V-195 Establishment of primary pleural mesothelium with 3D organotypic co-culture

Hintergrund

Pleural mesothelial cells (PMCs) lead the pleural immune response and serve as progenitor cells after pleural or lung damage. They therefore play a key role in pleural diseases such as bacterial infections, malignant pleural effusion, pleural carcinosis or pleural mesothelioma. All these diseases prove a significant healthcare burden, however, its scientific consideration is quite low. The aim of the present study was to develop a three-dimensional (3D) in vitro organotypic model for investigation of pathological conditions of the pleural mesothelium. 3D organotypic models are a promising approach to gain an in vivo like understanding of molecular disease development. While these models are well established for a variety of organs, such as lung, colon, intestine, liver, pancreas, esophagus, prostate, or omentum majus, a 3D organoid model of the pleura is still missing.

Material und Methode

To construct a 3D organotypic model, primary human PMCs and fibroblasts were isolated from human pleura biopsies. Purification of primary human PMCs and fibroblasts was verified by immunofluorescence staining. The 3D collagen gel culture was assembled by plating of human pleural fibroblasts inside the gel, followed by seeding of PMCs on the gel to construct the normal pleura.

Ergebnis

Isolated human PMCs showed cobblestone appearance and expressed mesenchymal characteristics: α-SMA, vimentin but not prolylhydroxylase 1 (PHD1). The extracted fibroblasts maintained their spindle cell appearance and were positive for PHD1 (fibroblast marker). Grown on top of matrix-embedded fibroblasts, the primary human PMCs establish a monolayer and have direct contact with the underlying fibroblasts. Forty-eight hours after attachment, PMCs had cobblestone appearance and intercellular junctions were present between the mesothelial cells as shown by immunostaining for ZO-1. The structural and functional phenotype of the PMCs in our 3D organotypic culture was preserved over six days of culture, as evidenced by the expression of mesenchymal (vimentin, α-SMA, ZO-1) and proliferation marker (Ki67).

Schlussfolgerung

The presented 3D organotypic model of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment and presents a novel tool for development of preventive and therapeutic enhancement of various pleural diseases.

Publication History

Article published online:
06 September 2021