# Generation of a human vascularized 3D airway model replicating native mucosal heterogeneity

## TL;DR

Researchers developed a 3D human airway model that mimics real mucosal structures and functions, offering a better alternative to traditional models for studying respiratory diseases.

## Contribution

A reproducible protocol for generating a vascularized 3D airway model with native mucosal heterogeneity and functional characteristics.

## Key findings

- The model develops a well-differentiated pseudostratified epithelium with basal, multiciliated, and secretory cells after 4 weeks.
- The model exhibits physiological ciliary beating frequency, mucus production, and functional particle clearance.
- Endothelial cells within the hydrogel form a 3D network of vascular structures.

## Abstract

In vitro models of the human airway are essential to study respiratory diseases and test potential therapeutics while reducing animal testing. Current models often use two-dimensional culture conditions rather than replicating the physiological 3D environment and do not allow direct cell-cell interactions between the diverse cell types found in the mucosa. Here, we provide a detailed step-by-step instruction for reproducibly generating a complex tri-culture model, which can be used to investigate the human airway environment in health and disease. The model is fabricated by preparing an epithelialized fibrin hydrogel with embedded endothelial and stromal cells. To assure complete differentiation into a mucociliary phenotype, samples are maintained at air-liquid interface (ALI) for 28 days. Afterwards, morphology and functionality can be validated using downstream analysis techniques such as immunohisto- and cytochemistry, electron microscopy, ciliary beating frequency analysis, measurement of mucociliary clearance and RNA isolation. After 4 weeks of maturation, a well-differentiated pseudostratified epithelium comprising basal, multiciliated and secretory cells is developed. We also observe a physiological ciliary beating frequency, mucus production and a functional particle clearance. Inside the hydrogel, endothelial cells form a three-dimensional network of vascular structures. These features make our model ideal for replicating human mucosal heterogeneity, especially compared to airway models using tumor-derived or immortalized cell lines, monocultures or rigid substrates. Hence, this protocol paves the way for fellow researchers to achieve robust airway in vitro modeling that can be performed in a standard cell culture lab without the need for extraordinary equipment or specialized expertise.