Cytoskeleton adaptation to stretchable surface relaxation improves adherent cryopreservation of human mesenchymal stem cells

Adherent cell systems are usually dissociated before being cryopreserved, as standard protocols are established for cells in suspension. The application of standard procedures to more complex systems, sensitive to dissociation, such as adherent monolayers, especially comprising mature cell types or tissues remains unsatisfactory. Uncontrolled cell detachment due to intracellular tensile stress, membrane ruptures and damages of adhesion proteins are common during freezing and thawing of cell monolayers. However, many therapeutically relevant cell systems grow adherently to develop their native morphology and functionality, but lose their integrity after dissociation. The hypothesis is that cells on stretchable substrates have a more adaptable cytoskeleton and membrane, reducing cryopreservation-induced stress. Our studies investigate the influence of stretchable surfaces on the cryopreservation of adherent cells to avoid harmful dissociation and expedite post-thawing cultivation of functional cells. A stretching apparatus for defined radial stretching, consisting of silicone vessels and films with specific surface textures for cell culture, was developed. Adherent human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultivated on a stretched silicone film within the vessel, forming a monolayer that was compressed by relaxation, while remaining attached to the relaxed film. Compressed hUC-MSCs, which were cryopreserved adherently showed higher viability and less detachment after thawing compared to control cells without compression. Within three to seven days post-thawing, the hUC-MSCs recovered, and the monolayer reformed. These experiments support the hypothesis that cryopreservation success of adherent cell systems is enhanced by improved adaptability of the cytoskeleton and cell membrane, opening up new approaches in cryobiotechnology.