Equine bronchial fibroblasts enhance proliferation and differentiation of primary equine bronchial epithelial cells co-cultured under air-liquid interface


Autoři: Vanessa Abs aff001;  Jana Bonicelli aff001;  Johannes Kacza aff002;  Claudia Zizzadoro aff003;  Getu Abraham aff001
Působiště autorů: Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken, Leipzig, Germany aff001;  Saxonian Incubator for Clinical Translation, University of Leipzig, Philipp-Rosenthal-Straße, Leipzig, Germany aff002;  Division of Veterinary Pharmacology and Toxicology, Department of Veterinary Medicine, University of Bari, SP 62 per Casamassima, km, Valenzano (BA), Italy aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225025

Souhrn

Interaction between epithelial cells and fibroblasts play a key role in wound repair and remodelling in the asthmatic airway epithelium. We present the establishment of a co-culture model using primary equine bronchial epithelial cells (EBECs) and equine bronchial fibroblasts (EBFs). EBFs at passage between 4 and 8 were seeded on the bottom of 24-well plates and treated with mitomycin C at 80% confluency. Then, freshly isolated (P0) or passaged (P1) EBECs were seeded on the upper surface of membrane inserts that had been placed inside the EBF-containing well plates and grown first under liquid-liquid interface (LLI) then under air-liquid interface (ALI) conditions to induce epithelial differentiation. Morphological, structural and functional markers were monitored in co-cultured P0 and P1 EBEC monolayers by phase-contrast microscopy, scanning and transmission electron microscopy, hematoxylin-eosin, immunocytochemistry as well as by measuring the transepithelial electrical resistance (TEER) and transepithelial transport of selected drugs. After about 15–20 days of co-culture at ALI, P0 and P1 EBEC monolayers showed pseudo-stratified architecture, presence of ciliated cells, typically honeycomb-like pattern of tight junction protein 1 (TJP1) expression, and intact selective barrier functions. Interestingly, some notable differences were observed in the behaviour of co-cultured EBECs (adhesion to culture support, growth rate, differentiation rate) as compared to our previously described EBEC mono-culture system, suggesting that cross-talk between epithelial cells and fibroblasts actually takes place in our current co-culture setup through paracrine signalling. The EBEC-EBF co-culture model described herein will offer the opportunity to investigate epithelial-mesenchymal cell interactions and underlying disease mechanisms in the equine airways, thereby leading to a better understanding of their relevance to pathophysiology and treatment of equine and human asthma.

Klíčová slova:

Cell differentiation – Epithelial cells – Epithelium – Equines – Fibroblasts – Fluorescence imaging – Permeability – Tight junctions


Zdroje

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Článek vyšel v časopise

PLOS One


2019 Číslo 11