Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids
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ORIGINAL PAPER
Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids Emma D. Wrenn 1,2 & Breanna M. Moore 1 & Erin Greenwood 1 & Margaux McBirney 1 & Kevin J. Cheung 1 Received: 3 August 2020 / Accepted: 9 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies nonlinearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between ~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material. Keywords Tumor organoids . Mouse mammary organoids . Organoid propagation . 3D culture . Optimal growth . CRISPR . Organoid suspension culture
Introduction A wealth of studies have demonstrated that 3D organotypic culture systems can faithfully recapitulate diverse aspects of the tissue architecture, heterogeneity, and spatial complexity of the in vivo microenvironment [30, 55, 57, 59, 60]. In recent years, organoids have been usefully applied to understanding diverse developmental and disease processes ranging from early embryogenesis [53, 62] to COVID-19 viral replication in different tissue types [64] to complex genotype-phenotype processes in degenerative brain diseases [19, 58].
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10911-020-09464-1) contains supplementary material, which is available to authorized users. * Kevin J. Cheung [email protected] 1
Translational Research Program, Public Health Sciences and Human Biology Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2
Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA 98195, USA
Organoids have also emerged as important models for cancer [15, 37, 55, 60]. Tumor organoids can be isolated from experimental mouse models as well as human patient primary and metastatic tumors, allowing for the representation of a wide range of tumor phenotypes and genotypes. To observe and perturb complex interactions amongst tumor cells, normal cells, and stroma, multicellular tumor organoids are embedded i
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