title: Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.
abstract: This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).
authors.affiliation: Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, 690041, Palchevsky st. 17, Vladivostok, Russia. Electronic address: nelodin54@yahoo.com.
authors.forename: Nelly A
authors.initials: NA
authors.lastname: Odintsova
authors.affiliation: Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, 690041, Palchevsky st. 17, Vladivostok, Russia.
authors.forename: Natalya V
authors.initials: NV
authors.lastname: Ageenko
authors.affiliation: Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, 690041, Palchevsky st. 17, Vladivostok, Russia.
authors.forename: Yulia O
authors.initials: YO
authors.lastname: Kipryushina
authors.affiliation: Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, 690041, Palchevsky st. 17, Vladivostok, Russia.
authors.forename: Mariia A
authors.initials: MA
authors.lastname: Maiorova
authors.affiliation: Laboratory of Cytotechnology, A.V. Zhirmunsky Institute of Marine Biology, The Far Eastern Branch of the Russian Academy of Sciences, 690041, Palchevsky st. 17, Vladivostok, Russia.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Boroda
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005615:Freezing; D011205:Povidone / Q000494:pharmacology; D012617:Sea Urchins / Q000166:cytology* / Q000196:embryology; D014199:Trehalose / Q000494:pharmacology; D014404:Tubulin / Q000502:physiology*
chemical_list: D003451:Cryoprotective Agents; D014404:Tubulin; D014199:Trehalose; D019855:Ethylene Glycol; D011205:Povidone; D004121:Dimethyl Sulfoxide
keywords: Cryoresistance; Cytoskeleton; Gene expression; Pigment differentiation; Sea urchin; Spiculogenesis