China Science

Bischof,JC; Mahr,B; Choi,JH; Behling,M; Mewes,D

Abstract The outcome of both cryopreservation and cryosurgical freezing applicationsis influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructiveagent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It alsodepends on the amount and type of crystalline, amorphous and/or eutectic phases formed duringfreezing which can differentially affect viability. This work describes the use of X-ray computertomography (CT) for non-invasive, indirect determination of the phase, solute concentration andtemperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation beforeand after freezing. Specifically, this work focuses on establishing the feasibility of CT (100-420kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPAand CDAs in unfrozen solutions and tissues at 20 degrees C, or the phase in frozen solutions andtissue systems at -78.5 and -196 degrees C. The solutions are composed of water with physiologicalconcentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco”s Modified Eagle”s Medium) with addedglycerol (0-8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cmdiameter cores which were either imaged fresh (3-4 h cold ischemia) or after loading with DMEM basedglycerol solutions (0-8 M) for times ranging from hours to 7 days at 4 degrees C. The X-rayattenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-rayattenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3%wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from0 to 155. At -196 degrees C the variation was from -80 to 95 HU while at -78.5 degrees C allreadings were roughly 10 HU lower. At 20 degrees C NaCl and DMEM solutions with 0-8 M glycerolloading show a linear variation from 0 to 145 HU. After freezing to -78.5 degrees C the variation ofthe NaCl and DMEMsolutions is more than twice as large between -90 and +190 HU and was distinctlynon-linear above 6 M. After freezing to -196 degrees C the variation of the NaCl and DMEM solutionsincreased even further to -80 to +225 HU and was distinctly non-linear above 4 M, which aftermodeling the phase change and crystallization process is shown to correlate with an amorphous phase.In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, aswell as showing some deviations at 0 M after storage, probably due to tissue swelling. The standarddeviations in all measurements were roughly 5 HU or below in all samples. In addition, two practicalexamples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue coresand, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure thepermeation of the glycerol into the sample at 20 degrees C, as well as the evolution of distinctamorphous vs. crystalline phases after freezing to -196 degrees C. In the mock cryosurgery example,the iceball edge was clearly visualized, and attempts to determine the temperature within theiceball are discussed. An added benefit of this work is that the density of these frozen samples, anessential property in measurement and modeling of thermal processes, was obtained in comparison toice.

Keywords Biocompatible Materials; Cryopreservation; Crystallography; Liver;Radiographic Image Interpretation, Computer-Assisted

Annals of Biomedical Engineering
0090-6964, Volume 35, Issue 2, 2007, Pages 3-304