DOI: 10.25824/redu/2brsm6
title: Hepatotoxicity assessment of microplastics in Huh7 and HepG2 cells through high-content analysis

description: Hepatotoxicity Assessment of Microplastics in Huh7 and HepG2 Cells through High-Content Analysis This repository contains the following resources produced during the Master’s research of Mariana Rodrigues da Silva at the NanoCell Interactions Lab, University of Campinas (UNICAMP), which focused on investigating the in vitro hepatotoxicity of microplastics (PS-MPs) using HepG2 and Huh7 cell lines, image-based assays (High-Content Analysis), and cellular profiling. The four main folders are organized as follows: ├── Cellpose_Models │ ├── Calcein-PI-Hoechst │ ├── Total-Association-of-PS-MPs │ ├── CellPainting │ └── LiveCellPainting ├── CellProfiler_Pipelines │ ├── Calcein-PI-Hoechst │ ├── Total-Association-of-PS-MPs │ ├── LiveCellPainting │ └── CellPainting ├── Images │ ├── Calcein-PI-Hoechst │ ├── Total-Association-of-PS-MPs │ ├── LiveCellPainting │ └── CellPainting └── Jupyter_Notebooks Description of Each Folder: Cellpose_Models: Contains trained models using Cellpose (version 2.2.3) for segmenting cellular structures (cells and/or nuclei) in Huh7 and HepG2 cell lines. Each subfolder corresponds to a specific imaging assay. CellProfiler_Pipelines: Contains pipelines developed in CellProfiler (version 4.2.5) to extract cellular features from Huh7 and HepG2 cell lines. Each subfolder corresponds to a specific imaging assay. Images: Contains raw image datasets for each imaging assay, organized into subfolders by assay name. Each subfolder contains two subfolders corresponding to the cell type (Huh7 or HepG2; except for CellPainting, which only contains Huh7). These folders are further subdivided into three independent replicate folders, each containing a subfolder named platemap with the plate layout for the 96-well plate used during image acquisition. The metadata specifies the compounds and concentrations used to treat each well. The cells were stained using six imaging panels: Calcein-PI-Hoechst (cell viability assay): Images acquired with a 10× objective on the Cytation 5 using the following LED cube channels: DAPI: nuclei of total cells (excitation/emission = 377/447 nm) GFP: live cells (excitation/emission = 445/510 nm) PI: dead cells (excitation/emission = 531/647 nm) Total Association of PS-MPs with Cells Over Time (interaction assay between cells and fluorescent PS-MPs over time): Images acquired with a 20× objective on the Cytation 5 using the following LED cube channels: DAPI: nuclei (excitation/emission = 377/447 nm) GFP: live cells (excitation/emission = 445/510 nm) PI: PS-MPs (excitation/emission = 531/647 nm) Live Cell Painting (cell profiling assay, Garcia-Fossa et al., 2024; DOI): Images acquired with a 20× objective on the Cytation 5 using the following LED cube channels: DAPI: nuclei (excitation/emission = 377/447 nm) GFP: nuclei, nucleoli, cytoplasm (excitation/emission = 445/510 nm) PI: acidic vesicles (excitation/emission = 531/647 nm) Cell Painting (cell profiling assay, Bray et al., 2016; DOI): Images acquired with a 20× objective on the Cytation 5 using the following LED cube channels: DAPI: plasma membrane and nuclei (excitation/emission = 377/447 nm) GFP: endoplasmic reticulum (excitation/emission = 445/510 nm) PI: actin cytoskeleton and Golgi complex (excitation/emission = 531/647 nm) CY5: mitochondria (excitation/emission = 628/685 nm) Jupyter_Notebooks: This folder contains: Data processing templates for normalization, aggregation, annotation, and feature selection. Templates for identifying cellular profiles using Linear Discriminant Analysis (LDA). Clustering methods, including Elbow Method, Silhouette Method, and K-means. Confusion matrix analysis to evaluate clustering quality. Feature importance identification scripts. Templates for statistical analysis, including Kruskal-Wallis tests with Bonferroni post hoc correction and boxplot visualization of individual features. These resources were developed to facilitate image-based workflows and advanced data analysis for understanding the hepatotoxic effects of microplastics in vitro.