Development and characterization of 2-dimensional culture for buffalo intestinal cells

Abstract

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/cm 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.

Aflatoxin M1 decreases the expression of genes encoding tight junction proteins and influences the intestinal epithelial integrity

“…Cytokeratins are intermediate lament protein family members that are expressed as a network radiating from the nucleus to the plasma membrane. Cytokeratins are intracytoplasmic cytoskeleton proteins that are commonly used to identify (Giotakis et al, 2021;Chaudhary et al, 2018;Coch and Leube, 2016). Cytokeratins play an important role in protecting epithelial cells from mechanical and non-mechanical stressors (Karantza, 2011).…”

Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

“…This may be attributed to the inability of BIEC-c4 cells to form tight junctions. The BIEC-c4 cells could be tested in further studies for the expression level of tight junction proteins (Anderson and Van Itallie 1995 ; Chaudhary et al 2018 ; McNeil et al 2006 ; Miyazawa et al 2010 ). In an earlier study, fetal-derived bovine duodenal epithelial cells not only showed high TEER values but also strongly expressed both zonula occludens-1 (ZO-1) and beta-catenin (Miyazawa et al 2010 ).…”

“…Bovine intestinal epithelial cell cultures have been established from the duodenum, jejunum, ileum, and colon of adult cattle and fetal calves (Birkner et al 2004 ; Kaushik et al 2008 ; Loret et al 2009 ; Rusu et al 2005 ; Yang et al 2015 ; Zhan et al 2017 ). Recently, intestinal epithelial cell culture established from the jejunum of buffalo has been reported (Chaudhary et al 2018 ).…”

Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves

“…Among the various fatty acid binding protein (FABP) isoforms, the fatty acid binding protein 2 (FABP2) is specific to the intestinal epithelium (Smathers and Petersen, 2011); thus, it can be considered a molecular biomarker of the small intestine (Thumser et al, 2014;Saqui-Salces et al, 2017;Chaudhary et al, 2018). Similar to FABP2, cytokeratins are proteins highly expressed in the epithelial cells.…”