title: Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.
abstract: 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.
authors.affiliation: School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.
authors.forename: Ka Yaw
authors.initials: KY
authors.lastname: Teo
authors.forename: Tenok O
authors.initials: TO
authors.lastname: DeHoyos
authors.forename: J Craig
authors.initials: JC
authors.lastname: Dutton
authors.forename: Frederick
authors.initials: F
authors.lastname: Grinnell
authors.forename: Bumsoo
authors.initials: B
authors.lastname: Han
mesh_terms: D002452:Cell Count; D002461:Cell Line, Transformed; D002470:Cell Survival; D024042:Collagen Type I / Q000737:chemistry; D015925:Cryopreservation / Q000379:methods*; D003599:Cytoskeleton / Q000473:pathology; D020405:Dermis / Q000166:cytology; D005109:Extracellular Matrix / Q000473:pathology* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000473:pathology*; D005615:Freezing; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D045663:Quantum Dots; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D024042:Collagen Type I