July 2002
Volume 43, Issue 7
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Cornea  |   July 2002
Recovery of Endothelial Function after Vitrification of Cornea at −110°C
Author Affiliations
  • W. John Armitage
    From the Division of Ophthalmology, University of Bristol, Bristol, United Kingdom.
  • Samantha C. Hall
    From the Division of Ophthalmology, University of Bristol, Bristol, United Kingdom.
  • Caroline Routledge
    From the Division of Ophthalmology, University of Bristol, Bristol, United Kingdom.
Investigative Ophthalmology & Visual Science July 2002, Vol.43, 2160-2164. doi:
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      W. John Armitage, Samantha C. Hall, Caroline Routledge; Recovery of Endothelial Function after Vitrification of Cornea at −110°C. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2160-2164.

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Abstract

purpose. To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at −110°C.

methods. Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7°C/min to −110°C, which was below the glass transition temperature (T g) of the solution. After rewarming at approximately 12°C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34°C.

results. Addition and removal of 6.8 M PROH without cooling to −110°C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to −110°C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 ± 2 μm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at −13 ± 2 μm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer.

conclusions. Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at −110°C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.

Cryopreservation is currently the only technique that offers the prospect of truly long-term storage of corneas for transplantation. The availability of a reliable method of corneal cryopreservation would confer significant logistic advantages and would be a valuable enhancement to eye-bank practice. Although cryopreserved corneas have been successfully grafted, 1 laboratory and clinical studies have both provided evidence of the significant endothelial damage that can occur as a result of freezing. 2 Cells in monolayers, such as corneal endothelium, are generally more susceptible to freezing injury than isolated cells frozen as suspensions. 3 Disruption of cells and tissue structure by ice during cryopreservation could be avoided by vitrifying instead of freezing, 4 but this approach poses considerable challenges. 
When an aqueous solution freezes, water crystallizes into ice, and the solutes become increasingly concentrated in the diminishing liquid phase as the temperature decreases. Both of these changes are potentially harmful to tissues. With vitrification, there is no phase change of liquid into solid. Instead, crystallization (and the resultant concentration of solute) is suppressed by an extreme increase in viscosity of the solution, brought about by cooling. Ultimately, the viscosity becomes so high that the solution takes on the properties of a solid, yet the molecules remain randomly organized as in a liquid. This amorphous, glassy state is attained at the glass transition temperature (T g). 5  
To vitrify tissues at practicable cooling rates requires exposure to high, multimolar concentrations of solutes that readily form glasses. Typically, vitrification solutions comprise complex mixtures of solutes on the basis that none of the individual components exceeds its putative toxic concentration, yet the overall solute concentration is sufficient to vitrify at practicable cooling rates. 4 Although a variety of cells and, more recently, tissues have been vitrified using this approach, 6 7 8 9 attempts to vitrify cornea in these solutions have been unsuccessful. 10 11 12 In view of the difficulty of modifying these multicomponent solutions in a rational manner, we decided to investigate solutions containing a single major solute. We have shown that cornea tolerates exposure to 5.5 M propane-1,2-diol (PROH). 13 This solute has efficient glass-forming properties and, at this concentration, should vitrify when cooled at approximately 50°C/min. 14 15 However, when corneas were cooled in 5.5 M PROH, devitrification (i.e., crystallization of ice during warming) was the cause of substantial endothelial damage. Data reported by MacFarlane 5 suggest that during warming at even 300°C/min, a rate not attainable by convection heating of cornea, 65% of the water in this solution would crystallize. At concentrations of PROH higher than 6 M, however, less that 10% of water should crystallize during warming at rates lower than 50°C/min. It was clear, therefore, that solute concentrations substantially higher than those required to vitrify were required to minimize devitrification. 
Our purposes were twofold: to investigate whether cornea could tolerate sufficiently high concentrations of PROH, not only to vitrify at low cooling rates, but to avoid significant devitrification during warming and to determine whether corneal endothelial function would be retained after vitrification. In this study, we showed that rabbit cornea exposed to 6.8 M PROH could be cooled to −110°C without visible crystallization of ice. Although corneas were thickened after warming and removal of PROH, an endothelial monolayer was retained. There was, moreover, clear evidence of endothelial bicarbonate pump activity, which is crucial for controlling stromal hydration and, hence, corneal transparency. To our knowledge, vitrification of cornea with a functioning endothelium was therefore achieved for the first time. A preliminary experiment with human cornea also showed retention of endothelium after vitrification. These results have implications for both tissue transplantation and the storage of tissue-engineered products that contain living cells. 16  
Methods
These experiments required the use of rabbit corneas and abided by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Human corneas were obtained from the CTS Bristol Eye Bank (Bristol, UK). Specific consent for their use in research had been obtained in accordance with the Human Tissues Act of 1961, and their use corresponded with the provisions of the Declaration of Helsinki. Only corneas that were unsuitable for transplantation were used. 
Experimental Protocol
Rabbit corneas, suspended on support rings, 17 were exposed to a series of solutions (50-mL volumes) of increasing PROH concentration according to the protocol in Table 1 . The carrier solution for PROH was a modified Ringer’s solution, comprising 128.3 mM NaCl, 4.8 mM KCl, 0.8 mM MgCl2 · 6H2O, 0.7 mM Na2PO4 · 2H2O, 5 mM glucose, 20 mM HEPES buffer, 6% wt/vol polyethylene glycol, 2.5% wt/vol chondroitin sulfate, and 0.25 M sucrose to partially dehydrate the stroma. The molar concentrations of these constituents were maintained constant in all the PROH solutions. Initially, the exposure temperature was 4°C. Above 25% vol/vol PROH, however, the exposure temperature was lowered to reduce chemical toxicity (Grant LTD6 and LTD20 constant temperature baths; Camlab, Cambridge, UK). After the final step in 50% vol/vol PROH, corneas were transferred to 30-mL glass vials containing 8.5 mL 50% vol/vol PROH at −20°C. The vials were suspended above the surface of liquid nitrogen and cooled to −110°C, which is below the glass transition temperature of this solution (i.e., T g = −107°C). 15 After reaching −110°C, the corneas were warmed by placing the vials in a constant-temperature bath at −20°C. The cooling and warming temperature profiles were measured with type T thermocouples in dummy samples. The changes in temperature were nonlinear, but the approximate rates of cooling and warming averaged between the temperature limits of −20°C and −110°C were, respectively, −7°C/min and +12°C/min. During removal of PROH in steps according to the protocol in Table 1 , the sucrose concentration was increased to 1 M to act as an osmotic buffer. Endothelial function was then assessed by corneal perfusion. 
Corneal Perfusion
Corneas, still on their support rings, were mounted against a plastic backing plate with inlet and outlet ports so as to create an artificial anterior chamber that allowed endothelial perfusion in a manner similar to that described by Dikstein and Maurice. 17 The endothelial surface was perfused with medium at 2 mL/hour, 15 cm H2O pressure, and 34°C. Corneal thickness was measured by ultrasonic pachymetry (assumed speed of sound through rabbit stroma, 1580 m/sec; DGH 500; DGH Technology, Inc., Exton, PA). For an individual cornea, each thickness measurement was the mean of four readings taken from the central corneal area. Endothelial function was assessed by monitoring changes in corneal thickness during perfusion with either a bicarbonate-free solution, M199 Hanks’ (Medium 199 with Hanks’ salts, M-3274; Sigma, Poole, UK), to suppress the endothelial pump, or with a solution containing 26 mM NaHCO3, M199 Earle’s (Medium 199 with Earle’s salts, M-3769; Sigma), to support the endothelial pump. The osmolalities of these perfusates, measured by freezing-point depression (Roebling Osmometer; Camlab), were, respectively, 300 ± 10 mOsmol/kg (SD; n = 11) and 301 ± 10 mOsmol/kg (n = 10). When the switch was made between perfusates, approximately 5 mL of the new solution was flushed gently through the perfusion chamber (dead space, including tubing, <0.5 mL) to ensure a complete exchange of solutions. The epithelial surface of the cornea was covered with silicone oil to prevent corneal thinning by evaporative loss of fluid. 
Endothelial Morphology
Corneas were stained with trypan blue to reveal cells with damaged plasma membranes and with alizarin red S to stain cell borders. 18 Before the staining procedure, vitrified corneas, which had been perfused with M199 Earle’s for 1.5 hours after removal of the PROH, were placed in 5% dextran T500 in Eagle’s minimum essential medium for 2 hours at ambient temperature to reverse stromal edema. Without this step, individual endothelial cells could not be distinguished. The corneas that had merely been exposed to the vitrification solution (i.e., without vitrification) were stained on completion of the PROH removal protocol (Table 1) . The endothelial surface was first covered with 0.2% trypan blue in 0.9% NaCl for 90 seconds. The trypan blue was gently rinsed away with 0.9% NaCl and the endothelium covered with 0.2% alizarin red S in 0.9% NaCl (pH 4.2) for 90 seconds. After rinsing with 0.9% NaCl to remove excess dye, the corneas were fixed for 10 minutes in 2.5% glutaraldehyde. The corneas were then removed from their supporting rings and trimmed of scleral and conjunctival tissue, and a 7.5-mm button was trephined from the central region. The button was placed on a cavity slide and mounted under a coverslip with a drop of 0.9% NaCl. The coverslip was taped to the slide to flatten the cornea. The endothelium was viewed by transmitted light with a microscope (Dialux 22EB; Leica, Milton Keynes, UK). Photographs were taken of the endothelium and of a calibrated stage micrometer to allow cell density to be determined from the photomicrographs. 
Human Cornea
Corneas from a 70-year-old human donor were also vitrified using the same protocol developed for rabbit cornea. Death-to-enucleation time was 21 hours, and the eyes were in moist chambers at 4°C for a further 21 hours before the corneoscleral discs were excised and placed in organ culture at 34°C. 19 The corneas remained in organ culture for only 3 days. They were then placed in 5% dextran medium for 24 hours at 34°C before undergoing the vitrification protocol. After vitrification and removal of the PROH, the corneas were placed in 5% dextran medium for 2 hours at ambient temperature before staining with trypan blue and alizarin red S. 
Results
Untreated Control Corneas
Freshly isolated corneas (n = 10) were perfused for 6 hours: the first 2 hours with M199 Earle’s (i.e., with bicarbonate ions), followed by 2 hours with M199 Hanks’ (bicarbonate-free), and then a return to M199 Earle’s for the final 2 hours (Fig. 1) . The initial corneal thickness was 365 ± 14 μm (SD) and, during the first 2 hours of perfusion, the corneas thinned slightly at −4 ± 3 μm/hour to 357 ± 13 μm. The switch to bicarbonate-free perfusate induced swelling at +39 ± 5 μm/hour, the corneas attaining a thickness of 435 ± 16 μm after 2 hours. Restoring bicarbonate to the perfusate resulted in corneal thinning at −29 ± 3 μm/hour, and a final thickness of 377 ± 17 μm. 
Exposure to PROH
Addition and removal of 6.8 M PROH according to the protocols in Table 1 , but without cooling to −110°C, resulted in a thickening of corneas during subsequent perfusion with M199 Earle’s compared with treated control corneas that had merely been held in the carrier solution without PROH (Fig. 1) . Swelling rates during M199 Hanks’ perfusion were +40 ± 4 μm/hour (n = 8) and +37 ± 4 μm/hour (n = 6), respectively, for corneas exposed to PROH and the treated control. Thinning rates during the final 2 hours of perfusion with M199 Earle’s were −29 ± 3 and −26 ± 2 μm/hour, respectively. One-way ANOVA, including the untreated control values, showed no differences at the 5% level of significance (swelling rates, P = 0.3; thinning rates, P = 0.2). Normal corneal thickness in rabbits is approximately 350 to 400 μm, but treated control corneas exposed to the carrier solution were initially much thinner than this, owing to the presence of the 0.25 M sucrose (Fig. 1) . The calculated osmolality of 0.25 M sucrose in phosphate-buffered saline is 570 mOsmol/kg. Mishima and Hedbys 20 derived an equation for corneal thickness as a function of osmolality, which is similar to the Boyle-van’t Hoff equation used to describe osmotically induced changes in cell volume. When adjusted for an isotonic corneal thickness of 365 μm (untreated control thickness at start of perfusion), this equation, q = 0.073 + 0.292 · π0/π (where q is corneal thickness, π0 is isotonic osmolality, and π is the osmolality of the sucrose solution), gives a corneal thickness in the sucrose solution of 227 μm. This is somewhat less than the thickness of the treated control corneas observed at the start of perfusion (272 ± 12 μm, n = 6), but this is not unexpected, because these corneas had already been returned to isotonic medium for 15 minutes before the start of perfusion (i.e., equivalent to the final step of the PROH removal protocol in Table 1 ). Indeed, back-extrapolation of the treated control line in Figure 1 , 15 minutes before the start of perfusion, produced a corneal thickness of approximately 230 μm, which agrees well with the calculated value. This explains, therefore, the low starting thickness and the phase of rapid swelling of the treated control corneas during the initial 2-hour perfusion with M199 Earle’s (Fig.1)  
Vitrification
There was no visible crystallization of ice when corneas were cooled to −110°C in 6.8 M PROH. Cracking of the aqueous glass was seen in half of the samples. During warming, a few small, discrete sites of crystallization were observed, but this potentially harmful devitrification appeared to be away from the endothelial surface. After removal of the PROH, the corneas swelled during the first 3 hours of perfusion with M199 Earle’s, almost doubling in thickness, but they began to thin at −10 ± 5 μm/hour (n = 5), suggesting endothelial function (Fig. 2) . To rule out the possibility that this thinning was a purely passive osmotic effect, a second series of cooling experiments was performed in which the corneas were switched to perfusion with M199 Hanks’ after 3 hours, the point at which the initial swelling phase had virtually ceased. This removal of HCO3 induced a second phase of swelling at +58 ± 2 μm/hour (n = 4), suggesting that inhibition of the endothelial HCO3 pump had occurred. Moreover, when after 2 hours HCO3 was restored to the perfusion solution, the corneas began to thin again at a rate of −13 ± 2 μm/hour (Fig. 2) . Because the osmolalities of M199 Earle’s and M199 Hanks’ were virtually the same (see the Methods section), these thickness changes were not a result of differences in osmolality between the two perfusates. This thinning rate was, however, lower than that observed after corneas had been merely exposed to PROH but not vitrified (cf. Fig. 1 : unpaired t-test, P < 0.001). 
Morphologically, vitrified cornea retained an apparently intact and continuous endothelial monolayer as revealed by the trypan blue–alizarin red S staining (Fig. 3a) . The cells appeared somewhat rounded and the image was generally hazy, most probably due to persistent edema despite the period of incubation in 5% dextran. After simply adding and removing 6.8 M PROH (i.e., without vitrification), occasional dark red spots were evident where cells had been lost from the mosaic (Fig. 3b) . These features were not seen in vitrified cornea, which had been perfused for 1.5 hours before staining, allowing time for cells to migrate and spread to fill any gaps in the mosaic. The cell densities were within the normal range for rabbit cornea stained and fixed by this method (i.e., 4673 cells/mm2, 95% CI 4255–5181, n = 6), 21 which causes shrinkage and thus an apparent increase in cell density when compared with specular microscopy of untreated cornea. 
Human Cornea
After removal of the PROH, the endothelium of the vitrified human cornea showed a virtually continuous mosaic with only the occasional missing or blue-stained cell (Fig. 4)
Discussion
The high concentration of PROH required to vitrify cornea at practicable cooling rates could have damaged endothelial cells through osmotic stress and/or chemical toxicity. To lessen osmotic stress, PROH was added and removed in steps and, during the removal of PROH, the concentration of sucrose in the bathing medium was increased to 1 M to act as an osmotic buffer. To reduce chemical toxicity, the temperature of exposure was decreased as PROH concentration increased. This lowering of temperature would also have reduced solute permeability, and it is highly likely that the stromal concentration of PROH at the lower exposure temperatures increased more through water efflux than PROH influx. 
Exposure to 6.8 M PROH using the described protocol did not markedly impair endothelial function, although the corneas were somewhat thicker than the control. This may have been a reflection of increased passive permeability of the endothelial layer, reduced pump activity, or simply a result of retention of PROH in the stroma. If the corneas had continued to thin at the observed rate, a further 6 hours of perfusion would have been required for a return to normal thickness. This concentration of PROH is considerably higher than the 1 to 1.5 M concentrations of cryoprotectants used for protecting cells against freezing injury, and exposure to such a high concentration represented the first major hurdle in our attempt to cryopreserve cornea by vitrification. 
More significantly, however, our experiments demonstrated that endothelial function could be retained after vitrification of intact cornea, as shown by the thinning of corneas during subsequent perfusion. The corneal swelling that occurred when endothelial pump function was suppressed by the removal of bicarbonate from the perfusate, and the resumption of thinning when bicarbonate was restored, suggested active hydration control rather than a purely passive osmotic response. 22 23 Because, to our knowledge, similar in vitro functional results have not been reported after the cryopreservation of rabbit cornea by freezing, our results support the contention that the avoidance of ice crystallization by vitrification may be advantageous for this tissue. 
An apparently intact endothelial mosaic was evident after vitrification and perfusion. Rabbit corneal endothelial cells readily undergo mitotic division, but this was unlikely to be a significant factor in the present study, because little cell division would be expected during the 6-hour assessment perfusion, which was performed with a culture medium that does not support endothelial cell proliferation (i.e., M199 without serum). The morphologic observations suggested, however, that cell migration and spreading to fill gaps left by detached cells occurred during perfusion. 
Theoretically, there should be no additional damage caused by cooling to below T g because there is no phase change during vitrification. The vitrified corneas, however, swelled substantially more during subsequent perfusion than those merely exposed to the PROH without further cooling. Whether this represents additional damage, perhaps through toxicity as a result of longer exposure times to the 6.8 M PROH during the cooling to −110°C, or simply reflects a higher tissue concentration of PROH, again because of the longer exposure time, remains to be resolved. Other questions that remain to be resolved include whether additional solutes known to suppress crystallization, such as antifreeze proteins from fish 24 or polyvinyl alcohol, 25 would prevent the small amount of observed devitrification, or even permit a lower PROH concentration and whether more rapid warming by electromagnetic irradiation would be advantageous. 26 Finally, there is the question of whether cooling the vitrified corneas below T g to temperatures typically used for cryopreservation (i.e., below −140°C) would cause potentially damaging cracking in the corneal glass. 
One major difference between our approach and that of others was the use of a single major solute rather than a complex mixture of components. 6 7 8 9 We demonstrated that, for rabbit cornea at least, this approach can be successful and that endothelial cells can tolerate sufficiently high concentrations of a single solute to vitrify. Rabbit cornea, however, differs from human in a number of important respects, notably thickness, stromal properties, and the fact that rabbit corneal endothelium readily undergoes mitotic division. Preliminary experiments with human cornea using this protocol showed retention of endothelium after vitrification, but these results now should be confirmed by longer-term functional and morphologic studies. 
 
Table 1.
 
Protocols for Stepwise Addition and Removal of PROH
Table 1.
 
Protocols for Stepwise Addition and Removal of PROH
Step PROH (% vol/vol) Sucrose (M) Duration (min) Temperature (°C)
Addition of PROH
 1 0 0.25 10 4
 2 5 0.25 10 4
 3 10 0.25 10 4
 4 15 0.25 10 4
 5 25 0.25 10 −5.5
 6 35 0.25 20 −10
 7 45 0.25 10 −15
 8 50 0.25 10 −15 to −20
Removal of PROH
 1 25 1.0 15 −15
 2 10 1.0 15 −7.5
 3 0 1.0 15 0
 4 0 0 15 25
Figure 1.
 
Changes in thickness (mean, SD) of corneas after addition and removal of 6.8 M PROH (▪, n = 8) according to the protocol in Table 1 or after exposure only to the PROH carrier solution, which contained 0.25 M sucrose, for an equal period (treated control: □, n = 6). Sucrose was not present for the final 15 minutes of the protocol in either group. The thickness changes in untreated control corneas are also shown (•, n = 10). For all groups, the first 2 hours of perfusion were with M199 Earle’s (26 mM HCO3 ), followed by 2 hours with M199 Hanks’ (bicarbonate-free), with a return to M199 Earle’s for the final 2 hours.
Figure 1.
 
Changes in thickness (mean, SD) of corneas after addition and removal of 6.8 M PROH (▪, n = 8) according to the protocol in Table 1 or after exposure only to the PROH carrier solution, which contained 0.25 M sucrose, for an equal period (treated control: □, n = 6). Sucrose was not present for the final 15 minutes of the protocol in either group. The thickness changes in untreated control corneas are also shown (•, n = 10). For all groups, the first 2 hours of perfusion were with M199 Earle’s (26 mM HCO3 ), followed by 2 hours with M199 Hanks’ (bicarbonate-free), with a return to M199 Earle’s for the final 2 hours.
Figure 2.
 
Changes in thickness (mean, SD) of corneas cooled to −110°C in 6.8 M PROH. After warming and removal of PROH, corneas were either perfused continuously with M199 Earle’s for 5 hours (○, n=5) or switched to M199 Hanks’ (bicarbonate-free) after the first 3 hours and then back to M199 Earle’s after a further 2 hours (• n = 4).
Figure 2.
 
Changes in thickness (mean, SD) of corneas cooled to −110°C in 6.8 M PROH. After warming and removal of PROH, corneas were either perfused continuously with M199 Earle’s for 5 hours (○, n=5) or switched to M199 Hanks’ (bicarbonate-free) after the first 3 hours and then back to M199 Earle’s after a further 2 hours (• n = 4).
Figure 3.
 
Endothelial morphology after staining with trypan blue and alizarin red S. (a): Cornea after vitrification; cell density, 4600 cells/mm2. Following removal of the PROH, the cornea was perfused for 1.5 hours and then thinned in 5% dextran for 2 hours. Although edema was still evident, the endothelial mosaic was continuous, with little evidence of missing cells. (b) Cornea after addition and removal of 6.8 M PROH but without vitrification; cell density, 4500 cells/mm2. The cornea was stained immediately after removal of the PROH, and occasional random isolated cell loss was observed. Bars, 50 μm.
Figure 3.
 
Endothelial morphology after staining with trypan blue and alizarin red S. (a): Cornea after vitrification; cell density, 4600 cells/mm2. Following removal of the PROH, the cornea was perfused for 1.5 hours and then thinned in 5% dextran for 2 hours. Although edema was still evident, the endothelial mosaic was continuous, with little evidence of missing cells. (b) Cornea after addition and removal of 6.8 M PROH but without vitrification; cell density, 4500 cells/mm2. The cornea was stained immediately after removal of the PROH, and occasional random isolated cell loss was observed. Bars, 50 μm.
Figure 4.
 
Endothelial morphology of a vitrified human cornea after staining with trypan blue and alizarin red S. The cornea was vitrified in 6.8 M PROH according to the protocol used for rabbit cornea. Only occasional missing or blue-stained cells were observed. Cell density, 2300 cells/mm2. Bar, 50 μm.
Figure 4.
 
Endothelial morphology of a vitrified human cornea after staining with trypan blue and alizarin red S. The cornea was vitrified in 6.8 M PROH according to the protocol used for rabbit cornea. Only occasional missing or blue-stained cells were observed. Cell density, 2300 cells/mm2. Bar, 50 μm.
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Figure 1.
 
Changes in thickness (mean, SD) of corneas after addition and removal of 6.8 M PROH (▪, n = 8) according to the protocol in Table 1 or after exposure only to the PROH carrier solution, which contained 0.25 M sucrose, for an equal period (treated control: □, n = 6). Sucrose was not present for the final 15 minutes of the protocol in either group. The thickness changes in untreated control corneas are also shown (•, n = 10). For all groups, the first 2 hours of perfusion were with M199 Earle’s (26 mM HCO3 ), followed by 2 hours with M199 Hanks’ (bicarbonate-free), with a return to M199 Earle’s for the final 2 hours.
Figure 1.
 
Changes in thickness (mean, SD) of corneas after addition and removal of 6.8 M PROH (▪, n = 8) according to the protocol in Table 1 or after exposure only to the PROH carrier solution, which contained 0.25 M sucrose, for an equal period (treated control: □, n = 6). Sucrose was not present for the final 15 minutes of the protocol in either group. The thickness changes in untreated control corneas are also shown (•, n = 10). For all groups, the first 2 hours of perfusion were with M199 Earle’s (26 mM HCO3 ), followed by 2 hours with M199 Hanks’ (bicarbonate-free), with a return to M199 Earle’s for the final 2 hours.
Figure 2.
 
Changes in thickness (mean, SD) of corneas cooled to −110°C in 6.8 M PROH. After warming and removal of PROH, corneas were either perfused continuously with M199 Earle’s for 5 hours (○, n=5) or switched to M199 Hanks’ (bicarbonate-free) after the first 3 hours and then back to M199 Earle’s after a further 2 hours (• n = 4).
Figure 2.
 
Changes in thickness (mean, SD) of corneas cooled to −110°C in 6.8 M PROH. After warming and removal of PROH, corneas were either perfused continuously with M199 Earle’s for 5 hours (○, n=5) or switched to M199 Hanks’ (bicarbonate-free) after the first 3 hours and then back to M199 Earle’s after a further 2 hours (• n = 4).
Figure 3.
 
Endothelial morphology after staining with trypan blue and alizarin red S. (a): Cornea after vitrification; cell density, 4600 cells/mm2. Following removal of the PROH, the cornea was perfused for 1.5 hours and then thinned in 5% dextran for 2 hours. Although edema was still evident, the endothelial mosaic was continuous, with little evidence of missing cells. (b) Cornea after addition and removal of 6.8 M PROH but without vitrification; cell density, 4500 cells/mm2. The cornea was stained immediately after removal of the PROH, and occasional random isolated cell loss was observed. Bars, 50 μm.
Figure 3.
 
Endothelial morphology after staining with trypan blue and alizarin red S. (a): Cornea after vitrification; cell density, 4600 cells/mm2. Following removal of the PROH, the cornea was perfused for 1.5 hours and then thinned in 5% dextran for 2 hours. Although edema was still evident, the endothelial mosaic was continuous, with little evidence of missing cells. (b) Cornea after addition and removal of 6.8 M PROH but without vitrification; cell density, 4500 cells/mm2. The cornea was stained immediately after removal of the PROH, and occasional random isolated cell loss was observed. Bars, 50 μm.
Figure 4.
 
Endothelial morphology of a vitrified human cornea after staining with trypan blue and alizarin red S. The cornea was vitrified in 6.8 M PROH according to the protocol used for rabbit cornea. Only occasional missing or blue-stained cells were observed. Cell density, 2300 cells/mm2. Bar, 50 μm.
Figure 4.
 
Endothelial morphology of a vitrified human cornea after staining with trypan blue and alizarin red S. The cornea was vitrified in 6.8 M PROH according to the protocol used for rabbit cornea. Only occasional missing or blue-stained cells were observed. Cell density, 2300 cells/mm2. Bar, 50 μm.
Table 1.
 
Protocols for Stepwise Addition and Removal of PROH
Table 1.
 
Protocols for Stepwise Addition and Removal of PROH
Step PROH (% vol/vol) Sucrose (M) Duration (min) Temperature (°C)
Addition of PROH
 1 0 0.25 10 4
 2 5 0.25 10 4
 3 10 0.25 10 4
 4 15 0.25 10 4
 5 25 0.25 10 −5.5
 6 35 0.25 20 −10
 7 45 0.25 10 −15
 8 50 0.25 10 −15 to −20
Removal of PROH
 1 25 1.0 15 −15
 2 10 1.0 15 −7.5
 3 0 1.0 15 0
 4 0 0 15 25
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