Intestinal epithelial organoids established from resected gut tissue have become a widely used research tool. However, it remains unclear how environmental cues and other sources of variation before, during and after establishment impact on organoid properties. Divergent microbiota composition in separately held mouse lines has been identified as a confounding factor in murine in vivo studies. To probe the effect of donor microbiota on organoids, we analyzed the proteomes and transcriptomes of primary organoid cultures established from colonized and germ-free mice, and further compared them to their tissue of origin and to commonly used cell lines. The long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts. This signature, which closely mimicked expression patterns in the gut epithelium, included high levels of the inflammasome scaffold protein ASC in organoids which was lacking in commonly used cell lines. Mining of the transcriptome dataset uncovered similar high expression of also other inflammasome components, including Naip1-6, Nlrc4, and Caspase-1 in all organoids compared to the reference cell lines. Taken together, these results reveal a robust global expression pattern in organoids established from colonized and germ-free animals and suggest that organoids represent a more suitable culture model than immortalized cell lines for studies of intestinal epithelial inflammasomes.