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

This research article discusses the creation and testing of a co-culture model, specifically focusing on the interaction between primary equine bronchial epithelial cells (EBECs) and equine bronchial fibroblasts (EBFs). The findings indicate that the communication between these two types of cells through paracrine signalling plays a significant role in their growth, adhesion, and differentiation—crucial characteristics to understand and address the pathophysiology and treatment of human and equine asthma.

Establishment of Co-Culture Model

• The researchers first built a unique co-culture model involving EBECs and EBFs, designed to elucidate the role of intercellular interaction in epithelial wound repair processes.
• EBFs (between their 4th and 8th passage) were initially placed at the bottom of 24-well plates and treated with Mitomycin C when reaching an 80% confluence or coverage.
• Following this, freshly-isolated EBECs (P0) or slightly older, passaged EBECs (P1) were introduced to the top of membrane inserts within the fibroblast-populated well plates.

Growth and Differentiation under Different Conditions

• Two successive configurations were used for the growth and development of these cells: initially under a liquid-liquid interface (LLI), then under an air-liquid interface (ALI) to trigger epithelial differentiation.
• Different indicators of progress—morphology, structure, functional markers—were tracked in P0 and P1 EBEC monolayers as they grew along with EBFs.
• Observations were assisted by techniques such as phase-contrast microscopy, scanning and transmission electron microscopy, and hematoxylin-eosin immunostaining.
• Moreover, the study gauged the viability of selective barrier functions in treated monolayers through the measurement of transepithelial electrical resistance (TEER), along with the transport of select drugs across this boundary.

Notable Observations and Possible Implications

• After approximately 15-20 days of ALI co-culture, both P0 and P1 EBEC monolayers exhibited a pseudo-stratified architecture—a form of layering seen in tissues facing significant mechanical stress—with the presence of ciliated cells and a honeycomb-like pattern of tight junction protein 1 (TJP1) expression.
• Intriguingly, the study found marked differences in the behaviour of co-cultured EBECs when compared to the previously well-known mono-culture system. This pointed to potential paracrine cross-talk, or communication mediated through cellular secretions, between the two cell types under study.
• These findings point to this EBEC-EBF co-culture model as an important platform to facilitate future investigations into cell interactions, especially those between epithelial and mesenchymal components. This can provide more insights into the way diseases evolve in the equine airways, helping us in better managing and treating conditions like equine and human asthma.