Tupitsyna AV et al. (2023), Isolation of Extracellular Vesicles of Holothur...

ECB-ART-51686

Int J Mol Sci 2023 Aug 17;2416:. doi: 10.3390/ijms241612907.

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Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix).

Tupitsyna AV , Grigorieva AE , Soboleva SE , Maltseva NA , Sedykh SE , Poletaeva J , Dmitrenok PS , Ryabchikova EI , Nevinsky GA .

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Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

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	Figure 1. Typical profile of gel filtration of supernatant obtained using whole holothurian body extracts previously partially purified by several different centrifugations (sample I); (__), absorbance at 280 nm (mA280). The first peak in terms of the elution volume (mL) corresponds to those in the case of “typical” mammalian exosomes with molecular weights 1000 ± 100 kDa [15,28,29,30,31].
	Figure 2. Representative images of structural components in the preparation obtained by centrifugation and ultracentrifugation of holothurian whole-body extract (sample I). (A)—general view of the preparation, white arrows show electron-dense spherical particles, black arrows—particles of irregular shape; (B)—ribbon-like structures (arrows); (C)—membrane-like fragments (arrows); (D)—“non-vesicle”, note small “grains” and “chains” around; (E)—vesicle (<100 nm). Asterisks show structureless impurities. TEM, negative staining with uranyl acetate. The length of the scale bars corresponds to 100 nm.
	Figure 3. Representative images of structural components in preparation (sample II) obtained by gel filtration of sample I. (A)—general view of the preparation, white arrows show ribbon-like structures; black arrows—various membrane profiles; (B)—irregularly shaped membrane-bordered structures (arrows) filled with fine granular material; (C)—rounded vesicle with uneven edges; (D)—spherical non-vesicles (black arrows) and elongated membrane particles (white arrows). TEM, negative staining with uranyl acetate. The length of the scale bars corresponds to 100 nm.
	Figure 4. Representative images of holothurian cells. Coelomic epithelial cells covering: (A,B)—respiratory tree, insert shows formation of coated vesicle; (C–E)—gonad; (F)—cell of respiratory epithelium. (G)—myoepithelial cell; (H)—smooth muscle cell. Late endosomes containing intraluminal vesicles are shown by asterisks (A,B) and circles (C–H). Note fusion of late endosome with plasma membrane evidencing for “a birth of exosomes” in (F), arrowheads show sites of contact of two membranes. 1—nucleus; 2—lipid droplet; arrows show apical plasmalemma; short arrows—tight junctions; thick arrow shows cross-section of cilia in scalloped membrane sack; broken arrows show myofibrils. TEM, ultrathin sections. The length of the scale bars corresponds to 500 nm.
	Figure 5. Typical profile of gel filtration of supernatant obtained using mixture of intestines, gonads, and lungs (previously partially purified by several different centrifugations); (__), absorbance at 280 nm (mA280). Red marks the seven fractions of the first peak. The first peak in terms of the elution volume (mL) corresponds to those in the case of “classical” mammalian exosomes with molecular weights of mammalian exosomes, while second peak—different admixtures [15,28,29,30,31].
	Figure 6. Representative images of structural components in preparation obtained by gel filtration of pooled holothurian respiratory tree, digestive tract, and gonads (sample III), preliminary purified by ultracentrifugation. Upper row—ELVs; (A)—vesicles with thick envelope (white arrows); (B)—non-vesicles (black arrows) and small particles, insert enclosed an aggregate of small particles; (C)—deposits of structureless pollution. TEM, negative staining with uranyl acetate. The length of the scale bars corresponds to 100 nm.

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