Long term low temperature storage of living tissue is an active area of research. Cryoprotectant perfusion allows tissues to vitrify on cooling, minimizing ice crystal formation and thus preserving the small scale structure that is vital to tissue function. The challenge of cooling to vitrification is largely the challenge of obtaining good perfusion of cryoprotectant throughout the tissue, something that is much less of an issue for an isolated organ or tissue sample than it is for an entire animal or human. The more significant challenges are those related to the goal of warming vitrified tissue while retaining full function.
The near term goal for reversible vitrification is to enable more cost-effective organ donation and transplantation. That organ transplantation is a very expensive, uncertain process, and the available supply of organs very limited, is due in large part to the inability to keep an organ alive for long outside the body. In the mid-term, the transplantation industry will expand to include the manufacture of universal off-the-shelf tissues and organs that can be transplanted into any individual, grown from stocks of engineered cells. The logistics of that industry will be greatly aided by the ability to indefinitely store manufactured tissues, rather than making them to order. In the longer term, reversible vitrification will be used to preserve people who are close to death, in the hopes that future medical technology will allow their repair and revival.
At present vitrification of a patient at death by the still small cryonics industry is a one-way trip; the hope for future medical technology includes the development of means to safely warm the patient as well as repair other issues. It is not unreasonable to think that a future in which aging can be reversed at a very late stage is also a future in which vitrified tissues can be warmed. Unfortunately, the most successful of early approaches to warming vitrified tissues, discussed in today's research materials, won't help the cryopreserved patients already vitrified in past years. It requires nanoparticles to be perfused into tissues with cryoprotectant at the time of cooling. It will, however, help to start the organ transplantation industry on the road towards the use and refinement of vitrification technologies, still an important goal.
To extend the preservation of donor hearts beyond the current 4-6 hours, 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.
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.