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

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.

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions

“…The amount of CHOL was increased up to about 34 mol% in 3D plasma membrane lipid extract vs. 21 mol% in 2D. Our previous studies revealed a similar trend for SM [18]. All these results suggest that the differences in structural components of 2D and 3D plasma membranes as well as in their ratios could underlie eventual specificities in their phase behavior.…”

“…These in vitro studies are consistent with in vivo conditions in which a deep DPH fluorescent probe was used. The fluidity of the outer and the inner plasma membrane monolayers was lower in 3D (outer 0.2351 ± 0.0025; inner 0.2229 ± 0.0017) than 2D cells (outer 0.2423 ± 0.0019, inner 0.2208 ± 0.0021) [18]. Overall, these data demonstrate a generally lower order of the ESR spin-probe in 3D plasma membrane lipids in the deep hydrophobic core of the bilayer compared to 2D.…”

“…The organization of the lipid molecules in domains is considered as an important factor for protein sorting, dynamically regulating the association of protein targeting and so would support different biochemical functions and regulate whole cell physiology. Indeed, our group was able to establish differences in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and ''matrix'' cell cultures [18]. Thus, the reported differences in the physicochemical properties of plasma membranes in 2D and 3D cells imply that investigations of any membrane-related processes should be cautiously interpreted when using cells cultured as conventional monolayers.…”

Deconstructing Signaling in Three Dimensions

“…54 This membrane reorganization can alter the distribution of lipids and lipid rafts in the membrane. 55,56 These differences can then affect lipid-dependent signaling, such as signaling through the PI3K–Akt–mTOR pathway, which regulates cell metabolism, migration, and apoptosis.…”

Effect of Inulin Intake on the Content and Susceptibility to Oxidative Damage of Cholesterol in Rat Liver Plasma Membranes

“…The fluorescence of NBD-cholesterol was measured at 502 (excitation beam) and 520 (emission beam). Calculation of the amount of NBD-cholesterol accessible to dithionite quenching was performed as described previously [ 7 ].…”