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The therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date.


To explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs.


We generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids.


We discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 μm, smaller than that on PVA substrates (approximately 27 μm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids.


Changes in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

Key Words: Cell membrane fluidity, Cell-cell interaction, Cellular spheroids, Chitosan, Hyaluronan


Core Tip: The fluidity of the cell membrane is an important physical property for the regulation of cell behavior. To date, the changes in cell membrane fluidity are not fully understood in spheroid-forming cells. In this study, various substrates were utilized to grow mesenchymal stem cells and to form homotypic or heterotypic cellular spheroids on the substrates. During the spheroid-forming process, vesicle-like bubbles were randomly generated on the cell membrane. The evidence suggests that the physiological alterations of cells after they form cellular spheroids may be associated with changes in membrane fluidity during the spheroid-forming process.

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