023 Adaptation of a commercial diathermy machine for radiofrequency warming of vitrified organs

Abstract

Electromagnetic warming of vitrified tissue is superior to external conduction for rapidly and uniformly traversing the temperature zone of maximum ice growth where risk of devitrification is greatest. The temperature zone where previously-nucleated ice crystals grow most rapidly typically spans tens of degrees Celsius below the melting temperature, above which ice cannot exist, and below which ice growth is kinetically inhibited by viscosity. The frequency used for electromagnetic warming should be chosen to couple well to the cryoprotectant solution in this temperature zone, while coupling poorly at higher temperatures. Such a frequency will cause rapid passage through the ice growth temperature zone and then cause warm spots to loiter above the melting temperature while colder parts of the organ catch up, thereby avoiding thermal runaway. For the M22 vitrification solution that has been used for rabbit kidney vitrification in our laboratory, the temperature zone of rapid ice growth is approximately −75 °C to −60 °C. The solution viscosity in this temperature zone is very high, with a Debye dipole relaxation frequency on the order of 10 MHz. This suggested an electromagnetic warming frequency much lower than the hundreds of megahertz typically studied in cryobiology. A Magnatherm 1000 (International Medical Electronics, Ltd.) diathermy machine providing ∼200 W of RF power at 27 MHz was modified for dielectric warming of 20 mL samples. The diathermy treatment head consisting of a resonant series capacitor and wire coil for induction heating was disassembled and modified by replacing the capacitor with a custom fabricated sample holder composed of two opposed aluminum plates (5 cm × 4 cm curved plates making a cylindrical space of 4.5 cm diameter and 4 cm height between them), and by adding delay lines and variable inductance for tuning purposes. The Magnatherm control console conveniently includes a net forward voltage display that allowed manual tuning of the variable inductance during sample warming without any other instrumentation. Samples of vitrification solution, or rabbit kidneys in vitrification solution, were placed in 20 mL glass vials or 20 mL polyethylene specimen containers of 3 cm diameter surrounded by foam insulating sleeves of 1.25 cm thickness. Temperatures during warming were measured by a Luxtron 790 Fluoroptic Thermometer, and temperature uniformity after warming was assessed by measurements at multiple positions inside and outside kidneys using a thermocouple needle probe. M22 solutions used during temperature uniformity measurements were gelled by the addition of 5% hydroxyethyl cellulose to inhibit convection during warming. During warming of vitrified 20 mL samples of M22 in LM5 (low ionic carrier solution), a peak warming rate of 160 °C/min was observed at −55 °C (the melting point), declining to only 20 °C/min at −25 °C. Warming time between −90 °C and −40 °C was 30 s. At the end of warming the coldest point in the solution was −28 °C (bottom) and warmest point was−25 °C (middle). Vitrified kidneys were found to warm approximately half as fast as solutions of the same volume, with temperatures of 39 °C in the medulla and −21 °C in the cortex after 3 min of warming from −100 °C. Source of funding: This research was supported by 21st Century Medicine, Inc. and University of Seville. Conflict of interest: None declared. wowk@21cm.com