The overall goal of this explant culture is to facilitate the culture of embryonic epicardial cells to facilitate exploration of the role of specific genes in epicardial cell biology. This method can help answer key questions in the cardiac field such as how do epicardial cells contribute to cardiovascular development, disease, and regeneration? The main advantage of this technique is that it enables the long-term culture of a highly pure population of epicardial cells.
Generally, individuals new to this method will struggle because isolating the ventricles takes the kind of precision that comes only with practice. To harvest the embryonic ventricles, begin by placing a timed pregnant mouse at embryonic day 12.5 in the supine position on a dissecting table, and disinfect the abdomen with 70%ethanol. Lifting the skin over the belly, make a one to two centimeter incision along the midline.
Then, pull both sides of the incision to reveal the abdominal wall, and cut the wall to expose the uterine horn. Next, use sterile forceps to lift up the horn and carefully cut away the fat tissue and blood vessels attached to the horn. Make a final cut at the cervix to retrieve the uterus, and place the uterine horn in a petri dish containing cold, sterile PBS.
Rinse the tissue gently, and place the petri dish on ice. Using scissors, cut through the midline of the uterine horn on the side opposite to the placenta to expose the embryos. Then, use sterile forceps to cut open the embryonic yolk sac to free the embryos, placing each embryo into a new petri dish containing cold, sterile PBS as it is harvested.
After decapitation, for each embryo in turn, cut open the chest wall to expose the heart. Using sterile forceps, lift the heart and cut away the vessels attaching the heart to the chest well. Then, trim the outflow tract and both atria from each heart, and place the ventricles in a new dish containing cold PBS on ice.
To culture the epicardial explants on glass chamber slides, add 500 microliters of epicardial explant culture medium to each well of an eight well chamber slide and seed one excised ventricle dissected side down into the center of each well. Then, gently place the plate in a cell culture incubator for 48 to 72 hours to allow the explants to adhere to the chamber slide surfaces. For differentiation of the epicardial cells into smooth muscle cells, culture the epicardial monolayer in differentiation medium for another six days.
To culture the epicardial cells on a collagen gel, first mix the appropriate volume of collagen solution with five x DMEM by pipetting. The solution will turn yellow. Next, add a corresponding volume of neutralization solution with mixing.
The solution should turn pink. Now, transfer 100 microliters of the collagen solution into each well of a 96 well plate and place the plate into the cell culture incubator to allow the gel to polymerize. After 30 to 60 minutes, add 200 microliters of epicardial explant culture medium into each well and transfer an excised ventricle, dissected side down, into the center of each well.
Gently place the plate into the cell culture incubator for three days. On the third day, remove the ventricles and return the plate to the incubator for two more days. After 48 to 72 hours of culture, the superimposition of red fluorescent protein immunofluorescents and bright field images demonstrates that a majority of the cells that migrate from the ventricles are of epicardial origin.
qPCR analysis of RNA isolated from these primary epicardial cell cultures also reveals a robust expression of epicardial-specific genes, with very little cardiomyocyte marker gene expression. Here, the localization of ZO-1 to the plasma membrane indicates that the cells have yet to undergo epithelial to mesenchymal transition. Further, immunostaining for alpha Tubulin demonstrates a polarized microtubule alignment of the epicardial cells, facilitating the directional migration of the cells during their transition.
After six days of culture and differentiation medium, the presence of smooth muscle actin evinces the successful differentiation of the epicardial cells into smooth muscle cells. Indeed, phalloidin immunostaining of a collagen gel invasion assay reveals the ability of epicardium-derived cells to migrate. To determine the depth of cell penetration into the collagen matrix, a 3D reconstruction can be generated from the confocal images for a z-stack visualization of the epicardium-derived cell penetration.
While harvesting the ventricles, it's crucial to avoid damaging the epicardium. Following this procedure, other methods to assess the differentiation, migration, and cell surface protein expression of the epicardial cells can be performed to answer additional questions about their function. This method can help researchers in the cardiac field to explore epicardial biology using mouse models.
