(582) Cystic Fibrosis Porcine Pancreatic Duct Cell Culture Models to Study Disease Pathophysiology

Background: Cystic fibrosis (CF) is caused by mutations in the gene that encodes cystic fibrosis transmembrane conductance regulator (CFTR). Despite significant progress made in treating the life-threatening lung disease in CF, there has been little development in understanding the CF pancreatic disease. One major barrier has been the lack of in vitro cell cultures developed from relevant animal models of CF.

Methods: Pancreatic duct epithelial cells (PDEC) and pancreatic duct organoids were grown from newborn CF (CFTR^-/-) and non-CF pigs (CFTR^+/+ and CFTR^+/-). Gene expression was determined using RT-PCR. Brightfield microscopy images were obtained using Olympus CKX53 microscope to evaluate pancreatic duct organoid and PDEC morphology. Cells were dispersed using Trypsin-EDTA and counted using a Beckman Coulter Z1 Coulter Particle Counter to determine cell proliferation over time.

Results: Porcine PDEC cells were characterized in a previous report [1]. P<span style="mso-font-kerning: 12.0pt;">ancreatic duct organoid cultures expressed ductal (cytokeratin-8) and epithelial (E-cadherin, zona occludens-1) markers with no evidence of acinar (amylase) or islet (insulin, glucagon) markers confirming their ductal origin. Only non-CF organoids and PDEC expressed CFTR. Non-CF porcine pancreatic duct organoids developed a hollow center, which is a sign of maturity and fluid secretion, while CF porcine pancreatic duct organoids were smaller in size, with no easily appreciable hollow center and a thicker wall. Compared to non-CF, CF PDEC and organoids demonstrated increased cell proliferation.

Conclusions: This is the first PDEC and pancreatic duct organoid model generated from CF pigs, an animal model that recapitulates the human disease. Increased cell proliferation in CF PDEC and organoids needs further study.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation Second Year Clinical Fellowship Award [002012B121] and the Stead Family Children’s Hospital and Children’s Miracle Network Funding Grant.

References: [1] O'Malley Y, Rotti PG, Thornell IM, Vanegas Calderón OG, Febres-Aldana C, Durham K, Yao J, Li X, Zhu Z, Norris AW, Zabner J, Engelhardt JF, Uc A. Development of a polarized pancreatic ductular cell epithelium for physiological studies. J Appl Physiol (1985). 2018 Jul 1;125(1):97-106. doi: 10.1152/japplphysiol.00043.2018. Epub 2018 Mar 8. PMID: 29517421; PMCID: PMC6086968.