Improved tissue cryopreservation using inductive heating of magnetic nanoparticles

Abstract

Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes.

Fig. 1. Schematic illustrating tissue vitrification, convective warming, and nanowarming

(A) Tissues are harvested from a donor. Representative harvest of a blood vessel is shown. (B) Tissues are loaded in a vial with cryoprotectant (VS55) and msIONPs in a stepwise protocol, vitrified by standard convection, and stored at cryogenic temperatures. Warming by standard convection (C), leads to failure in larger 50 mL systems (D). Nanowarming in an alternating magnetic field, an inductive RF coil that stimulates nanoparticle heating (E), avoids warming failure and renders the tissue suitable for further testing or use (F).

Fig. 2. Iron oxide nanoparticle characterization

(A) Representative TEM images of IONPs and msIONPs. (B) Schematic detailing the synthesis of msIONPs. The IONPs were coated with mesoporous silica shell followed by co-modification of PEG and TMS on the surface of msIONPs. (C) Size distribution of IONPs and msIONPs quantified by dynamic light scattering (DLS) and by analyzing 500 − 1000 NPs from TEM images. (D) Table of parameters of IONPs and msIONPs. (E) Photographs depicting stability of msIONPs and IONPs in VS55 at room temperature over time.

Fig. 3. VS55 loading, vitrification, and nanowarming of porcine arteries

(A) Quantitation and distribution of VS55 loading over time in the wall of one out of three representative porcine arteries imaged in (B). The corresponding pseudocolor image shows green artery wall and red VS55 solution by μCT. The red dashed line represents the fully loaded HU. The green dashed line represents artery in PBS HU (n=3 for calibration curve). (B) Photographs and computed tomography images of three separate arteries in 1 mL vials demonstrating successful artery vitrification (left), failure due to cracking (middle), and failure due to crystallization (right). The density differences due to the cracking are noted by arrows in B. (C) Graph of temperature over time for convective cooling vitrification of arteries in 1 mL vials at 15 °C/min > CCR of VS55. (D) Graph of temperature over time for nanowarming at 20 kA/m and 360 kHz of the same arteries in (C) loaded with msIONP in VS55 in 1 mL vials. Nanowarming reached 130 °C/min which is > CWR of VS55.

Fig. 4. Nanowarming scale up from 1 kW to 15 kW inductive heating system

(A) Schematic of RF heating system scale up from 1 kW to 15 kW to enable heating up to 80 mL with an increased SAR. (B) The cross-sectional and 3D representations of the cylindrical system to be vitrified and convectively warmed or nanowarmed. (C) The limitations of gold standard convective cooling and rewarming vs. nanowarming on vitrification (success and failure) of 0.5 – 2.5 cm radius cylinders as reported numerically in table S2. Successful cooling (blue shading) and warming (red shading) are defined by the critical minimum cooling and warming rates for VS55 and thermal stress lower than 3.2 MPa. More details of this model are given in the Supplementary Materials.

Fig. 5. Nanowarming maintains viability of porcine carotid in 1 to 50 mL systems

(A) Viability of porcine carotid artery normalized to the control (fresh tissue in growth media) as measured by alamarBlue assay and TUNEL stain. The upper plot shows the cytotoxicity effect of adding VS55 and 10 mg Fe/mL msIONP to the artery (n = 4 − 7). The striped bars represent viability of fresh samples normalized to control (black control = 100%), samples that were maintained on ice for the same period of time (1 − 2 h) (red, 89 ± 2%), samples that were exposed to VS55 (blue, 100 ± 1.4%) or VS55 and 10 mg Fe/mL msIONP (brown, 128 ± 19%). The bottom plot shows the artery viabilities after nanowarming (solid purple, 1 mL: 87 ± 10%, patterned purple, 50 mL: 86 ± 3%), fast convective heating (solid green, 1 mL: 82 ± 10%, patterned green, 50 mL: 20 ± 6%) and slow warming (pink, 1 mL: 28 ± 9%). In 1 mL, the nanowarmed sample viability is comparable to the fresh control and slow warmed sample showed a decline of viability compared to fresh control (P < 0.0001). In 50 mL, the nanowarmed sample viability is slightly lower than the fresh control (P = 0.0275) but comparable to nanowarmed sample in 1 mL (P = 0.9996). The fast convective warmed sample viability is significantly reduced in 50 mL (P < 0.0001). The statistical analyses of multiple comparisons of other possible conditions are included in table S4. N = 3 − 6 for both 1 mL and 50 mL systems; n = 4 − 7 in 1 mL system; n= 3 − 5 in 50 mL. N = number of pigs; n = number of arteries (B) Histological images of H&E stained control, nanowarmed, convective (1 mL), and slow warmed arteries. Scale bar is 60 μm. Normalized tissue white space compared to control: 2 ± 0.3%, P = 0.09 for nanowarmed samples, 31 ± 1%, P < 0.0001 for slow warmed convective samples. See also fig. S3. (C) TUNEL stained images corresponding to the same histology samples in B. All data is presented as the mean ± standard deviation.

Fig. 6. Nanoparticle washout from porcine carotid arteries

Schematic and photograph of arteries loaded with 1.0 mg Fe/mL of msIONP compared to control (no msIONPs) in VS55 at room temperature during loading and washout (N = 2, n = 2). GRE and SWIFT MR images were acquired and the R₁ map was generated from the SWIFT data. The color bar indicates the R₁ in 1/s. Images were taken at 4 and 24 h after msIONP loading and post washout.

Fig. 7. Nanowarming maintains biomechanical properties of porcine carotid arteries

Arterial rings tested were taken from control (fresh artery) or from tissue that had been vitrified and convectively rewarmed, nanowarmed, or slow warmed. (A) Schematic illustrating how the biomechanical testing was conducted. Arteries were pulled in circumferential direction at a rate of 2 mm/min. The stress and strain was calculated by the equations shown in the figure, where F is the force measured at time t, A₀ is the initial cross sectional area of the artery ring, L₀ is the initial length of the artery, L(t) is the displacement at time t. (B) Representative stress strain curve. Elastic modulus is defined as the slope of linear region of the stress versus strain curve. The toe region is defined as the x-intercept of the stress versus strain curve. (C) Biomechanical properties of the rewarmed samples compared with fresh controls. All biomechanical properties of the rewarmed samples were normalized to control, which are fresh artery rings dissected from the same carotid artery to minimize the variances between the donor pigs. Red bars represent nanowarmed samples (elastic modulus = 98 ± 13%, toe region = 93 ± 11%, L₀ =102 ± 7%), yellow bars represent convective warmed samples (elastic modulus = 94 ± 13%, toe region = 90 ± 15%, L₀ = 99 ± 2%), blue bars represent slow warmed samples (elastic modulus = 71 ± 5%, toe region = 68 ± 16%, L₀ =120 ± 6%). All data is presented as the mean ± standard deviation from n = 4 − 7 samples from N = 2 pigs for each condition.