Abstract
Background
The epicardium, the most external layer of the heart, is composed of a layer of epithelial cells and underlying connective tissue. Following myocardial infarction, epicardial cells are activated and provide a source of paracrine factors and progenitor cells. In the border zone of the ischaemic tissue, the activated epicardial cells support cardiac and vascular regeneration by releasing pro-angiogenic and pro-survival factors, and by differentiating towards multiple cell lineages. During this process, activated epicardial cells migrate to the site of injury where they contribute to both post-ischemic remodelling and fibrosis. There is limited knowledge of the cellular and molecular regulation of these processes in large animals and humans, in part due to the lack of robust and representative models.
Purpose
In this project, we developed an ex vivo 3D organotypic model derived from porcine hearts, amenable to culture, which enables structural, molecular and cellular studies of the epicardium.
Methods
Thin epicardial/cardiac tissue slices (EpCardio-TS) were obtained by using a vibratome to cut the first layer of tissue from the epicardial side of porcine heart cubes. Slices were cultured for up to 72h in a bioreactor that uses a 3D printed chamber connected to a control system that allows maintenance and adjustment of culture conditions, and ensures continuous media flow. Local intracellular delivery of fluorescent quantum-dots (Qdots) was performed using nanoneedle chips to track epicardial cells, whilst cell fate is visualised in 3D by performing immunofluorescence on decolourised slices.
Results
Intact EpCardio-TS obtained from porcine heart included a viable epicardium, expressing typical epicardial markers (wt-1, mesothelin, uroplakin), and an electrically active myocardium. Live/dead staining showed epicardial (67.8±16.2%, N=5) and myocardial (40.8±28.6%, N=3) viability, and TUNEL assay confirmed low levels of apoptosis (6.3±5.1% of wt-1+ epicardial cells N=1). Moreover, the presence of proliferating epicardial cells (PCNA+), the increase in wt-1+ cells, and the increase in epicardial gene expression (Tbx18 and TCF21) suggested that cells maintain their progenitor phenotype and undergo activation in culture. Nanoinjection of fluorescent Qdots to EpCardio-TS localized them to the wt-1+ cells on the slice surface, presenting a strategy to mark the epicardial layer. This, combined with the successful decolourisation of the slices, provides an in vitro platform to track the role of epicardial cells in cardiac remodelling and fibrosis.
Conclusions
EpCardio-TS represents a robust ex vivo model merging the complexity of a 3D organotypic culture with the simplicity of the in vitro culture. EpCardio-TS are amenable to culture and cell tracking, and can therefore find application in toxicology and gene therapy screening for the modulation of epicardial interactions with myocardial and non-myocardial cells of the heart.
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