Nine-day post-confluent CACO-2 cell layers on Millipore PCF filters were exposed to varying concentrations of specific seminal plasma components in culture medium in the basal-lateral fluid compartment 24 hours prior to measurements of transepithelial electrical resistance (Rt) and 14C-D-mannitol permeability (Jm). Human recombinant proteins, TNF-α and IL1β, were obtained from PeproTech (Rocky Hill, NJ). IFN-γ was a product of Life Technologies (Frederick, MD). Zinc sulfate heptahydrate, histone from calf thymus, spermine, citric acid monohydrate, urea and fructose were products of Sigma-Aldrich (St. Louis, Mo.). Human recombinant EGF and TGF were products of Enzo Biochem (New York, NY) and PeproTech, respectively.
Whereas CACO-2 cultures 9 days post-seeding are not fully differentiated, barrier function is in fact near maximal.[18 (link)] Given our current study’s focus on exposure of the basal-lateral cell surface to SP, we are in fact modeling the effect of SP on an already compromised epithelium, one that allows SP penetration from the lumen into the interstitium, as would occur in an epithelium wounded mechanically or by proinflammatory cytokine damage. The epithelium in the immediate vicinity of that site of compromise would very likely not be in a completely differentiated state.
Whereas CACO-2 cultures 9 days post-seeding are not fully differentiated, barrier function is in fact near maximal.[18 (link)] Given our current study’s focus on exposure of the basal-lateral cell surface to SP, we are in fact modeling the effect of SP on an already compromised epithelium, one that allows SP penetration from the lumen into the interstitium, as would occur in an epithelium wounded mechanically or by proinflammatory cytokine damage. The epithelium in the immediate vicinity of that site of compromise would very likely not be in a completely differentiated state.
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