Spatial Distance Affects Hypoxia/Reoxygenation Injury in a Co‐Culture of Separate Endothelial Cell and Cardiomyocyte Layers

Abstract

Heart disease is the leading cause of death and disability worldwide, with significant burden attributed to myocardial ischemia. Reperfusion has been shown to cause additional injury beyond the ischemic insult itself. There is still no effective remedy for reperfusion injury. Endothelial cells (ECs) have been suggested to protect cardiomyocytes (CMs) from reperfusion injury through cell-to-cell interactions, such as paracrine signaling. Co-culture models of ECs and CMs have been used to investigate the role of cell-cell interactions, autocrine and/or paracrine, in cell functions and differentiation. Among these models, mixed co-culture is the simplest to perform: two different cell types are in direct contact within a single culture compartment at the desired cell ratio. However this model is limited as isolated treatments and downstream analysis of single cell types are not feasible. Previous studies in ECs and CMs measuring release of lactate dehydrogenase indicated that hypoxia caused significant damage to the integrity of cell membranes, which importantly was increased upon reoxygenation. To investigate if ECs can attenuate cell membrane leakage of CMs and if the protective effect of ECs can be further optimized by varying the contact distance between the two cell lines, we tested three types of cell culture inserts: Falcon Corning, Millicell Milipore, and Greiner Bio-One. The inserts varied in their inter-cell layer distance at 0.5, 1, and 2 mm, respectively. We found that, when ECs and CMs were placed in nearly direct contact conditions (0.5 mm), there was a slight abrogation of the reoxygenation injury in co-cultured medium. This abrogation was significantly increased when the spatial distance was increased to 1 mm. Most interestingly, when distance was further increased to 2 mm, we observed a significant increase of LDH release during hypoxia as well as a further increase upon reoxygenation. This observation indicates that a sufficient culture spatial area is necessary for ECs to crosstalk with CMs, so that the secreted signal molecules can circulate and fully stimulate the protective pathways. Our study suggests for the first time that optimizing the ECs and CMs co-culture spatial environment is necessary to provide a promising in-vitro model of testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury.