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 2D 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. While both cell culture models yielded epithelial-like growth with barrier formation (TER > 400 Ω×cm 2 ), transcriptomic analysis revealed higher expression of fibroblast-/mesenchymal stromal ECM markers ( COL1A1, COL3A1, POSTN, MFAP5) in the inserts from the primary cultures. Additionally, extracellular matrix (ECM)-remodeling genes ( TGFB1, TIMP1, TIMP2, TIMP3, MMP2, MMP7, MMP12, MMP13) were upregulated in primary culture inserts, suggesting increased tissue remodeling activity in conjunction with the expression of inflammatory mediators ( ICAM1, IL6, CXCL12). Organoid-derived inserts showed less affected mitochondrial pathways (Cytochrome c oxidase ( COX1, COX2), NDUFA, NDUFB, ATP synthase genes) and ribosomal machinery ( RPL, RPS families), while maintaining epithelial purity with no detectable fibroblast or immune cell contamination. The expression of mRNA for numerous short-chain fatty acid (SCFA) transporters is confirmed, including MCT1, MCT4, DRA, PAT, SMCT1, AE2, and SLCO2A1. This study establishes organoids as a physiologically relevant model for studying rumen epithelial physiology.