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Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol

Authors

  • Nadezhda Stefanova,

    1. Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 8 Dragan Tzankov Str., Sofia 1164, Bulgaria
    2. Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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  • Galya Staneva,

    1. Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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  • Diana Petkova,

    1. Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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  • Teodora Lupanova,

    1. Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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  • Roumen Pankov,

    1. Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 8 Dragan Tzankov Str., Sofia 1164, Bulgaria
    2. Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
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  • Albena Momchilova

    Corresponding author
    1. Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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Tel.: +359 2 979 2686. E-mail addresses: albena@obzor.bio21.bas.bg

Abstract

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and “matrix” cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.

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