Cryopreservation of cells (mouse embryonic fibroblasts) is a fundamental task for wide range of applications. In practice, cells are protected against damage during freezing by applications of specific cryoprotectants and freezing/melting protocols. In this study by using AFM and fluorescence microscopy we showed how selected cryoprotectants (dimethyl sulfoxide and polyethylene glycol) affected the cryopreserved cells mechanical properties (stiffness) and how these parameters are correlated with cytoskeleton damage and reconstruction. We showed how cryopreserved (frozen and thawed) cells' stiffness change according to type of applied cryoprotectant and its functionality in extracellular or intracellular space. We showed that AFM can be used as technique for investigation of cryopreserved cells surfaces state and development ex vivo. Our results offer a new perspective on the monitoring and characterization of frozen cells recovery by measuring changes in elastic properties by nanoindentation technique. This may lead to a new and detailed way of investigating the post-thaw development of cryopreserved cells which allows to distinguish between different cell parts.

1st Department of Internal Medicine Cardioangiology Masaryk University Brno Czechia

Central European Institute of Technology Masaryk University Brno Czechia

Department of Analysis of Functional Materials Institute of Physics Academy of Sciences Czech Republic Prague Czechia

Department of Biology Faculty of Medicine Masaryk University Brno Czechia

International Clinical Research Center St Anne's University Hospital Brno Czechia

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Alessandrini A., Facci P. (2005). AFM: a versatile tool in biophysics. Meas. Sci. Technol. 16, R65–R92. 10.1088/0957-0233/16/6/R01 DOI

Cartagena-Rivera A. X., Wang W. H., Geahlen R. L., Raman A. (2015). Fast, multi-frequency, and quantitative nanomechanical mapping of live cells using the atomic force microscope. Sci. Rep. 5:11692. 10.1038/srep11692 PubMed DOI PMC

Chen S. W., Odorico M., Meillan M., Vellutini L., Teulon J.-M., Parot P., et al. . (2013). Nanoscale structural features determined by AFM for single virus particles. Nanoscale 5, 10877–10886. 10.1039/c3nr02706f PubMed DOI

Chinnadurai R., Garcia M. A., Sakurai Y., Lam W. A., Kirk A. D., Galipeau J., et al. . (2014). Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo. Stem Cell Rep. 3, 60–72. 10.1016/j.stemcr.2014.05.003 PubMed DOI PMC

Dimitriadis E. K., Horkay F., Maresca J., Kachar B., Chadwick R. S. (2002). Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810. 10.1016/S0006-3495(02)75620-8 PubMed DOI PMC

Ding Y., Xu G. K., Wang G. F. (2017). On the determination of elastic moduli of cells by AFM based indentation. Sci. Rep. 7:45575. 10.1038/srep45575 PubMed DOI PMC

Dokukin M. E., Guz N. V., Sokolov I. (2013). Quantitative study of the elastic modulus of loosely attached cells in AFM indentation experiments. Biophys. J. 104, 2123–2131. 10.1016/j.bpj.2013.04.019 PubMed DOI PMC

Dokukin M. E., Sokolov I. (2017). Nanoscale compositional mapping of cells, tissues, and polymers with ringing mode of atomic force microscopy. Sci. Rep. 7:11. 10.1038/s41598-017-12032-z PubMed DOI PMC

Dong J., Malsam J., Bischof J. C., Hubel A., Aksan A. (2010). Spatial distribution of the state of water in frozen mammalian cells. Biophys. J. 99, 2453–2459. 10.1016/j.bpj.2010.08.035 PubMed DOI PMC

Eiselleova L., Peterkova I., Neradil J., Slaninova I., Hampl A., Dvorak P. (2008). Comparative study of mouse and human feeder cells for human embryonic stem cells. Int. J. Dev. Biol. 52, 353–363. 10.1387/ijdb.082590le PubMed DOI

Fekete L., Kusova K., Petrak V., Kratochvilova I. (2012). AFM topographies of densely packed nanoparticles: a quick way to determine the lateral size distribution by autocorrelation function analysis. J. Nanoparticle Res. 14:10 10.1007/s11051-012-1062-7 DOI

Gavara N. (2016). A beginner 's guide to atomic force microscopy probing for cell mechanics. Microsc. Res. Tech. 1, 1–10. 10.1002/jemt.22776 PubMed DOI PMC

Gavara N., Chadwick R. S. (2012). Determination of the elastic moduli of thin samples and adherent cells using conical atomic force microscope tips. Nat. Nanotechnol. 7, 733–736. 10.1038/nnano.2012.163 PubMed DOI PMC

Guz N., Dokukin M., Kalaparthi V., Sokolov I. (2014). If cell mechanics can be described by elastic modulus: study of different models and probes used in indentation experiments. Biophys. J. 107, 564–575. 10.1016/j.bpj.2014.06.033 PubMed DOI PMC

Hermanowicz P., Sarna M., Burda K., Gabrys H. (2014). AtomicJ: an open source software for analysis of force curves. Rev. Sci. Instr. 85:063703. 10.1063/1.4881683 PubMed DOI

Hofer M., Falk M., Komurkova D., Falkova I., Bacikova A., Klejdus B., et al. . (2016). Two new faces of Amifostine: protector from DNA damage in normal cells and inhibitor of DNA repair in cancer cells. J. Med. Chem. 59, 3003–3017. 10.1021/acs.jmedchem.5b01628 PubMed DOI

Hubálek Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46, 205–229. 10.1016/S0011-2240(03)00046-4 PubMed DOI

Kasas S., Longo G., Dietler G. (2013). Mechanical properties of biological specimens explored by atomic force microscopy. J. Phys. D Appl. Phys. 46:133001 10.1088/0022-3727/46/13/133001 DOI

Kratochvílová I., Golan M., Pomeisl K., Richter J., Sedlakova S., Sebera J., et al. . (2017). Theoretical and experimental study of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability. RSC Adv. 7, 352–360. 10.1039/C6RA25095E PubMed DOI PMC

Lampugnani M. G., Pedenovi M., Niewiarowski A., Casali B., Donati M. B., Corbascio G. C., et al. . (1987). Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion, and growth of cultured mouse B16 melanoma cells. Exp. Cell Res. 172, 385–396. 10.1016/0014-4827(87)90396-X PubMed DOI

Levental I., Levental K. R., Klein E. A., Assoian R., Miller R. T., Wells R. G., et al. . (2010). A simple indentation device for measuring micrometer-scale tissue stiffness. J. Phys. Condens. Matter 22:194120. 10.1088/0953-8984/22/19/194120 PubMed DOI PMC

Lin C., Tsai S. (2012). The effect of cryopreservation on DNA damage, gene expression and protein abundance in vertebrate. Ital. J. Anim. Sci. 11:e21 10.4081/ijas.2012.e21 DOI

Loparic M., Wirz D., Daniels A. U., Raiteri R., Vanlandingham M. R., Guex G., et al. . (2010). Micro- and nanomechanical analysis of articular cartilage by indentation-type atomic force microscopy: validation with a gel-microfiber composite. Biophys. J. 98, 2731–2740. 10.1016/j.bpj.2010.02.013 PubMed DOI PMC

Mandumpal J. B., Kreck C. A., Mancera R. L. (2011). A molecular mechanism of solvent cryoprotection in aqueous DMSO solutions. Phys. Chem. Chem. Phys. 13, 3839–3842. 10.1039/c0cp02326d PubMed DOI

Mašek J., Bartheldyova E., Korvasova Z., Skrabalova M., Koudelka S., Kulich P., et al. . (2011a). Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: preparation and study of their structure. Anal. Biochem. 408, 95–104. 10.1016/j.ab.2010.08.023 PubMed DOI

Masek J., Bartheldyova E., Turanek-Knotigova P., Skrabalova M., Korvasova Z., Plockova J., et al. . (2011b). Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90. J. Control. Release 151, 193–201. 10.1016/j.jconrel.2011.01.016 PubMed DOI

Ofek G., Wiltz D. C., Athanasiou K. A. (2009). Contribution of the cytoskeleton to the compressive properties and recovery behavior of single cells. Biophys. J. 97, 1873–1882. 10.1016/j.bpj.2009.07.050 PubMed DOI PMC

Ogden R. W. (1972). Large deformation isotropic elasticity - correlation of theory and experiment for compressible rubberlike solids. Proc. R. Soc. Lond. Ser. 328, 567–583. 10.1098/rspa.1972.0096 DOI

Parsons J. T., Horwitz A. R., Schwartz M. A. (2010). Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 11, 633–643. 10.1038/nrm2957 PubMed DOI PMC

Pesl M., Pribyl J., Acimovic I., Vilotic A., Jelinkova S., Salykin A., et al. . (2016). Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing. Biosens. Bioelectron. 85, 751–757. 10.1016/j.bios.2016.05.073 PubMed DOI

Pogoda K., Jaczewska J., Wiltowska-Zuber J., Klymenko O., Zuber K., Fornal M., et al. . (2012). Depth-sensing analysis of cytoskeleton organization based on AFM data. Eur. Biophys. J. 41, 79–87. 10.1007/s00249-011-0761-9 PubMed DOI

Ragoonanan V., Hubel A., Aksan A. (2010). Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses. Cryobiology 61, 335–344. 10.1016/j.cryobiol.2010.10.160 PubMed DOI

Rajan R., Hayashi F., Nagashima T., Matsumura K. (2016). Toward a molecular understanding of the mechanism of cryopreservation by polyampholytes: cell membrane interactions and hydrophobicity. Biomacromolecules 17, 1882–1893. 10.1021/acs.biomac.6b00343 PubMed DOI

Sneddon I. N. (1965). The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int. J. Eng. Sci. 3, 47–57. 10.1016/0020-7225(65)90019-4 DOI

Touhami A., Nysten B., Dufrene Y. F. (2003). Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir 19, 4539–4543. 10.1021/la034136x DOI

Wang J., Wan Z. F., Liu W. M., Li L., Ren L., Wang X. Q., et al. . (2009). Atomic force microscope study of tumor cell membranes following treatment with anti-cancer drugs. Biosens. Bioelectron. 25, 721–727. 10.1016/j.bios.2009.08.011 PubMed DOI

Wolfram I. (2017). “Mathematica,” Wolfram Research.

Woods E. J., Thirumala S., Badhe-Buchanan S. S., Clarke D., Mathew A. J. (2016). Off the shelf cellular therapeutics: factors to consider during cryopreservation and storage of human cells for clinical use. Cytotherapy 18, 697–711. 10.1016/j.jcyt.2016.03.295 PubMed DOI

Xu X., Liu Y., Cui Z., Wei Y., Zhang L. (2012). Effects of osmotic and cold shock on adherent human mesenchymal stem cells during cryopreservation. J. Biotechnol. 162, 224–231. 10.1016/j.jbiotec.2012.09.004 PubMed DOI

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