems and animal ex-perimentation. By better recapitulating tissue architecture, cell-cell interactions, and func-tional heterogeneity, 3D platforms offer improved physiological relevance and transla-tional potential. In detail, we discuss the strengths and limitations of each experimental approach and highlight the role of advanced 3D models as complementary tools that may facilitate more accurate investigations of hepatic IRI and accelerate the development of therapeutic strategies.