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.     

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.     

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.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.     

Tolerance of human corneal endothelium to glycerol

As an initial step in the development of a method for corneal cryopreservation by vitrification, we attempted to establish the maximum concentration of glycerol to which human corneal endothelium could be exposed at 4 degrees C for 15 min without damage. Damage was defined as an increase in mean endothelial cell size or the inability to maintain corneal thickness for 1 week after exposure to glycerol. Using a system for long-term corneal perfusion, we perfused 24 paired human corneas with glycerol at 4 degrees C. The concentration of glycerol increased at a rate of 20% (w/v) (2.2 M) per hour until the desired maximum concentration was reached for that cornea, stabilized for 15 min, and then decreased at the same rate. The corneas were then perfused at 37 degrees C with Dulbecco's medium at a rate of 5 microliters/min under 18 mm Hg intracameral pressure for 7 days with daily measurements of corneal thickness. Endothelial morphology was examined by specular microscopy and by scanning electron microscopy. After 7 days of perfusion at 37 degrees C, there was a statistically significant direct relationship between the maximum concentration of glycerol to which the experimental eyes had been exposed and the increase in mean endothelial cell size. The mean endothelial cell size increased in corneas exposed to glycerol concentrations of 40, 50, and 60% (w/v), but did not differ significantly from baseline measurements in the corneas exposed to 30% glycerol or less. Thus, there was no detectable damage to human corneas exposed to 30% (w/v) (3.3 M) glycerol in this system. Tolerance of higher concentrations may be achieved by changes in the rates of addition and removal of glycerol or in the composition of the perfusate.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.     

Corneal tolerance of vitrifiable concentrations of glycerol.

Equilibration of corneas with sufficiently high concentrations of cryoprotectants to inhibit potentially damaging ice formation during cryopreservation has not yet been achieved. This study examined the effects on the structure and function of rabbit corneal endothelium of the low toxicity cryoprotectant glycerol. Corneas were exposed to concentrations ranging from 2.0 to 6.8 M glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mM 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 h. Endothelial structure was observed using specular microscopy during perfusion and scanning electron microscopy after perfusion. Corneas tolerated exposure to 2.0 and 3.4 M glycerol for 20 min at 4 and -5 degrees C, respectively. Tolerance of 4.8 M glycerol for 10 min at -10 degrees C was improved by decreasing the dilution temperatures. Ten-minute exposure to 6.1 and 6.8 M glycerol was tolerated at -15 degrees C. In all cases corneas initially showed signs of damage but endothelial function was regained following structural repair. Corneas exposed to 6.8 M glycerol and cooled below the glass transition temperature were nonfunctional after warming. Ice formation during warming was believed to be the cause of injury.     

Some effects of propane-1,2-diol on human platelets

The Kedem-Katchalsky equations and permeability data previously reported (F. G. Arnaud and D. E. Pegg. Permeation of glycerol and propane-1,2-diol into human platelets. Cryobiology 27, 130-136, 1990) have been used to design methods for adding and removing propane-1,2-diol (propylene glycol, PG) with human platelets. Mean platelet volume was kept within the tolerated range of 60 to 120% of normal. PG concentrations of 0.5, 1.0, 2.0, 2.5, and 3.0 M were studied at 2, 21, and 37 degrees C. PG was removed only at 21 degrees C. The effects of concentration of PG, temperature, and duration of exposure on the hypotonic stress response and ADP-induced aggregation were measured. It was found that platelets would tolerate exposure to PG concentrations up to 2 M at 21 or 2 degrees C for up to 15 min. The extent of damage increased considerably at higher temperatures and concentrations. These data provide the necessary basis for experiments to cryopreserve platelets with PG.     

Cryopreservation of rat hippocampal slices by vitrification

Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2 degrees C/min to -20 degrees C or below using 10 or 30% v/v glycerol or 20% v/v dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0 degree C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM vitamin C, the use of a more "intracellular" version of aCSF having reduced sodium and calcium levels and higher potassium levels, and the presence of a 25% w/v mixture of dimethyl sulfoxide, formamide, and ethylene glycol ("V(EG) solutes"; Cryobiology 48, pp. 22-35, 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. 260-273, 1984) was used as a carrier solution for up to 50% w/v V(EG) solutes, 0 degree C was more protective than 10 degrees C. Raising V(EG) concentration to 53% w/v allowed slice vitrification without injury from vitrification and rewarming per se, but was much more damaging than exposure to 50% w/v V(EG). This problem was overcome by using the analogous 61% w/v VM3 vitrification solution (Cryobiology 48, pp. 157-178, 2004) containing polyvinylpyrrolidone and two extracellular "ice blockers." With VM3, it was possible to attain a tissue K(+)/Na(+) ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric drug evaluation and development and the transplantation of integrated brain regions to correct brain disease or injury.     

Vitrification of posterior corneal lamellae

Cryopreservation of corneas has not yet been established as a routine method. Unsatisfactory experimental results with conventional techniques prompted us to explore the possibilities of vitrification. The aim of the present study was to optimize the heat exchange between the corneal tissue and cooling medium by reducing the corneal tissue volume and using a suitable sample container. A further objective was to promote vitrification by developing a new device for rapid cooling to -140 degrees C, just below the vitrification temperature of the cryopreservation medium. Experiments were done using posterior lamellar discs from pig corneas with a diameter of 7.5 mm and a thickness of 250-350 microm. The volume of tissue to be vitrified was 88% lower with posterior corneal lamellae than with the previously used corneoscleral discs. A very thin-walled (0.05 mm), teflon-coated bag served as the sample container. Immersed in only 0.1 ml of the vitrification solution VS41a, the lamellae were cooled to a final storage temperature of -196 degrees C. After warming and organ-culturing for 24h, the endothelium was stained with trypan blue and alizarin red, to determine cell viability. Vitrification of corneal lamellae without apparent ice formation or cracking of the specimen was achieved. Despite the successful vitrification, only a maximum of 10% of the endothelial cells was vital after warming. Thus, the toxicity of the cryoprotective agents and the devitrification that occurred during the heating process require further optimization of the method.     

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.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Clinical cryobiology of tissues: preservation of corneas

It is well recognized that the clarity of the cornea is a function of its hydration, and that this hydration is controlled by a "pump-and-leak" mechanism operating across the posterior monolayer of cells called the endothelium. A breakdown of the endothelium through disease or injury causes a marked increase in corneal thickness as the stroma imbibes fluid from the aqueous humor in the anterior chamber of the eye. This thickened, edematous condition of the stroma results in a cloudy cornea with an associated marked decrease in visual acuity. Treatment for this condition is usually by full-thickness corneal transplantation (penetrating keratoplasty), the success of which is dependent upon the donor cornea having an intact and healthy endothelium. It is essential, therefore, that any method of corneal storage for penetrating keratoplasty should protect and preserve the endothelium in a viable state. Current clinical practice relies upon short-term methods of preservation by two principal methods. Moist Chamber Storage is the time-honored corneal preservation method; it consists of keeping enucleated eyes at 0-4 degrees C in a sealed jar containing a pad of cotton gauze soaked in saline to provide a humid environment. The time limit placed upon this method of storage is 24-48 hr after which the viability of the endothelium deteriorates rapidly. Storage in M-K (McCarey-Kaufman) Medium involves excision of the corneoscleral segment from the donor eye and immersing it, endothelial side uppermost, in a medium consisting of tissue culture medium, 5% Dextran 40, and antibiotics. Laboratory and clinical studies indicate that storage in M-K medium at 4 degrees C preserves human endothelial cells for up to 4 days when the eye has been removed from the cadaver in less than 10 hr postmortem. Long-term preservation of corneas by freezing has long been a major goal in eye banking because indefinite storage by cryopreservation offers significant advantages for the quality and the quantity of material for use in keratoplasty, as well as for its distribution. However, procedures that have been developed for the cryopreservation of corneas have not been widely used, and a number of studies have shown that these procedures are inadequate for maintaining the integrity of the corneal endothelium. Not surprisingly, clinicians are now reluctant to accept corneas that have been frozen by these methods, though the clinical need is now greater than ever.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification

Nucleation and crystal growth are investigated for vitrification solution VS41A (dimethyl sulfoxide, formamide, and 1,2-propanediol) in an aqueous carrier solution giving, when added to these three cryoprotectants, a concentration of other solutes in the whole solution the same as that in Euro-Collins, with a 55% (w/v) cryoprotectant concentration. This concentration is assumed to achieve physical properties under 1 atmosphere similar to those of solution VS4 used under 1000 atmospheres. The thermal range and the kinetics of nucleation and crystal growth are investigated by DSC through different thermal treatments. It is found that the nucleation thermal range is below -90 degrees C and that of crystal growth is above -85 degrees C for a relatively long experimental time. The nucleation density is also studied through direct observations by cryomicroscopy and is related to the amount of crystallization calorimetrically recorded. The effect of storage below the glass transition shows the possibility of a slow increase in nucleation below the glass transition, as already observed by other authors for different aqueous solutions. Isothermal crystallization is analyzed within the Johnson-Mehl-Avrami model for temperatures above -75 degrees C. The corresponding samples have been cooled and warmed at the same rate of 40 degrees C/min and calculations give, at constant nuclei numbers, an activation energy of 9.3 +/- 0.3 kcal/mol and the Avrami exponent n = 2.2 +/- 0.05. This shows a two-dimensional crystal growth as observed by cryomicroscopy. The estimated critical warming rate relevant to the preservation of rabbit kidneys by vitrification is 270 degrees C/min with or without an increase in the nucleus density during storage. The present results support the possibility of using VS4 solution for vitrification of rabbit kidneys if pressure is not a limiting factor. Copyright 1993, 1999 Academic Press.     

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.     

Osmotic parameters of cells from a bioengineered human corneal equivalent and consequences for cryopreservation

A human corneal equivalent is under development with potential applications in pharmaceutical testing, biomedical research, and transplantation, but the ability to distribute this engineered tissue, depends on successful cryopreservation. Tissue recovery after exposure to conditions during cryopreservation depends on the response of its constituent cells to the changing environment as ice forms and solutes concentrate. This study defines the osmotic properties that define the rate of water movement across the plasma membrane of isolated human corneal endothelial, stroma, and epithelial cells. Cells were transferred from an isotonic (300 mosm/kg) to an anisotonic (150-1500 mosm/kg) solution at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after anisosmotic exposure allowed calculation of hydraulic conductivity (L(p)) and osmotically inactive volume fraction (V(b)). Experimental values for L(p) at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (E(a)). The L(p) for endothelial, stroma, and epithelial cells at 37 degrees C was 1.98+/-0.32,1.50+/-0.30, and 1.19+/-0.14 microm/min/atm, and the V(b) was 0.28, 0.27, and 0.41, respectively. The E(a) for endothelial, stroma, and epithelial cells was 14.8, 12.0, and 14.1 kcal/mol, respectively, suggesting the absence of aqueous pores. These osmotic parameters and temperature dependencies allow simulation of osmotic responses of human corneal cells to cryopreservation conditions, allowing amount of supercooling to be calculated to indicate the likelihood of intracellular freezing. Simulations show that differences in the osmotic parameters for the constituent cells in the bioengineered cornea result in significant implications for cryopreservation of the engineered corneal equivalent.     

Freezing monolayers of cells without gap junctions

Cells in monolayers have been reported to be more susceptible to freezing injury than the same cell type frozen in dispersed suspensions. There appears to be an enhanced susceptibility to intracellular freezing in the monolayers, which is thought to be facilitated by the presence of gap junctions allowing the spread of ice between neighbouring cells. MDCK Type II cells do not form gap junctions in monolayer culture. When frozen at rates of 0.2 to 10 degrees C/min, monolayers in 10% (v/v) propane-1,2-diol or dimethyl sulphoxide showed little influence of cooling rate on survival. This suggested that, in the absence of gap junctions, cells in monolayers did not display enhanced susceptibility to intracellular freezing. In contrast, however, monolayers frozen in glycerol showed a marked increase in cell damage when cooled at rates higher than 0.5 degrees C/min. This does not necessarily counter the suggestion that lack of gap junctions mitigates intracellular freezing as there is evidence that glycerol may itself promote intracellular freezing.