In order to cryopreserve functional engineered tissues (ETs), the microstructure of the extracellular matrix (ECM) should be maintained as well as the cellular viability since the functionality is closely related to the ECM microstructure. Since the post-thaw ECM microstructure is determined by the deformation of ETs during cryopreservation, freezing-induced deformation of ETs was measured with a newly developed quantum dot (QD)-mediated cell image deformetry system using dermal equivalents as a model tissue. The dermal equivalents were constructed by seeding QD-labeled fibroblasts in type I collagen matrices. After 24 hour incubation, the ETs were directionally frozen by exposing them to a spatial temperature gradient (from 4 °C to −20 °C over a distance of 6 mm). While being frozen, the ETs were consecutively imaged, and consecutive pairs of these images were two-dimensionally cross-correlated to determine the local deformation during freezing. The results showed that freezing induced the deformation of ET, and its magnitude varied with both time and location. The maximum local dilatation was 0.006 s −1 and was always observed at the phase change interface. Due to this local expansion, the unfrozen region in front of the freezing interface experienced compression. This expansion-compression pattern was observed throughout the freezing process. In the unfrozen region, the deformation rate gradually decreased away from the freezing interface. After freezing/thawing, the ET experienced an approximately 28% decrease in thickness and 8% loss in weight. These results indicate that freezing-induced deformation caused the transport of interstitial fluid and the interstitial fluid was extruded. In summary, the results suggest that complex cell-fluid-matrix interactions occur within ETs during freezing, and these interactions determine the post-thaw ECM microstructure and eventual post-thaw tissue functionality.

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.

Mass transport within collagen-based matrices is critical to tissue development, repair, and pathogenesis as well as the design of next generation tissue engineering strategies. This work shows how collagen precursors, specified by intermolecular cross-link composition, provide independent control of collagen matrix mechanical and transport properties. Collagen matrices were prepared from tissue-extracted monomers or oligomers. Viscoelastic behavior was measured in oscillatory shear and unconfined compression. Matrix permeability and diffusivity were measured using gravity-driven permeametry and integrated optical imaging, respectively. Both collagen types showed an increase in stiffness and permeability hindrance with increasing collagen concentration (fibril density); however, different physical property-concentration relationships were noted. Diffusivity wasn’t affected by concentration for either collagen type over the range tested. In general, oligomer matrices exhibited a substantial increase in stiffness and only a modest decrease in transport properties when compared to monomer matrices prepared at the same concentration. The observed differences in viscoelastic and transport properties were largely attributed to increased levels of interfibril branching within oligomer matrices. The ability to relate physical properties to relevant microstructure parameters, including fibril density and interfibril branching, is expected to advance the understanding of cell-matrix signaling as well as facilitate model-based prediction and design of matrix-based therapeutic strategies.

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