The freezing and preservation of bovine erythrocytes in liquid nitrogen

Bovine erythrocytes can be preserved for long periods of time by freezing and storing in liquid nitrogen. Blood group determinations, titrations, and isoimmunizations indicated that there were no detectable alterations in antigenic reactivities of preserved erythrocytes when compared with fresh samples of blood from the same animal. In addition, consistent percentages of recovery within a frozen mixture could be obtained, indicating that the deterioration of erythrocytes with time was not significant. Variations in the percentage of recovery of erythrocytes with different concentrations of sucrose (the cryoprotective agent) were significant at the 0.01 level of probability. The highest average percentages of recovery of erythrocytes from whole blood and the cellular fraction of blood (blood from which plasma was removed before freezing) were obtained with 45 and 40% sucrose, respectively. Results also indicated that whole blood gave a slightly higher percentage of recovery, than the cellular fraction. The individual donor effect, storage time in liquid nitrogen, and various saline concentrations of the thawing and washing solutions had no significant effects upon percentage of recovery.     

Preservation of erythrocytes by freezing in liquid nitrogen. Use of an I.B.M. blood regenerator]

The freezing of blood permits preservation of red cells over long periods of time, several months or years. Leucocyte and platelet contamination of red cell concentrates to be frozen is negligible. The amount of the various red cell metabolites (2.3 D.P.G., A.T.P., etc.) is maintained. Washing of thawed red cells removes the remaining plasma proteins and cell residues. The freezing method employed is that of Row et al. The protector used is 28% glycerol added in equal amounts to red cell concentrate to be frozen. The blood bag is kept in liquid nitrogen at -- 196 degrees C. Thawing takes place in a water bath at 45 degrees C. Wash solution is the IBM Blood regenerator. The solution used for removing glycerol is hypertonic natrium chloride. The following parameters have been investigated: --hemoglobin level; --osmotic fragility; --the amount of 2.3 D.P.G.; --residual glycerol after thawing; and clearance of leucocytes and platelets following each step of the protocol. Preliminary data regarding these features and therapeutic efficiency of processed blood are satisfactory.     

Interaction between advancing ice fronts and erythrocytes. Mechanism of erythrocyte destruction upon freezing and influence of cryoprotective agents.

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) are excluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells are engulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated.     

Mechanism of freezing injury to erythrocytes: Effect of initial cell concentration on the post-thaw hemolysis

It has been previously reported that the post-thaw hemolysis of erythrocytes, frozen under various conditions, depends upon the initial cell concentration; increasing the cell concentration decreases the proportion of intact cells after freeze-thawing. In the present study, the effect of cell concentration upon post-thaw hemolysis, examined mainly by the morphological observation of freezing patterns in specimens with or without cryoprotectant glycerol, was most marked in concentrated cell suspensions in which the cells had become shrunken as a result of extracellular freezing. The addition of glycerol lessened the packing effect progressively as the concentration was increased. The results thus obtained may be explained by assuming that cells, deformed in the freezing process, and rigid at low temperatures, might undergo mechanical damage when subjected to compression and abnormal contact.     

Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

High-presssure shift freezing. Part 2. Modeling of freezing times for a finite cylindrical model

A comprehensive vision of the heat transfer process involved in high-pressure shift freezing (HPSF) is shown in comparison to the process at atmospheric pressure. In addition, a mathematical model to predict the freezing times is presented. This model takes into consideration the dependence of the thermophysical properties relating to temperature and pressure and the supercooling reached by liquid water at atmospheric pressure after adiabatic expansion in the HPSF process. Experimental and theoretical data appear to agree.