Advances in cancer modeling: fluidic systems for increasing representativeness of large 3D multicellular spheroids

The representativeness of a cellular model is fundamental in pre-clinical cancer studies. Size, heterogeneity and perfusion are three key aspects characterizing the behavior of the tumor and driving its progression. In vitro resemblance of in vivo tumor conditions can be maximized by: (a) using heterogeneous large-sized three-dimensional (3D) multicellular models; (b) utilizing fluidic systems to modulate the culture microenvironment. This work discusses the benefits of using large-sized spheroids as 3D pre-clinical culture models, besides analyzing the microfluidic systems that permit their cultivation and manipulation in dynamic controlled conditions

ReViMS: Software tool for estimating the volumes of 3-D multicellular spheroids imaged using a light sheet fluorescence microscope

Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates. Here, we present Reconstruction and Visualization from Multiple Sections (ReViMS), an open-source, user-friendly software for automatically segmenting z-stacks of fluorescence images and estimating the volumes of 3-D multicellular spheroids. To assess the precision and accuracy of the volume estimates obtained with ReViMS, we used several cancer spheroids imaged with LSFM. Both the precision and accuracy were >95%, demonstrating the effectiveness of ReViMS

Open-Source Tools for Volume Estimation of 3D Multicellular Aggregates

Featured Application The main goal of this work is to provide an overview of open-source tools available for researchers interested in estimating the volume of 3D multicellular aggregates (e.g., spheroids, organoids), besides introducing a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS) tool (http://sourceforge.net/p/revims). Abstract The volume is one of the most relevant features that define the treatment of an in vivo tumour. When using cancer 3D in vitro models in pre-clinical studies, it becomes important to evaluate the macroscopic effects of drugs and radiotherapy treatments. Depending on the nature of the 3D in vitro model used, different open-source solutions can be used for measuring the volume by starting from microscope-acquired images. In this work, we introduced several open-source tools today available for estimating the volume of 3D multicellular aggregates (e.g., spheroids, organoids), also giving hints for defining the best software by analysing characteristics of 3D in vitro models and limits of the tools. Finally, using several cancer organoids imaged by a fluorescent microscope, we compared volume estimations obtained with different tools, besides presenting a new version of the Reconstruction and Visualization from Multiple Sections (ReViMS version 2.0) tool. This work aims to be the reference for researchers interested in estimating the volume of 3D multicellular aggregates through an open-source tool.Peer reviewe

Fault Control on a Thermal Anomaly: Conceptual and Numerical Modeling of a Low\u2010Temperature Geothermal System in the Southern Alps Foreland Basin (NE Italy)

The interest on low\u2010temperature geothermal resources is progressively increasing since their renewability and widespread availability. Despite their frequency, these resources and their development have been only partially investigated. This paper unravels the major physical processes driving a low\u2010temperature geothermal resource in NE Italy (Euganean Geothermal System) through conceptual and numerical modeling. Dense fracturing associated to regional fault zones and a relay ramp enhances regional to local flow of thermal waters. Their rapid upwelling in the Euganean Geothermal Field is favored by open extensional fractures deforming the relay ramp. The water (65\u201386 \ub0C) is intensively exploited for balneotherapy, rendering it a profitable resource. Three\u2010dimensional coupled flow and heat transport numerical simulations based on this conceptual model are performed. Despite the presence of a uniform basal heat flow, a thermal anomaly corresponding to field observations develops in the modeling domain reproducing the relay ramp. Intensive fracturing extending across a wide area and a slightly anomalous heat flow favors a local increase in convection that drives the upwelling of deep\u2010seated hot waters. The simulations corroborate and refine the conceptual model, revealing that water of up to 115 \ub0C is likely to be found in the unexplored part of the thermal field. This study furthers knowledge on fault\u2010controlled low\u2010temperature geothermal resources where the geological setting could enhance local convection without anomalous heat flows, creating temperatures favorable for energy production. Conceptual and numerical modeling based on solid geological and hydrogeological reconstructions can offer a support tool for further detailed explorations of these prominent resources

Nucleus segmentation : towards automated solutions

Single nucleus segmentation is a frequent challenge of microscopy image processing, since it is the first step of many quantitative data analysis pipelines. The quality of tracking single cells, extracting features or classifying cellular phenotypes strongly depends on segmentation accuracy. Worldwide competitions have been held, aiming to improve segmentation, and recent years have definitely brought significant improvements: large annotated datasets are now freely available, several 2D segmentation strategies have been extended to 3D, and deep learning approaches have increased accuracy. However, even today, no generally accepted solution and benchmarking platform exist. We review the most recent single-cell segmentation tools, and provide an interactive method browser to select the most appropriate solution.Peer reviewe

defining the hydrogeological behavior of karst springs through an integrated analysis a case study in the berici mountains area vicenza ne italy

Knowledge of the hydraulic and geological properties of karst systems is particularly valuable to hydrogeologists because these systems represent an important source of potable water in many countries. However, the high heterogeneity that characterizes karst systems complicates the definition of karst hydrogeological properties, and their estimation involves complex and expensive techniques. In this study, a workflow for karst spring characterization was used to analyze two springs, Nanto spring and Mossano spring, located in the Berici Mountains (NE Italy). Based on the data derived from 4 years of continuous hourly monitoring of discharge, water temperature and specific electrical conductivity, a hydrogeological conceptual model for the monitored springs was proposed. Flow rate measurements, which combined recession curve, flow duration curve and autocorrelation function techniques, were used to evaluate the spring discharge variability. Changes in spring discharge can be related both to the degree of karstification/permeability and to the size of the karst aquifer. Moreover, combining monitored parameters and rainfall—analyzed by the cross-correlation function and VESPA (Vulnerability Estimator for Spring Protection Areas) index approach—permitted assessment of the spring response to recharge and the behavior of the drainage system. Although the responses to the recharge events were quite similar, the two springs showed some differences in terms of the degree of karstification. In fact, Mossano spring showed a more developed karst system than Nanto spring. Three systems (two karsts and one matrix/fractured) are outlined for Mossano spring, while two systems (one karst and one matrix/fractured) are outlined for Nanto spring

A new digital divide threatening resilience: exploring the need for educational, firm-based, and societal investments in ICT human capital

The knowledge, skills, and abilities that human capital offers create tangible and intangible assets that equip organizations to thrive. In particular, in today’s Industry 4.0 environment, training, recruiting, and retaining highly qualified ICT-ready professionals remains a problem for many organizations including educational, governmental, healthcare, and business organizations. The COVID-19 pandemic revealed the importance of digital assets to our economies, and it is also demonstrating that there is potentially a new digital divide with even worse implications for companies, economies, and society, which is threatening the resilience of business, governance, and society. In this paper, we respond to the question “how can we develop ICT human capital in our global economy in an equitable, inclusive, and purposeful manner such that not organizations thrive, but also to promote social justice and equity in our global economy?

A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroinalginate microcarriers using light sheet fluorescent microscopy

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device

Vignetting and photo-bleaching correction in automated fluorescence microscopy from an array of overlapping images

We propose a novel acquisition scheme and non-parametric multi-image based method for correcting illumination in fluorescence images. Our approach measures changes in intensity of the subject by moving the microscope stage at regularly spaced intervals, and exploits this information to learn the correction function. The acquisition process and learning are performed prior to imaging, and take only a few minutes. Afterwards, images can be corrected for vignetting and photo-bleaching effects on the fly. Our approach can be implemented in any microscope with a motorized stage, and does not require a reference calibration slide. Experiments demonstrate that our method outperforms standard approaches to illumination correction

Fluid flow-induced modulation of viability and osteodifferentiation of periodontal ligament stem cell spheroids-on-chip

Developing physiologically relevant in vitro models for studying periodontitis is crucial for understanding its pathogenesis and developing effective therapeutic strategies. In this study, we aimed to integrate the spheroid culture of periodontal ligament stem cells (PDLSCs) within a spheroid-on-chip microfluidic perfusion platform and to investigate the influence of interstitial fluid flow on morphogenesis, cellular viability, and osteogenic differentiation of PDLSC spheroids. PDLSC spheroids were seeded onto the spheroid-on-chip microfluidic device and cultured under static and flow conditions. Computational analysis demonstrated the translation of fluid flow rates of 1.2 μl min-1 (low-flow) and 7.2 μl min-1 (high-flow) to maximum fluid shear stress of 59 μPa and 360 μPa for low and high-flow conditions, respectively. The spheroid-on-chip microfluidic perfusion platform allowed for modulation of flow conditions leading to larger PDLSC spheroids with improved cellular viability under flow compared to static conditions. Modulation of fluid flow enhanced the osteodifferentiation potential of PDLSC spheroids, demonstrated by significantly enhanced alizarin red staining and alkaline phosphatase expression. Additionally, flow conditions, especially high-flow conditions, exhibited extensive calcium staining across both peripheral and central regions of the spheroids, in contrast to the predominantly peripheral staining observed under static conditions. These findings highlight the importance of fluid flow in shaping the morphological and functional properties of PDLSC spheroids. This work paves the way for future investigations exploring the interactions between PDLSC spheroids, microbial pathogens, and biomaterials within a controlled fluidic environment, offering insights for the development of innovative periodontal therapies, tissue engineering strategies, and regenerative approaches.</p