Novel 3D organotypic co-culture model of pleura

Abstract

Pleural mesothelial cells are the predominant cell type in the pleural cavity, but their role in the pathogenesis of pleural diseases needs to be further elucidated. 3D organotypic models are an encouraging approach for an in vivo understanding of molecular disease development. The aim of the present study was to develop a 3D organotypic model of the pleural mesothelium. Specimens of human pleura parietalis were obtained from patients undergoing surgery at the University Hospital Leipzig, Germany. 3D co-culture model of pleura was established from human pleural mesothelial cells and fibroblasts. The model was compared to human pleura tissue by phase-contrast and light microscopy, immunochemistry and -fluorescence as well as solute permeation test. Histological assessment of the 3D co-culture model displayed the presence of both cell types mimicking the morphology of the human pleura. Vimentin and Cytokeratin, PHD1 showed a similar expression pattern in pleural biopsies and 3D model. Expression of Ki-67 indicates the presence of proliferating cells. Tight junctional marker ZO-1 was found localized at contact zones between mesothelial cells. Each of these markers were expressed in both the 3D co-culture model and human biopsies. Permeability of 3D organotypic co-culture model of pleura was found to be higher for 70 kDa-Dextran and no significant difference was seen in the permeability for small dextran (4 kDa). In summary, the presented 3D organoid of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment.

A Platform for Testing the Biocompatibility of Implants: Silicone Induces a Proinflammatory Response in a 3D Skin Equivalent

“…Based on our experience in establishing a 3D pleura equivalent, which proved to be applicable for investigating pleural infections, we aimed to engineer a 3D skin equivalent [31,32]. To verify the aforementioned goal of close resemblance to human skin, we compared our 3D skin equivalent histologically to specimens of human skin and were able to demonstrate a skin-typical layered structure (Figure 2).…”

“…Our group previously established an organotypic 3D equivalent of human pleura and successfully mimicked pleural empyema [31,32]. The aim of the present study was to establish a 3D skin equivalent, which would allow for investigations of interactions between human skin cells (keratinocytes and fibroblasts) and implants of different materials, starting with silicone, to assess biocompatibility.…”

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development

“…The cells obtained during each disaggregation step were collected in 25 cm 2 culture flasks with 5 mL of Roswell Park Memorial Institute (RPMI) 1640 medium, 20% fetal bovine serum (FBS), and 100 U/mL penicillin, as well as 100 µg/mL streptomycin. The protocol for isolation of primary cells was previously described in detail by Metelmann et al [25]. The primary cultures were incubated at 37 • C and 5% CO 2 in 5 mL of RPMI 1640, 20% FBS, and 100 U/mL penicillin/100 µg/mL streptomycin in 25 cm 2 culture flasks for 7 days with medium changed every 72 h. Isolated HPMCs and HPFs were purificated by immunofluorescence staining (previously described in detail by Metelmann et al [25]).…”

“…The protocol for isolation of primary cells was previously described in detail by Metelmann et al [25]. The primary cultures were incubated at 37 • C and 5% CO 2 in 5 mL of RPMI 1640, 20% FBS, and 100 U/mL penicillin/100 µg/mL streptomycin in 25 cm 2 culture flasks for 7 days with medium changed every 72 h. Isolated HPMCs and HPFs were purificated by immunofluorescence staining (previously described in detail by Metelmann et al [25]).…”

“…We introduced a 3D organotypic co-culture model of pleura to imitate the distinct pleural microenvironment. This mesothelial 3D model is derived from human pleura specimens and effectively reproduces the in vivo morphology as well as the microenvironment of human pleura [25]. Thus, it serves as an in vitro assay for pleural research.…”