Page 117 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

ENDOTHELIAL FUNCTION AFTER EXPOSITION OF MAGNESIUM DEGRADATION PRODUCTS
used.
2.3.3 Mitochondrial activity of cells after exposition of materials extracts
Indirect test by using material extracts of the samples were used to evaluate the mitochondrial activity of HUVEC and SMC. For this, monolayer of cells were exposed to serial dilutions (8) of the extracts for 48h. MTT assay was performed according the previous protocol described.
2.4 Effect of Mg on endothelial cells under flow
As a flow chamber, μ-Slide I 0.4 Luer (Ibidi, Planegg/Martinsried, Germany) was used. Slides were pre-coated with 1% of gelatin and HUVEC were seeded and incubated overnight. A spring of Mg with and without coating of about 4mg of weight and about 5.8mm2 of surface area were independently placed in the inlet tube of the system and the slides were attached to a fluidic unit, connected to the pump. Parameters were programed by using the ibidi Pump System (Ibidi, Planegg/Martinsried, Germany). Fluid shear stress of 20 dyne/cm2 was applied to the cells. Units and slides were placed in an incubator of 37OC at 5%CO2. Every 24 h and for 7 days, 1.5mL was removed and replaced for fresh medium. Changes in pH was measured and pictures were taken daily.
2.5 Functional assays
2.5.1 Sprouting assay
HUVEC at a concentration of 15.000 cells/well were cultured in μ-Slide Angiogenesis (Ibidi, Martinsried, Germany) containing 10μl of MatriGel (Becton Dickinson, Bedford, MA, USA) and cultured in ECM, overnight. Formation of sprouts was analyzed by conventional light microscopic analysis after 6 hours.
2.5.2 Paracellular permeability
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Trans-endothelial electric resistance (TEER) was measured by using ECIS, (Applied BioPhysics, NY, USA). HUVEC were grown in an array holder 8W10E (Ibidi, Martinsried, Germany) previously cleaned with L-cysteine (C7352-25, Sigma- Aldrich, St. Louis, Missouri, USA) for 15 min and coated with 1% gelatin for 30 min. The electrical impedance of the monolayer was measured at 1 V, 2000 Hz. When cells reached confluence and the resistance curve was constant, me- dium was removed and replaced by Mg extracts. The behavior of the monolayer was registered for 48h. Resistance was obtained and experimental conditions were compared after normalization of the data to the baseline resistance.
2.5.3 Monolayer integrity for HUVEC to Mg extracts
HUVEC were grown until confluence and culture medium was replaced for the Mg extracts. Cell behavior was re- corded every 10 min for 48h by a Solamere Nipkow confocal live cell imaging (Solamere, USA) system mounted to a Leica DM IRE2 inverted microscope at a magnification of 10X.
2.6 Vasomoter function
Extracts of the Mg materials were obtained in Krebs buffer (pH 7,4; 120 mmol/L NaCl, 5.9 mmol/L KCl, 25.2 mmol/L NaHCO3, 1.2 mmol/L NaH2PO4, 10.4 mmol/L glucose, 1.21 mmol/L MgCl2·6H2O, and 2.52 mmol/L CaCl2) as detailed above. Right coronary arteries (RCA) were obtained from two adults and healthy pigs and cut in multiple and compa- rable vascular rings (~2mm thick). Vasoreactivity was evaluated by the measurement of the endothelium-dependent relaxation to bradykinin (BK). For this, rings were randomly exposed to c.p Mg, HMT and MAN extracts and kept at 37oC, under constant agitation during 2h. As control, RCA in standard Krebs solution was used. Rings were mounted in organ baths containing Krebs solution at 37°C and gassed with 95% CO2 and 5% O2). The rings were equilibrated for 30 min and vasoreactivity was assessed by the addition of KCl (60mM). Subsequently, the rings were washed with fresh Krebs solution and maximally constricted with U46619. The vasorelaxation response against cumulative
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