Small intestinal epithelial cells (IEC) play a major role in the absorption of 
nutrients and toxins. Due to the similarity of genome-wide single copy protein 
orthologues between cattle and human, establishment of ruminant's primary small 
IEC culture could be a valuable tool for toxicity studies. Therefore, the 
current study focused on the development and characterization of buffalo IEC 
culture, as cattle slaughter is banned in India. The buffalo jejunum fragments 
were washed consecutively several times in saline, warm phosphate buffered 
saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol 
in PBS. The cells were cultured on 17 µg/cm2 collagen coated plates and 
transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and 
serum-free culture conditions. The cells were differentiated into typical 
epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. 
The cultured IEC were characterized by gene expression of epithelial cell 
markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula 
occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal 
peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has 
been recommended for culturing primary buffalo IEC on collagen coated plates for 
10 days. However, 50% of the successful cultures could not show epithelial 
phenotype on 10% FBS culture conditions even on collagen coated plates. 
Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP 
and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on 
collagen plates. Therefore, future studies are needed to understand the role of 
FABP2, IP and ZO1 in differentiation of buffalo IEC.