Evaluation of Smooth Muscle Cell Function in the Rat Mesentery Culture Model
Authors: Motherwell Jessica , Azimi Mohammad , Spicer Kristine , Alves Natascha , Breslin Jerome , Katakam Prasad , Murfee Walter 
Published: November 2023

An emerging challenge in tissue engineering biomimetic models is recapitulating the physiological complexity associated with real tissues. Recently, our laboratory introduced the rat mesentery culture model as an ex vivo experimental platform for investigating the multi‐cellular dynamics involved in angiogenesis within an intact microvascular network using time‐lapse imaging. A critical question remains whether the vascular smooth muscle cells maintain their functionality. The objective of this study was to determine if vascular smooth muscle cells along arterioles in cultured microvascular networks maintain the ability to constrict. Adult Wistar rat mesenteric tissues were harvested, rinsed in sterile DPBS and media, then placed underneath a commercially available polycarbonate membrane insert. Tissues were cultured up to 3 days with either minimum essential media (MEM) or MEM supplemented with 10% serum. Tissues were maintained at 37°C and 7.4 pH while exposed to topically administered vasoconstrictors (50mM KCl or 20nM Endothelin‐1 (ET‐1)) for 5 minutes; arterioles were observed. After 3 days in culture, tissues were viable and arterioles maintained their ability to constrict in response to both KCl and ET‐1. The percent arteriolar constriction in ex vivo tissues cultured in 10% supplemented media was decreased at 3 days for both constrictors compared to the responses in pre‐culture ex vivo control tissues. Day 3 serum stimulated microvascular networks were angiogenic characterized by increased capillary sprouting and vessel density. The mechanism of action for the receptor‐mediated vasoconstriction induced by ET‐1 was confirmed using antagonists BQ‐123 and BQ‐788. Day 0 ex vivo tissues were incubated with BQ‐123 (3μM) and BQ‐788 (3μM) for 30 minutes prior to exposure to 20nM ET‐1; control tissues were incubated without the antagonists. The combined presence of BQ‐123 and BQ‐788 resulted in significantly reduced (p<0.05) arteriole constriction (23 ± 5.4%; n=12) compared to control tissues (41 ± 4.1%; n=9). Interestingly, the arteriole constriction response to KCl in day 3 tissues cultured in media without serum was not significantly different than the uncultured group suggesting that angiogenesis versus culture conditions might influence smooth muscle cell function. The functionality in cultured tissues and association of impaired vasoconstriction during angiogenesis were confirmed with intravital microcopy experiments. No significant difference was observed for arteriole constriction response to 50mM KCl between the freshly harvested ex vivo Day 0 tissues (33 ± 3.1%; n=10) and the unstimulated Day 0 tissues evaluated intravitally (35 ± 4.3%; n=13). Intravital comparison of unstimulated and tissues stimulated by an in vivo wound healing stimulus also confirmed impaired vasoconstriction in angiogenic networks. This study demonstrates functional constriction of vessels within intact microvascular networks ex vivo and supports the novelty of the rat mesentery culture model as a biomimetic tool for microvascular research.Support or Funding InformationNIH R01AG049821, NIH P20GM103629