Improved Cell Line IPEC-J2, Characterized as a Model for Porcine Jejunal Epithelium Silke S. Zakrzewski 1 , Jan F. Richter 1¤ , Susanne M. Krug 1 , Britta Jebautzke 2 , In-Fah M. Lee 1 , Juliane Rieger 3 , Monika Sachtleben 3 , Angelika Bondzio 4 , Jo ¨ rg D. Schulzke 2 , Michael Fromm 1 , Dorothee Gu ¨ nzel 1 * 1 Institute of Clinical Physiology, Charite ´ – Universita ¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany, 2 Department of Gastroenterology, Division of Nutritional Medicine, Charite ´, Universita ¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany, 3 Institute of Veterinary Anatomy, Freie Universita ¨t Berlin, Berlin, Germany, 4 Institute of Veterinary Biochemistry, Freie Universita ¨t Berlin, Berlin, Germany Abstract Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species- specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line’s initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function. Citation: Zakrzewski SS, Richter JF, Krug SM, Jebautzke B, Lee I-FM, et al. (2013) Improved Cell Line IPEC-J2, Characterized as a Model for Porcine Jejunal Epithelium. PLoS ONE 8(11): e79643. doi:10.1371/journal.pone.0079643 Editor: James F. Collins, University of Florida, United States of America Received July 10, 2013; Accepted October 3, 2013; Published November 15, 2013 Copyright: ß 2013 Zakrzewski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Deutsche Forschungsgemeinschaft (DFG, Collaborative Research Center (SFB 852/1) ‘‘Nutrition and Intestinal Microbiota – Host interactions in the pig’’; http://www.dfg.de/en/) and by the Sonnenfeld-Stiftung Berlin (http://www.sonnenfeld-stiftung.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: correspondence: dorothee.guenzel@charite.de ¤ Current address: Institute of Anatomy II, Universita ¨tsklinikum Jena, Jena, Germany Introduction In intensive pig farming, a significant fraction of piglets die after weaning, in many cases due to infectious diarrhea [1]. Intense research efforts are therefore made to reduce mortality in animal breeding. For molecular studies on mechanisms and signaling pathways between germ exposure and diarrheal effect, porcine cell cultures are highly desirable. However, these cultures are only suitable if they closely match the properties of pig small intestinal epithelium. Thus, for research on intestinal barrier function, cell models have to meet specific physiological requirements: reflecting epithelial architecture, displaying adequate transepithelial resis- tance (TER) and transport properties, reacting to secretagogues, and expressing bowel-relevant tight junction (TJ) proteins. If these prerequisites have been achieved, the model system will be potentially suitable for studying effects of e.g. nutritional factors. Non-transformed continuous epithelial cell lines of only few species and gut sections are available so far, e.g. IEC-6 from rat small intestine [2], IEC-18 from rat ileum [3], IPEC-1 from pig ileum and jejunum [4], IPEC-J2 from pig jejunum [4], and PSI from pig small intestine [5]. In contrast to cultures of rodent cells, a unique side aspect of porcine cell culture models is the potential application for human purposes because the pig gastrointestinal tract physiology is highly comparable to that of humans [6]. It immediately stands out, compared to other commonly used intestinal cell lines (CMT-93, TER: 400 V?cm 2 [7]; HT-29/B6, TER: 500 V?cm 2 [8]) and pig bowel mucosa (R epi : 55 V?cm 2 , [9]), that all porcine cell lines mentioned above exhibit extraordinarily high TER values (1 to 15 kV?cm 2 ) when believed to be fully differentiated by the respective author [5,10-12]. TER is a key parameter of epithelial tightness and is determined by para- and by transcellular processes [13]. The paracellular pathway between enterocytes is limited by the TJ which is formed by opposing transmembrane TJ proteins and mediates different degrees of tightness. The TJ is of central interest as it forms a barrier against uptake of putatively immunogenic macromolecules and an excessive passage of water, small ions, and other solutes [14]. The transcellular pathway PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79643