Julie Venter1, Heather Francis2, Fanyin Meng2, Sharon DeMorrow2, Lindsey Kennedy3, Holly Standeford3, Laura Hargrove4, Nan Wu1, Ying Wan4, Gabriel Frampton1, Matthew McMillin1, Marco Marzioni5, Eugenio Gaudio6, Paolo Onori6, Shannon Glaser2, Gianfranco Alpini7. 1. Department Medicine, Division Gastroenterology, S&W and Texas A&M HSC, Temple, TX, United States. 2. Research, Central Texas Veterans Health Care System, United States; Scott & White Digestive Disease Research Center, Academic Operations, Scott & White, United States; Department Medicine, Division Gastroenterology, S&W and Texas A&M HSC, Temple, TX, United States. 3. Research, Central Texas Veterans Health Care System, United States. 4. Scott & White Digestive Disease Research Center, Academic Operations, Scott & White, United States. 5. Universita' Politecnica delle Marche, Ospedali Riuniti, Ancona, Italy. 6. Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, "Sapienza", Rome, Italy. 7. Research, Central Texas Veterans Health Care System, United States; Scott & White Digestive Disease Research Center, Academic Operations, Scott & White, United States; Department Medicine, Division Gastroenterology, S&W and Texas A&M HSC, Temple, TX, United States. Electronic address: galpini@tamu.edu.
Abstract
BACKGROUND: Since limited in vitro tools exist for evaluating the pathophysiology of extrahepatic bile ducts, we aim to develop an extrahepatic cholangiocyte culture system from normal rats. METHODS: Extrahepatic ducts were dissected from rats, cut in half length-wise and cultured on collagen-I coated plates. Transepithelial electrical resistance was measured. At ∼85% confluence, in extrahepatic cholangiocytes we measured: (i) cell size and distribution, and expression for cytokeratin-19, secretin, secretin receptor and somatostatin receptor type II (SSTR2), cystic fibrosis transmembrane conductance regulator (CFTR), chloride bicarbonate anion exchanger 2 (AE2), vascular endothelial growth factor-A (VEGF-A) and nerve growth factor (NGF); and (ii) the effect of secretin and/or somatostatin on 3'-5'-cyclic adenosine monophosphate (cAMP) levels and proliferation. RESULTS: Cytokeratin-positive extrahepatic cholangiocytes were cultured for 6 passages to form a cell monolayer. Cholangiocytes proliferated to confluence over a 2-week period. The size of extrahepatic cholangiocytes averaged ∼16 μm. Extrahepatic ducts and cholangiocytes were positive for secretin, secretin receptor and SSTR2, CFTR, AE2, VEGF-A and NGF. In extrahepatic cholangiocyte cultures, secretin increased cAMP (prevented by somatostatin), chloride efflux and proliferation. CONCLUSIONS: Extrahepatic cholangiocyte cultures may be important for studying diseases targeting extrahepatic cholangiocytes such as biliary atresia. Published by Elsevier Ltd.
BACKGROUND: Since limited in vitro tools exist for evaluating the pathophysiology of extrahepatic bile ducts, we aim to develop an extrahepatic cholangiocyte culture system from normal rats. METHODS: Extrahepatic ducts were dissected from rats, cut in half length-wise and cultured on collagen-I coated plates. Transepithelial electrical resistance was measured. At ∼85% confluence, in extrahepatic cholangiocytes we measured: (i) cell size and distribution, and expression for cytokeratin-19, secretin, secretin receptor and somatostatin receptor type II (SSTR2), cystic fibrosis transmembrane conductance regulator (CFTR), chloride bicarbonateanion exchanger 2 (AE2), vascular endothelial growth factor-A (VEGF-A) and nerve growth factor (NGF); and (ii) the effect of secretin and/or somatostatin on 3'-5'-cyclic adenosine monophosphate (cAMP) levels and proliferation. RESULTS: Cytokeratin-positive extrahepatic cholangiocytes were cultured for 6 passages to form a cell monolayer. Cholangiocytes proliferated to confluence over a 2-week period. The size of extrahepatic cholangiocytes averaged ∼16 μm. Extrahepatic ducts and cholangiocytes were positive for secretin, secretin receptor and SSTR2, CFTR, AE2, VEGF-A and NGF. In extrahepatic cholangiocyte cultures, secretin increased cAMP (prevented by somatostatin), chloride efflux and proliferation. CONCLUSIONS: Extrahepatic cholangiocyte cultures may be important for studying diseases targeting extrahepatic cholangiocytes such as biliary atresia. Published by Elsevier Ltd.
Entities:
Keywords:
Epithelia; Extrahepatic bile duct; Gastrointestinal hormones; Proliferation
Authors: Junya Shiota; Nureen H Mohamad Zaki; Juanita L Merchant; Linda C Samuelson; Nataliya Razumilava Journal: J Vis Exp Date: 2019-04-23 Impact factor: 1.424
Authors: Lindsey L Kennedy; Fanyin Meng; Julie K Venter; Tianhao Zhou; Walker A Karstens; Laura A Hargrove; Nan Wu; Konstantina Kyritsi; John Greene; Pietro Invernizzi; Francesca Bernuzzi; Shannon S Glaser; Heather L Francis; Gianfranco Alpini Journal: Lab Invest Date: 2016-10-24 Impact factor: 5.662