Single versus repeated exposure to human polarized intestinal epithelial monolayers for in vitro protein hazard characterization.

Recent studies suggest human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on permeable Transwell® filters are effective at differentiating between hazardous and non-hazardous proteins following a single exposure. In this study, IEC polarized monolayers were subjected to hazardous or non-hazardous proteins in nine exposures over 30 days and compared to a single exposure of the same protein. The objective was to evaluate whether repeated exposures to a protein differently alter barrier integrity or compromise cell viability compared to single exposures. Proteins tested included Clostridium difficile toxin A, Streptolysin O, Wheat Germ Agglutinin, Phaseolus vulgaris Hemagglutinin-E, bovine serum albumin, porcine serum albumin, and fibronectin. Evidence of diminished barrier integrity and/or cell viability following exposure to hazardous proteins was more pronounced in magnitude when IECs were subjected to multiple rather than single exposures. In some cases, an effect on IEC monolayers was observed only with repeated exposures. In general, IEC responses to non-hazardous proteins following either single or repeated exposures were minimal. Results from these studies support the utility of using cultured human IEC polarized monolayers to differentiate between hazardous and non-hazardous proteins and suggest that repeated exposures may reveal a greater magnitude of response when compared to single exposures.

Extended exposure duration of cultured intestinal epithelial cell monolayers in characterizing hazardous and non-hazardous proteins.

Recent studies suggest that human derived intestinal epithelial cells (IECs) cultured as polarized monolayers on Transwell® filters may respond differently when exposed to hazardous and non-hazardous proteins. This experimental platform was based on apical exposure of IEC monolayers to test proteins for 24 h followed by assessment of barrier integrity and cell viability. In this study, Caco-2 and T84 IEC polarized monolayers were evaluated for barrier integrity and cytotoxicity following exposure to hazardous and non-hazardous proteins for 24, 48 and 72 h. Hazardous proteins included Clostridium difficile toxin A (ToxA), Streptolysin O (SLO), Wheat Germ Agglutinin (WGA), and Phaseolus vulgaris haemagglutinin-E (PHA-E). Non-hazardous proteins included bovine serum albumin (BSA), porcine serum albumin (PSA), and fibronectin (Fbn). In general, evidence of diminished barrier integrity or cell viability observed following exposure to hazardous proteins for 24 h was more pronounced after 48 and 72 h for both IEC monolayers. Non-hazardous proteins exhibiting no impact following 24 h of exposure elicited minimal effects over longer exposure durations. These results support the utility of using cultured human IEC polarized monolayers to differentiate between hazardous and non-hazardous proteins and suggest that longer durations of exposure may further improve the ability to distinguish between them.