PSXIV-28 - Post-confluence cell age affects LPS induced interleukin-8 secretion in IPEC-J2 cells.

Abstract: Porcine intestinal epithelial cell line IPEC-J2 is routinely used to investigate epithelial cell interactions with common enteric porcine antigens and pathogens. Lipopolysaccharides (LPS) are bacterial antigens metabolized from Gram negative bacteria that can induce a cellular inflammatory cascade, resulting in changes to cell structure and integrity, and are often used to challenge IPEC-J2 cells. Although numerous investigators employ the IPEC-J2 LPS model, consistent methodology regarding cell culture conditions and treatment times is not well defined. The objective of the current study was to determine the optimal post-confluence cell age for LPS induction of IL-8 secretion and related cell integrity changes. For this study, cells were seeded at 2x10⁴/cm² in 24 well plates on day zero and incubated at 37˚C, 5% CO₂ for 3 days (confluence) prior to treatment in complete growth media (CGM) containing 5% fetal bovine serum. One day prior to treatment, CGM was removed and replaced with serum free (SF), phenol red-free media. On days 0, 5, 7, 12, 14 and 18 post confluence, cells were treated with LPS (0 or 10μg/ml) in SF and phenol red-free media for 24 hours. There were 5 replicates per treatment per timepoint. Lactate dehydrogenase (LDH) release, trans-epithelial electrical resistance (TEER), and Interleukin-8 (IL-8) secretion were measured at each experimental timepoint. Data were statistically analyzed for the effect of time, treatment, and the subsequent interactive effect. There was no effect of treatment, time or their interaction on LDH release (P > 0.05), indicating cellular health was maintained over time and 10μg/ml LPS did not influence cellular toxicity. There was an effect of treatment, time and the respective interaction that showed IL-8 secretory responsiveness was increased with LPS challenge (P < 0.01), was increased on d7 regardless of LPS challenge (P < 0.01) and peaked on d5 post-confluence (P < 0.01). Furthermore, TEER was maximized at d5 post-confluence and remained consistently higher relative to the d0 baseline throughout the experiment (P < 0.01). These data indicate that LPS-induced IL-8 secretion and related cellular integrity are optimized at d5 post-confluence in IPEC-J2 cells. In conclusion, in-vitro research utilizing the IPEC-J2 LPS model should allow cells to age 5 days post-confluence to maximize responses.