Biotransport approaches to improving tissue and organ cryopreservation

Abstract

Vitrification, as a means of ice-free cryopreservation, is a long-term preservation approach that can extend the preservation time to years, which can solve the dilemma of the tissue and organ grafts (e.g., arteries and kidneys) shortage for transplantation. Vitrification requires a high concentration of cryoprotective agents (CPA) to replace the water in cells and fast cooling to reach the vitrified state. Moreover, rewarming from vitrification requires a faster rate and uniformity, which is harder to achieve. Understanding the physical properties of CPA is fundamental before studying biological systems. Critical cooling rate (CCR) and critical warming rate (CWR) are the most important properties of a CPA, which refer to the minimum rates to suppress ice formation during cooling and warming processes, respectively. CWR is at least one or two orders of magnitude higher than CCR, therefore achieving a uniform and fast rewarming is the most critical to success. CPA cocktails (e.g., DP6 (6 M), VS55 (8.4 M), and M22 (9.3 M)) are always selected for vitrification since they have low CWRs and acceptable toxicity. However, we lack complete thermophysical data for CPA cocktails, especially for dilute concentration, which is important in tissue and organ cryopreservation since tissues normally aren’t equilibrated to the base CPA concentration. Therefore, we summarized the available data of CCRs and CWRs for a variety of CPAs and suggested a convenient mathematical expression for CCR and CWR that can guide general use for predicting the diluted CPA cocktails properties. Then, we used differential scanning calorimetry (DSC) and cryomicroscopy to experimentally study the phase diagrams of the CPA cocktails (at all concentrations) to depict the ice formation kinetics during a practical cooling and warming rate in organ preservation. Applying the vitrification and rewarming in biological systems is more complex since we need to consider the CPA transport, toxicity, and heat transfer in the tissue and organs. My projects in this dissertation focus on building up the models of heat and mass transfer and applying the model to the vitrification and rewarming experiments of tissues and organs. As for tissue, the barrier that’s hampering successful vitrification and rewarming is the lack of CPA transport. CPA is usually transported in tissue by passive diffusion, and the equilibration time hugely depends on the thickness of the tissue. Thicker tissues are usually poorly equilibrated with CPA, which requires a high warming rate for successful rewarming. Taking arterial tissue as an example, researchers have successfully vitrified up to 1 mm-thick carotid arteries and rewarmed them using water bath convection heating (~200 °C/min). Thicker tissues (>1 mm-thick) couldn’t be successfully rewarmed due to the lack of heating ability. We developed a model to predict the required heating rates based on the diffusion simulation for tissues with different thicknesses and applied an ultra-fast heating approach, metal form heating (~2000 °C/min) to rewarm thicker tissues. Guided by the model prediction and with the help of metal form warming, we were able to, for the first time, successfully rewarm up to 2 mm-thick aortae after vitrification. Organ cryopreservation is more complicated than tissues since not only the viability needs to be preserved, but also the functionality needs to be restored after vitrification and rewarming. Therefore, both the CPA transport and the rewarming need to be refined compared to those in the tissue system. Transport of CPA in organs is achieved by perfusion instead of diffusion, which is more efficient and causes less osmotic damage to the cells. Rewarming is achieved by radiofrequency heating of iron oxide nanoparticles (IONP) or “nanowarming”, which is a fast and uniform approach. Using a rat kidney model, we demonstrate the first reproducibly successful vitrification, long-term storage (up to 100 days), rewarming, and transplantation of organs that restore full renal function and solely sustain the life of nephrectomized transplant recipients. We further developed a model that studied the transport and toxicity kinetics of CPA during perfusion into organs to find an optimal loading protocol of CPA to organs, which can be used in large-scale organ vitrification. Because nanowarming is not dependent on system size or boundary conditions, this approach should successfully scale to human-sized organs and work with non-renal organs. Upon scaling, this technology has the potential to enable clinical organ banking and a new transplantation paradigm in which kidneys and other organs wait for patients rather than patients waiting for organs.