Development of a polarized pancreatic ductular cell epithelium for physiological studies

Abstract

Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate ([Formula: see text]) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer [Formula: see text] solution at 37°C gassed with 5% CO to measure short-circuit currents ( I). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO. Cells demonstrated a baseline I (12.2 ± 3.2 μA/cm) that increased significantly in response to apical forskolin-IBMX (∆ I: 35.4 ± 3.8 μA/cm, P < 0.001) or basolateral secretin (∆ I: 31.4 ± 2.5 μA/cm, P < 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆ I: 20.4 ± 3.8 μA/cm, P < 0.01). Extracellular pH and [Formula: see text] concentration increased significantly after forskolin-IBMX (pH: 7.18 ± 0.23 vs. 7.53 ± 0.19; [Formula: see text] concentration, 14.5 ± 5.9 vs. 31.8 ± 13.4 mM; P < 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent [Formula: see text] secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans. NEW & NOTEWORTHY Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion.

Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks

“…The conditional reprogramming method is applicable to many epithelial tissues including skin, prostate, lung, breast, kidney, salivary gland and liver cell types and it has recently been extended to neuroendocrine and endocrine tissues. Interestingly, the method seems generally applicable to many mammalian species such as mouse, rat, dog, ferret, horse, and cow [52,53,54,55,56,57]. An important feature is that these cultures can be used for establishing xenografts [12,50] and patient-derived xenograft cell lines [58] and can also be used to generate cell cultures from PDX and organoid cultures.…”

Mouse pancreatic ductal organoid culture as a relevant model to study exocrine pancreatic ion secretion

“…Whereas intracellular acidification caused by forskolin administration in HCO 3 /CO 2 − buffered solution was created by CFTR and electrogenic NBC1. In 2018, O'Malley et al reported the establishment of a culture system of differentiated pancreatic ductal epithelial cells in a polarized monolayer [32]. In this 2D air-liquid interface culture system, cells were grown on semipermeable filters and were reported to develop epithelial cell morphology.…”

Conditional reprogramming: next generation cell culture

“…The eligibility of CR to immortalize a wide range of epithelial cells of varied origins has been proved by many researches. Primary cells from both normal and pathological tissues can be proliferated under CR conditions ( Table 2 12 , 25 , 69 , 83 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 ). Of particular note, not only human tissues but also tissues from other origins such as mouse, horse, dog and fish have been used for CR culture.…”

“… Establishment of in vitro and in vivo models for drug screen and drug target identification 128 PDX tumor tissue (first passage) 3 Pig normal pancreatic tissue (newborn pig pancreata) 1 Pancreatic epithelial cells are expanded under CR conditions and have the characteristics of a ductal epithelium, which can differentiate into functional cells at the gas-liquid interface. A model for studying pancreas physiology and mechanisms of bicarbonate secretion 129 Liver Human liver tissue (freshly resected from patients with cirrhosis, hepatitis C, maple syrup urine disease, or citrullinemia type 1 disease) 11 Primary hepatocytes are grown from 6 out of 11 specimens under CR condition, which are genetically identical with original tissues and retain strong CYP3A4, 1A1 and 2C9 activities. Long term culture of patient-derived primary hepatocytes 130 Human tumor tissue (freshly resected) 20 Primary hepatocellular carcinoma cells continuously expand under CR condition and express tumor-specific marker.…”

“…In another study, CR enabled continuous expansion of C57BL mouse primary oral mucosal epithelial cells in vitro and maintains stem cell characteristics, which may be used as a source of seed cells for tissue engineered teeth 145 . In addition, several researchers have applied CR for establishment of stable cell lines from fish 143 , pig 127 , horse 129 , 139 , rabbit 127 and dog 144 . For instance, Gardell et al.…”