Figure 1.
PS reduces transepithelial, transcellular, and paracellular resistance of IPEC-J2.
(A) Time course of transepithelial resistance (TER) of IPEC-J2, which were cultured using different culture media (FBS, fetal bovine serum; ABS, adult bovine serum; GS, adult goat serum; PS, adult porcine serum; ‘+’ indicates supplementation with ITS and EGF) as indicated in the key. PS prevented cells from developing extremely high TER values over a time period of at least three weeks. Note the TER time course of PS (5% +), which developed a maximum around day 7 and reached a lower plateau level at day 14 post seeding. (n = 4–5 each) (B) TER values of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) were corrected for surface enlargement by villi and crypts of porcine jejunum (factor 10) (TERcorr) for comparison with pig jejunal values (dark grey bar). IPEC-J2/FBS exhibited higher TERcorr values (n = 20) than pig jejunum (n = 15; ***, p<0.001), whereas TERcorr of IPEC-J2/PS (n = 20) was not significantly (n.s.) different from porcine values. (C) TER time courses of IPEC-J2/FBS and IPEC-J2/PS were monitored two weeks before and after culture conditions were exchanged as indicated by ‘change’. IPEC-J2 immediately started to develop respective serum-typical TER values. (n = 4–5 each) (D) Two-path impedance spectroscopy was employed to determine Rpara (light grey bars) and Rtrans (dark grey bars) of IPEC-J2/FBS and IPEC-J2/PS. Significant reduction of both, Rpara (*, p<0.05, n = 6) as well as Rtrans (**, p<0.01, n = 6) occurred in PS when compared to FBS culture. Repi (white bars) is calculated as Rpara·Rtrans/Rpara+Rtrans.
Figure 2.
PS elevates active ion transport and membrane capacitance of IPEC-J2.
(A) Forskolin-induced short-circuit current (▵ISC) of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) were corrected for surface enlargement by villi and crypts of porcine jejunum (factor 10) (▵ISCcorr) for comparison with pig jejunal values (dark grey bar). Stimulation by forskolin resulted in an increased chloride secretory response in IPEC-J2/PS (n = 11; **, p<0.01) compared to IPEC-J2/FBS (n = 10), which almost reached porcine values (n = 9; n.s.). (B) Epithelial capacitance (Cepi) of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) was determined via impedance spectroscopy. In IPEC-J2/PS, Cepi was increased (n = 15; ***, p<0.001) compared to IPEC-J2/FBS (n = 22).
Figure 3.
PS optimizes cell dimension of IPEC-J2.
(A) Horizontal and (B) vertical aspects of IPEC-J2 were visualized by live cell imaging using FITC-dextran 4000 (FD4), those of pig jejunocytes by immunofluorescence staining of ZO-1 (A) and of E-cadherin (E-cad, B). Scale bar: 20 µm.
Figure 4.
PS approximates tight junction ultrastructure of IPEC-J2 to pig jejunum.
(A) Freeze-fracture images of IPEC-J2/FBS, IPEC-J2/PS, and pig jejunal tissue (scale bar: 200 nm). (B) Morphometric analysis of the TJ meshwork depth, (C) the number of horizontal strands, and (D) the TJ strand type (continuous vs. particle type). IPEC-J2/PS, n = 22; IPEC-J2/FBS, n = 23; pig jejunum, n = 20; n.s., not significant; *, p<0.05; **, p<0.01.
Figure 5.
PS affects permeability to fluorescein but not charge selectivity of IPEC-J2.
(A) Paracellular permeability to fluorescein (PFLU) of IPEC-J2/FBS (n = 7) and IPEC-J2/PS (n = 8). (B) Permeability ratio for sodium and chloride (PNa/PCl), indicating paracellular charge selectivity, as determined by dilution potentials measurements. The broken line indicates no charge selectivity. Pig jejunal tissue (n = 12), IPEC-J2/PS (n = 13), IPEC-J2/FBS (n = 11); n.s., not significant; ***, p<0.001.
Figure 6.
PS does not affect junctional protein localization within cell-cell contacts but controls tight junction protein quantity of IPEC-J2.
(A) Confocal immunofluorescence images of IPEC-J2/FBS, IPEC-J2/PS, and cryosectioned pig jejunal mucosae. Cldn5, tric, and occl are presented in green, counterstain in red, as indicated. Nuclei are presented in blue (DAPI). The broken line indicates different counterstain between IPEC-J2 and pig jejunum. Scale bar: 20 µm. (B) Tight junction proteins of IPEC-J2/FBS and IPEC-J2/PS (n = 3 to 4 different cell passages) were analyzed by Western blotting and were subsequently densitometrically quantified. To allow for different cell architecture, values were normalized to E-cadherin. All signals of IPEC-J2/PS are given in relation to IPEC-J2/FBS values (100%). *, p<0.05.
Figure 7.
PS approximates porcine jejunal marker patterns of IPEC-J2 to that of jejunum.
(A) Confocal immunofluorescence images of IPEC-J2/FBS, IPEC-J2/PS, and cryosectioned pig jejunal mucosae. Porcine jejunocyte marker proteins are presented in green, counterstain in red, as indicated. Nuclei are presented in blue (DAPI). In IPEC-J2, GLUT2 and Na/K-ATPase could be detected within the basolateral membrane, whereas ezrin and SGLT1 could be verified within the apical membrane of IPEC-J2/PS only. In addition, IPEC-J2 were positive for vimentin. The broken line indicates that counterstain choice differed between IPEC-J2 and pig jejunum. Scale bar: 20 µm. (B) Marker proteins of IPEC-J2/FBS and IPEC-J2/PS (n = 3 to 4 different cell passages) were analyzed by Western blotting and were subsequently densitometrically quantified. To allow for different cell architecture, values were normalized to E-cadherin, with the exception of vimentin, which was normalized to β-actin. All signals of IPEC-J2/PS are given in relation to IPEC-J2/FBS values (100%). *, p<0.05; **, p<0.01.