Affiliations: Laboratory of Genetics and Molecular Cardiology, Heart Institute, School of Medicine of São Paulo University, São Paulo, Brazil | Laboratory of Microrheology and Molecular Physiology, Instituto de Física da Universidade de São Paulo, São Paulo, Brazil | Program in Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA | Interventional Radiology, Vascular and Endovascular Surgery-Diagnostic Imaging Sector, School of Medicine of São Paulo University, São Paulo, Brazil
Note: [] Co-senior authors.
Note: [] Address for correspondence: Dr. Alexandre da Costa Pereira, Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo, Avenida Doutor Eneas de Carvalho Aguiar, 44, 10th floor (Building II), Cerqueira César, São Paulo 05403-000, SP, Brazil. Tel.: +55 11 3069 5579; E-mail: [email protected]
Abstract: One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.
