Vitrification can dramatically increase the storage of viable biomaterials in the cryogenic state for years. Unfortunately, vitrified systems ≥3 mL like large tissues and organs, cannot currently be rewarmed sufficiently rapidly or uniformly by convective approaches to avoid ice crystallization or cracking failures. A new volumetric rewarming technology entitled "nanowarming" addresses this problem by using radiofrequency excited iron oxide nanoparticles to rewarm vitrified systems rapidly and uniformly. Here, for the first time, successful recovery of a rat kidney from the vitrified state using nanowarming, is shown. First, kidneys are perfused via the renal artery with a cryoprotective cocktail (CPA) and silica-coated iron oxide nanoparticles (sIONPs). After cooling at −40°C min −1 in a controlled rate freezer, microcomputed tomography (μCT) imaging is used to verify the distribution of the sIONPs and the vitrified state of the kidneys. By applying a radiofrequency field to excite the distributed sIONPs, the vitrified kidneys are nanowarmed at a mean rate of 63.7°C min −1 . Experiments and modeling show the avoidance of both ice crystallization and cracking during these processes. Histology and confocal imaging show that nanowarmed kidneys are dramatically better than convective rewarming controls. This work suggests that kidney nanowarming holds tremendous promise for transplantation.

To extend the preservation of donor hearts beyond the current 4–6 h, this paper explores heart cryopreservation by vitrification—cryogenic storage in a glass-like state. While organ vitrification is made possible by using cryoprotective agents (CPA) that inhibit ice during cooling, failure occurs during convective rewarming due to slow and non-uniform rewarming which causes ice crystallization and/or cracking. Here an alternative, “nanowarming”, which uses silica-coated iron oxide nanoparticles (sIONPs) perfusion loaded through the vasculature is explored, that allows a radiofrequency coil to rewarm the organ quickly and uniformly to avoid convective failures. Nanowarming has been applied to cells and tissues, and a proof of principle study suggests it is possible in the heart, but proper physical and biological characterization especially in organs is still lacking. Here, using a rat heart model, controlled machine perfusion loading and unloading of CPA and sIONPs, cooling to a vitrified state, and fast and uniform nanowarming without crystallization or cracking is demonstrated. Further, nanowarmed hearts maintain histologic appearance and endothelial integrity superior to convective rewarming and indistinguishable from CPA load/unload control hearts while showing some promising organ-level (electrical) functional activity. This work demonstrates physically successful heart vitrification and nanowarming and that biological outcomes can be expected to improve by reducing or eliminating CPA toxicity during loading and unloading.

Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24–48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.

Cryopreserved tissues are increasingly needed in biomedical applications. However, successful cryopreservation is generally only reported for thin tissues (≤1 mm). This work presents several innovations to reduce cryoprotectant (CPA) toxicity and improve tissue cryopreservation, including 1) improved tissue warming rates through radiofrequency metal form and field optimization and 2) an experimentally verified predictive model to optimize CPA loading and rewarming to reduce toxicity. CPA loading is studied by microcomputed tomography (µCT) imaging, rewarming by thermal measurements, and modeling, and viability is measured after loading and/or cryopreservation by alamarBlue and histology. Loading conditions for three common CPA cocktails (6, 8.4, and 9.3 m) are designed, and then fast cooling and metal forms rewarming (up to 2000°C min −1) achieve ≥90% viability in cryopreserved 1-2 mm arteries with various CPAs. Despite high viability by alamarBlue, histology shows subtle changes after cryopreservation suggesting some degree of cell damage especially in the central portions of thicker arteries up to 2 mm. While further studies are needed, these results show careful CPA loading and higher metal forms warming rates can help reduce CPA loading toxicity and improve outcomes from cryopreservation in tissues while also offering new protocols to preserve larger tissues ≥1 mm in thickness.

Cryopreservation by vitrification has far-reaching implications. However, rewarming techniques that are rapid and scalable (both in throughput and biosystem size) for low concentrations of cryoprotective agent (CPA) for reduced toxicity are lacking, limiting the potential for translation. Here, we introduce a joule heating–based platform technology, whereby biosystems are rapidly rewarmed by contact with an electrical conductor that is fed a voltage pulse. We demonstrate successful cryopreservation of three model biosystems with thicknesses across three orders of magnitude, including adherent cells (~4 µm), Drosophila melanogaster embryos (~50 µm) and rat kidney slices (~1.2 mm) using low CPA concentrations (2–4 M). Using tunable voltage pulse widths from 10 µs to 100 ms, numerical simulation predicts that warming rates from 5 × 104 to 6 × 108 °C/min can be achieved. Altogether, our results present a general solution to the cryopreservation of a broad spectrum of cellular, organismal and tissue-based biosystems.