P043 ALI culture derived from human organoids recapitulates cell composition during intestinal crypt formation

Dotti, I.(1)*;Álvarez, I.(1);Esteller, M.(1);Ricart, E.(1);Salas, A.(1);

(1)IDIBAPS- Hospital Clínic- CIBER-EHD, Department of Gastroenterology, Barcelona, Spain;


The ALI (air liquid-interface) culture derived from human primary intestinal epithelium is generating considerable interest for closely reproducing the mature cell features of the differentiated epithelium in a 2D configuration. However, the phenotypic changes occurring in the human ALI culture during cell differentiation have thus far not been explored in depth. Here, we aimed to characterize the cell composition of human ALI culture over time.


3D epithelial organoids (n=3) were expanded from human colonic samples and organoid-derived single cells were seeded on a Transwell. After reaching confluence, the submerged 2D was switched to ALI and maintained for 7 days. The ALI culture was analysed every 24h by qPCR, membrane staining (H&E and immunofluorescence) and trans-epithelial resistance (TER) (Figure 1).



The confluent pre-ALI (TER>200 Ω*cm2) showed high mRNA expression of stem cell markers (i.e., LGR5, AXIN2), while several differentiation markers (i.e., ZG16, CLCA1, ANPEP, AQP8, BEST4, CHGA) were almost undetectable, thus resembling the stem cell niche compartment. Air exposure of the 2D culture induced an early and transient (limited to ALI-day 1) transcriptional up-regulation of proliferation markers (i.e., MKI67, CDCA7) and OLFM4, followed by a concomitant marked downregulation of stem cell markers, thus mimicking the Transit Amplifying compartment. OLFM4 protein, as observed by immunofluorescence, showed an early cytoplasmic accumulation followed by secretion at later time points (from ALI-day 5). Differentiation towards absorptive and secretory lineages increased gradually from ALI-day 5 and ALI-day 7 onward, respectively, at both the transcriptional and protein level. Expression changes were accompanied by the progressive transition of the ALI monolayer from a squamous to a columnar morphology. In parallel, TER promptly increased upon ALI switch, thus indicating the early establishment of cell-cell junctions.


By recapitulating the cell composition identified along the crypt, the ALI culture holds promise to be a versatile in vitro model for studying how the different epithelial cell compartments respond to specific environmental stimuli, like those present during an inflammatory flare.