Cryopreservation of network bioactivity and multi-lineage stromal cell differentiation potential within three-dimensional synthetic hydrogels.

Despite progress in many aspects of controlling cell behavior within synthetic three-dimensional hydrogels, approaches to cryopreserve these systems - encompassing the protection of both encapsulated cell viability and network bioactive functions - are lacking. Here, we demonstrate the retention of encapsulated human mesenchymal stromal cell (hMSC) viability following in situ cryopreservation regardless of cell line, material system, or storage duration. Further, the preservation extends to network bioactive functions, with hMSCs cryopreserved within degradable and adhesive hyaluronic-acid (HA) based hydrogels exhibiting degradation-mediated spreading within the gels equivalent to their non-frozen counterparts. Finally, the platform cryopreservation protocol preserves multi-lineage cellular differentiation capacity, with encapsulated hMSCs in non-degradable and adhesive/degradable HA-based hydrogels undergoing rates of adipogenesis and osteogenesis, respectively, equivalent to those in non-frozen gels on a per-cell basis. Collectively, these findings indicate a versatile platform technology that contributes to an increased understanding of three-dimensional cell-matrix interactions, and which may enable the indefinite cryopreservation of tissue engineering constructs for clinical applications.