Identification of an Epithelial Cell Line for Two- and Three- Dimensional Studies of Intestinal Ion Transport

Abstract

Cystic fibrosis (CF) is an inherited disease caused by mutations in the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Many individuals with CF experience gastrointestinal tract complications, caused by impaired salt and water secretion across the intestinal epithelium. Intestinal organoids provide a powerful tool to investigate CFTR dysfunction in CF, aiding the identification of small molecule CFTR modulators for personalised medicine. The work in this thesis sought to identify an intestinal epithelial cell line suitable for studies of CFTR-mediated ion transport in the intestine, as an alternative to primary organoid models. The cell line should grow both as a 2D epithelium for electrical measurements of ion transport with the Ussing chamber technique, and as 3D spheroids to optically measure lumen expansion driven by fluid secretion.

Studies using the human colonic cell line LIM1863, which expresses CFTR and forms spheroids that swell in response to cAMP-mediated salt and water secretion, revealed that this cell line was unable to form polarised epithelia with a transepithelial resistance. Subsequently a panel of 15 human colorectal adenocarcinoma cell lines was screened to identify a cell line suitable for Ussing chamber and spheroid swelling studies.

Immunoblotting revealed CFTR expression in 7 of the 15 cell lines screened. When the CFTR-expressing cell lines were seeded onto permeable filter supports, only HCA7 cells developed a transepithelial resistance. To investigate transepithelial ion transport by HCA7 cells, Ussing chamber experiments were performed. Forskolin stimulated a small increase in current that was potentiated by genistein and inhibited by CFTR_inh-172. Complementary to these studies, when HCA7 cells were grown in Matrigel they formed spheroids with a lumen; lumen expansion was stimulated by forskolin and reduced by CFTR_inh-172. Thus, HCA7 cells are a useful model of CFTR-mediated ion transport, which might be used to identify new modulators of intestinal ion transport.

Date of Award	21 Jun 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	David N Sheppard (Supervisor)