Survival of frozen-thawed human red cells as a function of the permeation of glycerol and sucrose

Previous studies have demonstrated that glycerol does not have to permeate bovine red cells to protect them against subsequent freezing and thawing. The present study is concerned with the relation between solute permeation and freezing injury of human red cells. Cells were held in 2 m glycerol for 30 sec to 10 min at 0 °C and then frozen to ?196 °C at 60 °C/min. Cells cooled at this rate have a very low probability of undergoing intracellular freezing. Percent survivals (≡percent unhemolyzed) increased by 21% (from 66 to 80%) over the first 3-min period. Extrapolation to zero time (and zero glycerol permeation) yields a survival of 57%. Between 30 sec and 3 min the calculated osmolal ratio of intracellular glycerol to other solutes increased 240% (from 2.5 to 5.7). The human red cell is impermeable to sucrose at 0 °C. Cells suspended in 1.40 m sucrose (equiosmolal to 2.0 m glycerol) for 0.5 to 10 min prior to freezing yielded as high survivals after thawing as did cells in glycerol.These data indicate that prior permeation of additive is not a prerequisite for the survival of red cells subjected to subsequent freezing and thawing. Although sucrose and glycerol protect equally well to this point, differences appear when attempts are made to remove the additive. Over 90% of the cells survive the removal of glycerol. Only some 30% survive the removal of sucrose. Cells frozen in an equisomolal solution of sodium chloride do not even survive the initial freezing and thawing.The findings indicate that slow freezing injury cannot be accounted for in terms of the attainment of a critical minimum volume, nor can it be considered to be equivalent to posthypertonic hemolysis.     

Survival of frozen-thawed fetal rat pancreases as a function of the permeation of dimethylsulfoxide and glycerol,warming rate,and fetal age

It was reported several years ago that frozen-thawed fetal rat pancreases can reverse diabetes when transplanted into afflicted rats. The cooling rate was shown to be critical on the basis of an in vitro assay involving the incorporation of isotopic amino acids into protein. The present paper reports experiments concerned with the influence of other cryobiological variables on the in vitro survival of frozen-thawed fetal rat pancreases (permeation of additive, warming rate, and dilution rate) and experiments concerned with the influence of the age of the fetal pancreases at the time of freezing.Warming rate and dilution rate had no observable effect on the survival of 17-day fetal pancreases, but the permeation of additive was critical. Survival of 17-day fetal pancreases was near zero when they were less than half equilibrated with glycerol, but it approached 100% when they were fully equilibrated. Our interpretation of these data is that permeation of the intercellular space is not sufficient to protect; permeation of additive into the intracellular space is required.Also critical was the age of the fetal pancreas. Survival of 18- and 19-day fetal pancreases after freezing and thawing (~30 and 10%) was far lower than that of 17-day fetal pancreases (~100%). The increased sensitivity of the older organs may be associated with their much greater size.     

Cryopreservation of human peripheral blood stem cells: optimal cooling and warming conditions

Peripheral blood stem cells are being used to reconstitute human bone marrow function after ablative therapy of blast transformation of chronic myelogenous leukemia. Studies were undertaken to establish the optimum cooling and warming conditions of the preservation of colonyforming activity in the peripheral blood of patients with CML.The results show that maximum recovery of CFU-c activity occurs after cooling at 3 °C/min, an average of 50% better than the recovery following cooling at 1 °C/min. CFU-c recovery decreased with decreasing warming rate, but high recovery was obtained with warming rates as low as 10 °C/ min. Viable cell count did not correlate with CFU-c recovery, therefore it represents a poor index for quality control.These results suggest that for clinical purposes bulk samples in flat bags with high surface area to volume ratios, frozen at a rate of 3 °C/min and thawed as rapidly as possible, should give maximum recovery of stem cell activity.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)     

The shape of red blood cells as a function of membrane potential and temperature

Summary It is well known that a pH shift of the outside medium from 5 to 9 produces a shape transformation of washed human red blood cells from stomatocytes to echinocytes in isotonic salt solutions. In addition, a stomatocytogenic effect is demonstrated here due to solutions of low ionic strength (below 70mm). An analysis of the true cell state in these situations, proved by measurements of predicted volume changes, indicates a good correlation between transmembrane potential and cell shape. The fact that amphotericin B acts as echinocytogenic agent in low ionic strength medium at pH 7.4 but not at pH 5.1 underlines this explanation. Therefore, a transmembrane potential positive inside produces stomatocytes, slightly negative inside (below–10 mV), normocytes, and strongly negative, echinocytes. The temperature dependence of this process underlines the rigidity-pattern hypothesis of red blood cell shape (Glaser & Leitmannová, 1975, 1977).     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Computation of the average shear-induced deformation of red blood cells as a function of osmolality

A model was developed for computing the average deformation of red cells as a function of suspending medium osmolality. It assumes a population of red cells characterized by a single value for surface area and for isotonic volume, but having a Gaussian distribution in mean intracellular hemoglobin concentration (MCHC). The ability of cells of a given hemoglobin concentration to deform is assumed to be limited by either the amount of redundant surface area available or the intracellular viscosity, determined by the intracellular hemoglobin concentration. The surface area limitation is calculated by finding the dimensions of a prolate ellipsoid having the volume and surface area of the red cell. The viscosity limitation is incorporated in two ways. First, the ratio of intracellular to extracellular viscosity must lie below a certain threshold to permit deformation, and second, its magnitude determines the extent of cell elongation. This model gave a reasonable fit to experimental data for a threshold viscosity ratio close to 1. Extension to cell populations for which either mean cell hemoglobin concentration or surface area had been modified also provided a close reproduction of the experimental curves.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

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.     

Autoxidation as a cause of altered lipid distribution in extracts from human red cells

A characteristic alteration in the distribution of human red cell phospholipids represents an artifact due to autoxidation of the lipid extract. This alteration is manifested on silicic acid chromatography by a decrease mainly in the phosphatidyl ethanolamine and phosphatidyl serine fractions (probably because of their abundance of highly unsaturated fatty acids) and an increase in the phospholipid recovered with the more polar fractions, sphingomyelin and lysolecithin. No evidence was found for "lysocephalin" formation or plasmalogen breakdown in dry lipid extracts after autoxidation by exposure to air at room temperature for 24-35 hr. On thin-layer chromatography, however, the ninhydrin-positive streaking in the autoxidized samples may be erroneously attributed to the presence of "lyso" derivatives. When the alterations in lipid distribution described above are found, the possibility of this artifact should be considered.     

Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle

The relative roles of cooling and warming rates on cell survival during a freeze-thaw cycle were investigated. Basically the faster the warming rate, the better the cells survive. One of the factors influencing this is the extended phase transition period at the slower thawing rates. The warming rate had a significant effect on cell damage and recovery, but this was not as great as comparative changes in the cooling rate were. This investigation also showed that under certain freeze-thaw conditions there was a lack of correlation between the two methods used for quantifying cell recovery (RI) and cell damage (PCR) as measured by radiochromate release. The analysis of the relationship between RI and PCR showed that PCR could be used to measure both lethal and nonlethal damage and enabled a clearer interpretation of cellular damage during cooling and thawing to be made.     

Viability of human diploid cells as a function of in vitro age

The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.