P511Immunomagnetic purification of cardiomyocytes, cardiac fibroblasts and endothelial cells from neonatal mouse and rat hearts

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Abstract

Pure cardiovascular cell lineages are a requisite for an unbiased and cell type specific investigation of (patho-) physiological mechanisms involved in heart failure. Moreover pure heart cells are needed as building blocks for engineered heart mimetics. The latter offers the possibility to replace infarcted myocardium and to investigate heart development and cardiomyogenesis in a simplified in vitro model. However, it has to be acknowledged that strategies capable to specifically purify heart cells are not well established. To overcome these experimental limitations we developed a rapid and automated neonatal heart dissociation protocol enabling an immunomagnetic enrichment of the three major heart cell types, namely cardiomyocytes (CMs), cardiac fibroblasts (Fibs) and endothelial cells (Endos) from both, mouse and rat neonatal hearts.

First, we established an enzyme mix optimal for the dissociation of neonatal mouse and rat hearts (P0-P3) by screening our enzyme library. Next, we optimized the dissociation by utilizing our gentleMACS technology resulting in a fast (1h), robust, and fully automated heart dissociation protocol. Analysis of the dissociated mouse and rat heart cells showed: (i) high cell vitalities (>90%), (ii) high frequencies of α-Actinin-positive CMs (>60%) and (iii) vimentin-positive non-CMs with a frequency of >40% (P2). In order to selectively enrich various cell-types from these heterogenous cell populations, we performed a cell-surface marker screen. We identified several candidates for the composition of antibody cocktails enabling selective enrichment of mouse and rat CMs, Fibs or Endos with purities of up to 97%. CMs grown in 2D cultures showed spontaneous beating activity and the expression of sarcomeric proteins in a cross-striated pattern (α-Actinin and cTnT). Additionally, heart cells were cultured on organotypic decellularized (1% SDS) mouse heart slices. Analysis of the slice cultures after recellularization showed (i) partial restoration of synchronous contractions and (ii) orientated and elongated heart cells along the microfibrillar heart structure. Purified Fibs were highly proliferative and could be expanded 34-fold over an analyzed culture period of 15 days. Finally, functionality of the purified endothelial cells was proven by Dil-LDL endocytosis.

In summary, we established an automated protocol for the dissociation of neonatal mouse and rat hearts, enabling the subsequent immunomagnetic enrichment of CMs, Fibs and Endos which can readily be utilized for 2D cell culture assays or to generate in vitro heart muscle models and surrogate tissue for myocardial repair.

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