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Spheroids, a part of 3D cell culture systems, are crucial models for bridging in-vitro and in-human studies. However, achieving reliable standardization remains difficult, even when comparing spheroids of similar diameters. The challenge arises due to their cross-sectional architecture, which increases heterogeneity and affects biological outcomes. Here we present a novel solution that integrates Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) with Biophysical Characterization (PCA-BC). This approach allows for the identification and classification of variability within and across spheroid populations, offering insights into factors that contribute to heterogeneity. Additionally, it highlights the impact of different operators on spheroid development. The PCA-BC method enables real-time analysis of spheroid samples, facilitating the identification of variability across 3D populations. The integration of PCA and HCA with biophysical characterization provides a clear and efficient means to monitor sample heterogeneity. It also helps track how different operators influence the results, improving overall standardization in 3D cell cultures. By offering structural insights into spheroid heterogeneity, the PCA-BC approach supports more informed decision-making. This significantly improves workflow efficiency, conserving both time and resources, and enhances the reliability of 3D cell culture experiments.

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

Three-dimensional (3D) culture systems like tumor spheroids represent useful in vitro models for drug screening and more broadly for cancer biology research, but the generation of uniform populations of spheroids remains challenging. The possibility to properly characterize spheroid properties would increase the reliability of these models. To address this issue different analysis were combined: i) a new device and relative analytical method for the accurate, simultaneous, and rapid measurement of mass density, weight, and size of spheroids, ii) confocal imaging, and iii) protein quantification, in a clinically relevant 3D model. The LoVo colon cancer cell line forming spheroids, treated with crizotinib (CZB) an ATP-competitive small-molecule inhibitor of the receptor tyrosine kinases, was employed to study and assess the correlation between biophysical and morphological parameters in both live and fixed cells. The new fluidic-based measurements allowed a robust phenotypical characterization of the spheroids structure, offering insights on the spheroids bulk and an accurate measurement of the tumor density. This analysis helps overcome the technical limits of the imaging that hardly penetrates the thickness of 3D structures. Accordingly, we were able to document that CZB treatment has an impact on mass density, which represents a key marker characterizing cancer cell treatment. Spheroid culture is the ultimate technology in drug discovery and the adoption of such precise measurement of the tumor characteristics can represent a key step forward for the accurate testing of treatment’s potential in 3D i n vitro models.

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models.

Articular cartilage lacks intrinsic regenerative capabilities, and the current treatments fail to regenerate damaged tissue and lead only to temporary pain relief. These limitations have prompted the development of tissue engineering approaches, including 3D culture systems. Thanks to their regenerative properties and capacity to recapitulate embryonic processes, spheroids obtained from mesenchymal stromal cells are increasingly studied as building blocks to obtain functional tissues. The aim of this study was to investigate the capacity of adipose stromal cells to assemble in spheroids and differentiate toward chondrogenic lineage from the perspective of cartilage repair. Spheroids were generated by two different methods (3D chips vs. Ultra-Low Attachment plates), differentiated towards chondrogenic lineage, and their properties were investigated using molecular biology analyses, biophysical measurement of mass density, weight, and size of spheroids, and confocal imaging. Overall, spheroids showed the ability to differentiate by expressing specific cartilaginous markers that correlate with their mass density, defining a critical point at which they start to mature. Considering the spheroid generation method, this pilot study suggested that spheroids obtained with chips are a promising tool for the generation of cartilage organoids that could be used for preclinical/clinical approaches, including personalized therapy.

(1) Background: Several studies have reported associations between maternal diet in terms of single foods or nutrients and human milk compounds, while the overall role of maternal diet and related dietary patterns has rarely been investigated. (2) Methods: Between 2012 and 2014, we enrolled 300 healthy Italian mothers, who exclusively breastfed their infant. During a hospital visit at 6 weeks postpartum, a sample of freshly expressed foremilk was collected and information on maternal dietary habits in the postpartum period was obtained through an interviewer-administered food frequency questionnaire. We applied principal component factor analysis to selected nutrients in order to identify maternal dietary patterns, and assessed correlations in human milk macronutrients and fatty acids across levels of dietary patterns. (3) Results: Five dietary patterns were identified, named “Vitamins, minerals and fibre”, “Proteins and fatty acids with legs”, “Fatty acids with fins”, “Fatty acids with leaves”, “Starch and vegetable proteins”. These dietary patterns were correlated with some milk components, namely fatty acids, and in particular ω-3 and its subcomponents. (4) Conclusions: This study showed that overall maternal dietary habits during breastfeeding may influence human milk composition, suggesting the importance of adequate maternal nutrition during lactation not only for the mother herself but also to provide the infant with milk containing adequate amount and quality of nutrients for a balanced nutrition.

Botrytis cinerea , responsible for grey mold diseases, is a pathogen with a broad host range, affecting many important agricultural crops, in pre and post harvesting of fruits and vegetables. Commercial fungicides used to control this pathogen are often subjected to photolysis, volatilization, degradation, leaching, and runoff during application. In this context, the use of a delivery system, based on poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) represents an innovative approach to develop new pesticide formulations to successfully fight B. cinerea infections. In order to study NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. The observations revealed that 50 nm Cu6-PLGA NPs penetrated into B. cinerea conidia and hyphae, as early as 10 min after administration. Pterostilbene, a natural compound, and fluopyram, a synthetic antifungal, were entrapped in PLGA NPs, added to B. cinerea conidia and mycelium, and their antifungal activity was tested. The results revealed that the compounds loaded in NPs exhibited a higher activity against B. cinerea. These results lay the foundations for the use of PLGA NPs as a new strategy in plant pest management.

Poly-(lactic-co-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The Aspergillus section Nigri causes otomycosis localized in the external auditory canal. In this research, Aspergillus brasiliensis, a species belonging to the Nigri section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-co-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against A. brasiliensis biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the A. brasiliensis infection was tested using Galleria mellonella larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside A. brasiliensis hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in A. brasiliensis biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in Aspergillus section Nigri biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.

Poly-(lactic-co-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The Aspergillus section Nigri causes otomycosis localized in the external auditory canal. In this research, Aspergillus brasiliensis, a species belonging to the Nigri section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-co-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against A. brasiliensis biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the A. brasiliensis infection was tested using Galleria mellonella larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside A. brasiliensis hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in A. brasiliensis biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in Aspergillus section Nigri biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.

Background The issue of retreatment with surfactant of infants with respiratory distress syndrome (RDS) has been poorly investigated. Our aim was to identify possible clinical predictors of the need for multiple doses of surfactant in a large cohort of very preterm infants. Methods Data were analyzed from three previous studies on infants born between 25 + 0 and 31 + 6 weeks of gestation with RDS who were treated with surfactant. Results We studied 448 infants. Among them 306 (68%) were treated with a single dose of surfactant and 142 (32%) were treated with multiple doses. Multivariable mixed effects logistic regression analysis showed that the odd of requiring multiple doses of surfactant was significantly lower in patients with higher gestational age (27–28 vs. 25–26 wks: OR 0.46, 95% C.l. 0.26–0.79; ≥ 29 vs. 25–26 wks: OR 0.34, 95% C.l. 0.13–0.85; overall P  = 0.013), while it increased in infants born to mothers with hypertensive disorders of pregnancy (OR 2.53, 95% C.l. 1.49–4.31; P  < 0.001) and with hemodynamically significant PDA (OR 2.74, 95% C.l. 1.66–4.53, P  < 0.001). Conclusions Gestational age, hypertension in pregnancy, and hemodynamically significant PDA can predict the need for multiple doses of surfactant. Further investigation is needed to evaluate if these sub-groups of preterm infants represent specific phenotypes of RDS who deserve a peculiar surfactant treatment.