Reseeding Decellularized Porcine Ventricular Extracellular Matrix with Isolated Primary Human Atrial Cells

As a result of pioneering research unveiling the influence of extracellular environment in determining stem/progenitor cell differentiation, a myriad of cellular scaffolds have been developed in order to optimize the delivery of these cells in a diseased environment as a technique to improve their efficacy in stem cell therapy. Although a variety of scaffolds are being studied in different contexts, the most physiologically relevant ones are those that most closely resemble the environment to be treated. Our lab has a keen interest in cardiovascular disease and has recently optimized a technique that decellularizes and reseeds isolated sections of healthy porcine ventricular tissue with primary human atrial cells. Decellularization involved a multi‐step, multi‐reagent washing process over a period of four days, after which confirmation of a lack of cells was determined by DAPI staining and subsequent western blotting looking for the absence of β‐tubulin. After decellularization, our objective was to successfully seed the decellularized tissue slices via co‐incubation of isolated primary human atrial fibroblasts (hAFs) or human c‐kit (+) cardiac progenitor cells (hCCs). These two cell types were chosen to compare how hAFs and hCCs respond differently to healthy extracellular matrix (ECM). Cells were isolated from the atrial appendage of patients undergoing open heart surgery by mincing followed by collagenase digestion and subsequent isolation of hCCs was performed using a c‐kit (+) magnetic bead isolation procedure. HAFs and hCCs were incubated separately on sections of decellularized porcine ventricular ECM over a period of 24 hours and then visualized using immunofluoresence. Collagen‐1, fibronectin, and versican antibodies were used to image the ECM structure and DAPI was used to identify cells embedded in the recellularized matrix. Successful seeding of cells was also evaluated by western blot detection of β‐tubulin in samples not prepared for immunofluorescence. In conclusion, our technique was successful in reseeding decellularized porcine ventricular extracellular matrix and can be extrapolated to detect other proteins in these tissues without having to reseed an entire decellularized heart. This can be used to compare differences among cell types and their responses between healthy and damaged or diseased cardiac ECM among different hearts or even within the same heart. Support or Funding Information Mazankowski Alberta Heart Institute University of Alberta Hospital Foundation Alberta Innovates Health Solutions