Increment of intracellular cAMP enhances the osmotic resistance of human polymorphonuclear cells and monocytes

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freeze etch electron microscope observations showed that there was no ice detectable extracellularly and only very small amounts detectable intracellularly in cells suspended in DAPP and quickly cooled to LN,. However, if cell suspensions in LN, were warmed rapidly to - 60°C and then held for 5 min. they showed massive amounts of both extracellular and intracellular ice. Biological data show that this devitrification was associated with complete loss of cell function. 31. Polymers Protect Monoc,ytes ,fiom Freezing Injwy through High Temperature Extracellular Glass Formation. T. TAKAHASHI, A. HIRSH,

E. E ERBE,*M. E HAMMETT,R. L.STEERE,* AND R. J. WILLIAMS (American Red Cross Biomedical Research & Development Laboratories, Bethesda, Maryland, and *Plant Virology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland). To understand the mechanism of cryoprotection by polymer solutions, we cryopreserved human monocytes in various polymer solutions without penetrating cryoprotectants. Hydroxyethyl starch (HES), dextran (MW lO,OOO-500,000). Ficoll, and polyvinylpyrroiidone (PVP) protect cells during slow freezing (2.X/ min) to -80°C at initial concentrations above 15% (w/w) only if followed by rapid warming. Under these conditions, about 60-70% of the cells retained normal membrane integrity and normal phagocytosis and chemotaxis. When cells suspended in 20% HES were extracellularly frozen, cooled to various temperatures slowly, and then plunged into LN2, survival after rapid warming increased to the maximum whenever slow cooling was extended below -20°C. By comparing freezing, osmotic stress, and calorimetric results. we have determined that cells in HES solution may in part be protected by an equilibrium glass transition of the extracellular medium at -20°C. Freeze etch electron microscope studies of monocytes frozen at 2.X/ min to -80°C in Hanks’ balanced salt solution without cryoprotectant showed that none of the cells developed intracellular ice despite 100% lethality. In contrast, 30% of cells froze massively during 2.5”Ci min cooling to -80°C in HES and 30% were also found to be dead after fast warming from that temperature. In addition, slow rewarming of cells suspended in HES to any temperature 2 - 60°C produced massive intracellular freezing in all cells and 100% lethality. This motivated the following general model of extracellular protection: (1) protective polymers allow the cells to moderately supercool between 0°C and - 2O”C, reducing osmotic stress; (2) below - 20°C. injurious water loss from the cells is eliminated by the solid amorphous solution (glass) between the extracellular ice and the cells which maintain osmotic stress at a tolerable level; (3) at - 70°C a small amount of intracellular ice forms in 70% of the cells but the viscosity of the moderately concentrated intracellular contents

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is such that, unless the system is slowly warmed through the temperature range between -60 and -2O”C, further ice growth is prevented: (4) during cooling from 0 to - 2o”C, some 30% of cells supercool considerably more than average for the population, probably due to an overly long pathlength to the nearest ice crystal. This produces massive intracellular freezing during cooling resulting in the 30% lethality seen even after fast warming. (Supported in part by NIH Grants BSRG RR05373 and HL 27537). 32. Comparison qf Firle Techniques ,for the Clyopreservation of Human Polymorphonuclear Cells.

T.TAKAHASHI,K.OHURA,* M. E HAMMETT, S. M. WAHL,~ AND E. J. BRoWNt (American Red Cross Biomedical Research & Development Laboratories, Bethesda, Maryland 20814; *Laboratory of Microbiology and Immunology, and *Laboratory of Clinical Investigation, National Institutes of Health, Bethesda, Maryland). Five selected methods (I) of cryopreservation purported to be successful for preserving human PMNs were compared. After cryopreservation, PMNs were assessed for numerical cell recovery, morphology (blister formation), fluorescein dye staining using fluorescein diacetate and ethidium bromide (FDAIEB), adhesion of latex particles, phagocytosis of bacteria, microbicidal activity, and chemotaxis. Among the five methods of cryopreservation, the method reported by Zaroulis and Leiderman resulted in the best “viability” (FDAIEB) and morphology. However, none of the methods preserved phagocytosis, microbicidal ability or chemotaxis. In regard to phagocytosis. although previously frozen cells had the capacity to adhere to particles, they lost the ability to engulf them. It was found that cryopreserved PMNs lose receptors for IgG, C3b, and chemoattractant. No difference in functional recovery was observed between cells collected with different anticoagulants (CPD vs heparin) or separated by different techniques (counterflow centrifugation elutriation vs Ficoll-Paque). We conclude that none of the reported techniques for the cryopreservation of human PMNs yields functional cells. REFERENCES 1. Absolom, D. R., and van Oss, C. J. Cvqohiology 17, 287-296 (1980), Lionetti, F. J. et a/. Cryobiology 17, 297-310 (1980), Zaroulis, C. G., and Leiderman, I. Z. Cryobiology 17, 311-317 (1980), Graham-Pole, J. et al. J. Clin. Put/lo/. 30, 758-762 (1977), and Rowe, A. W., and Cohen, E. VOX Sang. 10, 382-384 (1965). 33. Increment of Intracellular c,AMP Enhances the Osmotic Resistance of Human Poiymorphonuclear CellJ and Monocytes. T. TAKAHASHI.

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T. F. BUSBY, H. KON, AND M. E HAMMETT (American Red Cross Biomedical Research & Development Laboratories, Bethesda, Maryland). Human polymorphonuclear cells (PMNs) are quite sensitive to osmotic stress. This is believed to be the main reason for the difficulty of successful cryopreservation. For the purpose of making PMNs more resistant to the osmotic stress, we have treated cells with various chemicals including free radical scavengers and microtubule stabilizers before exposing them to the osmotic stress. Among the chemicals, theophylline, PGE,, and hydrocortisone were the most effective at improving PMNs resistance to osmotic stress. The common character among them is the ability to enhance the amount of intracellular CAMP and cell membrane permeable dibutyryl CAMP indeed made the cells more resistant. Though the increment of increased osmotic tolerance was statistically significant, it was not nearly good enough to make cells resistant to deep freezing. This explains the failure to cryopreserve PMNs treated by these chemicals reported by Ashwood-Smith and Kelley (I). PMNs enriched with CAMP could tolerate osmotic stresses of 900 and 75 mOsm, and retained both phagocytic and chemotactic abilities. CAMP may stabilize the plasma membrane in addition to the lysosomal menbrane because Fc receptors of plasma membrane were held well after osmotic stress. Using these chemicals the same effect was also observed in monocytes which are quite resistant to the osmotic stress without artificial protection.

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5°C. However, synthesis of these proteins was abruptly shut off by I day of deacclimation when freezing tolerance was declining. These results suggest that the low-temperature-induced proteins are involved or closely associated with freezing tolerance in spinach. 35. Fructure Damage to the Zonae of Mammaiiun Embryos during Crqlopreservation and Its Avoidunce. W. F. RALL AND T. K. MEYER (Rio

Vista Laboratories, Inc., Route 9, Box 242, San Antonio, Texas 78227).

Although fracture damage to the zonae pellucida and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism. Previous reports suggested that the incidence of fracture damage depended on the type of sample container and the cooling and/or warming conditions. Cryomicroscopical observations of embryo suspensions indicated that under some conditions a series of interconnecting fracture planes form in the extracellular ice matrix during rapid cooling below - 110°C(J. Reprod. Fert. 70,285 (1984)). Damage was observed on warming only in those places where the fracture planes crossed the zonae. The fracturing of samples was attributed to stresses caused by differences in the thermal expansion of the ice crystals, residual “glassy” solution and container walls during cooling. We have examined the incidence of zona damage when bovine ova with normal intact zonae are frozen by standard bovine embryo procedures. Suspensions of ova in I.5 M glycerol were placed into either IO x IOO-mmglass test tubes or 0.25-cc plastic REFERENCE insemination straws. and then frozen slowly at 0.4”Ci min to -35°C before rapid cooling in liquid nitrogen. I. Ashwood-Smith. M. J., and Kelley, N. fNSERM Ova were examined for zona damage after warming by 62, 151-156 (1976). procedures that ought to produce little or no thermal stress (slow warming in 20°C air) or high levels of 34. Indrtction ofFreezing Tolerunc~e in Spinac~h is ASstress (rapid warming in liquid baths). Ova frozen in sociuted tl,ith the Synthesis of Low Temperustraws (N = 206) exhibited no zona damage after slow twrc Indwed Proteins. C. L. GUY AND DALE HASKELL (Ornamental Horticulture, Univer- warming at -15O”Cimin. 24% (N = 159) when the straws were warmed in 20°C water (-1500”CYmin) or sity of Florida, Gainesville. Florida 3261I). 35°C water (-2000”C/min), respectively. Ova in straws When young spinach seedlings are grown at 25°C warmed in nonaqueous liquids exhibited lower rates of and then transferred to a constant low temperature zona damage: 5% (N = 184), 2% (N = IO@, and 3% (YC), they become acclimated to the cold and more (N = 100) in 20°C ethylene glycol, 20°C silicone oil, tolerant of intercellular freezing. We have studied the and 35°C silicone oil, respectively. Ova frozen in glass role of protein synthesis in the induction and loss of tubes exhibited a much higher incidence of zona freezing tolerance during cold acclimation and deac- damage than those frozen in straws, regardless of the climation. Leaf proteins were in l+vo radiolabeled warming conditions. That is, 27% of 55 ova warmed with [%]methionine at 0, 1, 2, 3. 4, 7, and 14 days of slowly at 25”Cimin exhibited damage, and 52% of 56 cold acclimation and at I, 3. and 7 days of deacclima- ova warmed rapidly in 35°C water (-5OO”Cimin) tion and subjected to 2D-gel electrophoresis. We showed zona damage. These results demonstrate that found two polypeptides were synthesized by the (I) ova frozen in straws exhibit considerably less zona second day of cold acclimation when freezing toler- damage than ova frozen in glass tubes. (2) The amount ance was also increasing. The two low-temperatureof zona damage depends on the warming conditions. induced polypeptides were continuously synthesized (3) Ova frozen in glass tubes may suffer zona damage during prolonged exposure (2 weeks) of spinach to during rapid cooling since slow warming did not pre-