Light microscope image of the two main cell types used and the different cultures.

<p><b>A:</b> Monolayers of NRVM cultured on multi-electrode arrays, <b>B:</b> Porcine ASC in culture, <b>C:</b> Monolayers of NRVM cultured together with microspheres, <b>D:</b> Co-cultures of NRVM and porcine ASC on a multi-electrode array and <b>E:</b> Co-cultures of NRVM and porcine ASC loaded on microspheres. Original magnification 10x. Black dots represent electrode terminals in the multi-electrode arrays. Abbreviations; ASC: adipose tissue-derived stromal cells, MS: microspheres and NRVM: neonatal rat ventricular myocytes.</p

Scanning electron microscope micrographs of the recombinant human collagen-based microspheres loaded with porcine ASC.

<p>Original magnification is indicated underneath the image. Abbreviation: ASC: adipose tissue-derived stromal cells.</p

Schematic illustration of how the recombinant human collagen-based microspheres mitigate the conduction slowing induced indirectly by porcine adipose tissue-derived stromal cells.

<p>(<b>1</b>) pASC secrete (a) paracrine factor(s) that induce NRVM to produce (a) secondary autocrine factor(s) that is responsible for the observed heterogeneous conduction slowing (<b>2</b>). When the microspheres are added to monolayers of NRVM, either bare or loaded with pASC, the secondary autocrine myocardial factor is buffered by the microspheres and the conduction slowing is mitigated (<b>3</b>). Abbreviations; NRVM: neonatal rat ventricular myocytes and pASC: porcine adipose tissue-derived stromal cells.</p

Influence of recombinant human collagen-based microspheres on conduction slowing mediated by a paracrine mechanism.

<p><b>A:</b> Activation map of a monolayer of NRVM co-cultured with pASC (left), and an activation map of a monolayer of NRVM cultured with pASC loaded microspheres (right). Conduction velocity is determined along the white arrows perpendicular to isochronal (black) lines. Lower panels: the effects on <b>B:</b> conduction velocity and <b>C:</b> conduction heterogeneity. * indicates p< 0.001 compared to the NRVM + pASC. Number of replicates per experimental condition is indicated in the bar-graphs. Abbreviations; MS: microspheres, NRVM: neonatal rat ventricular myocytes, and pASC: porcine adipose tissue-derived stromal cells.</p

The relationship between RMP and Vmax.

<p><b>A:</b> The effect of conditioned medium and pASC loaded microspheres on the RMP. <b>B:</b> The relationship between RMP and Vmax in the different cultures * indicates p≤ 0.05. Number of cells analyzed is indicated in the bar-graphs. Abbreviations; Cme: conditioned medium, MS: microspheres, NRVM: neonatal rat ventricular myocytes, pASC: porcine adipose tissue-derived stromal cells, and RMP: resting membrane potential.</p

Schematic overview of the hypothesis that recombinant human collagen-based microspheres mitigate the conduction slowing induced by pASC.

<p>From previous work we know that rASC and hASC induce conduction slowing through direct-electrotonic-coupling (<b>1</b>) and that pASC paracrine factors alone can induce conduction slowing in NRVM monolayers (<b>2</b>). We hypothesize that the microspheres will buffer the pASC paracrine factors, and mitigate the conduction slowing (<b>3</b>). The mitigating effect can also be achieved when the pASC are loaded onto the microspheres (<b>4</b>). When rASC or hASC are loaded onto the microspheres conduction slowing will still occur as these cells induce conduction slowing through direct-electrotonic-coupling(<b>5</b>). Abbreviations: hASC: human adipose tissue-derived stromal cells, NRVM; neonatal rat ventricular myocytes, pASC: porcine adipose tissue-derived stromal cells, and rASC: rat adipose tissue-derived stromal cells.</p

Effects of conditioned medium.

<p>The effects on <b>A:</b> conduction velocity and <b>B:</b> conduction heterogeneity of monolayers of NRVM cultured in various conditioned mediums. * indicates p≤ 0.05. Number of replicates per experimental condition is indicated in the bar-graphs. Abbreviations; Cme: conditioned medium, MS: microspheres, NRVM: neonatal rat ventricular myocytes and pASC: porcine adipose tissue-derived stromal cells. See text for details.</p

Influence of recombinant human collagen-based microspheres on conduction slowing mediated by a contact based mechanism.

<p>Activation maps of <b>A:</b> monolayers of NRVM cultured with rASC and rASC loaded microspheres, respectively. The effect on conduction velocity (<b>B</b>) and conduction heterogeneity (<b>C</b>) are determined along the white arrows perpendicular to isochronal (black) lines. Activation maps of <b>D</b>: monolayers of NRVM cultured with hASC and hASC loaded microspheres, respectively. The effect on conduction velocity (<b>E</b>) and conduction heterogeneity (<b>F</b>) are determined along the white arrows perpendicular to isochronal (black) lines.* indicates p< 0.001 compared to the monolayers of NRVM. Number of replicates per experimental condition is indicated in the bar-graphs. Abbreviations; hASC: human adipose tissue-derived stromal cells, MS: microspheres, NRVM: neonatal rat ventricular myocytes, and rASC: rat adipose tissue-derived stromal cells.</p

Effects of recombinant human collagen-based microspheres on NRVM monolayers.

<p><b>A:</b> An activation map of a monolayer of NRVM (left) and a monolayer of NRVM cultured with microspheres (right). Conduction velocities are determined along the white arrows perpendicular to isochronal (black) lines. The effects of culturing bare microspheres on monolayers of NRVM on <b>B:</b> conduction velocity and <b>C:</b> conduction heterogeneity. Number of replicates per experimental condition is indicated in the bar-graphs. Abbreviations; MS: microspheres and NRVM: neonatal rat ventricular myocytes.</p

Immunofluorescence micrographs of cultures stained with N-cadherin and Cx43.

<p><b>A:</b> A monolayer of NRVM, a monolayer of NRVM cultured in pASC conditioned medium and a monolayer of NRVM co-cultured with pASC loaded microspheres are stained for N-Cadherin and Cx43. <b>B:</b>The ratio of Cx43: N-Cadherin in the various cultures, determined by the number of pixels. Ratios are based on 5–10 images taken in each of three independent cultures. * indicates p< 0.05. Abbreviations; Cme: conditioned medium, Cx43: connexin 43, MS: microspheres, NRVM: neonatal rat ventricular myocytes and pASC: porcine adipose tissue-derived stromal cells.</p