Research ArticleNanotechnology

Improved tissue cryopreservation using inductive heating of magnetic nanoparticles

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Science Translational Medicine  01 Mar 2017:
Vol. 9, Issue 379, eaah4586
DOI: 10.1126/scitranslmed.aah4586
  • 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 CPA (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. IONP 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 comodification 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 to 1000 nanoparticles 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 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 phosphate-buffered saline (PBS) HU (n = 3 for calibration curve). (B) Photographs and CT 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, which is higher than the 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 higher than the CWR of VS55.

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

    (A) Schematic of RF heating system scale-up from 1 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 versus nanowarming on vitrification (success and failure) of cylinders with a radius of 0.5 to 2.5 cm 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 of Fe per milliliter of msIONP to the artery (n = 4 to 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 to 2 hours) (red, 89 ± 2%), samples that were exposed to VS55 (blue, 100 ± 1.4%), or samples that were exposed to VS55 and 10 mg of Fe per milliliter of 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 the slow-warmed sample showed a decline of viability compared to the 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 the 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 to 6 for both 1- and 50-ml systems; n = 4 to 7 in the 1-ml system; n = 3 to 5 in the 50-ml system. N = number of pigs; n = number of arteries. (B) Histological images of H&E-stained control and nanowarmed, convective-warmed (1 ml), and slow-warmed arteries. Scale bars, 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 are presented as the means ± SD.

  • Fig. 6. Nanoparticle washout from porcine carotid arteries.

    Schematic and photograph of arteries loaded with 1.0 mg of Fe per milliliter 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 hours after msIONP loading and after 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 were 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, and 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-strain curve. The toe region is defined as the x intercept of the stress-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%), and blue bars represent slow-warmed samples (elastic modulus, 71 ± 5%, toe region, 68 ± 16%; L0 = 120 ± 6%). All data are presented as the means ± SD from n = 4 to 7 samples from N = 2 pigs for each condition. Comparing to the control, the elastic modulus and toe region are declined (***P < 0.001) and the L0 is increased (**P = 0.0042) in the slow warmed samples.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/379/eaah4586/DC1

    Materials and Methods

    Fig. S1. Nanowarming scale-up in field and system size.

    Fig. S2. Nanowarming of cells.

    Fig. S3. Viability of carotid artery rewarmed on ice.

    Fig. S4. Quantitative analysis of H&E-stained artery cross section.

    Fig. S5. Impact of increased SAR on artery wall thickness.

    Fig. S6. Characterization of nanowarming in a 50-ml system.

    Fig. S7. Viability assessment of rewarmed heart valve leaflets in 1 ml.

    Fig. S8. Nanowarming of porcine femoral arteries in a 50-ml system.

    Fig. S9. Characterization of nanowarming in an 80-ml system (nonbiological).

    Fig. S10. Weight changes of artery rings before and after treatments.

    Table S1. Properties of CPA vitrification solutions.

    Table S2. Convective cooling and warming success and failure in cylindrical volumes.

    Table S3. Normalized viability.

    Table S4. P values for all significant findings.

    Table S5. Primary data.

  • Supplementary Material for:

    Improved tissue cryopreservation using inductive heating of magnetic nanoparticles

    Navid Manuchehrabadi, Zhe Gao, Jinjin Zhang, Hattie L. Ring, Qi Shao, Feng Liu, Michael McDermott, Alex Fok, Yoed Rabin, Kelvin G. M. Brockbank, Michael Garwood, Christy L. Haynes, John C. Bischof*

    *Corresponding author. Email: bischof{at}umn.edu

    Published 1 March 2017, Sci. Transl. Med. 9, eaah4586 (2017)
    DOI: 10.1126/scitranslmed.aah4586

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Nanowarming scale-up in field and system size.
    • Fig. S2. Nanowarming of cells.
    • Fig. S3. Viability of carotid artery rewarmed on ice.
    • Fig. S4. Quantitative analysis of H&E-stained artery cross section.
    • Fig. S5. Impact of increased SAR on artery wall thickness.
    • Fig. S6. Characterization of nanowarming in a 50-ml system.
    • Fig. S7. Viability assessment of rewarmed heart valve leaflets in 1 ml.
    • Fig. S8. Nanowarming of porcine femoral arteries in a 50-ml system.
    • Fig. S9. Characterization of nanowarming in an 80-ml system (nonbiological).
    • Fig. S10. Weight changes of artery rings before and after treatments.
    • Table S1. Properties of CPA vitrification solutions.
    • Table S2. Convective cooling and warming success and failure in cylindrical volumes.
    • Table S3. Normalized viability.
    • Table S4. P values for all significant findings.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5. (Microsoft Excel format). Primary data.

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