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.

Bioinspired Preservation of Natural Killer Cells for Cancer Immunotherapy.

The ability to cryopreserve natural killer (NK) cells has a significant potential in modern cancer immunotherapy. Current cryopreservation protocols cause deterioration in NK cell viability and functionality. This work reports the preservation of human cytokine-activated NK cell viability and function following cryopreservation using a cocktail of biocompatible bioinspired cryoprotectants (i.e., dextran and carboxylated ε-poly-L-lysine). Results demonstrate that the recovered NK cells after cryopreservation and rewarming maintain their viability immediately after thawing at a comparable level to control (dimethyl sulfoxide-based cryopreservation). Although, their viability drops in the first day in culture compared to controls, the cells grow back to a comparable level to controls after 1 week in culture. In addition, the anti-tumor functional activity of recovered NK cells demonstrates higher cytotoxic potency against leukemia cells compared to control. This approach presents a new direction for NK cell preservation, focusing on function and potentially enabling storage and distribution for cancer immunotherapy.

Natural killer-92 cells maintain cytotoxic activity after long-term cryopreservation in novel DMSO-free media.

Natural killer (NK) cells are a critical part of the innate immune system, and have emerged as attractive targets for immunotherapies for various malignancies. Alongside the need for expansion of NK cells to reach clinically useful numbers, a critical component in the availability of NK cells for allogeneic therapy is cryopreservation. While a continuously-growing cell line such as NK-92 can avoid issues associated with isolating, activating, expanding, and manufacturing large numbers of peripheral blood-derived NKs, cryopreservation of these cells has not made much progress. NK cells are highly sensitive to freezing and thawing, while the use of DMSO during cryopreservation raises serious safety concerns. In this work, we evaluated a number of cryoprotectants that do not contain DMSO for their capacity to cryopreserve NK-92 cells over long-term while retaining their cytotoxic activity and viability, with the aim of identifying potential replacements to DMSO for safe clinical use of these cells.