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This technical paper introduces a novel organ preservation system based on isochoric (constant volume) supercooling. The system is designed to enhance the stability of the metastable supercooling state, offering potential long-term preservation of large biological organs at subfreezing temperatures without the need for cryoprotectant additives. Detailed technical designs and usage protocols are provided for researchers interested in exploring this field. The paper also presents a control system based on the thermodynamics of isochoric freezing, utilizing pressure monitoring for process control. Sham experiments were performed using whole pig liver sourced from a local food supplier to evaluate the systems ability to sustain supercooling without ice nucleation for extended periods. The results demonstrated sustained supercooling without ice nucleation in pig liver tissue for 24 and 48 h. These findings suggest the potential of this technology for large-volume, cryoprotectant-free organ preservation with real-time control over the preservation process. The simplicity of the isochoric supercooling device and the design details provided in the paper are expected to serve as encouragement for other researchers in the field to pursue further research on isochoric supercooling. However, final evidence that these preserved organs can be successfully transplanted is still lacking.

This study was motivated by the increasing interest in finding ways to preserve organs in a supercooled state for transplantation. Previous research with small volumes suggests that the isochoric (constant volume) thermodynamic state enhances the stability of supercooled solutions. The primary objective of this study was to investigate the feasibility of storing a large organ, such as the pig liver, in a metastable isochoric supercooled state for clinically relevant durations. To achieve this, we designed a new isochoric technology that employs a system consisting of two domains separated by an interior boundary that can transfer heat and pressure, but not mass. The liver is preserved in one of these domains in a solution with an intracellular composition, which is in osmotic equilibrium with the liver. Pressure is used to monitor the thermodynamic state of the isochoric chamber. In this feasibility study, two pig livers were preserved in the device in an isochoric supercooled state at -2°C. The experiments were terminated voluntarily, one after 24 h and the other after 48 h of supercooling preservation. Pressure measurements indicated that the livers did not freeze during the isochoric supercooling preservation. This is the first proof that organs as large as the pig liver can remain supercooled for extended periods of time in an isotonic solution in an isochoric system, despite an increased probability of ice nucleation with larger volumes. To serve as controls and to test the ability of pressure monitoring to detect freezing in the isochoric chamber, an experiment was designed in which two pig livers were frozen at -2°C for 24 h and the pressure monitored. Histological examination with H&E stains revealed that the supercooled liver maintained a normal appearance, even after 48 h of supercooling, while tissues in livers frozen to -2°C were severely disrupted by freezing after 24 h.

ObjectivePreservation of biological materials at subzero Centigrade temperatures, cryopreservation, is important for the field of tissue engineering and organ transplantation. Our group is studying the use of isochoric (constant volume) systems of aqueous solution for cryopreservation. Previous studies measured the pressure-temperature relations in aqueous isochoric systems in the temperature range from 0°C to - 20°C. The goal of this study is to expand the pressure-temperature measurement beyond the range reported in previous publications.Materials and methodsTo expand the pressure-temperature measurements beyond the previous range, we have developed a new isochoric device capable of withstanding liquid nitrogen temperatures and pressures of up to 413 MPa. The device is instrumented with a pressure transducer than can monitor and record the pressures in the isochoric chamber in real time. Measurements were made in a temperature range from - 5°C to liquid nitrogen temperatures for various solutions of pure water and Me2SO (a chemical additive used for protection of biological materials in a frozen state and for vitrification (glass formation) of biological matter). Undissolved gaseous are is carefully removed from the system.ResultsTemperature-pressure data from - 5°C to liquid nitrogen temperature for pure water and other solutions are presented in this study. Following are examples of some, temperature-pressure values, that were measured in an isochoric system containing pure water: (- 20°C, 187 MPa); (-25°C, 216 MPa); (- 30°C, 242.3 MPa); (-180°C, 124 MPa). The data is consistent with the literature, which reports that the pressure and temperature at the triple point, between ice I, ice III and water is, - 21.993°C and 209.9 MPa, respectively. It was surprising to find that the pressure in the isochoric system increases at temperatures below the triple point and remains high to liquid nitrogen temperatures. Measurements of pressure-temperature relations in solutions of pure water and Me2SO in different concentrations show that, for concentrations in which vitrification is predicted, no increase in pressure was measured during rapid cooling to liquid nitrogen temperatures. However, ice formation either during cooling or warming to and from liquid nitrogen temperatures produced an increase in pressure.ConclusionsThe data obtained in this study can be used to aid in the design of isochoric cryopreservation protocols. The results suggest that the pressure measurement is important in the design of "constant volume" systems and can provide a simple means to gain information on the occurrence of vitrification and devitrification during cryopreservation processes of aqueous solutions in an isochoric system.