Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models.

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.

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480.

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells.

Comparative evaluation of percutaneous laser and radiofrequency ablation in patients with HCC smaller than 4 cm.

OBJECTIVE: This study was done to compare percutaneous laser ablation (PLA) and radiofrequency thermoablation (RFA) for the treatment of hepatocellular carcinoma (HCC) ≤ 4 cm, in patients with liver cirrhosis. MATERIALS AND METHODS: Thirty patients with single HCC ≤ 4 cm in diameter were randomly assigned to one of two treatments: 15 patients were treated with PLA, using a multifibre system connected to a neodymium yttrium-aluminium-garnet laser source; 15 patients were treated with RFA, using an expandable needle electrode. Patients were followed up for up to 12 months. RESULTS: A complete response was obtained in 87 % lesions treated with PLA and in 93 % lesions treated with RFA (p = ns). The overall local recurrence-free survival rates at 3, 6 and 12 months were comparable. However, a higher rate of recurrence was observed in the PLA group for lesions ≥ 21 mm (p = 0.0081). A postablation syndrome was documented in 13 patients (1 PLA; 12 RFA). Tumour necrosis factor-α was significantly higher in the RFA group (p < 0.05). CONCLUSIONS: RFA is more effective in the treatment of HCC compared to PLA for lesions ≥ 21 mm. However, PLA should be considered a viable treatment option for HCC ≤ 20 mm, in view of the lower incidence of complications.

Citral-Enriched Fraction of Lemon Essential Oil Mitigates LPS-Induced Hepatocyte Injuries.

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.

Radiofrequency thermoablation of HCC larger than 3 cm and less than 5 cm proximal to the gallbladder without gallbladder isolation: a single center experience.

Radiofrequency ablation (RFA) is an effective minimally invasive treatment for nonsurgical hepatocellular carcinoma (HCC), but ablation of tumors close to the gallbladder could be associated with several complications. We report our experience on the treatment of HCC close to the gallbladder with RFA. Eight RFA procedures were performed in eight patients with HCC larger than 3 cm and less than 5 cm close to the gallbladder. In all cases, a percutaneous approach was used. There were no major complications. Only in two patients a minimal wall thickening of the gallbladder was observed. Contrast enhanced computed tomography carried out after 30 days from the first procedure showed complete necrosis in seven patients (87%). Only one patient had local recurrence at 11 months of followup. Although limited, our experience suggests that, after careful preprocedural planning, in experienced hands and with appropriate technology, percutaneous RFA could be safely performed even for lesions larger than 3 cm located in close adjacency to the gallbladder.

Kidney transplant: usefulness of real-time elastography (RTE) in the diagnosis of graft interstitial fibrosis.

The aim of this study is to evaluate the usefulness of real-time elastography (RTE) in the diagnosis of graft interstitial fibrosis. We prospectively enrolled 50 patients clinically suspected of graft fibrosis. RTE was performed with a broadband linear transducer using a dedicated ultrasound machine. Tissue mean elasticity (TME) was calculated by two blinded operators. All patients underwent biopsy after RTE. To determine cortical fibrosis Banff score was used. The receiver operating characteristic curves analysis was performed to evaluate the accuracy of TME to discriminate between patients with mild fibrosis (F1) versus patients with moderate to severe fibrosis (F2-F3). Inverse correlation between TME values and the degree of fibrosis has been shown (p < 0.05). Patients with F1 had mean TME values significantly higher compared with TME in patients with F2 (p = 0.005) and F3 (p = 0.004). The diagnostic accuracy of TME measurement for F2-F3 evaluated by area under the curve-receiver operating characteristic analysis was 0.95. RTE was able to evaluate kidney fibrosis in a non-invasive way and could be used as complementary imaging during follow-up of renal transplant patients.

Malignant Peripheral Nerve Sheath Tumour: CT and MRI Findings.

Malignant peripheral nerve sheath tumour (MPNST) is extremely rare malignancy in the general population, occurring more frequently in patients with Neurofibromatosis type 1 (NF1). In the literature five cases of MPNST arising from the parapharyngeal space (PPS) in patients without neurofibromatosis have been reported. We report imaging techniques in a patient with MPNST in the PPS, who had neither a family history nor sign of NF1. Computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed for a correct therapeutic planning. CT and MRI findings were correlated with hystopathological diagnosis.

Liver elasticity in NASH patients evaluated with real-time elastography (RTE).

Liver elasticity as assessed by real-time elastography (RTE) has been shown to be correlated to liver fibrosis in various chronic liver diseases. The aim of our study was to assess the RTE performance in the evaluation of liver fibrosis in nonalcoholic steatohepatitis (NASH), as well as the histopathologic variables determining the eventual discordance between the RTE-predicted and the biopsy-proven fibrosis. Fifty-two consecutive biopsy proven NASH patients and 20 controls were studied. Liver tissue elasticity measurements were performed using the Hitachi EUB-8500 sonographer and the EUP-L52 Linear (3-7 MHz) probe. RTE liver tissue mean elasticity (TME) values were calculated and correlated to the histologic fibrosis, activity and steatosis scores. A decrease in TME was observed with increasing fibrosis (r = -0.75). Similarly, TME varied together consistently with steatosis (r = -0.3). In contrast, TME did not show any correlation with the severity of inflammation. Multiple regression analysis showed that fibrosis was the only variable able to significantly (p < 0.0001) modify TME values. The diagnostic accuracy of TME measurement for F > 0 evaluated by AUC-ROC analysis was 0.86. The diagnostic accuracy of TME measurement for F ≥ 2 was 0.92. We suggest that RTE could be used as a complementary imaging method to evaluate liver fibrosis in NASH patients. Future studies of larger patient cohorts are necessary for the validation of the technique.