DOI: 10.6084/m9.figshare.27890316.v1
title: 2024_StellatoRydzykEtAl_SupplementaryMaterial5

description: COLLECTION TITLE:2024_StellatoRydzykEtAl_SupplementaryMaterials<br>ARTICLE (when using these files, please, cite the following article):Mariachiara Stellato, Martyna Malgorzata Rydzyk, ..., and Filippo Piccinini. Radiomic analysis of 3D spheroids using 2D brightfield images, 2024.<br>ABSTRACT OF THE ARTICLE:Radiomics is a field of quantitative imaging involving the extraction of numerous features from medical images that become mineable data for diagnostic, prognostic, and predictive purposes. In this work, we focused on the analysis of 2D brightfield images acquired with standard microscopes available in all biological laboratories today. In particular, we analysed images of 3D multicellular spheroids, an in vitro model commonly used in regenerative medicine and oncology. Besides describing Analysis of Spheroid version 3.0 (AnaSP 3.0), a new release of an open-source software suite specifically designed for segmenting and extracting features from 2D brightfield images of multicellular spheroids. In this paper, we demonstrated how advanced features like sphericity, volume, and image gradient can be used for indirectly assessing the spheroidization of the spheroids over time, the number of cells composing the spheroids, and the extension of their central necrotic core. AnaSP 3.0 is freely available at: https://sourceforge.net/p/anasp. Concerning the previous versions, it presents new modules for extracting advanced features using grey-level images and not just binary masks. For validating the tool, we used spheroids generated with U-shape bottom multi-well plates and different concentrations of mesenchymal stem cells and osteosarcoma cell lines.<br>DESCRIPTION OF THE FILES IN THE COLLECTION:Files, images and data used in the above-mentioned scientific work.- Supplementary Material 1: Original images and masks related to Saos-2/MSC co-culture spheroids (generated with 5 various mixtures of cells, i.e. 0%-100%, 25%-75%, 50%-50%, 75%-25%, 100%-0%) obtained using an IncuCyte S3 microscope that can automatically acquire time-lapse images over time.- Supplementary Material 2: Fluorescent images related to Saos-2/MSC co-culture spheroids (generated with 5 various mixtures of cells, i.e. 0%-100%, 25%-75%, 50%-50%, 75%-25%, 100%-0%), obtained with a confocal microscope (10x magnification factor, conversion coefficient of 1.7 μm/pixel) for the Propidium Iodide (PI, red signal) and Hoechst stainings (blue signal).- Supplementary Material 3: Images and masks of MSC spheroids (3 different cell densities were seeded: 3000, 4000, and 5000 cells per well, in 100 μL of MEM alpha medium, supplemented with 20% of FBS, 1% of Gibco GlutaMAX Supplement, and 1% of Penicillin-Streptomycin antibiotic) analysed with a brightfield microscope (20x magnification factor, 0.297 μm/pixel conversion coefficient) and mesenchymal stem single-cells analysed with a confocal microscope (40x magnification factor, 0.62 μm/pixel conversion coefficient, staining used: red signal = DIL; blue signal = Hoechst).- Supplementary Material 4: Images and masks of 143B 3D spheroids (generated with three different seeding densities: 5000, 7500, and 10000 cells per well in 100 μL of DMEM) and 2D sections (obtained by embedding the spheroid in paraffin and sectioning them at a thickness of 5 µm using a rotary Leitz Sledge Microtome 1400), both analysed with a brightfield microscope (10x magnification factor, 0.41 µm/pixel conversion coefficient).- Supplementary Material 5: Tables containing the cell counting results (CellCount_Tables.pdf) and the necrotic percentage results (NecroticPercentage_Tables.pdf). The cell counting results are extracted from MSC spheroids described in Supplementary Material 3 with two methods. The first based on a classical cell counting approach and the second based on AnaSP. The classical cell counting approach was obtained disaggregating the spheroids with a solution composed of Gibco™ TrypLE™ Express Enzyme (1X), without phenol red mixed with 0.2 mg/mL of Gibco™ Collagenase, Type I, powder. The disaggregated spheroids' cells were then counted with a Countess® II FL Automated Cell Counter. The cell counting with AnaSP was performed starting from brightfield images. The necrotic percentage results are extracted from MSC spheroids described in Supplementary Material 4 with two methods. The first was a classical method based on spheroid slicing and the second a method based on gradient analysis of brightfield images. The classical method was performed by slicing the spheroids, staining them using Hematoxylin and Eosin staining to visualise nuclei and cytoplasm of the single cells. Then, brightfield images of the slices were acquired as described in Supplementary Material 4. The acquired images were finally analysed by two experts to determine the necrotic region. The gradient based method was performed by acquiring the brightfield images of the spheroids as described in Supplementary Material 4. The images were then analysed using AnaSP with a custom gradient-based method to estimate the necrotic percentage in each spheroid.<br>MAIN CONTACT FOR THIS WORK:Dr. Filippo Piccinini, PhD, University of Bologna, Italy, IRST IRCCS Meldola Italy.Emails: f.piccinini@unibo.it or filippo.piccinini@irst.emr.it<br>CATEGORIES:Bioinformatics and computational biology<br>KEYWORDS:Radiomic analysisMicroscopy image dataSpheroid culture systemsOncologiaOsteosarcomasRegenerative medicine approachMesenchymal cells<br>COPYRIGHT:* Copyright (C) 2024, * Filippo Piccinini (E-mail: filippo.piccinini85@gmail.com)* Mariachiara Stellato (E-mail: mariachiarastellato@gmail.com)* Martyna Malgorzata Rydzyk (E-mail: marti960118@gmail.com)* University of Bologna, Italy.* All rights reserved.* This material is free; you can redistribute it and/or modify it under the terms of the CC BY 4.0.* This material is distributed WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.