Basic structure and cytocompatibility of giant membrane vesicles derived from paraformaldehyde-exposed human cells.

Exposure of cultured mammalian cells to paraformaldehyde (PFA) is an effective approach to induce membrane blebs, which is followed by their detachment from the cellular cortex to yield giant membrane vesicles in extracellular spaces. Although PFA-induced giant vesicles have attracted significant interest in the field of cell membrane dynamics, their biochemical components and cytocompatibility remain largely unknown. In this report, we exposed human cervical cancer HeLa cells to PFA under metal-free buffer conditions to produce giant vesicles. We analyzed the components and structure of the purified PFA-induced giant vesicles. Co-culturing PFA-induced giant vesicles with exponentially growing HeLa cells resulted in docking of a significant number of the giant vesicles to the cell surface with seemingly no cytotoxicity. Intriguingly, we found that pre-treatment of HeLa cells with peptide-N-glycosidase and neuraminidase was effective in facilitating cellular uptake of constituents residing inside the vesicles. The results revealed further details about the effect of PFA on cell membranes and provide insights for studying the interaction between PFA-induced giant vesicles and cultured cells.

Chemical manipulations to facilitate membrane blebbing and vesicle shedding on the cellular cortex.

OBJECTIVES: Most attention has been focused on physiologically generated membrane blebs on the cellular cortex, whereas artificial membrane blebs induced by chemicals are studied to a lesser extent. RESULTS: We found that exposure of HeLa human cervical cancer cells to paraformaldehyde (PFA), followed by incubation in phosphate-buffered saline (PBS) efficiently induced large membrane blebs on the cellular cortex. Intriguingly, sequential exposure of the PFA-treated cells to PBS containing dimethyl sulfoxide (DMSO) facilitated shedding of the blebs from the cellular cortex, yielding a high quantity of large extracellular vesicles in the supernatant, which was applicable to assess the potentials of compounds and proteins as membrane influencers. Similar effects of PFA and DMSO were detected on the cellular cortex of other human, mouse, and fish cells. CONCLUSIONS: Our procedure to facilitate membrane blebbing and vesicle shedding by chemicals may be practical for the manipulation of membrane dynamics and the development of vesicle-inspired technologies using a wide variety of cell types.

Wash-free instant detection of giant plasma membrane vesicles.

Giant plasma membrane vesicles (GPMVs) are large extracellular vesicles produced by the exposure of cells to paraformaldehyde or other stresses, providing an experimental system to elucidate cell surface dynamics. Here we show that addition of the membrane permeable fluorescent RNA-indicators, acridine orange and thioflavin T, to GPMV-containing solutions prepared from cultured HeLa cells was sufficient for the fluorescent visualization of seemingly all GPMVs. Our findings provide a wash-free instant method using non-lipid-type fluorescent dyes for GPMV detection, which should be useful for researchers interested in studying cell membrane dynamics and biochemistry using GPMVs.