Cryopreservation of human mesenchymal stem cells in covalently crosslinked polymeric hydrogels

Tissue engineering combines material properties as well as biological factors to produce viable tissue for medical and therapeutic purposes. The ability to preserve tissue engineering scaffolds indefinitely may be useful in cases where the need for replacement is deferred (i.e., for individuals at hereditary risk for musculoskeletal disorders later in life). Cryopreservation, the long-term storage of specimens in liquid N₂ using dimethyl sulfoxide (DMSO) as a cryoprotective agent, is a widely used and relatively inexpensive method of preserving mammalian cells. Past reports have leveraged the efficacy of this approach to cryopreserve cell-laden physically crosslinked 3D hydrogels – water-swollen networks of crosslinked polymers – with good retention of encapsulated cell viability and scaffold structure. However, the effectiveness of the approach has not been assessed for cells encapsulated within covalently crosslinked hydrogels; such a technique could be valuable for regenerative applications, as a wide body of research has demonstrated the control of advanced cell behaviors including stem cell differentiation specifically within covalently crosslinked hydrogels. The main interest in this research is the freezing, and later thawing, of various cell types in biodegradable gels while maintaining viability. Beyond retention of viability, it is important that tissue and cells preserved maintain their function post cryopreservation. Preliminary investigation by our lab indicates the efficacy of the basic approach - hydrogels made from two different polymers containing encapsulated human mesenchymal stem cells were subjected to an iterative cooling/freezing process with or without pre-incubation in media containing DMSO. Retention of cell viability upon thawing was significantly greater in (+) versus (-) DMSO hydrogels for all groups investigated. Further, the cryopreserved cells were shown to retain their 'stemness', i.e., their differentiation potential. These results indicate the potential of the approach as a platform technology that may be a valuable tool in the development of cell-based regenerative strategies.