Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro

Abstract

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acidsupplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor β (FGF-β) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-β concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Preliminary Study on the Development of In Vitro Human Respiratory Epithelium Using Collagen Type I Scaffold as a Potential Model for Future Tracheal Tissue Engineering

“…Tracheal ciliated epithelial cells are located only at the luminal surface of the tracheal epithelium and they are specialized cells that are responsible for transporting secretions across the airway [50] and maintaining the airway's immunoprotection [29]. Whether achieved via transdifferentiation of primary cells or through ciliogenesis in undifferentiated mesenchymal stem cells, having epithelial cells with intact cilia is a critical factor for the successful engineering of tracheal epithelial tissue.…”

“…Therefore, supplementing culture media with retinoid as a ciliogenic regulatory element [60] can improve the ciliation of seeded RECs on the collagen scaffold. Moreover, Luengen et al showed in 2020 that a balanced combination of RA and vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor β in culture medium plays a critical role in the differentiation status of respiratory epithelium; it needs to be considered in future studies [29].…”

“…Proper epithelialization of tissue-engineered airway constructs, poor vascularization, and less-than-favorable mechanical properties are the current challenges. Complex and often non-reproducible differentiation procedures and difficulty in maintaining the mucocilated phenotype of the primary airway epithelial cells are the major complications persisting in the field [29]. Hence, in this study, we introduced a three-dimensional (3D) model of an in vitro respiratory epithelium (RE) construct comprising RECs and fibroblasts from human nasal turbinate, which were incorporated into a freeze-dried ovine collagen scaffold and grown in an air-liquid interface culture condition.…”

Might Routine Vitamin A Monitoring in Cystic Fibrosis Patients Reduce Virus-Mediated Lung Pathology?

“…Apart from affecting immune responses, vitamin A also supports the growth and integrity of epithelial cells ( 14 ), features associated with wound prevention and repair. When introduced into in vitro systems, vitamin A assists the establishment of epithelial monolayers and organ culture systems ( 56 ). Epithelial cells of the respiratory tract express retinaldehyde dehydrogenase 2 (RALDH2 or ALDH1A2), an enzyme necessary for conversion of retinaldehyde to its end-stage metabolite, retinoic acid ( 48 ).…”

Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models

“…We here describe a protocol for the generation of reliable 3D in vitro models of upper respiratory tract, namely, nose and trachea. We propose the use of a synthetic hydrogel-based 3D culture system that is able to reproduce in vitro the complex biochemical and mechanical cues deriving from the native extracellular matrix [ 6 , 7 ] as well as several aspects of the native cellular microenvironment, mimicking the original conditions of the airway epithelia [ 8 ].…”