Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells
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Evaluation of in vitro cytotoxicity and parac
Article history:Received 1 August 2007Accepted 28 February 2008Available online 29 March 2008
Keywords:In vitro cytotoxicityMouse embryonic stem cellsParacellular permeabilityTransepithelial electrical resistanceMTT assay
Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, using
toxicity data to determine the starting dose for in vivo testing, by cal-culating the standard regression between mean IC50 values andcorresponding acute oral LD50 data (Registry of Cytotoxicity, RC; ICC-VAM publication 01-4499, 2001a). The regression could then be usedto estimate the LD50 value of a new compound as the in vivo startingdose of a study. The application of this technique, however, is limitedby the lack of information on in vitromodels for gastrointestinal, der-
primers in lower case), alpha fetoprotein; AR, amphiregulin; Arnt gene, arylhydrocarbon receptor nuclear translocator; C-I, collagen type I; C-IV, collagen typeIV; ECM, extracellular matrix; EGF, epidermal growth factor; Egfr gene, epidermalgrowth factor receptor; FITC, uorescein isothiocyanate; FN, bronectin; LN,laminin; GFs, growth factors (upper case); IC50, inhibitory concentration 50%; IS,internal standard; KGF, keratinocyte growth factor; LIF, leukemia inhibitory factor;LY, lucifer yellow; MA, multicellular aggregates; MEF, mouse embryonic broblasts;mES, mouse embryonic stem (cells); Oct-4 gene, Oct-4/POU domain transcriptionfactor; PP, paracellular permeability; RC, Registry of Cytotoxicity; TEER, transepi-thelial electrical resistance or transmonolayer specic electrical resistance; Tgfbr2gene, transforming growth factor beta-receptor II; TJ, tight junction.* Corresponding author. Tel.: +1 718 990 2640; fax: +1 718 990 1877. 1 Center for Documentation and Evaluation of Alternative Methods to Animal
Toxicology in Vitro 22 (2008) 12731284
Contents lists available at ScienceDirect
journal homepage: www.elsE-mail address: email@example.com (F.A. Barile).1. Introduction
The Interagency Coordinating Committee on the Validation ofAlternative Methods (ICCVAM) and the National Toxicology Pro-gram (NTP) Interagency Center for the Evaluation of AlternativeToxicological Methods (NICEATM) recommended the development
and submission of novel in vitro methods as simple predictivemodels for human acute cytotoxicity (AC). Optimization of thesesystems should also yield models that mimic gastrointestinalabsorption, dermal toxicity, and acute lethality in vivo, as well aspredict rodent LD50 values (ICCVAM publications 01-4499 and01-4500, 2001a,b). To address this problem, several investigatorshave recommended the ZEBET1 approach as a strategy to reducethe number of animals required for acute oral toxicity testing (Halleet al., 2000; Spielmann et al., 1999). The method uses in vitro cyto-Abbreviations: 3H-D, [3H]-D-mannitol; AC, acute cytotoxicity; Afp gene (gene0887-2333/$ - see front matter 2008 Elsevier Ltd. Adoi:10.1016/j.tiv.2008.02.023combinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formationof intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage ofparacellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouseembryonic broblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% conuence, cells were pas-saged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA).After 4 days, cultures containing MA were transferred to culture inserts coated with ECM componentsonly, and grown in the presence of selected individual GFs. An additional 1014 days revealed conuentmonolayers with TEER values of 500700 ohms cm2 (X cm2). Monolayers grown on inserts coated withECM components, such as bronectin or collagen-IV, in the presence of epidermal growth factor or kerat-inocyte growth factor in the medium, yielded the highest TEER measurements when compared to cul-tures grown without GFs or ECM. Acute cytotoxicity (AC) studies with conuent monolayers of mEScells in 96-well plates indicated that there is a high correlation (R2 = 0.91) between cell viability and TEERfor 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. Incomparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC50 valuesfor mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreasesbefore the integrity of the monolayer is compromised. This system represents a novel approach for themanipulation of mES cells toward specic intact monolayers, as an in vitro model for biological mono-layer formation, and most importantly, for applications to cytotoxicity testing.
2008 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tmonolayers with mouse embryonic stem
Anthony R. Calabro, Roula Konsoula, Frank A. BarileSt. Johns University College of Pharmacy and Allied Health Professions, Department of PParkway, Queens, NY 11439, United Statesll rights reserved.cellular permeability of intactells
aceutical Sciences, Toxicology Division, 8000 Utopia
evier .com/locate / toxinvi t
components in human ES cells. Based on these reports, we ex-plored the possibility that mES cells could be stimulated to differ-entiate and form conuent monolayers with high transmonolayerresistance when grown on porous inserts coated with ECM sub-strata. Moreover, the inclusion of mitogenic GFs in the media, inthe absence of MEF layers or LIF, could further promotedifferentiation.
Thus, two objectives directed our experimental plan. First, wesystematically designed a series of experiments aimed at deter-mining which combination of GFs and ECM coatings would guidemES cells toward the formation of intact cultured monolayers, asdetermined by TEER measurement and passage of PP markers. Sec-ond, we used the culture model to develop an in vitro system formeasurement of AC and PP. The contention is that the mES modelimproves the predictive ability of in vitro acute toxicity testing as-says for in vitro/in vivo correlations over Caco-2 cells because thelatter are a differentiated immortal cell line with higher TEER val-ues and greater resistance to chemical insults than mortal contin-uous cells. And, although IEC-18 cells are mortal diploid intestinalmouse cells, they do not form intact monolayers with sufcientTEER registry (150200 X cm2). Finally, our mES cell model followson the successful development of prediction models using cultures
y in Vitro 22 (2008) 12731284mal and bloodbrain barrier passage and biotransformation (Currenet al., 1998). In fact, monolayers of intestinal and colonic epithelialcells have been used as cell culture models for detecting transepithe-lial transport of drugs, paracellular permeability (PP) and otherintestinal responses to xenobiotics (Carriere et al., 2001). The effectof chemicals on the relationship between PP and AC testing in anin vitro model, however, is not well established.
We recently used Caco-2 monolayers (Konsoula and Barile,2005) and IEC-18 cells (Konsoula and Barile, 2007) as in vitro mod-els for comparing PP with AC of 20 chemicals. Caco-2, an immortalcell line originating from human colon, demonstrate transepitheli-al electrical resistance (TEER) values that are relatively higher thanIEC-18 monolayers cultured from the small intestine. The resultsfrom these studies revealed that nite IEC-18 cells grown on cellculture inserts were sensitive to toxic insult as immortal Caco-2cells, and cell viability decreased before membrane integrity wascompromised. IEC-18 cells, however, registered signicantly lowerabsolute TEER values than Caco-2 cells. The lower resistance, aswell as the higher transport of chemicals in IEC-18 cells in culture,is analogous to the lower resistance of epithelial barriers withinthe small intestine (He et al., 1998). The diminished expressionof junctional proteins (ZO-1, occludin, e-cadherin) by IEC-18 sug-gests that junctional complexes are less tightly organized than inCaco-2 (Quaroni and Hochman, 1996). These features also explainthe higher TEER resistance of Caco-2 cells and prompt the need todevelop uniform, predictable in vitro models using diploid cells formeasuring AC and PP.
Mouse embryonic stem (mES) cells are derived from pluripotentcells of the early mammalian embryo and are capable of unlimited,undifferentiated proliferation in vitro (Evans and Kaufman, 1981).In chimeras with intact embryos, culturing of mES cells provide apowerful approach for introducing specic genetic changes intothe mouse cell line (Bradley et al., 1984). Pluripotency allows forthe ability of the cells to differentiate to the three embryonic germlayers. However, there are few reports demonstrating the differen-tiation of any ES cells to epidermal or epithelial structures. For in-stance, Yamada et al. (2002) describe the differentiation of mEScells into a functional gut-like organ in vitro that exhibits morpho-logical and physiological properties characteristic of the gastroin-testinal tract. Kuwahara et al. (2004) report that mES cellsundergo in vitro organogenesis by forming contracting gut-like or-gans from embryoid bodies (EBs). These structures are surroundedby epithelium, lamina propria, and muscularis. Ishikawa et al.(2004) further characterize interstitial cells of Cajal and smoothmuscle cells arising from EBs derived from mES cells. All of thesestudies were prompted by the developmental understanding thatin vivo epithelium undergoes continuous renewal by multipotentstem cells, which remain anchored to the crypt base. Thus a singleembryonic stem cell has potential to migrate and commit to all ofthe epithelial lineages (Kirkland and Henderson, 2001).
Several growth factors, particularly IL-3, (Wiles and Keller,1991), retinoic acid (Bain et al., 1995), and TGFb1 (Rohwedelet al., 1994), have been shown to direct linear specic differentia-tion of mouse stem cells. Epidermal growth factor (EGF), and therelated EGF family member, amphiregulin (AR), are mitogenicpolypeptides that induce differentiation into ectoderm and meso-derm (Gritti et al., 1999). Keratinocyte growth factor (KGF) is anepithelial cell-specic mitogen responsible for normal proliferationand differentiation of epithelial cells (Visco et al., 2004). In mostcases, the growth factors were applied to aggregates of ES cellsafter removal of leukemia inhibitory factor (LIF), a cytokine thatinhibits differentiation. In the absence of LIF, mES cells create intra-cellular contacts and initiate signaling and spontaneous differenti-
1274 A.R. Calabro et al. / Toxicologation (Furue et al., 2005). Keller et al. (2004) describe the inductionof epithelial- or epidermal-specic gene expression and differenti-ation using a combination of GFs plus extracellular matrix (ECM)of a pluripotent mouse embryonic stem cell line (embryonic stemcell test) for embryotoxicity testing (Genschow et al., 2000). Thus,the mES cells can be manipulated to form specic intact monolay-ers with critical tight junction (TJ) formation with signicant TEERvalues, and can be applied to the development of AC testingmodels.
2. Materials and methods
2.1. Cell culture and chemicals
Cell culture liquid and powder media, and cell culture gradechemicals and supplements were obtained from Invitrogen Corp.(Carlsbad, CA, USA). Biocoat cell culture inserts, Falcon tissueculture multiwell plates, asks, and other sterile disposable sup-plies were obtained from Becton Dickinson Labware (Bedford,MA, USA). All other test chemicals, including those listed in Table1, were obtained from SigmaAldrich (Sigma Life Sciences, St.Louis, MO, USA; Aldrich Chemical Co., Inc., Allentown, PA, USA).
Table 1Comparison of IC50 data (mmol/l) for mES cells using the MTT assay and TEERmeasurements, with IC50 calculations (mmol/l) and LD50 values (mmol/kg) from theRC database for the same chemicals
Chemicals MTT 24-h TEER 24-h *RC IC50*RC LD50
Acrylamide 5.03 21 1.61 2.39Actinomycin 0.0009 0.08 0.000008 0.0057Antipyrine 13 130 11.6 9.56Cadmium chloride 0.1 0.14 0.0064 0.48Cupric sulfate 0.4 0.5 0.33 1.2Dimethyl formamide 69.9 700 114 38.3Doxorubicin 0.008 0.05 0.00033 1.2Glycerol 34.68 588 624.0 137Ibuprofen 0.145 0.1 0.52 4.89Lithium sulfate 9 97 34 10.8Manganese chloride 1.1 7 0.13 7.5Niacinamide 25.1 158 44 28.7Nickel chloride 0.277 1.0 0.27 0.81Propranolol 0.056 0.08 0.12 1.59Quinine HCl 0.084 0.11 0.075 1.72Salicylic acid 0.95 0.74 3.380 6.45Sodium dichromatea 0.075 0.048 0.00093 0.19Trichlorforon 0.96 2.08 0.27 1.75Verapamil HCl 0.09 0.44 0.10 0.22*From the Registry of Cytotoxicity (RC) database (ICCVAM, 2001).a Dihydrate salt.
Chemicals used in the studies were suggested by the Registry ofCytotoxicity (RC; Halle, 2003); they were selected based on the
without extracellular matrix (ECM) component coating (Biocoat
inserts, Becton Dickinson Labware, Bedford, MA, USA). These com-ponents included: collagen type I (C-I), collagen type IV (C-IV),bronectin (FN), and laminin (LN). In addition, cells were grownin the presence of one of the following human growth factors (Sig-ma Life Sciences, St. Louis, MO, USA): EGF, 400 ng/ml; AR, 200 ng/ml; transforming growth factor-b1 (TGFb1), 0.4 ng/ml; and, kerat-inocyte growth factor (KGF), 2 ng/ml. The differentiated MA cul-tures were grown for another 1016 days, during which timedaily transmonolayer specic resistance (X cm2, see Section2.2.3) was measured using the Millicell-ERS resistance system(Millipore Corp., Temecula, CA, USA) before and after incubationwith test chemicals.
2.2.2. MTT cell viability assayThe acute cytotoxic effects of 19 chemicals (see Table 1) on cell
viability were measured in conuent monolayers in 96-well plates,using the MTT assay (Dolbeare and Vanderlaan, 1994). This assaywas originally described by Mosmann (1983), and modied as pre-viously described (Schmidt et al., 2004; Konsoula and Barile, 2007).The tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
Fig. 2. Further culture in the absence of feeder layers and LIF induce MA to losetheir aggregate morphology, as cells migrate to the periphery with continuedproliferation.
A.R. Calabro et al. / Toxicology inverication of the data set (RC-II), and for their validity in estab-lishing a regression model between oral LD50 and IC50 values froma single mammalian cell line (ICCVAM publication 01-4500,2001b).
Mouse ES cells were propagated in culture with and withoutmitomycin-C treated mouse embryonic broblast layers [MEFs,3T3 Swiss mouse broblasts, CCL-92; American Type Culture Col-lection (ATCC), Rockville, MD, USA]. MEFs were seeded at104 cells/cm2 in T-75 culture asks and grown in Dulbeccos mod-ied Eagles medium supplemented with 10% newborn calf serum(DMEM-10), 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 4 mM glu-tamine, and 5% antibiotic/antimycotic solution, in an atmosphe...