The Scar-in-a-Jar: Studying Antifibrotic Lead Compounds from the Epigenetic to the Extracellular Level in One Well

Abstract

Fibrosis represents an excessive accumulation of collagen in tissues. The resultant consequences range from cosmetic disfiguration, impairment of implants to organ failure. The development of effective anti-fibrotics is therefore an important clinical goal. Unfortunately, screening for antifibrotics has been limited by incomplete procollagen processing and poor matrix formation in vitro and the inability to normalise the amount of deposited collagen for the cell number in the same culture well. The "scar-in-a-jar" concept is based on advanced cell culture technology making use of macromolecular crowding. This system ensures that the complete biosynthetic and depositional cascade of collagen matrix formation is in place. We have developed a rapid (2 days) and an accelerated matrix formation protocol (6 days) with the latter allowing for crosslinking of the deposited matrix in vitro. Automated adherent cytometry and in situ quantitation of immunofluorescence intensity of extra- and intracellular antigens is combined with automated image acquisition and semi-automated image processing, This discovery tool allows for a novel in situ assessment of the area of deposited collagen(s) normalised to cell number thereby simultaneously monitoring cell proliferation/cytotoxicity. In addition, multiplexing enables quantitation to include noncollagenous ECM proteins. Bioimaging data obtained with known and novel antifibrotic compounds at the epigenetic (histone deacetylase), post-transcriptional (miRNA), posttranslational (Prolyl-4-hydroxylase) and post-secretional level (C-proteinase, lysysl oxidase, matrix metalloproteinase-1) correlated excellently with biochemical analyses.