M.W. Scheiwe

Basic investigations on the freezing of human lymphocytes

Human lymphocytes were frozen at constant cooling rates in the range 2.4 to 1000 degrees K/min without cryoadditive on the cold stage of a thermally defined cryomicroscope. The volume loss due to water efflux was quantified optically for the cooling rates 2.4, 12, 48, and 120 degrees K/min. The likelihood of the formation of intracellular ice was determined as function of the cooling rate. Intracellular crystallization temperatures were obtained for ice formation during both cooling and rewarming. A theoretical analysis of the cell volume loss during freezing was compared to the experimental data and used for an indirect determination of the water permeability of the cells. A relative optimum of the cooling rate is predicted theoretically under the assumption of a critical level of intracellular salt concentration near the eutectic temperature. The dependence of survival and cooling rate was determined cryomicroscopically by simultaneously applying the FDA/EB fluorescence viability test. The optimal cooling rate of about 35 degrees K/min was also found for 2-ml samples frozen within the range of cooling rates of interest. The results show that for freezing in physiological saline solution (1) the optimum of the cooling rate is theoretically predictable, (2) cryomicroscopical data are significant for freezing of samples of larger volume, and (3) the lethal type of intracellular crystallization is cooling rate dependent and distinguishable from innocuous types.

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Abstract Red blood cells were frozen in small capillaries down to −196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration CHES reduces the optimum cooling rate Bopt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.

The cryoprotective properties of hydroxyethyl starch investigated by means of differential thermal analysis.

Abstract Phase transition temperatures of the water-rich part of the H 2 O-NaCl-HES ternary system have been studied by means of differential thermal analysis. The experimental data indicate that the protective action of the macromolecular agent HES is different from that of DMSO and glycerol which seems essentially to be due to colligative properties. As expected, there is no evidence of a significant freezing-point depression caused by HES concentrated up to 40 wt%. The occurrence of a ternary eutectic point may therefore be excluded. Instead, an isothermal eutectic trough is observed. The extrapolated course of its projection onto the basal composition triangle indicates that a certain portion of water is absorbed by HES and kept from freezing, i.e., appears to be thermally inert within the range of temperatures studied. The protective action of HES against solution effects, therefore, is attributed to its water-absorptive capacity and kinetics instead of a postponement of lethal salt enrichment to lower temperatures as caused by DMSO or glycerol. Consequently, it is possible to determine a minimum initial HES concentration that completely prevents lethal salt enrichment during cooling. For the case of red blood cells, the derived algebraic expression yields an initial HES fraction of 11 wt% if the respective values are inserted.

A cryomicroscope for the analysis of solute polarization during freezing

Abstract A better understanding of the freezing process in the extracellular suspension medium implies the consideration of deviations from equilibrium, i.e., unsteady diffusion of heat and mass with a moving phase boundary. Such phenomena, especially solute redistribution in front of the advancing phase interface, can readily be investigated with a special cryomicroscope equipped with a spectrophotometer. A major advantage of this method is the combination of quantitative measurements in conjunction with visual observations, allowing a control of the solid-liquid interface morphology (planar-cellular-dendritic) which is crucial to the solidification process. The freezing stage designed for this purpose produces a temperature field in the sample layer resembling that within a large plate-shaped container, and hence well-defined thermal gradients (having a dominant effect on the shape of the interface). Aqueous solutions of NaMnO 4 , exhibiting a maximum absorption at 525 nm and a phase diagram as well as diffusive properties very similar to NaCl in water, turned out to be a particularly suitable model for simulating of solidification of biological solutions. As long as freezing is unidimensional (planar), the concentration profiles can be scanned on-line, while multidimensional (cellular, dendritic) structures require off-line densitometric determination from photomicrographs. The experimental results agree quite well with mathematical models for both types of solidification. The observed transition points between planar freezing and higher-order structures correspond to those resulting from constitutional supercooling, a criterion roughly indicating the conditions for interface instability based on temperature and concentration gradients at the phase boundary.

Thermally defined cryomicroscopy and thermodynamic analysis in lymphocyte freezing

A cryomicroscope is described which provides the possibility of quantifying the volume loss of cells during freezing, detection of intracellular ice formation during cooling and warming, as well as the determination of viability as function of (constant) cooling rates. The basic mechanisms occurring in cryopreservation have been studied with this system using the human lymphocyte suspended in pure saline as a biological model system; experimentally observed exosmosis during freezing is compared to predictions from a thermodynamic model. Cell volume loss during freezing has been determined experimentally for cooling rates of 2.4, 12, 48, and 120 degrees K/min. Exosmosis also was calculated corresponding to various assumptions regarding the concentration dependence of the hydraulic permeability of the cells. Further calculations of exosmosis are performed for determining the effects of the initial cell volume. The temperatures and transition cooling rate ranges of intracellular ice formation have been determined. On the basis of exosmosis and a lethal level of intracellular salt concentration, a hypothetical relative optimum of the cooling rate is theoretically predicted and compared to the experiments.

Fc receptors and human T lymphocytes in frozen-thawed peripheral blood cells

The present study was carried out to investigate the influence of cryopreservation on human T-cell subsets defined by their membrane receptors for Fc IgM (TM) and Fc IgG (TG) and by their membrane antigens. For this purpose isolated T cells, obtained by neuraminidase-treated sheep erythrocyte (E-N) rosetting, and enriched mononuclear cells were cryopreserved using a programmed freezing procedure. A significant decrease of the TM and TG cells was found whereas the proportion of T cells and their subsets determined by monoclonal antibodies seemed not to be influenced. The effectiveness of T-cell separation by E-N rosetting of frozen lymphocytes demonstrated no impairment of the E-receptor binding capacity of T cells. The PHA reactivity of separated T cells was maintained after cryopreservation; however, the spontaneous blastogenesis was reduced significantly. The selective loss of the TM and TG cells seemed to be dependent on the length of the phase transition time; over 90 sec the capacity of the expression of Fc receptors was profoundly affected. Neither an additional 20 hr incubation after hypotonic shock prior to cryopreservation nor incubation after thawing could repair this function of T cells. The data suggest irreversible damage of the Fc receptor expression capacity on the cell membrane as a result of a disturbance of metabolic pathways rather than a preferentially greater sensitivity of these cells to cryopreservation.

The Loss of Intracellular Water during Freezing in Presence of Hydroxyethyl Starch

According to Mazur’s two-factor-hypothesis (Mazur 1965), the kinetics of the loss of intracellular water during freezing play a governing role concerning the survival of biological cells in suspensions exposed to low temperatures. If cooling is too slow or too fast, the cell may either be damaged by osmotical effects or intracellular ice formation, respectively, while an intermediate range of cooling rates generally yields a relative maximum in the survival curve. The absolute value of the corresponding “optimal” cooling rate is closely related to the hydraulic membrane permeability (being the limiting factor in volume shrinkage), and hence varies — in some cases orders of magnitude — from one particular type of cell to another (Rapatz et al., 1968). This qualitative interpretation of freezing injury has become widely accepted, and Mazur was also the first to give a quantitative description of the shrinkage of the cell volume due to the osmotically induced water loss resulting from freezing (Mazur 1963). In the meantime, the model has been picked up by many authors who modified and refined it in order to take into account particular aspects (e.g. Ling and Tien, 1969; Mansoori, 1975; Silvares et al., 1975; Levin et al., 1978; Hua et al, 1982). More recently, experimental results obtained by means of cryomicroscopy have been correlated with the model (e.g., Knox and Diller, 1978; Diller, 1982; Scheiwe and Korber, 1982 and 1983). The agreement is not always satisfactory, among other reasons possibly because of the rather rough approximation of the extracellular medium obeying phase diagram conditions during the freezing process while considerable deviations from equilibrium have been shown experimentally (Korber et al.,1982 and 1983).