The fundamentals of isochoric freezing and its role in the cryopreservation of biological materials.

摘要

Efforts to preserve tissues and organs for transplantation are limited by the time they can be preserved for transport. However, traditional isobaric cryopreservation (preservation of biological materials at subzero °C temperatures and constant pressure), while indispensable for biological and medical applications, has limitations with many types of biological materials. The results of this report suggest that isochoric (constant volume) cryopreservation, which takes advantage of the thermodynamics of water solidification, is a promising new concept for long term preservation of certain biological cells and tissues. Verification studies were performed on the isochoric pressure vessel and showed that the experimental data from the device and method correlate well with the fundamental thermodynamic analyses. Further, isochoric preservation was shown to be an effective method for determining the pressure-temperature phase diagrams of various solutions. Cell preservation experiments in the isochoric chamber showed that while mammalian cells and E. Coli survived well as temperatures as low as -15°C and 159MPa after an exposure time of 120 minutes, their survival dropped (and precipitously so, for E. Coli) at a temperature of -20°C and pressure of 200 MPa, suggesting that a pressure/temperature threshold causing irreversible damage occurs in this range. In the isochoric pressure vessel yeast decreased in volume with increased pressure, and became irreversibly damaged at pressures beyond 100 MPa, while LDH enzymes was found to be deactivated at temperatures and pressures as low as -10°C and 100 MPa, which indicates that it would be an unsuitable measure of tissue health following preservation in the isochoric vessel. Overall, these results suggest that isochoric preservation is a good option for the cryopreservation of cells and tissues, particularly as these studies were performed without the use of any type of cryoprotectants, and that future studies should focus on optimizing the conditions under which isochoric preservation is carried out.