Common Culture Mediums Skew Patient-Derived Small Intestine and Colonic Organoid Identity

Human patient-derived organoids from the small intestine and colon have become commonplace in research focusing on cellular function, metabolism, and cancer progression. Many studies using organoids will build upon literature that uses organoids cultured in different base mediums, either commercial-generated or lab-generated. Little to no research has been undertaken to evaluate whether organoids cultured from different medium can be interchangeably analyzed and if varying mediums yield genetically, morphologically, transcriptionally comparable organoids. This study aims to evaluate small intestine and colonic organoids under differing medium sources, both to determine the best culture condition for maintaining origin tissue identity whilst determining if culturing medium affects the ability to cross-analyze mediums. We have directly compared organoids derived from the small intestine and colon in multiple mediums including the Organoid Growth Medium (OGM) and Organoid Differentiation Medium (ODM) from StemCell Technologies, as well as multiple lab-generated mediums including those using conditioned medium from either individual Wnt3a, R-Spondin1, and Noggin (WRN) or from the combination cell line, L-WRN. Organoids grown in OGM and ODM maintained more cystic structures regardless of origin or culture duration whereas organoids in lab-generated mediums were slightly more budded and darker – classically interpreted as more differentiated. Utilizing immunofluorescence on whole-mount and paraffin-embedded organoids, we found that organoids grown in OGM and ODM have a higher number of MKI67-positive cells compared to organoids grown in lab-generated mediums. This is consistent with both flow cytometry and single cell RNA-sequencing (scRNA-seq) analysis, where commercial medium consistently produces more proliferative organoids than lab-generated mediums. Further scRNA-seq analysis highlights that organoids grown in OGM and ODM are highly skewed toward stem and progenitor cell types, while lab-generated mediums produce more differentiated cell types, while maintaining a small population of progenitors. Lastly, gene set enrichment analysis of organoids grown in commercial and lab-generated mediums highlights that organoids grown in commercial mediums, such as OGM, have a high correlation with mitotic fetal cells, rather than either the small intestinal or colonic origin. Yet, organoids grown in lab-generated mediums can both unbiasedly-identify their tissue of origin and region-specific cell types. Overall, these results convey that commercial mediums derive more stem-like organoids both morphologically and transcriptionally, concordant with multiple techniques suggesting these organoids are highly proliferative and revert to a more fetal-like state. In contrast, lab-generated mediums derive organoids with more differentiated cell types that maintain more of their tissue identity both morphologically and transcriptionally. Together these suggest that commercial and lab-generated mediums must not be used interchangeably and highlight the key need to standardize organoid culturing conditions when utilizing comparative analyses. This research is supported by grant 2444112 from the NSF GRFP, grant AI125181 from the National Institutes of Health, and grant U54CA274367 from the National Cancer Institute. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.