Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol

Polymer has been used as substitute to replace glycerol for cryopreservation of red blood cells (RBCs). But polymer can not penetrate cell membrane, it can not efficiently protect the inner membrane. In this study, RBCs were incubated with glucose, fructose, galactose or trehalose and frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. The postthaw quality was assessed by RBC hemolysis, RBC morphology, PS distribution, osmotic fragility, and the 4 °C stability. The results indicated the loading efficiency of monosaccharide was significantly higher than that of trehalose. Adding trehalose and 40% dextran caused more serious hemolysis before freezing. The percent hemolysis of RBCs loaded with high concentration of trehalose was approximately 16% and significantly more than that of RBCs loaded with glucose (approximately 5%, P < 0.05). Intracellular trehalose can not increase the postthaw recovery of RBCs compared with cells frozen without sugar. However, low concentration of intracellular glucose or galactose can reduce the percent hemolysis to less than 5% and significantly less than that of RBCs frozen without sugar (P < 0.05). Finally, the ability of galactose or fructose to maintain the 4 °C stability was significantly more than that of glucose. In conclusion, the injuries caused by trehalose loading may directly lead to postthaw hemolysis and poor quality of RBCs. However, monosaccharide can enhance the recovery of frozen RBCs. The cryoprotective effect of galactose may be better than that of glucose or fructose. In the future, we will continue to look for a safe and efficient trehalose loading process and try to decrease the osmotic fragility of RBCs frozen with polymers and sugars.     

Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes

Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.     

海藻糖载入红细胞及其冷冻干燥的实验研究

冷冻干燥保存是长期保存人体红细胞的理想方案之一。冻干保护剂海藻糖渗入细胞内后,对细胞膜和细胞内物质有保护作用,其中的一个作用是增加细胞质的浓度,使冻干过程容易形成稳定的玻璃态。应用高渗法处理红细胞,通过考察胞内海藻糖含量、红细胞冻干后的存活率、腺苷三磷酸酶(ATPase)、超氧化物歧化酶(SOD)活力以及细胞形态变化,研究胞内海藻糖含量对红细胞冻干后活性的影响。结果显示:海藻糖对红细胞冻干具有明显的保护作用,随胞内海藻糖浓度升高,其保护性能逐渐增强;43.8mmol/L的胞内海藻糖浓度对红细胞保护最好,细胞存活率达到53.6%,形态保持良好,ATP和SOD活力均在正常的范围内。     

Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation

One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs.RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 °C or 37 °C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 °C was relatively innocuous; however, oxidative injury was evident at 37 °C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 °C, but seemed to have exacerbated the severity of cellular injury at 37 °C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation.The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 °C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.     

The Effect of Polymeric Nanoparticles on Biocompatibility of Carrier Red Blood Cells

Red blood cells (RBCs) can be used for vascular delivery of encapsulated or surface-bound drugs and carriers. Coupling to RBC prolongs circulation of nanoparticles (NP, 200 nm spheres, a conventional model of polymeric drug delivery carrier) enabling their transfer to the pulmonary vasculature without provoking overt RBC elimination. However, little is known about more subtle and potentially harmful effects of drugs and drug carriers on RBCs. Here we devised high-throughput in vitro assays to determine the sensitivity of loaded RBCs to osmotic stress and other damaging insults that they may encounter in vivo (e.g. mechanical, oxidative and complement insults). Sensitivity of these tests is inversely proportional to RBC concentration in suspension and our results suggest that mouse RBCs are more sensitive to damaging factors than human RBCs. Loading RBCs by NP at 1:50 ratio did not affect RBCs, while 10–50 fold higher NP load accentuated RBC damage by mechanical, osmotic and oxidative stress. This extensive loading of RBC by NP also leads to RBCs agglutination in buffer; however, addition of albumin diminished this effect. These results provide a template for analyses of the effects of diverse cargoes loaded on carrier RBCs and indicate that: i) RBCs can tolerate carriage of NP at doses providing loading of millions of nanoparticles per microliter of blood; ii) tests using protein-free buffers and mouse RBCs may overestimate adversity that may be encountered in humans.     

Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.     

Antioxidant Status and Susceptibility of Sickle Erythrocytes to Oxidative and Osmotic Stress

The purpose of this study was to determine if differences in antioxidant status between the red blood cells (RBCs) of sickle cell anemia (SCA) patients and controls are responsible for the differential responses to oxidative and osmotic stress-induced hemolysis. Susceptibility to hemolysis was examined by incubating oxygenated and deoxygenated RBCs at 37°C with 73 mM 2,2' azobis (2-amidinopropane) HC1 (AAPH), a peroxyl radical generator, for up to 3.5 hours. The ability of RBCs to maintain membrane integrity under osmotic stress was determined over a range of diluted saline-phosphate buffer. Sickled RBCs showed a lesser degree of AAPH-induced hemolysis than control groups and were more resistant to osmotic stress-induced hemolysis. SCA patients had higher levels of RBC vitamin E and RBC lipids, but lower RBC GSH, plasma lipids and plasma carotenes than those of the hospital controls. No significant differences were observed in the levels of retinol, vitamin C, vitamin E, MDA and conjugated dienes in plasma, or the levels of MDA and conjugated dienes in RBCs. The results obtained suggest that the differences in antioxidant status between sickled RBCs and controls do not appear to be responsible for their different susceptibility to oxidative or osmotic stress-induced hemolysis observed.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.     

Effect of hydration on the water content of human erythrocytes.

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.     

Intracellular delivery of trehalose into mammalian cells by electropermeabilization

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.