Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

Influence of various cooling and warming temperatures on survival after thawing of cryopreserved Plasmodium berghei

Heavy concentrations of viable P. berghei in the natural milieu [20% ($\text{v}\text{v}$) parasitized red blood cells, or 20% ($\text{w}\text{v}$) homogenate of splenic tissue in which malarial cells sequestered wer suspended in a serum-free, protective medium. Various rates of cooling are designated as low (1.3 °C/min) and intermediate (4 °C/ min) on exposure in cold gas evolving from liquid nitrogen refrigerant to ?70 °C, and this followed by direct immersion in the low temperature refrigerant (?196 °C). Cooling designated high was accomplished by abrupt immersion of the sealed vials with the live malaria-bearing tissue in the liquid nitrogen refrigerant. Rates of warming and thawing were designated low (after slow rewarming of frozen tissue in air at 25.5 °C) and high (after rapid rewarming and thawing in a water bath at 40 °C). Strip chart recordings were made of the complete cooling and freezing wave patterns of the suspending medium to ?70 ° C. The functional survivals of the freeze-thaw P. berghei malaria were measured by a special infectivity titration method.None of the cooling and freezing treatments adversely influenced the parasite survivals. Our data showed the optimum cooling velocity that maximally protected this highly lethal P. berghei strain within the host erythrocyte matrix was 1.3 ° C/min to ?70 to ?196 ° C. The functional survivals of two RBC stabilates with P. berghei, after retrieval from 25 days storage in the liquid nitrogen refrigerant, excelled by more than 100-fold the infectivity titer found by viability assay in the pool of the 0-days nonfrozen infected RBC.The precise factors favoring the maximal survivals of the freeze-thaw P. berghei are unclear. Several factors, singly or in combination, may have played key roles in protecting the living P. berghei from the freeze-thaw damage. These factors are: The composition of the suspending medium fortified by additions of bicarbonate, glucose, lactalbumin hydrolysate and yeastolate; the presence of naturally occurring peptide-containing materials surrounding the parasites in the host red cell milieu; and the protective glycerol agent. Any of these constituents singly or combined possess potential for reducing freeze-thaw injury to the parasites to produce maximal survivals.     

Liquid scintillation counting of aqueous samples using Triton-containing scintillants

Markedly unstable count rates were observed using a toluene-Triton (2:1, $\text{v}\text{v}$) scintillant during counting of water-soluble radioactive compounds when < 5% ($\text{v}\text{v}$) water was present, because of the separation of phases. Efficiency correction in these instances could not be made by using ³H₂O as internal standard, because under the same conditions count rates with tritiated water were stable. Increasing water to ≥6% stabilized the count rates. With toluene-Triton (2:1, $\text{v}\text{v}$) scintillant, the water level should preferably be maintained between either 6 and 12 or 18 and 24% for ¹⁴C- and ³H-labeled compounds for counting at 6°C or at ambient temperature (but only between 6 and 12% for ³H counting at room temperature). With a “Tritosol” (Anal. Biochem.63, 555 (1975) modified to contain 35 ml of ethylene glycol, 140 ml of ethanol, 250 ml of Triton X-100, 575 ml of xylene, 3 g of PPO, and ±200 mg of POPOP, water levels of up to 23% were acceptable for ¹⁴C and ³H for counting at room temperature or at 6°C. Within these limitations, with the toluene-Triton or with the modified Tritosol as scintillant, both polar and apolar radioactive compounds exhibited similar efficiencies and gave quench-correction curves, based on the external standard ratio, that were linear for both ¹⁴C and ³H-labeled compounds.     

Histological studies on cultured canine heart valves recovered from −196 °C

Adult canine heart valves have been frozen to ?196 °C, (0.5 to 0.7 °C/min from 0 to ?100 °C) with 10% DMSO ($\text{v}\text{v}$), stored, thawed at ~150 °C/min, and then cultured for 9 to 12 days. A histological analysis of sections derived from several valves indicates viability, but with a not inconsiderable loss of stromal fibrocytes and some damage to the endothelial lining. The practicality of freezing valve tissue for banking will have to be looked at critically, before valve transplants can be considered as a possible alternative to the well established use of mechanical valve prosthesis. However, demonstrating viability of heart valve tissue extends the range of tissues that are amenable to cryopreservation.     

Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, v_j, within the lipid matrix. At T = 35°C a value of v_j = 1.6 · 10³ s^?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 10⁸ s^?1 and 1.5 · 10⁸ s^?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{min at}\text{T = 37°}\text{C}$) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 100}\text{min at}\text{T = 37°}\text{C}$.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.     

Some combined effects of hypertonic solutions and changes in temperature on posthypertonic hemolysis of human red blood cells

The combined effects of hypertonic solutions and temperature changes on the posthypertonic hemolysis of human red blood cells have been investigated. Cells were exposed to hypertonic solutions of sodium chloride and also to hypertonic solutions of the extracellular cryoprotective additive sucrose, such as would occur during the freezing of cells in an isotonic salt solution to which 15% $\text{w}\text{v}$ sucrose had been added. In both cases the extent of posthypertonic hemolysis was increased by temperature reduction per se when the osmolality of the extracellular solution exceeded about 1400 mOsm/kg water. The posthypertonic hemolysis of cells exposed to a hypertonic solution at 0 °C was reduced with the temperature of the resuspension solution up to 35 °C.     

Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.     

Survival of electrical activity of deep frozen fetal mouse hearts after microwave thawing

Hearts removed from 17–19 day fetal mice were frozen in liquid nitrogen and tested for electrical activity after rewarming. After exposure to various cryoprotective agents, hearts were cooled at 0.5–0.7 °C/min. to ?100 °C and then stored in liquid nitrogen for periods between 72 and 216 hr. Exposure to controlled microwaves at 2450 MHz or immersion in a water bath at 25 C was used in thawing. Electrical activity was studied for periods as long as 90 days after subcutaneous implantation into the ear of syngeneic adult mice. Overall, 59% of 54 frozen-thawed fetal hearts showed strong electrical activity after 30 days when the cryoprotective solution that had been used contained 10% ($\text{v}\text{v}$) dimethylsulfoxide (DMSO) and 10% ($\text{v}\text{v}$) fetal calf serum in Hepes buffer. This system consists of a multicellular structure that is nourished by diffusion; it is well suited for the evaluation of different cryoprotective agents and for various thawing techniques.     

Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates

Callow L. L. and Farrant J. 1973. Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates. International Journal for Parasitology, 3: 77–88. An investigation of the optimal conditions for the preservation of Leishmania tropica var major by freezing was undertaken because it was important to obtain a high yield when the thawed organisms were cultured. As a prerequisite for comparing different conditions, assay methods were devised. One method was based on the reduced growth of promastigotes in diphasic medium that was found to follow inoculation of relatively few organisms. The other employed serial ten-fold dilutions of suspensions of organisms, and the inoculation of medium at each dilution stage. Viability was related to the time taken for flagellates to be found in the medium. A 1·0 m solution of glycerol in the flagellate suspension inhibited growth when diphasic medium was inoculated. This effect was removed by separating the organisms from the glycerol before inoculation, or by diluting the suspension approximately one hundred-fold. A similar inhibition was not observed for dimethylsulphoxide (DMSO). Glycerol (1·0 m), DMSO (1·5 m), polyvinylpyrrolidone (10% w/v) and sucrose (0·3 m) were not obviously detrimental to the organisms. Normal growth in diphasic medium resulted when these additives were removed after being in contact with organisms for 5 h in an ice bath. In freezing experiments, flagellates survived freezing and thawing while they were still in contact with their nutrient medium, and also after they had been separated, washed and resuspended in isotonic saline with 10 mm of glucose. The survival rate was much greater when either 1·5 m DMSO or 1·0 m glycerol was added. These additives were compared at one rate only, $\text{0·3°C}\text{min}$, and DMSO gave better protection. With 1·5 m DMSO, maximal survival was obtained at a cooling rate of $\text{1·9°C}\text{min}$. Relatively high rates of cooling, that is, over $\text{400°C}\text{min}$ were detrimental to the organisms.     

Alteration of Mycobacterium phlei membrane structure by freezing and thawing: reversal by heating.

Membrane vesicles from Mycobacterium phlei which contain the electron transport chain, when subjected to freezing to ?70 °C followed by slow thawing, exhibited a decreased level of phosphorylation coupled to the oxidation of substrates. This loss in oxidative phosphorylation was restored following heat treatment (50 °C for 10 min) of the membranes. Freeze treatment (?70 °C for 10 min) of membrane vesicles also resulted in a decrease in membrane bound coupling factor-latent ATPase activity. The soluble coupling factor(s) or cryoprotective agents (i.e., glycerol or dimethyl sulfoxide) were found to protect the membrane vesicles from the effects of freezing. Membrane vesicles depleted of particulate bound coupling factor were sensitive to exposure to low temperatures; however, complete protection was afforded by the addition of coupling factor. In addition, prolonged sonication of electron transport particles resulted in lowered $\text{P}\text{O}$ ratios, and heat treatment of these sonicated particles restored $\text{P}\text{O}$ ratio. Therefore, it appears that the effects of heat treatment and freeze treatment on membrane vesicles are reversible. The steady state level of reduced cytochrome b was considerably higher (40%) in heat-treated electron transport particles as compared to untreated particles (28.5%); electron transport particles subjected to freeze treatment showed a lower steady state level of cytochrome b (16.6%) as compared to electron transport particles. The steady state level of cytochrome b in freeze-treated particles returned to the original level (27.5%) for electron transport particles when subjected to heat treatment. Nevertheless, the total amount of enzymatically reducible cytochrome b was the same for all membranes after subjection to the various types of treatment. In contrast to cytochrome b, the reduced steady state levels of cytochrome c, and a + a₃ were not altered by heat or freeze treatment.     

Effect of protected protein supplements on some testicular traits in Brahman cross bulls

Differences in a number of testicular traits were examined in 12 Brahman cross (F₂ generation $\text{1}\text{2}$ and $\text{3}\text{4}$ BX) bulls fed either poor-quality native pasture (NP) hay or NP hay with a protected protein supplement. Supplementation for 60 days significantly (P < 0.05) increased roughage dry matter intake (7.7 $\text{v}$ 5.6 kg/head/day), enabling maintenance of liveweight, whereas control animals lost 40 kg. There were significant (P < 0.05) decreases in scrotal circumference (1.5cm) and testicular consistency (0.8 score) in the control group, in which testes weights at slaughter were significantly (P < 0.05) less (373 g $\text{v}$ 459 g), with corresponding lower epididymal weights (37.4 g $\text{v}$ 43.5 g). Estimates of daily sperm production per gram (DSPG) were similar for both groups, and testis daily sperm production (DSP) was somewhat but not significantly (P>0.05) lower in the control group (4.3 × 10⁹$\text{v}$ 6.0 × 10⁹) as a result of lower testis weights. Total epididymal sperm storage capacity was also lower in control bulls (17.2 × 10⁹$\text{v}$ 27.0 × 10⁹), but only significantly (P < 0.05) in relation to cauda sperm reserves (8.5 × 10⁹$\text{v}$ 13.6 × 10⁹). Luteinizing hormone (LH) and testosterone responses to gonadotrophin releasing hormone (GnRH) treatment were similar for both groups, although LH responses to GnRH were greater in $\text{1}\text{2}$ BX than in $\text{3}\text{4}$ BX bulls.     

Kinetics of monensin complexation with sodium ions by 23Na NMR spectroscopy

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by ²³Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na⁺ + Mon^? ? MonNa. The dissociation rate constant at 25°C is 63 s^?1, with an activation enthalpy of 10.3 $\text{kcal}\text{mol}$ and activation entropy of ?15.8 $\text{cal}\text{mol}$ deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.