title: Cell blebbing upon addition of cryoprotectants: a self-protection mechanism.
abstract: In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.
authors: Center for Biomedical Engineering, University of Science and Technology of China, Hefei, Anhui 230027, China; Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors: Renquan
authors: R
authors: Ruan
authors: Center for Biomedical Engineering, University of Science and Technology of China, Hefei, Anhui 230027, China; Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors: Lili
authors: L
authors: Zou
authors: Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, United States of America.
authors: Sijie
authors: S
authors: Sun
authors: Center for Biomedical Engineering, University of Science and Technology of China, Hefei, Anhui 230027, China; Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors: Jing
authors: J
authors: Liu
authors: School of Life Science, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors: Longping
authors: L
authors: Wen
authors: Department of Mechanical Engineering, University of Washington, Seattle, WA, 98195, United States of America.
authors: Dayong
authors: D
authors: Gao
authors: Center for Biomedical Engineering, University of Science and Technology of China, Hefei, Anhui 230027, China; Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors: Weiping
authors: W
authors: Ding
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000648:ultrastructure; D000222:Adaptation, Physiological; D001343:Autophagy / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D009994:Osmolar Concentration; D009997:Osmotic Pressure
chemical_list: D000199:Actins; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide