Widhe, M. | Search bioRxiv

bioRxiv2024

A Fibronectin (FN)-Silk 3D Cell Culture Model as a Screening Tool for Repurposed Antifibrotic Drug Candidates for Endometriosis

This study advances sustainable pharmaceutical research for endometriosis by aligning with the UN Sustainable Development Goals on health, gender equality, and responsible consumption in developing in vitro 3D cell culture models of endometriotic pathophysiology. Fibrosis is a key aspect of endometriosis, yet current models to study it remain limited, especially in 3D. This work aims to bridge the translational gap between in vitro fibrosis research and preclinical testing of non-hormonal drug candidates. When grown in a 3D matrix of sustainably produced silk protein functionalized with a fibronectin-derived cell adhesion motif (FN-silk), endometrial stromal and epithelial cells respond to transforming growth factor beta-1 (TGF-{beta}1) in a physiological manner as probed at the mRNA level. For stromal cells, this response to TGF-{beta}1 is not observed in spheroids, while epithelial cell spheroids behave similarly to epithelial cell FN-silk networks. Pirfenidone, an antifibrotic drug approved for the treatment of idiopathic pulmonary fibrosis, reverses TGF-{beta}1-induced upregulation of mRNA transcripts involved in fibroblast-to-myofibroblast transdifferentiation of endometrial stromal cells in FN-silk networks, supporting the drugs potential as a repurposed non-hormonal therapy for endometriosis. This study demonstrates how a sustainable approach - from project conceptualization to material selection - can be integrated into pharmaceutical research for womens health. Table of contentsThis paper presents in vitro 3D cell culture models of fibrosis in endometriosis. Endometrial stromal and epithelial cells cultured in networks of silk protein functionalized with a fibronectin-derived cell adhesion motif showed physiological-like fibrotic behavior. Pirfenidone was able to reverse fibrosis of endometrial stromal cells in vitro, demonstrating this models suitability as a screening tool for antifibrotic drugs for endometriosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/616776v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12727c3org.highwire.dtl.DTLVardef@1a39292org.highwire.dtl.DTLVardef@1ebdb2aorg.highwire.dtl.DTLVardef@181d6ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2018

Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) fibrillar network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into fibrillar networks under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed fibrils. In the resulting 3D scaffold, the cells proliferate and spread out with tissue-like morphology. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk fibers with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables both differentiation of stem- or precursor cells in 3D and facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a fibrillary ECM-like network, with potential as base for engineering of functional tissue.

bioengineering↗