Abstract 5: Novel Tools for Identification and Purification of Cardiomyocytes, Fibroblasts and Endothelial Cells From Neonatal Mouse and Rat Hearts

Abstract

Although isolation of vital cardiac cells from neonatal hearts is one of the most used experimental model in cardiac research, the manual dissociation of neonatal hearts is a laborious and difficult-to-standardize procedure. To shorten sample processing time and increase reproducibility, we developed a fully automated dissociation process followed by magnetic purification of cardiomyocytes (CMs), cardiac fibroblasts (CFs) and endothelial cells (ECs). Neonatal hearts were dissociated within 1 h resulting in high yields of single cells with excellent viability. Depending on age and species following cell ratios were recovered after dissociation: 50-70% CMs, 30-40% CFs, 10-20% ECs. In order to purify CMs from dissociated neonatal hearts, we defined antibody cocktails enabling non-myocyte removal. Our depletion strategy allowed for the enrichment of CMs from whole hearts or preparations of heart chambers with purities of up to 98%. To enable simultaneous detection of CMs and subtypes, we developed recombinant antibodies against general CM markers like alpha Actinin, Myosin Heavy Chain or cardiac Troponin T as well as CM subtype-specific antibodies against MLC2a and MLC2v distinguishing between atrial and ventricular CMs respectively. Besides, we developed magnetic enrichment strategies for the purification of CFs and ECs. Current purification strategies that are solely based on antibodies against EC surface markers do not result in sufficiently pure cardiac ECs. Therefore, we established a 2-step enrichment protocol allowing for the complete removal of contaminating fibroblasts and leukocytes. Our data indicate that this protocol significantly improves the purity of primary cardiac ECs. Functionality of ECs before and after enrichment was proven by Dil-Ac-LDL endocytosis. Similarly, our magnetic enrichment protocol for cardiac fibroblasts revealed homogenous expression of fibroblast markers like Vimentin and Prolyl-Hydroxylase, but virtually no contaminations with CMs or ECs. In summary, we established an automated protocol for dissociation of neonatal hearts enabling subsequent purification, characterization and cultivation of CMs, CFs and ECs which can readily be used for cell culture assays or to generate heart muscle models.