Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels

While significant progress has been made in directing the behavior of cells encapsulated within three-dimensional (3D) covalently crosslinked hydrogels, the capacity of these materials to support in situ cryopreservation of cells directly within the gels has not been assessed. Here, we demonstrate the retention of human mesenchymal stem cell (hMSC) viability within hyaluronic acid (HA) and polyethylene glycol based hydrogels via a facile gradual cooling and freezing protocol. Encapsulated cell viability was retained at similar rates in both materials systems regardless of initial duration in culture or adhesive ligand incorporation, indicating the versatility of the approach. Additionally, the cryopreservation protocol maintains stem cell differentiation potential; incubation in adipogenic differentiation media induced equal rates of hMSC adipogenesis in freeze-thawed and non-frozen HA based hydrogels on a per-cell basis. Collectively, these findings highlight the cryopreservation protocol as a platform technology that, in addition to contributing to an increased understanding of three-dimensional cell-matrix interactions, could enable the long-term preservation of tissue engineering constructs for clinical applications.