Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids

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.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion

“…2b), in line with previous reports using a 4T1 breast cancer cell line 20 . The detection of basal-like cells during the monoclonal MMTV-PyMT organoid formation and under non-invasive conditions in BME is consistent with recent studies 21 , and indicates that the presence of basal-like luminal cells is not dependent on pro-invasive stimuli from the ECM (for example from Collagen I).…”

Generation and functional characterization of murine mammary organoids

“…Also see troubleshooting -Problem 4 for inconsistent results across technical replicates. Alternatives: To test response of mammary organoids to drug treatments, reduced Matrigel concentration; 2%–5% Matrigel in growth medium can be used to limit usage of reagents ( Wrenn et al., 2020 ; Zhang et al., 2016 ).

Figure 11 Cell growth assay of PTEN-wild-type and PTEN-mutant mammary organoids PTEN wild-type (PTEN +/+ ) and PTENC124S mutant (PTEN CS/+ ) mammary organoids were plated in 96-well plates and grown for 6 days in Organoid culture medium.

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“…Also see troubleshooting -Problem 4 for inconsistent results across technical replicates. Alternatives: To test response of mammary organoids to drug treatments, reduced Matrigel concentration; 2%–5% Matrigel in growth medium can be used to limit usage of reagents ( Wrenn et al., 2020 ; Zhang et al., 2016 ). …”

“…The volume of Matrigel used to perform drug screen with organoids can scale up quickly and become costly. As an alternative, organoids suspensions in low Matrigel concentration (e.g., 2%), diluted in growth medium, have been used and could be considered as an option to reduce usage of reagents and associated costs ( Wrenn et al., 2020 ).…”

Unraveling the Breast: Advances in Mammary Biology and Cancer Methods

“…Compared to 2D cultures, one of the shortcomings of 3D cell cultures is that the 3D cultures are more expensive and laborious. To overcome these limitations, Wrenn and colleagues [ 9 ] developed a suspension culture method optimized for production of large numbers of organoids, enabling impressive expansion from one million to over 100 million cells from mouse-derived tumors within two weeks. Moreover, building on their protocol, they established conditions for efficient lentiviral transduction to generate organoids having genetic knockout, knockdown, or overexpression.…”