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High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Within the peri-portal region of the adult liver, portal fibroblasts exist in close proximity to epithelial ductal/cholangiocyte cells. However, the cellular interactions between them are poorly understood. Here, we provide two co-culture techniques to incorporate liver portal mesenchyme into ductal cell organoids, which recapitulate aspects of their cellular interactions in vitro. We integrate several techniques from mesenchyme isolation and expansion to co-culture by microfluidic cell co-encapsulation or 2D-Matrigel layer. The protocol is easily adaptable to other cells from other organs. For complete information on the generation and use of this protocol, please refer to Cordero-Espinoza et al. 1 ., Competing Interests: Declaration of interests M.H. is an inventor in several patents on organoid technology., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)