Interrupted slow cooling of mouse cardiac endothelial cell monolayers

Abstract

The increasing evidence of the role of the endothelium in cardiac physiology and the importance of a functional endothelium for vascular grafts necessitate in vitro models for research. Cryopreservation of cardiac endothelial cell monolayers will enable their on-demand availability. Here, we used interrupted slow cooling (graded freezing) to characterize the cryobiological response of a mouse cardiac endothelial cell (MCEC) line in a monolayer format and describe a procedure for its successful cryopreservation. MCEC monolayers on fibronectin-coated coverslips made of Rinzl, whose coefficient of thermal expansion is matched to ice, were equilibrated at −5 °C, ice-nucleated, cooled at 1 °C/min, and at various temperatures during cooling, samples were either directly-thawed or plunged into liquid nitrogen and then thawed. In the absence of cryoprotectants, direct-thaw samples revealed cryoinjury from solute effects, while plunge-thaw samples revealed damage from intracellular ice formation. MCEC monolayers were then loaded with 5 % dimethyl sulfoxide (Me₂SO), 6 % hydroxyethyl starch (HES), and 2 % chondroitin sulfate (CS) prior to interrupted slow cooling. The maximum immediate post-thaw relative viability (93.7 ± 5.5 %) and absolute viability (95.5 ± 21.1 %) were attained after plunge from −40 °C. Both fresh and cryopreserved MCEC monolayers exhibited metabolic activity, angiogenic potential through tube formation on Matrigel, intracellular calcium signaling, and expression of the tight junction protein ZO-1. Viability and functional assessments after post-thaw overnight culture did not show delayed-onset cryoinjury. Thus, the combination of Me₂SO, HES, CS, and Rinzl substrate resulted in viable and functional MCEC monolayers post thaw. This cryopreservation protocol will allow accessibility of cardiac endothelial monolayers for use in studying cardiac physiology, cardiovascular disease modeling, and drug-induced cardiotoxicity testing.