Cryopreserved Cells Regeneration Monitored by Atomic Force Microscopy and Correlated With State of Cytoskeleton and Nuclear Membrane.
IEEE Trans.on Nanobioscience. 2018-10-01; 17(4): 485-497
DOI: 10.1109/tnb.2018.2873425
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Atomic force microscopy (AFM) helps to describe and explain the mechanobiological
properties of living cells on the nanoscale level under physiological conditions.
The stiffness of cells is an important parameter reflecting cell physiology.
Here, we have provided the first study of the stiffness of cryopreserved cells
during post-thawing regeneration using AFM combined with confocal fluorescence
microscopy. We demonstrated that the nonfrozen cell stiffness decreased
proportionally to the cryoprotectant concentration in the medium. AFM allowed us
to map cell surface reconstitution in real time after a freeze/thaw cycle and to
monitor the regeneration processes at different depths of the cell and even
different parts of the cell surface (nucleus and edge). Fluorescence microscopy
showed that the cytoskeleton in fibroblasts, though damaged by the freeze/thaw
cycle, is reconstructed after long-term plating. Confocal microscopy confirmed
that structural changes affect the nuclear envelopes in cryopreserved cells. AFM
nanoindentation analysis could be used as a noninvasive method to identify cells
that have regenerated their surface mechanical properties with the proper
dynamics and to a sufficient degree. This identification can be important
particularly in the field of in vitro fertilization and in future cell-based
regeneration strategies.