Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.