Correlation of in vitro cytotoxicity with paracellular permeability using cultured cells

This work is divided into two parts. The first part is the development of in vitro models to differentiate between acute cytotoxicity (AC) and paracellular permeability of selected chemicals. The study compares the low resistance rat intestinal mortal cell line, IEC-18 (transepithelial electrical resistance, TEER = 160 +/- 10 O-cm 2) with the high resistance human intestinal cell line, Caco-2 (TEER = 900 +/- 100 O-cm2). The two cell lines differ in state of differentiation, TEER and paracellular permeability characteristics. Cytotoxicity was carried out using MTT cell viability assay in 96-well plates for 24h exposure time. PP was measured using TEER (membrane integrity indicator) and PP markers such as [3-H] D-mannitol, LY (lucifer yellow) and FITC-dextran (fluorescein-dextran) on cells grown on inserts. The data showed that there is a high correlation (R2=0.99) between MTT and TEER using IEC-18 cell for 24h exposure time. IEC-18 is as same sensitive as Caco-2 for both MTT and TEER measurements. Decrease in TEER is inversely proportional with increase in PP of tight junction indicators. There is a good correlation between IC50s MTT, TEER and Registry of Cytotoxicity (RC) data. The cytotoxicity data indicate, at equivalent concentrations, cell viability decreases before the integrity of the membrane is compromised. Based on the results from the experiments, both cell lines can be used as an in vitro model to differentiate between concentrations needed for AC and those required for PP.;The second part is the manipulation of Mouse embryonic pluripotent stem cells (mES, ES-D3) to form membrane structures in cell culture inserts, using combinations of extracellular matrix (EM) components and growth factors (GF). The ability of the stem cells to form intact membranes and construct tight junctions (TJ) is assessed by measuring transepithelial electrical resistance (TEER) and passage of TJ markers. Proof of differentiation and formation of epidermal-like membranes is supported by the expression of genetic markers for embryoid bodies (EB), differentiated cells, as well as undifferentiated cultures. Further evidence for formation of TJ in differentiated cultures is demonstrated by immunohistochemical localization of TJ markers, occludin and beta-actin. After 4 days EB were transferred to 24-well culture inserts coated separately with EM components (collagen I, collagen IV, fibronectin and laminin). Induction of TJ was further facilitated by treating EB on the inserts with 1 of 4 growth factors (amphiregulin, EGF, KGF and TGFbeta-1). After 10 to 14 days, TEER was measured at 500--700 O-cm 2, with the addition of fibronectin or collagen-IV, plus EGF or KGF yielding the highest values. The cytotoxicity data indicate that, at equivalent concentrations, cell viability is altered before PP is compromised. Also, TEER measurements inversely correlate with increased passage of TJ markers for most chemicals. We conclude that mES cells can be induced to form TJ in coated culture inserts after treatment with appropriate GF as evidenced by progression of high TEER values comparable to those seen previously with Caco-2 cells. Support for TJ formation is demonstrated by expression of TJ specific genes and immunohistochemical analysis for markers. The extent and direction of differentiation is time- and factor-dependent and can be accurately monitored. ES cells therefore can be induced to differentiate into intact confluent epidermal/epithelial-like membrane structures. This represents the first report describing the manipulation of mouse stem cells toward specific tissue organization, with the potential for use as an in vitro model for biological membrane formation and cytotoxicity testing.