Ken Muldrew, Kelli Novak, Chris Studholme, Greg Wohl, Ronald Zernicke, Norman S. Schachar, and Locksley E. McGann. Transplantation of Articular Cartilage Following a Step-Cooling Cryopreservation Protocol. Cryobiology, 43(3), 260-267, November 2001.
Abstract
Using a step-cooling cryopreservation protocol that held the tissue 60 min at -4 degrees C, 30 min at -8 degrees C, and 10 min at -40 degrees C before plunging into liquid nitrogen, we were able to get a substantial improvement in the magnitude and pattern of chondrocyte recovery following cryopreservation, achieving postthaw recoveries of 62 +/- 13%. These results are consistent with the hypothesis that ice growth within articular cartilage is planar, but they provide no direct support for that hypothesis. Transplanting (step-cooled) cryopreserved osteochondral allografts into adult Suffolk/Romanoff crossbred sheep for periods of 3 months and 1 year further tested the efficacy of the cryopreservation protocol. Unfortunately, the cryoinjury sustained by the chondrocytes during cryopreservation, although apparently nonlethal immediately after thawing in many cases, was not innocuous in the long term. The presence of large clusters of chondrocytes at 1 year after transplantation illustrates that cryoinjury not detectable with a membrane integrity assay can still have far-reaching effects on transplanted tissue.
Studholme, C.V., McGann, L.E. Phase Diagram Prediction for Solutions of Interest in Cryobiology. Advances in Cryogenic Engineering, 41, 47-54, 1996.
Abstract
Simulation of osmotic responses in cells and tissues at low temperatures requires descriptions of the composition of the extracellular solution and the osmotic responses of cells to the changing environment. The principles of irreversible thermodynamics have been used to describe osmotic water and solute flow across plasma membranes as the temperature and environment change. Insufficient experimental data is available for the empirical descriptions of phase diagrams now used to describe the composition of the extracellular solution. In order to describe the general phase behavior in binary mixtures of interest in cryobiology, a set of equations were derived from the Gibb’s free energy equation, using changes in enthalpy and entropy upon unmixing and freezing. The parameters required are: the molecular weights, molecular volumes, van der Waals gas constant, b, applied to liquids, and a solubility parameter, d. Where data was available, the excess enthalpy of mixing was used to determine d, otherwise parameters were derived from least-squares fitting to experimental phase diagram data from the literature. Predictions fitted experimental phase diagram data very well (within 0.1K in some cases) from 0C down to the eutectic point for solutions with various cryoprotectants. These equations, based on thermodynamics, provide an analytical tool for simulation and optimization of procedures used in cryopreservation, and can be generalized to mixtures with an arbitrary number of components.