Establishment of primary pleural mesothelium with 3D organotypic coculture
Background/Purpose
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.
Methods
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.
Results
Isolated human PMCs showed cobblestone appearance and expressed mesenchymal characteristics: αSMA, 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).
Conclusions
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.
Untersuchungen zur Pathogenese apikaler bullöser Veränderungen in den Lungenspitzen im Segment 1 & Segment 6
Das Auftreten des primären Spontanpneumothorax trifft vorwiegend jugendliche Patienten. Klinisch relevant ist dabei die hohe Rezidivrate die damit einhergeht. Zum jetzigen Zeitpunkt ist die Pathogenese dieser apikal bullösen Veränderungen der Lungenspitzen im Bereich Segment 1 und Segment 6 des jeweiligen Lungenflügels aufgrund der klinischen Situation von großem Interesse. Bei den hier geplanten Untersuchungen ist von Bedeutung, die Mechanismen der bullösen Veränderungen zu charakterisieren, die Lokalisation und die molekularen Ursachen im Defekt der Lungenstruktur zu identifizieren. Vorwiegend ist von Interesse die Untersuchung der idiopathischen juvenilen Pneumothoraces. Dabei werden in der Studie Gewebe von Lunge und Pleura von Patienten mit entsprechender Indikation für chirurgische Intenvention im Labor ex vivo histologisch und molekularbiologisch analysiert und in vitro kultiviert. Fokussiert wird bei den Analysen vor allem auf mögliche Veränderungen der extrazellulären Matrix, Veränderungen im Regenerationsverhalten, auf Veränderungen durch epitheliale-mesenchymale Transition der Epithelzellen und Apoptoseverhalten. Zusätzlich werden in vitro Studien mit der alveolarepithelialen Lungenadenokarzinomzelllinie A549 durchgeführt, um den Einfluss von mesenchymalen Stromazellen als mögliche zelltherapeutische Intervention zu erfassen.
