Abstract
To more closely mimic the native cellular microenvironment, 3D scaffolds derived from the extracellular matrix (ECM) are being developed as alternatives to conventional 2D culture systems. In the present study, we established methods to fabricate nonchemically cross-linked 3D porous foams derived entirely from decellularized porcine left ventricle (DLV) for use as an in vitro cardiac cell culture platform. Furthermore, we explored the effects of physically preprocessing the DLV through mechanical mincing versus cryomilling, as well as varying the ECM concentration on the structure, composition, and physical properties of the foams. Our results indicate that the less highly processed minced foams had a more cohesive and complex network of ECM components, enhanced mechanical properties, and improved stability under simulated culturing conditions. To validate the DLV foams, a proof-of-concept study was conducted to explore the early cardiomyogenic differentiation of pericardial fat adipose-derived stem/stromal cells (pfASCs) on the minced DLV foams relative to purified collagen I gel controls. Differentiation was induced using a modified cardiomyogenic medium (MCM) or through stimulation with 5-azacytidine (5-aza), and cardiomyocyte marker expression was characterized by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction. Our results indicate that early markers of cardiomyogenic differentiation were significantly enhanced on the DLV foams cultured in MCM, suggesting a synergistic effect of the cardiac ECM-derived scaffolds and the culture medium on the induction of pfASC differentiation. Furthermore, in analyzing the response in the noninduced control groups, the foams were observed to provide a mildly inductive microenvironment for pfASC cardiomyogenesis, supporting the rationale for using tissue-specific ECM as a substrate for cardiac cell culture applications.
Highlights
Mammalian cell culture studies for cardiac applications have conventionally been performed in 2D on tissue culture polystyrene (TCPS) coated with fibronectin,[1] laminin,[2,3] or fetal bovine serum (FBS).[4]
Scanning electron microscopy (SEM) analysis revealed that the cryomilled foams contained a more fragmented network compared with the minced foams, which was evident at the lower concentrations that were qualitatively more porous (Fig. 2)
In conclusion, we have successfully established straightforward methods to fabricate 3D porous foams derived entirely from decellularized left ventricular myocardium, which are stable in culture without the need for chemical cross-linking
Summary
Mammalian cell culture studies for cardiac applications have conventionally been performed in 2D on tissue culture polystyrene (TCPS) coated with fibronectin,[1] laminin,[2,3] or fetal bovine serum (FBS).[4].
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