Pitfalls in AR42J-model of cerulein-induced acute pancreatitis

Abstract

Background AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J are often used as a cell-culture model of cerulein (CN)-induced acute pancreatitis (AP). Nevertheless, it is controversial how to treat AR42J for reliable induction of AP-like processes. Gene knockout and/or overexpression often remain challenging, as well. In this study, we demonstrate conditions for a reliable induction of proinflammatory markers upon CN treatment in AR42J and high transfection efficacy using Glyoxalase-I (Glo-I) as a target of interest. Methods Effects of dexamethasone (dexa) and CN on cell morphology and amylase secretion were analyzed via ELISA of supernatant. IL-6, TNF-α and NF-κB-p65 were measured via qRT-PCR, ELISA and Western Blot (WB). Transfection efficacy was determined by WB, qRT-PCR and immune fluorescence of pEGFP-N1-Glo-I-Vector and Glo-I-siRNA. Results Treatment of AR42J with 100 nm dexa is mandatory for differentiation to an acinar-cell-like phenotype and amylase production. CN resulted in secretion of amylase but did not influence amylase production. High levels of CN-induced amylase secretion were detected between 3 and 24 hours of incubation. Treatment with LPS alone or in combination with CN did not influence amylase release compared to control or CN. CN treatment resulted in increased TNF-α production but not secretion and did not influence IL-6 mRNA. CN-induced stimulation of NF-κB was found to be highest on protein levels after 6h of incubation. Transient transfection was able to induce overexpression on protein and mRNA levels, with highest effect after 12 to 24 hours. Gene-knockdown was achieved by using 30 pmol of siRNA leading to effective reduction of protein levels after 72 hours. CN did not induce amylase secretion in AR42J cell passages beyond 35. Conclusion AR42J cells demonstrate a reliable in-vitro model of CN-induced AP but specific conditions are mandatory to obtain reproducible data.

Glyoxalase-I Is Upregulated in Acute Cerulein-Induced Pancreatitis: A New Mechanism in Pancreatic Inflammation?

“…Rat pancreatic AR42J acinar cells were purchased from ATCC (CRL1492, Manassas, VA, USA) and maintained in DMEM (high glucose, Biochrom/Merck, Berlin, Germany), supplemented with 10% fetal calf serum (FCS, Biochrom/Merck) and 1% penicillin/streptomycin (P/S, PAA, Pasching, Austria). AR42J cells derived from Wistar rats are adherent cells with secretory activity following induction with glucocorticoids [33]. Cells were kept at standardized conditions (37 • C with 5% CO 2 ) and medium was replaced every 48 h. AR42J cells were passaged once a week.…”

“…If cells were thawed from frozen stocks, they were initially supplemented with DMEM and 40% FCS. After thawing, cells were allowed to grow and acclimate for 4 to 6 weeks (4 to 6 passages) prior to performing experiments [33].…”

Derivation of pancreatic acinar cell carcinoma cell line HS‐1 as a patient‐derived tumor organoid

“… 18 , 19 Among many rat cell lines, the AR42J cell has been most widely used, presumably because of its dual properties in both exocrine and endocrine lineages. 20 It allows functional analysis as a substitute for normal islet cells and acinar cells, 21 which are still difficult to maintain in culture. Genetically engineered mice represent an alternative in vivo models, having pancreas‐specific overexpression of SV40 22 and ablation of Tsc1 , either alone 23 or in combination with Trp53 deletion.…”