The two most significant challenges for successful cryopreservation of engineered
tissues (ETs) are preserving tissue functionality and controlling highly tissue-type
dependent preservation outcomes. In order to address these challenges, freezing-induced
cell-fluid-matrix interactions should be understood, which determine the post-thaw
cell viability and extracellular matrix (ECM) microstructure. However, the current
understanding of this tissue-level biophysical interaction is still limited. In this
study, freezing-induced cell-fluid-matrix interactions and their impact on the cells
and ECM microstructure of ETs were investigated using dermal equivalents as a model
ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in
type I collagen matrices with varying cell seeding density and collagen concentration.
While these dermal equivalents underwent an identical freeze/thaw condition, their
spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular
level cryoresponse were characterized. The results showed that the extent and characteristics
of freezing-induced deformation were significantly different among the experimental
groups, and the ETs with denser ECM microstructure experienced a larger deformation.
The magnitude of the deformation was well correlated to the post-thaw ECM structure,
suggesting that the freezing-induced deformation is a good indicator of post-thaw
ECM structure. A significant difference in the extent of cellular injury was also
noted among the experimental groups, and it depended on the extent of freezing-induced
deformation of the ETs and the initial cytoskeleton organization. These results suggest
that the cells have been subjected to mechanical insult due to the freezing-induced
deformation as well as thermal insult. These findings provide insight on tissue-type
dependent cryopreservation outcomes, and can help to design and modify cryopreservation
protocols for new types of tissues from a pre-developed cryopreservation protocol.