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Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

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.

SUBMITTER: Zakrzewski SS

PROVIDER: S-EPMC3829867 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

Zakrzewski Silke S SS Richter Jan F JF Krug Susanne M SM Jebautzke Britta B Lee In-Fah M IF Rieger Juliane J Sachtleben Monika M Bondzio Angelika A Schulzke Jörg D JD Fromm Michael M Günzel Dorothee D

PloS one 20131115 11

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 ...[more]

PMID: 24260272

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Project description:The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.

Dataset Information

Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

Publications

Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

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