Evaluation of liquid versus air-liquid interface in in-vitro swine jejunum epithelial cell differentiation
Intestinal epithelial cells cultured in-vitro as organoids (enteroids) are not exposed to the luminal signals in the intestine, resulting in lack of differentiation. To promote differentiation, we tested two luminal culture conditions: conventional Liquid-Liquid (LL) and Air-Liquid (AL). Swine jejunum enteroids were plated in transwells, grown for ten days and then media in the insert was removed (AL) or kept (LL). Cells were harvested after 1, 3 and 5 days. Total RNA was extracted and 400 ng converted to cDNA. Gene expressions of Sodium-Glucose Cotransporter 1 (SGLT1), SRY-Related HMG Box 9 (SOX9), Villin (VIL1), Proliferating Cell Nuclear Antigen (PCNA), Peptide Transporter 1 (PEPT1), Mucin 2 and Chromogranin A were detected by qPCR and relative expressions calculated using the -2ΔΔCt method with Glyceraldehyde-3-Phosphate Dehydrogenase as reference. Treatment differences were detected by one-way ANOVA followed by multiple comparisons. SGLT1 (glucose absorption) and SOX9 (transient amplifying cells) did not change when AL and LL interfaces were compared. VIL1 (enterocytes), PCNA (proliferation), and PEPT1 (absorption of peptides) expressions were greater in AL compared with LL on days 1 and 3, with no differences on day 5. Mucin 2 (goblet cells) and Chromogranin A (endocrine cells) had greater expression in AL interfaces compared with LL on days 1, 3, and 5. The data showed increased expression of genes from day 1 to day 3 in AL interfaces while little to no difference from day 3 to 5. Therefore, differentiation in swine jejunum enteroids is favored when cultured in AL interface for 3 days.
