Single cell transcriptomics reveals that air-liquid interface culture promotes goblet cell differentiation and inhibits glycolysis in organoid cell monolayers

Faithfully recapitulating the cellular heterogeneity of the intestinal epithelium is essential when using organoid models. Air-liquid interface (ALI) culture has been shown to promote secretory cell dif-ferentiation but its impact on gene expression in each epithelial cell type remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize the cellular heterogeneity of rabbit caecum-derived organoid monolayers grown under immerged or ALI conditions. We then compared these organoid cell type-specific gene expression profiles to a scRNA-seq atlas of the rabbit caecal epithelium <i>in vivo</i>. We selected the rabbit model notably because, unlike mice, it pos-sesses BEST⁴⁺ epithelial cells, a newly discovered subset of mature absorptive cells. Our analysis revealed a high degree of transcriptomic similarity between <i>in vivo</i> and organoid-derived stem and transit-amplifying cells. ALI culture markedly enhanced the differentiation of the secretory lineage, especially goblet cells, which transcriptome closely resembled that of <i>in vivo</i> goblet cells. Further-more, ALI was the only condition allowing the detection of enteroendocrine cells. BEST⁴⁺ cells, however, were absent from organoids in immerged or ALI conditions despite their presence <i>in vivo</i>. In addition, ALI culture led to a consistent downregulation of hypoxia and glycolysis-associated genes across all cell types, which suggests a metabolic shift likely driven by increased oxygen avail-ability in ALI conditions. Cell-cell communication analyses further indicated that ALI more closely mirrored <i>in vivo</i> patterns than immerged condition. Altogether, these results demonstrate that ALI culture allows to better recapitulate the <i>in vivo</i> cellular heterogeneity and molecular signatures of the intestinal epithelium.