Cryopreservation of human platelets with propane-1,2-diol

The preceding papers in this series have described techniques that permit the introduction and removal of propane-1,2-diol (propylene glycol, PG) with human platelets, in concentrations up to 2 M, without producing serious damage. These methods have now been used in attempts to cryopreserve platelets, with assessment of survival by the hypotonic stress response and ADP-induced aggregation. PG concentrations of 0.5, 1.0, 2.0, and 2.5 M and cooling rates between 0.4 and 100 degrees C/min were studied. The maximum response in the hypotonic stress test was no better than 17% and the greatest ADP-induced aggregation was only 6%; these results were obtained with 0.5 M PG, a cooling rate of 14 degrees C/min, and rapid warming (approximately 150 degrees C/min). The failure of PG concentrations greater than 0.5 M to improve survival was unexpected. When cooling was interrupted at progressively lower temperatures and function assessed, it was possible to relate the extent of damage to temperature and then, with the aid of phase diagrams, it was possible to show that, irrespective of the initial concentration of PG, the extent of damage was closely correlated with the concentration of PG produced at the minimum temperature used. It is concluded that the toxicity of PG increases so steeply with the increasing concentration produced by the separation of ice during freezing that this effect is sufficient to counteract the cryoprotective action of this solute for platelets.     

Propane-1,2-diol as a potential component of a vitrification solution for corneas

Any method of cryopreservation of the cornea must maintain integrity of the corneal endothelium, a monolayer of cells on the inner surface of the cornea that controls corneal hydration and keeps the cornea thin and transparent. During freezing, the formation of ice damages the endothelium, and vitrification has been suggested as a means of achieving ice-free cryopreservation of the cornea. To achieve vitrification at practicable cooling rates, tissues must be equilibrated with high concentrations of cryoprotectants. In this study, the effects of propane-1,2-diol on the structure and function of rabbit corneal endothelium were studied. Corneas were exposed to concentrations of propane-1,2-diol ranging from 10 to 30% v/v in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 hr. Exposure to 10-15% v/v propane-1,2-diol was well tolerated for 20 min at 4 degrees C when the cryoprotectant was removed in steps or by sucrose dilution. However, exposure to 25% v/v propane-1,2-diol for 20 min at 0 or -5 degrees C was consistently tolerated only when 2.5% w/v chondroitin sulfate was included in the vehicle solution. Exposure to 30% v/v propane-1,2-diol was harmful at -5 and -10 degrees C. The endothelial damage following exposure to 30% v/v propane-1,2-diol was probably the result of a toxic effect rather than osmotic stress. Although 25% v/v propane-1,2-diol does not vitrify at cooling rates that are practicable for corneas, it could at this concentration form a major component of a vitrification solution comprising a mixture of cryoprotectants.     

Corneal tolerance of vitrifiable concentrations of propane-1,2-diol

The merit of corneal cryopreservation by vitrification as opposed to conventional freezing is the avoidance of ice damage which is believed to disrupt the integrity of the corneal endothelium resulting in loss of corneal transparency. The cornea must be equilibrated with high concentrations of cryoprotectant in order to achieve vitrification at practicable cooling rates. In an earlier study, corneas were exposed to 3.4 mol/liter propane-1,2-diol (Rich and Armitage (1990) Cryobiology 27, 42-54). The present study exposed rabbit corneas to concentrations of propane-1,2-diol between 3.4 and 5.4 mol/liter in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, 6% (w/v) bovine serum albumin, and 2.5% (w/v) dextran sulfate. Dextran sulfate was as effective as chondroitin sulfate at improving endothelial tolerance of 3.4 mol/liter propane-1,2-diol. This beneficial effect may be linked to the polyanionic nature of these molecules. Corneas exposed to 5.4 mol/liter propane-1,2-diol were cooled in liquid nitrogen vapor at a temperature of -140 degrees C for 2 h. Warming was achieved by direct transfer to a dilution solution at -10 degrees C. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed by specular microscopy during the perfusion and by scanning electron microscopy after perfusion. Corneas tolerated exposure to 3.4 mol/liter propane-1,2-diol for 20 min at 0 degrees C and to 4.1 mol/liter for 10 min at -10 degrees C. Exposure to 4.8 and 5.4 mol/liter for 10 min at -10 degrees C caused endothelial damage, although a degree of endothelial function was retained. Function following exposure to 5.4 mol/liter was improved by reducing the temperature of exposure to -15 degrees C. Corneas cooled after exposure to 5.4 mol/liter propane-1,2-diol for 10 min at -15 degrees C apparently vitrified, but devitrified on warming. The corneas swelled to such an extent during perfusion that the endothelium could not be viewed by specular microscopy, subsequent scanning electron microscopy showed a severely disrupted endothelium.     

Permeation of glycerol and propane-1,2-diol into human platelets

The permeability of human platelets to glycerol (GLY) and propane-1,2-diol (propylene glycol, PG) has been determined by measuring the time course of their change in volume following abrupt immersion in solutions of these solutes. A simple light-scattering method, and its calibration to measure mean platelet volume is described. The data are analyzed by means of the Kedem-Katchalsky (K-K) equations, modified to take into account the nonideal behavior of both intracellular and extracellular solutes. The values of the K-K parameters at 2, 21, and 37 degrees C, respectively, were as follows: the hydraulic conductivities (Lp) were 1 x 10(-7), 7 x 10(-7) and 3 x 10(-6) cm.sec-1.atm-1; the solute permeabilities for PG (omega RTPG) were 1.9 x 10(-6), 2.8 x 10(-5), and 1.3 x 10(-4) cm.sec-1; the solute permeabilities for GLY (omega RTGLY), at 21 and 37 degrees C only, were 2.6 x 10(-7) and 1.4 x 10(-6) cm.sec-1. The reflection coefficient (sigma) was 1 throughout. The relevant activation energies were -Lp, 16.5 kcal.mol-1; omega RTPG, 20.5 kcal.mol-1; and omega RTGLY, 17.9 kcal.mol-1. The use of these data is illustrated by computing schedules for the addition and removal of GLY and PG so that the amplitudes of changes in platelet volume are held within predetermined limits.     

Introduction and removal of cryoprotective agents with rabbit kidneys: assessment by transplantation

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.     

The potential of an equimolar combination of propane-1,2-diol and glycerol as a vitrification solution for corneas

Corneas must first be equilibrated with multimolar concentrations of cryoprotectants if the formation of ice during cryopreservation is to be avoided by vitrification at practicable cooling rates. Rabbit corneas were exposed to equimolar mixtures of the cryoprotectants propane-1,2-diol and glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring its ability to control stromal hydration during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial morphology was observed by specular microscopy during perfusion and by scanning electron microscopy after perfusion. Endothelial pump activity and structural integrity of the endothelial layer were demonstrated after 20 min exposure at 4 degrees C to a total concentration of 1.4 mol/liter cryoprotectant (i.e., 0.7 mol/liter propane-1,2-diol + 0.7 mol/liter glycerol). Exposure to 2.0 and 3.4 mol/liter cryoprotectant for 20 min at 4 degrees and -5 degrees C, respectively, resulted in initial endothelial damage; but this repaired and a functioning endothelial pump was subsequently demonstrated. Although exposure to 4.1 mol/liter cryoprotectant for 10 min at -10 degrees C caused irreparable damage to 2/4 corneas, reduced dilution temperatures together with increased dilution time allowed exposure to 4.8 and 5.5 mol/liter cryoprotectant with retention of endothelial pump activity. Exposure to 6.1 mol/liter cryoprotectant for 10 min at -15 degrees C caused endothelial damage which was not mitigated by the presence of 2.5% w/v chondroitin sulfate. Endothelial function may be improved by further modification of addition and dilution protocols or by exposure to the cryoprotectants at lower temperatures.     

Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.     

Effects of four cryoprotectants in combination with two vehicle solutions on cultured vascular endothelial cells.

The necessary first step in successful organ cryopreservation will be the maintenance of endothelial cell integrity during perfusion of high concentrations of cryoprotective agents (CPAs). In this report we compare the effects of incubation on cultured porcine endothelial cells at 10 degrees C for 1 h with the CPAs glycerol, dimethyl sulfoxide (Me2SO), ethanediol (EG), and propane-1,2-diol (PG) in the vehicle solutions RPS-2 (high potassium, high glucose) and HP-5NP (low potassium, high sodium), both with and without added colloids. Tritiated adenine uptake and acid phosphatase estimation of cell number were used as indicators of cell viability. HP-5NP was superior to RPS-2 except with Me2SO when the differences in viability were not significant. Adding Haemaccel to HP-5NP improved the results, but adding albumin to RPS-2 was of no significant benefit. Osmotic stress appeared to be the major problem with glycerols use. Beyond 3.0 M the toxicity of Me2SO increased dramatically but it could not be determined if this was osmotic or chemical toxicity. PG was remarkably well tolerated to 3.0 M but a sharp decrease in cell viability beyond this concentration suggests that PG may be most useful with mixtures of other CPAs. Overall, EG appeared to be the least toxic CPA and in the context of vascular preservation warrants further investigation.     

Ferrocene Compounds XXXVI. Lipase-catalyzed Enantioselective Acetylation of Prochiral 2-(ω-Ferrocenylalkyl)propane-1,3-diols

Enantioselective acetylation (desymmetrization) of prochiral 2-(ferrocenylmethyl)propane-1,2-diol (1), 2-(2-ferrocenylethyl)propane-1,2-diol (2) and 2-(3-ferrocenylpropyl)propane-1,2-diol (3) into chiral monoacetates [(+)-4-(+)-6], with a series of microbial lipases in benzene at 27°C, revealed the lipase from Pseudomonas sp (PSL) as the most selective. Acetylation was fastest and most enantioselective for conversion 1→(+)-4 by PSL (97% e.e.). By comparison of the compounds (+)-4-(+)-6 with their benzene analogues of the known (R) absolute configuration, on the basis of their elution orders on Chiracel OD, and the same direction of their optical rotations, an R-configuration is proposed for (+)-monoacetates 4-6.     

Ferrocene Compounds XXXVI. Lipase-catalyzed Enantioselective Acetylation of Prochiral 2-(ω-Ferrocenylalkyl)propane-1,3-diols

Enantioselective acetylation (desymmetrization) of prochiral 2-(ferrocenylmethyl)propane-1,2-diol (1), 2-(2-ferrocenylethyl)propane-1,2-diol (2) and 2-(3-ferrocenylpropyl)propane-1,2-diol (3) into chiral monoacetates [(+)-4-(+)-6], with a series of microbial lipases in benzene at 27°C, revealed the lipase from Pseudomonas sp (PSL) as the most selective. Acetylation was fastest and most enantioselective for conversion 1→(+)-4 by PSL (97% e.e.). By comparison of the compounds (+)-4-(+)-6 with their benzene analogues of the known (R) absolute configuration, on the basis of their elution orders on Chiracel OD, and the same direction of their optical rotations, an R-configuration is proposed for (+)-monoacetates 4–6.     

The effects of 1,2-propanediol as a cryoprotectant on the freezing of mouse oocytes

Cryopreservation of mammalian eggs has been successfully accomplished using 1,2-propanediol (PG). Effects of holding times of 0 and 30 min at -40 degrees C and storage times of 1 d and 1 mo at -196 degrees C were investigated in combination with various concentrations of PG (1.0, 1.5, and 2.0M) to determine the survival and fertilizability of mouse oocytes rapidly frozen and thawed in straws. A rapid one-step dilution using 0.5 M sucrose solution inside the straws was used following the thawing of oocytes. A significant effect of PG concentration was found between 1.0 M and 1.5 or 2.0 M (P<0.01), but no significance was discovered between 1.5 M and 2.0 M (P>0.05) on subsequent survival and fertilizability of frozen and thawed mouse oocytes. With 2.0 M PG, the best survival rate (58.3%) and fertilizability rate (19.0%) were obtained by holding at -40 degrees C for 30 min and by storage at -196 degrees C for 1 d. Thirty minutes of holding at -40 degrees C reduced oocyte damage during the procedure but not significantly (P>0.05). In addition, there was no significant difference in the various storage periods (P>0.05). This study demonstrated that mammalian oocytes can be cryopreserved in the presence of 1,2-propanediol by utilizing a rapid freezing and thawing procedure.     

Antioxidative phenylpropanoids from berries of Pimenta dioica

A phenylpropanoid, threo-3-chloro-1-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol, was isolated from the berries of Pimenta dioica together with five known compounds, eugenol, 4-hydroxy-3-methoxycinnamaldehyde, 3,4-dimethoxycinnamaldehyde, vanillin and 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol. In addition, the stereochemistry of 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol was determined. The phenylpropanoids inhibited autoxidation of linoleic acid in a water-alcohol system.     

Perfusion of rabbit kidneys with solutions containing propane-1,2-diol

Propane-1,2-diol (propylene glycol, PG) permeates more rapidly than glycerol, has a strong glass-forming tendency, and appears to have a low toxicity. It is therefore attractive as a potential cryoprotectant for renal preservation. In this paper we compared the effect on subsequent function, of exposing rabbit renal cortical slices to 1 M PG or glycerol in a range of vehicle solutions and we demonstrated a remarkably low toxicity of PG at this concentration. Rabbit kidneys were then perfused with solutions containing PG up to a maximum concentration of 3 M, after which the cryoprotectant was removed and the function of cortical slices prepared from the perfused kidneys was assessed. Marked differences in perfusion characteristics were found between PG and glycerol and between different vehicle solutions for PG, but the two most suitable perfusates, both containing about 100 mM mannitol, permitted normal function in slices prepared after removal of PG. These results indicate that, with an appropriate vehicle perfusate, exposure to PG up to a concentration of 3 mol/liter has remarkably little effect upon vascular resistance and the renal cortical functions measured.     

The effect of propane-diols on the intestinal uptake of nutrients and brush border membrane enzymes in the rat

The effect on rats of oral doses (38.66 mM/kg body wt) of propane-1,2-diol (PD) administered daily for 10 (Group 1), 20 (Group 2), and 30 days (Group 3) was investigated. Weight gain was initially retarded (P less than 0.05) in Group 1, but was later reversed and elevated significantly (P less than 0.05) in Groups 2 and 3 as compared with their respective controls receiving an equal volume of saline. PD showed a tendency toward enhancing the activities of various enzymes involved in terminal digestion, with the significant effect exerted in few groups on sucrase (P less than 0.05), lactase (P less than 0.05), and gamma-glutamyl transpeptidase (P less than 0.05) when compared with the respective controls. Absorption of D-glucose, glycine, L-aspartic acid, L-lysine, and calcium was elevated and was especially significant in Groups 2 and 3 (P less than 0.001). The structural integrity of the jejunal surface was retained for the most part. A similar examination of the effects of PD was also carried out in vitro to ascertain whether PD itself or its metabolites are involved in its action. The in vitro effects of propane-1,2-diol were compared with those of the more toxic compound propane-1,3-diol. The former exerted greater inhibitory action on the activities of the disaccharidases. The degree of inhibition was in the order sucrase much greater than lactase greater than maltase. The kinetic data revealed that inhibition by 1,2-diol in native and detergent solubilized sucrase is noncompetitive, with Ki values in the range of 0.35-0.41 M. The two diols did not alter the nutrient transport in the brush border membrane vesicles. The present work on rats indicates that PD may influence the intestinal digestive and absorptive functions in vivo and that this in vivo effect of PD is different from that observed in vitro suggesting that the nutritional and toxicological effect of PD may be mediated by different mechanisms.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO2 by the tricarboxylic acid cycle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Phospholipid metabolism in human platelets preserved at 22 degrees C: differential effects of storage on phospholipase A2- and C-mediated reactions

The effects of preservation at 22 degrees C on phospholipid metabolism were studied in human platelets. Stimulation of fresh platelets with thrombin caused a rapid and transient rise of 1,2-diacylglycerol (DG) which was derived from phosphatidylinositol (PI) by its strictly specific phospholipase C. Lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) were also accumulated as a result of the action of phospholipase A2. No significant changes in phospholipid metabolism were detected in platelets preserved at 22 degrees C up to 6 hr. However, platelets stored for more than 12 hr showed (1) an accumulation of both lysoPC and lysoPE before thrombin activation, (2) a subsequent decrease in the formation of lysoPC and lysoPE after thrombin activation when compared to fresh platelets, (3) a threefold lower rate of liberation of arachidonic acid than fresh platelets after activation, and (4) a lower rate and extent of aggregation than fresh platelets. Nevertheless, the amount of 1,2-DG produced during preservation up to 48 hr was similar to that observed in fresh platelets. The results indicate that the markedly enhanced activity of phospholipase A2, but not phospholipase C, that occurs during platelet storage leads to the deterioration of aggregation and arachidonic acid liberation in response to thrombin.     

Long-lived fluorescence lifetime from tyrosine in a peptide derived from S-100b.

It was shown in an earlier report (Turner et al., 1989, Biochem. Cell Biol. 67; 179-186) that the anomalous steady-state fluorescence emission spectra observed for the protein S-100b in aqueous solution at pH 7.5 contains a long-lived fluorescence decay component. In this study, a peptide consisting of residues 11 to 27 of the beta-subunit, was investigated. 11Ile-Asp-Val-Phe-His15-Gln-Tyr-Ser-Gly-Arg20-Glu-Gly- Asp-Lys-His25-Lys-Leu27 Fluorescence lifetimes were measured at the emission maximum and in the red edge of the spectrum. At wavelengths greater than 320 nm, the data was best fit with three exponentials. The third exponential gave lifetimes of 13.1 ns and 15.9 ns when the peptide was dissolved in the solvents propane-2-ol and propane-1,2-diol, respectively (lambda EX = 275 nm, lambda EM = 350 nm). These fluorescence lifetimes are similar to that observed for a decay component of native S-100b in the red edge of the emission, suggesting that the 1 degrees and 2 degrees features of a heptadecapeptide from S-100b protein has enough structural information when dissolved in solvents of intermediate polarity provide appropriate conditions for long-lived fluorescence from a tyrosine/tyrosinate species to occur.     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of corneal endothelium following exposure to a vitrification solution

Corneal endothelium, a monolayer of cells lining the inner surface of the cornea, is particularly susceptible to freezing injury. Ice formation damages the structural and functional integrity of the endothelium, and this results in a loss of corneal transparency. Instead of freezing, an alternative method of cryopreservation is vitrification, which avoids damage associated with ice formation. Vitrification at practicable cooling rates, however, requires exposure of tissues to very high concentrations of cryoprotectants, and this can cause damage through chemical toxicity and osmotic stress. The effects of a vitrification solution (VS1) containing 2.62 mol/liter (20.5%, w/v) dimethyl sulfoxide, 2.62 mol/liter (15.5%, w/v) acetamide, 1.32 mol/liter (10%, w/v) propane-1,2-diol, and 6% (w/v) polyethylene glycol were studied on corneal endothelium. Endothelial function was assessed by monitoring corneal thickness during 6 hr of perfusion at 35 degrees C with a Ringer solution supplemented with glutathione and adenosine. Various dilutions of the vitrification solution were introduced and removed in a stepwise manner to mitigate osmotic stress. Survival of endothelium after exposure to VS1 or a solution containing 90% of the cryoprotectant concentrations in VS1 (90% VS1) was dependent on the duration of exposure, the temperature of exposure, and the dilution protocol. The basic dilution protocol was performed at 25 degrees C: corneas were transferred from 90% VS1 or VS1 into 50% VS1 for 15 min, followed by 25% VS1 for 15 min and finally into isosmotic Ringer solution. Using this protocol, corneal endothelium survived exposure to 90% VS1 for 15 min at -5 degrees C, but 5 min in VS1 at -5 degrees C was harmful and resulted in some very large and misshapen endothelial cells. This damage was not ameliorated by using a sucrose dilution technique; but endothelial function was improved when the temperature of exposure to VS1 was reduced from -5 to -10 degrees C. Exposure to VS1 for 5 min at -5 degrees C was well tolerated, however, when the temperature of the first dilution step into 50% VS1 was reduced from 25 to 0 degree C. The large, misshapen cells were not observed under these conditions nor after exposure to VS1 at -10 degrees C. These results suggested that damage was the result of cryoprotectant toxicity rather than osmotic stress. Thus, corneal endothelium survived exposure to two solutions of cryoprotectants, namely, 90% VS1 and VS1, that were sufficiently concentrated to vitrify. Whether corneas can be cooled fast enough in these solutions to achieve vitrification and warmed fast enough to avoid devitrification remains to be determined.     

Osmotic response of mammalian cells: Effects of permeating cryoprotectants on nonsolvent volume

The nonsolvent volume, b, of a cell permits calculation of cell water volume from measurements of total cell volume, and, consequently, it is used extensively in the determination of membrane permeability coefficients for water and solutes and also in simulations of water and solute fluxes during freezing of cells. The nonsolvent volume is most commonly determined from the ordinate intercept of plots of cell volume as a function of the reciprocal of extracellular nonpermeating solute concentration (so-called Boyle-van't Hoff plots). Once derived, b is often assumed to be constant even under conditions that may differ markedly from those under which it was determined. Our aim was to investigate whether this assumption was valid when cells were exposed to the cryoprotectants glycerol, dimethyl sulphoxide (Me₂SO), or propane-1,2-diol. Rabbit corneal keratocytes, a fibroblastic cell type, were exposed to 10% (v/v) cryoprotectant for 30 min at 22°C in solutions containing a range of nonpermeating solute concentrations. Cell volumes were determined by an electronic particle sizer and mode volume plotted as an inverse function of the concentration of nonpermeating solute. The cells behaved as osmometers under all conditions studied, but we found no evidence to suggest that the nonsolvent volume of cells was altered by Me₂SO or propane-1,2-diol. Glycerol, however, reduced the slope of the Boyle-van't Hoff plot, but this could be ascribed to the failure of the cells to equilibrate fully with the glycerol over the 30 min exposure time; thus, b was unaffected by glycerol. It may be assumed, therefore, that the nonsolvent volume was not influenced by the presence inside cells of any of these nonelectrolyte cryoprotectants. © 1996 Wiley-Liss, Inc.