Model epithelia from rumen organoids.

The ruminal epithelium maintains livestock health by absorbing nutrients while maintaining a tight barrier between the lumen and the plasma space. This study demonstrates that rumen organoid-derived two-dimensional cultures approximate native tissue architecture and reach greater epithelial purity than primary cultures, particularly at later passages. For the initial transcriptomic and structural comparison, cells isolated from stratum basale by fractional trypsinization were used to generate model epithelia on cell culture inserts either from early-passage primary cultures (∼passage 3) or after organoid expansion (∼passage 10). Transcriptomic analysis was used to compare the primary culture inserts, the organoid-derived inserts, and the native tissues from which they had been derived. Although both cell culture models yielded epithelial-like growth with barrier formation (transepithelial electrical resistance > 400 Ω·cm2), transcriptomic analysis revealed higher expression of fibroblast/mesenchymal stromal extracellular matrix (ECM) markers (COL1A1, COL3A1, POSTN, and MFAP5) in the inserts from the primary cultures. In addition, ECM-remodeling genes (TGFB1, TIMP1, TIMP2, TIMP3, MMP2, MMP7, MMP12, and MMP13) were upregulated in primary culture inserts, suggesting increased tissue remodeling activity in conjunction with the expression of inflammatory mediators (ICAM1, IL6, and CXCL12). Organoid-derived inserts showed less affected mitochondrial pathways [Cytochrome c oxidase (COX1, COX2), NDUFA, NDUFB, and ATP synthase genes] and ribosomal machinery (RPL and RPS families), while maintaining epithelial purity with no detectable fibroblast or immune cell contamination. The expression of mRNA for numerous short-chain fatty acid transporters is confirmed, including MCT1, MCT4, DRA, PAT, SMCT1, anion exchanger 2, and SLCO2A1. This study establishes organoids as a physiologically relevant model for studying rumen epithelial physiology.NEW & NOTEWORTHY This study provides a comprehensive comparison between rumen tissue, primary culture, and organoid-derived cell culture inserts through detailed transcriptomic analysis. Cells grown over multiple passages in organoid culture differentiate into model epithelia that better approximate native tissue architecture and reach greater epithelial purity than primary cultures, particularly at later passages. The establishment of a sustainable organoid system with demonstrated molecular fidelity offers a valuable tool for studying rumen epithelial biology while reducing dependence on native tissue.