A Low-Cost, Tabletop LOD-EPR System for Nondestructive Quantification of Iron Oxide Nanoparticles in Tissues.

Iron oxide nanoparticles (IONPs) have wide utility in applications from drug delivery to the rewarming of cryopreserved tissues. Due to the complex behavior of IONPs (e.g., uneven particle distribution and aggregation), further developments and clinical translation can be accelerated by having access to a noninvasive method for tissue IONP quantification. Currently, there is no low-cost method to nondestructively track IONPs in tissues across a wide range of concentrations. This work describes the performance of a low-cost, tabletop, longitudinally detected electron paramagnetic resonance (LOD-EPR) system to address this issue in the field of cryopreservation, which utilizes IONPs for rewarming of rat kidneys. A low-cost LOD-EPR system is realized via simultaneous transmit and receive using MHz continuous-wave transverse excitation with kHz modulation, which is longitudinally detected at the modulation frequency to provide both geometric and frequency isolation. The accuracy of LOD-EPR for IONP quantification is compared with NMR relaxometry. Solution measurements show excellent linearity (R2 > 0.99) versus Fe concentration for both measurements on EMG308 (a commercial nanoparticle), silica-coated EMG308, and PEG-coated EMG308 in water. The LOD-EPR signal intensity and NMR longitudinal relaxation rate constant (R1) of water are affected by particle coating, solution viscosity, and particle aggregation. R1 remains linear but with a reduced slope when in cryoprotective agent (CPA) solution, whereas the LOD-EPR signal is relatively insensitive to this. R1 does not correlate well with Fe concentration in rat kidney sections (R2 = 0.3487), while LOD-EPR does (R2 = 0.8276), with a linear regression closely matching that observed in solution and CPA.

CPA toxicity screening of cryoprotective solutions in rat hearts.

In clinical practice, donor hearts are transported on ice prior to transplant and discarded if cold ischemia time exceeds ∼5 h. Methods to extend these preservation times are critically needed, and ideally, this storage time would extend indefinitely, enabling improved donor-to-patient matching, organ utilization, and immune tolerance induction protocols. Previously, we demonstrated successful vitrification and rewarming of whole rat hearts without ice formation by perfusion-loading a cryoprotective agent (CPA) solution prior to vitrification. However, these hearts did not recover any beating even in controls with CPA loading/unloading alone, which points to the chemical toxicity of the cryoprotective solution (VS55 in Euro-Collins carrier solution) as the likely culprit. To address this, we compared the toxicity of another established CPA cocktail (VEG) to VS55 using ex situ rat heart perfusion. The CPA exposure time was 150 min, and the normothermic assessment time was 60 min. Using Celsior as the carrier, we observed partial recovery of function (atria-only beating) for both VS55 and VEG. Upon further analysis, we found that the VEG CPA cocktail resulted in 50 % lower LDH release than VS55 (N = 4, p = 0.017), suggesting VEG has lower toxicity than VS55. Celsior was a better carrier solution than alternatives such as UW, as CPA + Celsior-treated hearts spent less time in cardiac arrest (N = 4, p = 0.029). While we showed substantial improvement in cardiac function after exposure to vitrifiable concentrations of CPA by improving both the CPA and carrier solution formulation, further improvements will be required before we achieve healthy cryopreserved organs for transplant.

Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model.

Banking cryopreserved organs could transform transplantation into a planned procedure that more equitably reaches patients regardless of geographical and time constraints. Previous organ cryopreservation attempts have failed primarily due to ice formation, but a promising alternative is vitrification, or the rapid cooling of organs to a stable, ice-free, glass-like state. However, rewarming of vitrified organs can similarly fail due to ice crystallization if rewarming is too slow or cracking from thermal stress if rewarming is not uniform. Here we use "nanowarming," which employs alternating magnetic fields to heat nanoparticles within the organ vasculature, to achieve both rapid and uniform warming, after which the nanoparticles are removed by perfusion. We show that vitrified kidneys can be cryogenically stored (up to 100 days) and successfully recovered by nanowarming to allow transplantation and restore life-sustaining full renal function in nephrectomized recipients in a male rat model. Scaling this technology may one day enable organ banking for improved transplantation.

Model-Guided Design and Optimization of CPA Perfusion Protocols for Whole Organ Cryopreservation.

Vitrification could enable long-term organ preservation, but only after loading high-concentration, potentially toxic cryoprotective agents (CPAs) by perfusion. In this paper, we combine a two-compartment Krogh cylinder model with a toxicity cost function to theoretically optimize the loading of CPA (VMP) in rat kidneys as a model system. First, based on kidney perfusion experiments, we systematically derived the parameters for a CPA transport loading model, including the following: Vb = 86.0% (ra = 3.86 µm), Lp = 1.5 × 10-14 m3/(N·s), ω = 7.0 × 10-13 mol/(N·s), σ = 0.10. Next, we measured the toxicity cost function model parameters as α = 3.12 and ß = 9.39 × 10-6. Combining these models, we developed an improved kidney-loading protocol predicted to achieve vitrification while minimizing toxicity. The optimized protocol resulted in shorter exposure (25 min or 18.5% less) than the gold standard kidney-loading protocol for VMP, which had been developed based on decades of empirical practice. After testing both protocols on rat kidneys, we found comparable physical and biological outcomes. While we did not dramatically reduce toxicity, we did reduce the time. As our approach is now validated, it can be used on other organs lacking defined toxicity data to reduce CPA exposure time and provide a rapid path toward developing CPA perfusion protocols for other organs and CPAs.

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

Vitrification and Rewarming of Magnetic Nanoparticle-Loaded Rat Hearts.

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.

Vitrification and Nanowarming of Kidneys.

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.

Preparation of Scalable Silica-Coated Iron Oxide Nanoparticles for Nanowarming.

Cryopreservation technology allows long-term banking of biological systems. However, a major challenge to cryopreserving organs remains in the rewarming of large volumes (>3 mL), where mechanical stress and ice formation during convective warming cause severe damage. Nanowarming technology presents a promising solution to rewarm organs rapidly and uniformly via inductive heating of magnetic nanoparticles (IONPs) preloaded by perfusion into the organ vasculature. This use requires the IONPs to be produced at scale, heat quickly, be nontoxic, remain stable in cryoprotective agents (CPAs), and be washed out easily after nanowarming. Nanowarming of cells and blood vessels using a mesoporous silica-coated iron oxide nanoparticle (msIONP) in VS55, a common CPA, has been previously demonstrated. However, production of msIONPs is a lengthy, multistep process and provides only mg Fe per batch. Here, a new microporous silica-coated iron oxide nanoparticle (sIONP) that can be produced in as little as 1 d while scaling up to 1.4 g Fe per batch is presented. sIONP high heating, biocompatibility, and stability in VS55 is also verified, and the ability to perfusion load and washout sIONPs from a rat kidney as evidenced by advanced imaging and ICP-OES is demonstrated.

Surgical treatment of mucin-producing cholangiocarcinoma arising from intraductal papillary neoplasm of the intrahepatic bile duct: a report of 2 cases

Intraductal papillary neoplasms of the bile duct (IPNB) leads to malignant transformation and mucin production. Herein, we presented two cases of mucin-producing IPNB with obstructive jaundice who underwent resection of the intrahepatic lesions and bypass hepaticojejunostomy. The first case was a 69 year-old male patient with 5-year follow up for gallstone disease. Imaging studies showed mucin-secreting IPNB mainly in the hepatic segment III bile duct (B3) and multiple intrahepatic duct stones for which, segment III resection, intrahepatic stone removal, end-to-side choledochojejunostomy and B3 hepaticojejunostomy were conducted. The second case was a 74 year-old female patient with 11-year follow up for gallstone disease. Imaging studies showed mucin-producing IPNB with dilatation of the segment IV duct (B4) and mural nodules for which, segment IV resection, partial resection of the diaphragm and central hepaticojejunostomy were conducted. Both patients recovered uneventfully from surgery. These cases highlight that in patients with IPNB, abundant production of highly viscous mucin inducing obstructive jaundice may be associated with malignant transformation.