The storage period of cryopreserved red blood cells (RBCs) is 10 years by regulation both in Japan and United States. The quality of RBCs frozen with a high glycerol concentration at -85℃ for as long as 20 years was studied. The post-thawed deglycerolized red blood cells (DRBCs) were resuspended either in saline or mannitol-adenine-phosphate (MAP) solution. The DRBCs were stored at 4±2℃ for up to one week, and value of adenosine triphosphate (ATP), 2, 3-diphospho-glycerate (2, 3-DPG), morphology scores, methemoglobin and P_<50> were measured at the time of 0, 4,12, 24, 72 hour (s) and one week after storage. The results of the DRBCs showed that the quality were maintained in good conditions. There was no significant difference from liquid preserved RBCs in MAP solution stored for 24 hours. After one week of post-thawed storage, the DRBCs resuspended in MAP solution showed better results than that of resuspended in saline. There was no significant difference from liquid preserved RBCs in MAP solution stored at 2 to 3 weeks. These results indicated the possibility of extending the period of frozen RBCs at least up to 20 years and, extending the storage period of DRBCs resuspended in MAP solution up to one week.

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To develop platelet substitutes, we prepared paraformaldehyde-fixed and lyophilized human platelets according to Read et al. Although rehydrated and lyophilized platelets (RL platelets) lost their ability of collagen-dependent aggregation, ristocetin-dependent aggregation remained 90% of intact platelets. Flow cytometric analysis demonstrated that membrane glycoprotein (GP) I b and GP III a were present on the surface of RL platelets. GMP-140, a marker of platelet acivation, was also detected. Our results showed that the RL platelets may substitute adhesion function, i.e., an important function of platelets to stop bleeding at injured vessel wall, but slight activation occurred during preparing them. We suggest that RL platelets may be hopeful as a platelet substitute.

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Cryopreservation of human placental/umbilical cord blood (PCB) is required for PCB banking. We collected and separated PCB following the New York Blood Center's protocol and cryopreserved them in liquid nitrogen (LN_2). Red cells were sedimented by addition of 1% hydroxyethyl starch with gentle centrifugation. Mean recovery of nucleated cells (NC) and colony-forming units (CPU) was 82.9% and 90.4%, respectively (n=25). Volume of the NC suspension was further reduced to 20 ml by centrifugation and dimethyl sulfoxide and Dextran40 solution were added slowly to a final concentration of 10% and 1%, respectively. Those units were cryopreserved in a -80℃ mechanical freezer slowly, and transferred to LN_2 storage tank. Thawing of PCB was done rapidly, and the frozen cells were washed with a solution containing 10% Dextran 40 and 5% human serum albumin. NC and CPU recovery was 88.8 and 81.2%, respectively. Thus, the HES/slow cooling method has proved to be quite useful for cell processing of cord blood. In the Tokyo Cord Blood Bank, we separate, concentrate and cryopreserve cord blood nucleated cells following this method, and the method seems to have become the standard method for other local cord blood banks in Japan.

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Placental/umbilical cord blood is a new source for hematopoietic stem cells that can be used for bone marrow reconstitution. More than 600 cases of unrelated allogeneic cord blood transplantation have been performed worldwide, and cord blood banks have been established in United States, Europe and Asia. In order to secure the safety and efficacy of cord blood transplantation, it is important to maintain not only the good recovery of cell number but also viability of hematopoietic progenitor cells. Cryopreservation is necessary for cord blood banking and that is one of the important processes to produce quality controlled products. In this study, we analyzed the characterization of cord blood progenitor cells during freezing and thawing by differential scanning calorimetry.

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Freezing behavior of xylem ray parenchyma cells in several hardwood and softwood species that inhabit in cold areas changes from supercooling in summer to extracellular freezing in winter. It was suggested that the seasonal changes in the freezing behavior were due to changes of the cell wall properties as a result of seasonal cold acclimation, although at ultrastructural level little changes of the cell walls were detected among seasons. The property of the cell walls may also have important roles for occurrence and/or inhibition of freezing injury in plant cells by extracellular freezing, in which close apposition of membranes as a result of cellular shrinkage and cellular deformation produces injury, because cellular deformation is mainly produced by deformation of cell walls. For understanding more precisely about the mechanisms of freezing adaptation of plant cells, it is necessary to clarify changes of the cell wall properties by cold acclimation.

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Spin-lattice relaxation times (T_1) and spin-spin relaxation times (T_2) of water in fig (Ficus carica L.) fruit with ripening were determined by inversion recovery (180°-τ-90° pulse sequence) method and CPMG method using ^1H-NMR, respectively. Spectral recovery observation showed that a spectrum in fig tissues consisted of several spectra having different chemical shifts and recovery times with ripening. Furthermore, semi-log plots of signal intensity of ^1H-NMR revealed that water in the tissues consisted of three water components with T_1 values ranging about below 0.1s (component I), between 0.6s and 1.0s (component II), and over 1.2s (component III), respectively. Furthermore, T_2 values showed long fraction ranging over 0.1s and short fraction. The highly mobile water could be estimated as free water derived from vacuoles, while water with restricted mobility was as loosely bound water and bound water from cytoplasm and the apoplastic region, respectively. In mature receptacle tissues especially, component n disappeared but component III appeared as major fraction with significant water uptake in the tissues. Disruption of the apoplastic region in receptacle tissues of mature fig fruit seems to promote increase in free water and decrease in bound water with cell enlargement. These results indicated that water compartments reflected water absorption and cellular heterogeneity in both receptacle and drupelet tissues.

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Transitional changes in cell volume under the external osmotic stress was measured by dielectric method to determine the water permeability of the plasma membrane, Lp, which were 0.015, 0.020, and 0.090, 0.033 [pm/(s・Pa)] at 25℃, for rice (Oryza saliva L. ssp. japonica), grape (Vitis sp.), CH27, and yeast (Saccharomyces cerevisiae) cells, respectively. The rate of water removal under osmotic stress was determined both by Lp and the cell diameter, D. The combined parameter, Lp/D was calculated to be 0.73, 0.58, 6.4 and 5.5 [10^<-9> (s・Pa)^<-1>] for rice, grape, CH27, and yeast cells, respectively. The smaller Lp/D for plant cells corresponds to the slower water removal under the freeze-induced osmotic stress, which seemed to explain the difficulty of plant cells in freeze preservation as compared with microorganisms and animal cells.

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The amount of bound water in yeast cells is estimated by TG (thermogravimetry) -DTA (differential thermal analysis) measurements. It is shown that when the trehalose content is beyond 2-3% (w/w), the amount of bound water is drastically decreased and simultaneously the viability of dried cells is increased. This suggests that the major part of bound water is replaced by trehalose. In addition, by the measurement of NMR spin-lattice relaxation time (T_1) for the intracellular water protons, it is shown that extra trehalose (> 3-4%) acts as a water structuring agent in yeast cells. In conclusion, such a dual role is essential in endowing yeast cells with high water-stress resistance.

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Glass-forming composition regions were determined by a simple DTA method for the aqueous glycerol solutions mixed with a second solute additive. The second solute additives used were DMSO, sucrose, trehalose, maltose, glucose, fructose, KCl, CH_3COOK and KH_2PO_4. It is shown that glass-forming composition region is expanded to the lower glycerol concentration side with addition of a second solute. The effect of solute addition on the Te value of the resultant solution is rather small.

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Double crystallizations during warming observed with the certain compositions of NaCl-glucose-water system solution, were investigated by X-ray diffraction. From the results a lower temperature crystallization is identified as ice formation. Viscosities of the ternary system solutions were measured at 30℃. Viscosities of NaCl-glucose-water system solutions are higher than those of KCl-glucose-water system solutions in the same compositions.

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Water in polymer gels remains sometimes unfrozen during cooling to turn into a glassy state, and to crystallise during subsequent warming. As change in the polymer network on freezing is seen to be a primary cause for the occurrence of a glass transition, and both freezing and drying are dehydration processes from around polymers, observation of structural change of crosslinked dextran gel beads was conducted during absorption/desorption or freezing by using an environmental SEM to investigate their dependency on the crosslink density. It was confirmed by the observation that the polymer gel beads with lower crosslink density are ready to change their bead structure during drying and freezing, and that they are more flexible than those with higher crosslink density.

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