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There is growing interest in using isochoric freezing and isochoric supercooling for the preservation of biological matter at subfreezing temperatures. Custodiol® is a commonly used intracellular composition type, subnormothermic preservation solution. It is anticipated that Custodiol® will also be used for isochoric freezing and isochoric supercooling preservation of biological matter. The thermodynamic properties of Custodiol® at subfreezing temperatures as well as the metastable behavior of the solution at subfreezing temperatures were not studied in the past. This study was designed to generate the thermodynamic data needed for the use of Custodiol® for the preservation of biological matter in isochoric systems at subfreezing temperatures. The experiments were performed in a specially designed isochoric chamber that can measure simultaneously the temperature and pressure in the isochoric chamber, and thereby correlate pressure and temperature at thermodynamic equilibrium in isochoric systems as well as the nucleation temperature in isochoric supercooling. The primary focus of this study is on determining the temperature at which nucleation is initiated and to identify the temperature threshold for nucleation due to its specific relevance to various applications in medicine. 

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 system’s 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.