The safety and therapeutic effectiveness of human red cells stored at -80 degrees C for as long as 21 years.

Human red cells frozen by various methods have been stored in the frozen state at -80 degrees C for as long as 21 years. This report discusses: red cells frozen with 42 percent weight per volume (wt/vol) glycerol in an ionic medium in a polyvinylchloride (PVC) plastic bag using the Cohn method; red cells frozen with 45 percent wt/vol glycerol in a low ionic medium in a PVC plastic bag using the Huggins method; red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a polyolefin plastic bag using the Meryman-Hornblower method; and red cells frozen with 40 percent wt/vol glycerol in an ionic medium in a standard 600-ml or an elongated 800-ml PVC plastic primary collection bag with an adapter port using the Naval Blood Research Laboratory (NBRL) method. After frozen storage for as long as 21 years by the four methods described above, the thawed red cells were deglycerolized with 50 to 150 ml of 12 percent sodium chloride and 1.5 to 2.0 l of sodium chloride-glucose or sodium chloride-glucose-phosphate solution. After washing and storage at 4 degrees C for 24 hour, the red cells had a mean freeze-thaw-wash recovery value of 90 percent, a mean 24-hour posttransfusion survival value of 85 percent, a mean index of therapeutic effectiveness of 75 percent, normal or slightly impaired oxygen transport function, and minimal hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.

We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.3% and 64.3% of biopsied and control embryos, respectively, implanted into the uterine wall, and 52.6% and 52.4% of biopsied and control embryos, respectively, developed into fetuses. In a separate series of experiments, we have demonstrated that biopsied mouse embryos can be successfully cryopreserved by ultrarapid freezing even though they have a punctured zona pellucida. Biopsied (frozen-thawed), control (frozen-thawed), and nonfrozen embryos had an implantation rate of 81.1%, 74.3%, and 74.1%, respectively, and a fetal formation rate of 62.2%, 62.9%, and 66.7%, respectively.

In situ freezing of the brain for metabolic studies: evaluation of the "box" method for large experimental animals.

The box method of freezing the brain in situ was assessed in baboons. The cooling rate of the tissue was monitored in several regions located at various depths from the skull surface. These measurements allowed us to examine the time required for the tissue to reach 0 degree C, in relation to its depth measured from the top of the skull. To define brain regions with proven ischaemia, frozen tissue sections were surveyed for areas of decreased pH. In addition, concentrations of ATP, phosphocreatine, and lactate were determined in gray matter located at various depths from the top of the brain surface. Normal tissue pH and low lactate concentration, without any significant decrease in high-energy phosphate levels, were found in regions at a depth less than approximately 10 mm from the brain surface. Deep structures including the inferiomedial aspect of the temporal lobe, the lateral geniculate body, and the limbic system (hippocampus) consistently showed mild tissue acidosis, indicating that these regions were subjected to some degree of ischaemia before they were reached by the freezing front. In some cases, acidosis was also detectable in the thalamus, basal ganglia, and in the deeper part of some sulci. We conclude that, with baboons, in situ freezing using the box method is valid for metabolic studies of the cerebral cortex and structures located at a depth less than approximately 10 mm from the top of the brain surface.

Safety considerations regarding the use of propane and other liquefied gases as coolants for rapid freezing purposes.

Liquid propane and similar coolants are used in the rapid freezing of biological specimens. These coolants form explosive gas mixtures with air, with a 14,000-fold increase in volume over that of the liquid. The liquefied gases have high vapour pressures and, unless they are maintained below their flashpoint, the vapour above them will reach ignitable concentrations. The flashpoint of liquid propane is -104 degrees C. Ethane has a higher vapour pressure, and vapour mixed with air above liquid ethane can be ignited at a coolant temperature of -130 degrees C. The danger is minimized if the coolant is maintained near its freezing point and under a nitrogen atmosphere, in a fume cupboard. Liquid nitrogen evaporates to a 690-fold increase in volume at room temperature. It is important to ventilate the working area, especially when cryo-sectioning in a small room, otherwise there is a possibility of asphyxiation.

Membrane structure in ultrarapidly frozen, unpretreated, freeze-fractured myocardium.

Ultrarapid freezing has been applied to monitor the structure of the freeze-fractured myocardial sarcolemma. Our two goals were to demonstrate that large areas of membrane can be preserved free of visible ice crystal damage and, thus, be amenable to quantitative analysis and to compare the structure of directly frozen myocardial membranes with conventionally prepared tissue. The E face was most affected by lack of chemical pretreatment. First, our laboratory reported an increase in E face particle density from 379 +/- 30/micron 2 in conventional fixed tissue to 489 +/- 18/micron 2 in unpretreated tissue. Discrete arrays of 12-15 nm particles on the E face were a striking feature of the unfixed sarcolemma. However, P face intramembrane particle (IMP) density remained unchanged from previous estimates in fixed tissue. Specialized regions of the sarcolemma were enhanced in ultrarapidly frozen tissue. Particle domains of the adherens junctions were very prominent in forming a cap alongside the gap junctions. Both the P and E faces of the gap junctions were highly ordered into hexagonal arrays. Caveolae in the membrane were infrequent in both P and E faces.

Selective killing of leucocytes by freezing: potential for reducing the immunogenicity of pancreatic islets.

Recent developments in transplantation immunobiology, concerning the mechanism of tissue rejection, clearly indicate that antigen recognition alone is not sufficient for lymphocyte activation. "Passenger" leucocytes (antigen presenting cells) carried in the donor tissue are now recognized as the major immunogenic stimulus, such that removal of these contaminating leucocytes, using a variety of procedures, has enabled the immunogenicity of allografts to be reduced and the survival of the graft to be significantly extended. Remarkable advances have been made in recent years in preventing rejection of islet allografts, and even xenografts, in experimental animals by using procedures which do not involve continuous immunosuppressive therapy. Cryopreservation offers not only the means by which donor tissue can be stored effectively during such procedures but also the possibility that, under appropriate conditions, the freezing and thawing process itself could modulate tissue immunogenicity by allowing the selective killing of immunocompetent leucocytes whilst preserving the function of parenchymal cells in the graft. In this preliminary study we have characterized the survival of leucocytes and islets from the same species (rat) after cryopreservation by the same technique using dimethyl sulphoxide (Me2SO) as the cryoprotectant. Optimum survival of rat lymphocytes and macrophages was found at cooling rates in the range of 0.3-5 degrees C/min, after cooling at rates greater than 75 degrees C/min, survival was reduced to a negligible level. On the other hand, recovery of islets was 73 +/- 9% at 75 degrees C/min, indicating that depletion of lymphoid cells, with satisfactory preservation of endocrine cells, should be obtainable at this cooling rate.

Recobrado del F VIII: C en el crioprecipitado; efecto del ph de la solución anticoagulante y el tiempo de descongelación / Recovery of F VIII: C in cryoprecipitate; effect of ph of anticoagulant solution and melting time

Se estudió el efecto de los anticoagulantes ACD-A y CPD así como los métodos de descongelación (rápida y lenta) sobre el recobrado de F VIII: C en el crioprecipitado. El recobrado fue siempre menor cuando se empleó el ACD-A y la descongelación lenta, metodologías utilizadas hasta ahora en nuestros bancos de sangre para la obtención de crioprecipitado. Este recobrado puede incrementarse hasta el 285

Cryopreservation of hematopoietic progenitor cells collected by hemapheresis.

Colony-forming units-granulocyte/monocyte (CFU-GM) harvested from normal donors as a byproduct of plateletapheresis can be cryopreserved successfully with 10 percent dimethyl sulfoxide with the use of standard protocols that have been developed for freezing bone marrow. Short-term storage of CFU-GM in polypropylene vials in the liquid phase of liquid nitrogen yielded a recovery rate of 88 +/- 5 percent (mean +/- SEM). Significant loss of committed progenitor cells was not detected until 1 year after freezing (65 +/- 5%). A comparison of CFU-GM recovery with the polyolefin bag technique (107 +/- 13%) was not statistically different from that obtained with polypropylene vials (78 +/- 7%). Although the freezing bags are more expensive and prone to fracture than the vials, they are easier to handle, store, and thaw in the laboratory, and reinfusion to the patient is more convenient. Cryopreservation of CFU-GM harvested from peripheral blood appears feasible in either of two freezing systems.

Acquired secretion defect in platelets after cryopreservation in dimethyl sulfoxide.

Despite the use of preservatives, platelets are severely damaged during cryopreservation and, following freezing, function poorly in a number of in vitro tests. We report here that cryopreserved platelets show diminished aggregation in response to collagen. This may be a consequence of a secretion defect as evidenced by a 20 to 30 percent loss of dense- and alpha-granule content (p less than 0.05) and an impaired secretion mechanism. Analysis of adenine nucleotides confirmed the defect in dense granule adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content (storage pool), but in addition revealed a 50 percent fall in cytosolic ATP (metabolic pool). In contrast, the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate), was normal. We concluded that platelet cryopreservation leads to a secretion defect, probably as a result of activation during freezing and thawing procedures.

A simple method for controlled freezing of human donor corneae.

A simple method for freezing cultured human donor material with dimethyl sulfoxide (DMSO) was developed empirically in a commercially available microcomputer-controlled apparatus allowing reproducible freezing curves to be made. The optimum cooling rate was found to be 1.0 degrees C/min down to -40 degrees C and then 5.0 degrees C/min to -80 degrees C. Total cell loss rates were 36.9 +/- (SD) 19.8% of postmortem endothelial cell counts. An improved method for evaluating endothelial viability was applied, which enabled us to observe definite cell survival after a postculture period.

[A brief review of the literature for deep-freezing cattle embryos]. / Een beknopt literatuuroverzicht voor het diepvriezen van runderembryo's.

Low temperature preservation of bovine embryos is briefly reviewed. The following subjects are discussed: the desired developmental stage of the embryo, the packaging system during freezing, the cryoprotective agents, the freezing and thawing methods and the results made possible by freezing bovine embryos.

[Cryogenic preservation of erythrocytes by a droplet freezing technic].

The droplet freezing technique has two major advantages: firstly the good recovery of frozen erythrocytes with low concentration of cryoprotectants, and secondly the simplification of the post-thaw treatment. However, this technique has not been utilized for blood preservation for transfusion due to difficulties of the sterile freeze-thawing operation. In this study, the technique was modified to perform the freeze-thawing in a closed system under sterile conditions. An apparatus with a new type of nozzle was devised for this purpose. Fifty milliliters of packed red blood cells mixed with an equal volume of additive solutions were frozen in liquid nitrogen in droplet form (0.7 mm in diameter). The frozen droplets were collected into an aluminium container (18 X 18 X 0.5 cm) and thawed by immersing the container into a water bath at 47 degrees C. After thawing, the red cells were washed with either normal saline or hypertonic sugar solutions and resuspended in normal saline. Although the washing procedure involving centrifugation was necessary at least once to remove free hemoglobin and fragile cells produced during freeze-thawing, the post-thaw treatment of the modified droplet freezing was much more simple than that of ordinary bulk freezing technique. Approximately 85% of the frozen cells were recovered with cryoprotective additive solutions consisting of 0.4-0.5 M sugar (maltose or glucose) and 0.4-0.5M glycerol in 0.03 M sodium chloride. The osmotic fragility and ATP level of the post-thawed red cells were little changed in comparison with that of unfrozen cells. These results suggests a possibility of this technique for practical application in the future.

[The morphological and functional integrity of cryo-preserved leukocytes]. / Untersuchungen zur morphologischen und funktionellen Integrität kryokonservierter Leukozyten

Human peripheral blood lymphocytes were frozen by a simplified method which is described. Light and electron microscopic investigations of the frozen-thawed cells showed no significant morphological changes compared to the fresh cells; comparison of E-rosette formation and nonspecific acid esterase activity before and after cryoconservation afford evidence of a possible slight change in the percent relationship of T- and B-cells, whereas thymidine uptake values within normal range after stimulation with PHA and homologous cells demonstrate the functional integrity of the lymphocytes.