Optimization of Primary Human Bronchial Epithelial 3D Cell Culture with Donor-Matched Fibroblasts and Comparison of Two Different Culture Media

Abstract

In vitro airway models are increasingly important for pathomechanistic analyses of respiratory diseases. Existing models are limited in their validity by their incomplete cellular complexity. We therefore aimed to generate a more complex and meaningful three-dimensional (3D) airway model. Primary human bronchial epithelial cells (hbEC) were propagated in airway epithelial cell growth (AECG) or PneumaCult ExPlus medium. Generating 3D models, hbEC were airlifted and cultured on a collagen matrix with donor-matched bronchial fibroblasts for 21 days comparing two media (AECG or PneumaCult ALI (PC ALI)). 3D models were characterized by histology and immunofluorescence staining. The epithelial barrier function was quantified by transepithelial electrical resistance (TEER) measurements. The presence and function of ciliated epithelium were determined by Western blot and microscopy with high-speed camera. In 2D cultures, an increased number of cytokeratin 14-positive hbEC was present with AECG medium. In 3D models, AECG medium accounted for high proliferation, resulting in hypertrophic epithelium and fluctuating TEER values. Models cultured with PC ALI medium developed a functional ciliated epithelium with a stable epithelial barrier. Here, we established a 3D model with high in vivo–in vitro correlation, which has the potential to close the translational gap for investigations of the human respiratory epithelium in pharmacological, infectiological, and inflammatory research.

Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells

“…This is important for electrophysiological studies that rely on the integrity of the epithelial barrier. Interestingly, we observed that PN-ALI differentiated epithelia had a lower TEER compared to AB-ALI differentiated epithelia, consistent with previously published studies and likely attributable to a combination of reduced expression of sealing claudins and increased electrogenic ion transport 12 , 13 , 22 , 23 . Conversely, the higher TEER in AB-ALI differentiated cultures could be due to a squamous epithelium phenotype, which has been shown to confer higher TEER values 12 , 24 .…”

Recent Advances in 3D Cultures

“…One of the most challenging organs to mimic is the lung [18,19]. Maurer J. et al [20] proposed a novel complex 3D airway model for the study of respiratory diseases. Primary human bronchial epithelial cells were cultured on a collagen matrix with donor-matched bronchial fibroblasts, and characterized for properties and functions of epithelial barrier and ciliated epithelium.…”