Pressure in isochoric systems containing aqueous solutions at subzero Centigrade temperatures

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.

研究目标
在零下摄氏度温度下保存生物材料（即低温保存（cryopreservation））对于组织工程与器官移植领域具有重要意义。本团队正研究等容（isochoric，恒容）水溶液体系在低温保存中的应用。既往研究已在0℃至-20℃的温度范围内测定了等容水溶液体系的压力-温度关系。本研究的目标是将压力-温度测量范围拓展至既往文献报道的范围之外。

材料与方法
为将压力-温度测量范围拓展至既往范围之外，我们研发了一款新型等容装置，可耐受液氮温度及最高413 MPa的压力。该装置配备压力传感器，可实时监测并记录等容腔体内的压力。本研究针对纯水及不同浓度的二甲基亚砜（Me2SO）溶液开展了测量，测量温度范围为-5℃至液氮温度，其中二甲基亚砜是一种用于冷冻状态下生物材料保护及生物物质玻璃化（vitrification，即形成玻璃态结构）的化学添加剂。实验过程中会仔细移除体系中未溶解的气体。

结果
本研究呈现了纯水及其他溶液在-5℃至液氮温度范围内的温度-压力数据。以下为等容体系中纯水的部分实测温度-压力值示例：(-20℃, 187 MPa)；(-25℃, 216 MPa)；(-30℃, 242.3 MPa)；(-180℃, 124 MPa)。该数据与文献报道的冰I相、冰III相与水三相点的压力及温度（分别为-21.993℃与209.9 MPa）相符。令人意外的是，在三相点以下的温度区间，等容体系内的压力会升高，并在液氮温度区间仍维持较高水平。针对不同浓度的纯水与二甲基亚砜溶液开展的压力-温度关系测量结果显示：对于理论上可发生玻璃化的浓度体系，在快速冷却至液氮温度的过程中未检测到压力升高；但在冷却过程中，或在升降温至液氮温度并返回的过程中出现结冰现象时，均会引发压力升高。

结论
本研究获取的数据可用于辅助等容低温保存方案的设计。研究结果表明，压力测量对于“等容”体系的设计具有重要意义，同时可为等容水溶液体系低温保存过程中玻璃化与反玻璃化的发生情况提供一种简便的信息获取手段。