Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Abstract

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

Fructooligosaccharide Intake Alters the Phospholipid and Fatty Acid Composition of Liver Plasma Membranes

“…our previous studies demonstrated the protective role of SM against oxidative damage of membrane phospholipids and cholesterol (17). Since the content of SM was higher in membranes of prebiotic-fed rats, we measured the level of membrane cholesterol, which could be readily oxidized by exposure to cholesterol oxidase.…”

Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell culture models

“…For example, it has been shown that antibodies against B1-integrins exhibit different behaviour when tested in 2D compared to 3D models (Wang et al, 1998); induced doxorubicin-resistance by the extra cellular matrix in human osteosarcoma and HT1080 cells has been demonstrated (Fourre et al, 2008;Harisi et al, 2007); reduced radiation induced toxicity when cells are grown in a 3D environment has also been reported (Sowa et al, 2010). The use of collagen matrices as 3-D cell culture matrices has greatly increased in the last few years and numerous other publications can be found in the literature describing modification of the cell phenotype, metabolism or composition when grown in a 3D matrix, compared to 2-D environments (Lupanova et al, 2010;Wu et al, 2009). …”

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

“…Reduced radiation induced toxicity when cells are grown in a 3D environment has also been recently demonstrated 11 . Numerous other publications can be found in the literature describing modification of the cell phenotype, metabolism or composition when grown in a 3D matrix, compared to 2-D environments 12,13 .…”