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(Investigative Ophthalmology and Visual Science. 2003;44:5110-5115.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.03-0370
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Cryopreservation of Human Donor Corneas with Dextran

Markus Halberstadt,1 Matthias Böhnke,2 Susanne Athmann,3 and Michael Hagenah3

1From the Department of Ophthalmology, Inselspital, Bern, Switzerland; 2Private Practice, Hamburg, Germany; and the 3Department of Ophthalmology, University of Hannover, Hannover, Germany.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To assess freeze-thaw–induced endothelial cell loss by using phase-contrast microscopy and early morphologic changes within each layer of human donor corneas by using confocal microscopy.

METHODS. Twenty-eight human corneas were cryopreserved in minimum essential medium containing 10% dextran with a molecular weight (MW) of 500,000 as an extracellular cryoprotectant, at a cooling rate of 1°C/min and stored in liquid nitrogen at -196°C. After thawing, the tissue was organ cultured to detect latent cell damage. In 22 of the corneas, the endothelial layer was subjected to routine phase-contrast microscopy after 24 hours of organ culturing. The other six specimens were evaluated layer by layer in a scanning slit confocal microscope after 6, 24, and 48 hours of organ culturing.

RESULTS. Before cryopreservation, the mean ± SD numerical density of endothelial cells was 1940 ± 220 cells/mm2. After cryopreservation and subsequent organ culturing, the endothelial cell density decreased to 1300 ± 360 cells/mm2, and two of the corneas had a completely necrotic endothelium (P = 0.001). Confocal microscopy revealed all corneal layers in each of the six specimens examined to be structurally integral after 48 hours of organ culturing. Although the reflectivity of some of the keratocytes was enhanced, there were no signs of keratolysis.

CONCLUSIONS. The present study demonstrates that each corneal layer is capable of regaining its structural integrity after cryopreservation in the presence of dextran. Because the freeze-thaw–induced endothelial cell loss is still highly variable, the technique must be further refined before it can be applied clinically.

Cryopreservation is the only available method permitting corneal storage for an indefinite period.1 2 The transplantation of human corneas that have been cryopreserved in the presence of dimethyl sulfoxide (DMSO) has been successful, in both the short-3 4 5 and long-term.6 However, with the introduction of short-term storage at 4°C, cryopreservation failed to gain wide acceptance, owing to its technically demanding nature, its costliness, and the variable clinical6 7 and experimental4 8 9 results achieved.

In experimental studies with rabbit and porcine corneas, the technical demands of cryopreservation have been somewhat lessened by substituting the intracellular cryoprotectant DMSO for an extracellular one, dextran10 (Halberstadt M, et al. IOVS 2001;42:ARVO Abstract 230).

Because survival of the endothelium is a crucial determinant of success after corneal preservation and transplantation, ophthalmologists usually restrict their appraisal of freeze–thaw damage in cryopreserved material to the monitoring of this layer’s viability and functionality.11 At one time, a more comprehensive view of the cornea’s condition was sought at by routine inspection with a phase-contrast microscope. However, in organ culture, the various layers of a perfectly transparent cornea are but poorly differentiated by this means, the endothelial layer alone being patent.12 Confocal microscopy, in contrast, clearly reveals also the upper layers when these are viewed tangentially. Furthermore, nonapplanatory confocal microscopy permits the direct and reproducible observation in organ culture of dynamic processes at the cellular level, such as wound healing, which is not feasible using standard clinical instruments. Repeated observations in the confocal microscope of organ cultured corneal tissue that has undergone freeze–thaw trauma would thus permit an assessment of early morphologic changes in each layer.

In the present study, we wanted to ascertain whether cryopreservation of corneoscleral discs in the presence of dextran is a viable option for human as well as for porcine and rabbit tissues. Our objectives were to determine by phase-contrast microscopy the extent of endothelial cell loss due to freezing and thawing and to assess the early morphologic changes within each corneal layer of cryopreserved tissue by scanning slit confocal microscopy.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Human Corneas
Twenty-eight corneoscleral discs were obtained from 24 donors with the consent of their relatives and stored in organ culture within a mean ± SD postmortem time of 21.8 ± 5.1 hours. The mean age of donors was 57.4 ± 13.9 years. Corneoscleral discs were excised after enucleation, as previously described13 and organ cultured at 37°C14 in Earle’s minimum essential medium (MEM; Biochrom, Heidelberg, Germany) containing 2% fetal calf serum (FCS; Biochrom). Before and after organ culturing, the corneal endothelium was evaluated in an inverted phase-contrast microscope (Diavert; Leitz, Wetzlar, Germany) after intercellular swelling of the monolayer in hypo-osmotic balanced salt solution.14 Four representative central areas of each endothelium were photographed, and the central numerical density of endothelial cells was calculated with the aid of a calibrated grid. Only corneas that were unsuitable for transplantation were used for our investigation, which complied with the policies of the review boards of the participating institutions and with the regulations laid down in the Declaration of Helsinki.

Twenty-two corneas incurred a loss of endothelial cells or displayed signs of degeneration during the mean organ culturing period of 15.4 ± 5.2 days, which rendered them unsuitable for penetrating keratoplasty. These corneas were organ cultured for a second time in the presence of 6% dextran (MW 500,000; Pharmacia, Uppsala, Sweden), which served as a deswelling agent, for a mean time of 7.5 ± 1.7 days, in readiness for tectonic (emergency) grafting. After this extended period in a dextran-containing medium, the mean endothelial cell density was 1940 ± 220 cells/mm2. These corneas were cryopreserved and examined in the phase-contrast microscope to assess the endothelial cell loss induced by freeze–thaw trauma.

The remaining six corneas were deemed to be unsuitable for transplantation, in that their donors registered serologically positive for hepatitis C within a mean time of 24 hours after enucleation. These corneas were organ cultured in the absence of dextran for a mean time of 32 ± 8.5 hours. After organ culturing, they were cryopreserved and analyzed in the confocal microscope.

Cryopreservation
The freezing medium consisted of MEM and 10% dextran. Before freezing, the corneoscleral discs were stored in 10 mL of the cryopreservation medium for at least 3 hours at 31°C to allow for osmotic equilibrium.10 They were then transferred to aluminum vials containing 0.5 mL of the cryopreservation medium within which they were maintained for 15 minutes at 3°C.10 The cryopreservation process was conducted in an automated freezing system (model BV 6; Cryson, Schoellkrippen, Germany) with an initial cooling rate of 1°C/min down to -40°C and then one of -5°C down to -80°C. The aluminum vials were then transferred to liquid nitrogen for storage at -196°C. After storage, the tissue within each vial was thawed in a water bath at 37°C.15

Study 1: Phase-Contrast Microscopy
After cryopreservation, 22 corneal discs were again organ cultured in Earle’s MEM containing 10% FCS and 6% dextran for 24 hours at 37°C. This second organ-culturing period was undertaken to allow wound-healing processes to be expressed, thereby permitting the detection of latent endothelial cell loss.15 16 To this end, the corneoscleral discs were stained first with alizarin red S at pH 4.2 for 2 minutes and then, after a rinse in 0.9% sodium chloride, with 0.25% trypan blue for 1 minute. In this mode of staining, all cell nuclei are stained by trypan blue, which aids in the identification of giant cells and mitotic figures. The presence of giant cells17 is indicative of severe cryodamage. Mitotic divisions may be triggered by the high concentration of FCS used and would mask a high endothelial cell loss.

Statistical Analysis
The percentage of endothelial cell loss after cryopreservation was calculated as the difference between the numerical density before and after cryopreservation and subsequent organ culturing. Parametrical data were compared by paired t-test, with the level of significance set at P = 0.05. Data are expressed as the mean ± SD.

Study 2: Confocal Microscopy
After cryopreservation and thawing, six corneas were evaluated by confocal microscopy. During confocal microscopy, the corneoscleral discs were maintained within tissue wells containing 10 mL of organ culture medium supplemented with 10% FCS and 6% dextran (Fig. 1) . Each cornea was evaluated immediately after thawing and then after 6, 24, and 48 hours of organ culturing at 37°C. Each cornea was positioned initially with its epithelial side uppermost and scanned from the epithelial to the endothelial layer. It was then turned over so that its endothelial side faced upward and was again scanned, with special attention being paid to the endothelium and posterior keratocytes. Confocal microscopy was performed with a prototype real-time, scanning slit confocal microscope (Hund Mikrophthal Wetzlar; Germany) as described in detail elsewhere.18 The microscope was equipped with nonapplanating, high-numerical-aperture (NA), water-immersion (40x/0.75 NA, 25x/0.65 NA; Leitz) objectives. To investigate the excised corneoscleral disc within the tissue well, we repositioned the objective from the horizontal to the vertical axis (Fig. 1) . Real-time sections of each corneal layer were recorded on S-VHS-videotape. Images of interest were digitized and then exposed to photographic film using a laser film printer (Optimas, Zürich, Switzerland).



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  		FIGURE 1. Confocal microscopy of a corneoscleral disc within a tissue well.

 
A cornea maintained for 4 days in organ culture and for 24 hours in deswelling medium served as the control.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Phase-Contrast Microscopy
Before cryopreservation, the mean numerical density of endothelial cells was 1940 ± 220 cells/mm2. After cryopreservation and subsequent organ culturing, the endothelial cell density declined by 33% to 1300 ± 360 cells/mm2, and two of the corneas had a completely necrotic endothelium (P = 0.001; Fig. 2 ). The other 20 corneas manifested a confluent monolayer that consisted of hexagonally shaped endothelial cells. No giant cells were apparent (Fig. 3) .



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  		FIGURE 2. Endothelial cell density before and after cryopreservation (n = 22; P = 0.001).

 


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  		FIGURE 3. Endothelial monolayer of a cryopreserved cornea 48 hours after the onset of organ culturing, stained with alizarine red and trypan blue. The cell pattern was fairly regular, there being only a few fusions and no giant cells. Inverted phase-contrast micrograph; scale bar, 10 µm.

 
Confocal Microscopy
Epithelium.
After up to 48 hours of organ culturing, each cornea manifested an intact Bowman’s membrane and a structurally integral layer of basal epithelial cells. Immediately after the corneas were thawed, basal cells displayed dark (nonreflective) cell bodies that were adumbrated by reflective borders, thereby rendering them clearly visible as individual units (Fig. 4A) . The nonreflective cells were irregularly interspersed with highly reflective bodies of similar size. Six hours after the onset of organ culturing, the basal cells detached from Bowman’s layer and differentiated into wing cells, the borders of which became more reflective (Fig. 4B) . After 24 hours, the wing cells were displaced progressively anteriorly into the organ culture medium. Individual wing cells were poorly visible, their nuclei being virtually nonreflective but their borders still reflective (Fig. 4C) . After 48 hours, the epithelium displayed multicornered superficial cells with high and low reflectivity (Fig. 4D) . The cryopreserved epithelium had a morphologic appearance similar to that of the control cornea.



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  		FIGURE 4. Confocal micrographs of the epithelial layer (objective: 25/0.65 NA). (A) Immediately after thawing, basal cells were visible; (B) 6 hours after thawing, the basal cells were beginning to detach from Bowman’s layer and to differentiate into wing cells; (C) 24 hours after thawing, epithelial cells were proliferating; the layer of wing cells was confluent; and (D) 48 hours after thawing, wing cells (poorly visible) were displaced progressively anteriorly. Scale bar, 10 µm.

 
Stroma.
Immediately after thawing and after up to 48 hours of organ culturing, keratocytes were clearly visible within the anterior, mid and posterior stroma (Fig. 5) . After 48 hours of culturing, some of the keratocytes had became highly reflective (Fig. 5D) . As in the control cornea, the stromal extracellular matrix and collagen fibrils were not visible. Within the posterior stroma, keratocytes exhibited a typically elongated appearance toward the intact and nonreflective Descemet’s membrane (Fig. 5) . With the exception of enhanced keratocyte reflectivity, the cryopreserved stroma had a morphologic appearance similar to that of the control cornea.



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  		FIGURE 5. Confocal micrographs of the posterior stromal layer (objective 40/0.75 NA). Appearance (A) directly after thawing and (B) 6, (C) 24, and (D) 48 hours after thawing. Keratocytes had a typically elongated appearance; the extracellular matrix was not visible. (D) Keratocyte with enhanced reflectivity. Scale bar, 10 µm.

 
Endothelium.
Immediately after thawing, the endothelial monolayer displayed randomly scattered bright areas corresponding in size to a single cell, but that were not clearly demarcated and lacked a clearly visible nucleus (Fig. 6A) . The three dimensional z-scan revealed these cells to be either completely detached from Descemet’s membrane or to be tenuously connected thereto by membranous structures. After 6 hours of culturing, attached endothelial cells were apparent as an array of hexagonal units that were demarcated by nonreflective borders (Fig. 6B) . Observations and photodocumentation were rendered difficult by the presence of posterior folds. Twenty-four hours after the onset of culturing, only a few endothelial cells detached sporadically. Fewer posterior folds existed and no denuded regions of Descemet’s membrane were visible. These changes were accompanied by an increase in cell polymorphism (Fig. 6C) . After 48 hours of culturing, the endothelial monolayer was confluent and comprised enlarged but still hexagonal cells. Their nuclei were poorly visible and the intercellular space was slightly widened. No further increase in cell polymorphism was apparent (Fig. 6D) .



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  		FIGURE 6. Confocal micrograph of the endothelial layer (objective: 40/0.75 NA). (A) Directly after thawing, randomly scattered bright areas, corresponding in size to a single cell, possibly represent degenerative cells leaving the monolayer; (B) 6 hours after thawing, hexagonally shaped endothelial cells were still detaching. Photodocumentation was difficult, owing to the presence of posterior folds. However, (C) 24 and (D) 48 hours after thawing, the endothelial monolayer was confluent. The hexagonal pattern of organization was still apparent, although there was an increase in cell polymorphism. Only a few cells were becoming detached. Scale bar, 10 µm.

 
In the control cornea, the endothelial monolayer was confluent and only a few sporadically cells detached. Individual cells maintained their hexagonal shape with but minor variations in size.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Little is known about the early cellular changes incurred by corneal tissue cryopreserved in the presence of intracellular19 20 or extracellular21 cryoprotectants. It is extremely difficult to compare the reported findings regarding cell viability after corneal cryopreservation, in that each study evaluates changes in a specific functional process or relies on the subjective assessment of cellular morphology.11 22 Another difficulty experienced in assessing the effectiveness of cryopreservation is that the deleterious changes noted using different viability assays may be reversed with time by repair. Similarly, latent injury which passes undetected immediately after thawing may lead to an underestimation of the true damage caused by freeze–thaw trauma.15 16 To reveal latent endothelial cell loss after freezing, cryopreserved corneoscleral discs were maintained in organ culture for at least 24 hours.13 15

The results of the present study demonstrate survival of human corneal endothelial cells, as judged by phase contrast and confocal microscopic evaluation, after cryopreservation of corneas in medium containing 10% dextran, but no penetrating cryoprotectants. Although their endothelial cell density was variable, only 2 of the 28 corneas displayed a completely necrotic endothelial layer. The mechanism whereby dextran protects against cryodamage is not well understood.10 23 In previous studies, the highly variable loss of endothelial cells from cryopreserved human corneas was attributed to differences in donor age and postmortem time, which led to the recommendation that only tissue derived from individuals below 50 years of age and obtained within 8 hours of death should be considered for cryopreservation.24 In the present study, endothelial cell density was determined by phase-contrast microscopy after corneal storage in dextran-containing medium for more than 1 week. There is evidence that storage in dextran for more than 4 days is harmful to corneas.25 26 Hence, in the present study, donor age, postmortem time, the prolonged incubation period in dextran-containing medium and the cell loss incurred during organ culturing before cryopreservation, may have predisposed corneal tissue to freeze–thaw injury and a high endothelial cell loss. The detachment of endothelial cells observed in the present study, as well in earlier investigations,9 27 is probably a result of osmotic or chemical damage or a combination of the two,28 and may involve disruption of the cytoskeleton and changes in cell-adhesion properties. This detachment phenomenon may thus be partially reversible.27 However, 2 of the 22 cryopreserved corneas displayed a completely necrotic endothelium. The reason for the highly variable cell loss remains unclear, but the absence of giant cells and the reestablishment of a confluent monolayer consisting of hexagonally shaped endothelial cells indicate that freeze–thaw trauma in 20 of the 22 corneas was of a moderate degree only.

Six hours after thawing, cryopreserved corneal discs manifested folding of Descemet’s membrane. This phenomenon is a consequence of overhydration and renders an evaluation by confocal microscopy more difficult. After 24 hours of organ culturing, cryopreserved corneal discs had undergone dehydration and only a few posterior folds remained, thereby improving contrast in the confocal microscope. The observed corneal dehydration could be attributable not only to an intact physical barrier function and a metabolically active pumping function of the endothelium, but also to a dextran-induced osmotic effect.29 Hence, it cannot be assumed that the physiological pump function of the endothelium was reestablished. However, previous perfusion studies have demonstrated that endothelial cells are indeed viable during this process of dehydration,13 although the possibility that the endothelium had undergone repair by mitotic division during the perfusion period cannot be categorically excluded.

In our study, the stroma of frozen corneas was morphologically well preserved. It has been postulated that the keratocytes may be protected from cryodamage and lysis by the surrounding collagenous lamellae.19 However, keratocytes within cryopreserved donor grafts fail to incorporate radiolabeled sulfate,30 and the incidence of apoptosis among this population of cells increases in such tissue,31 although most of the keratocytes contain normal-appearing organelles and are surrounded by collagen fibrils with the usual alignment.9 19 32 Given that apoptosis is not an immediate phenomenon, signs of its being underway may not be apparent immediately after thawing. Apoptotic keratocytes have been observed 24 hours after thawing,31 but we detected no evidence of keratolysis or stromal disorganization after up to 48 hours of organ culturing.

An evaluation of corneas by confocal microscopy after 48 hours of organ culturing revealed the reflectivity of some of the keratocytes to have increased. Similar changes have been reported in cases of herpetic keratopathy or after photorefractive keratectomy and have been interpreted as a sign of augmented keratocyte activity during wound healing.33 34 An increase in reflectivity may also result from the phagocytosis of dextran by keratocytes.35 Corneas used for confocal microscopy were exposed only once to dextran-containing medium for a maximum of 48 hours after thawing. However, keratocytes have been shown to contain dextranlike intracellular deposits, even after a single day of exposure to this agent.25 The presence of these intracellular vacuoles is not necessarily a sign of cell degeneration or death, but may be merely indicative of high endocytotic activity.29 36 However, there is strong evidence that, even after 4 days of exposure to this agent, endothelial cell viability is impaired.25 26 Whether dextran indeed affects cell viability is as yet unknown. Because we did not examine the cells by transmission electron microscopy or stain specifically for dextran, we were unable to demonstrate its intracellular presence.

The state of the corneal epithelium at the time of dissection is known to be quite variable. It is influenced not only by postmortem time but also by factors such as lid closure and disinfection. In organ culture containing 2% FCS the entire epithelial sheet may slough off and be regenerated from the limbal region of the corneoscleral discs.14 In our study, the epithelium was multilayered and differentiated immediately after thawing. The borders of the wing cells were high reflective, indicating a hypoxic state.12 However, up to 48 hours of organ culture it manifested a high proliferative activity, which indicates that the cells were viable. This finding reflects above all, the high concentration of FCS used (10%).

The present study demonstrates that each corneal layer is capable of regaining and maintaining its structural integrity for up to 48 hours after cryopreservation in the presence of 10% dextran, but no penetrating cryoprotectants. The endothelium in particular, being crucial for corneal transparency, maintained its capacity to undergo dynamic morphologic changes such as wound healing and re-established a confluent monolayer. Unlike other cryopreservation techniques, such as vitrification37 or freezing in the presence of DMSO,8 27 38 our method is easy to perform. It does not require many incubation steps and does not involve high concentrations of cryoprotectants or rapid cooling and thawing rates. This study represents the first attempt to cryopreserve human corneas in the presence of dextran using a technique that was developed for the freezing of porcine corneas.10 Since the results are still highly variable, the technique must be further refined before it can be considered for clinical application in humans. In particular, the phenomenon of delayed cryodamage8 31 must first be thoroughly investigated and understood.


   Footnotes
 
Supported by Grant HA 1526/3-1 from the German Research Foundation.

Submitted for publication April 11, 2003; revised July 13 and August 10, 2003; accepted August 12, 2003.

Disclosure: M. Halberstadt, None; M. Böhnke, None; S. Athmann, None; M. Hagenah, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Markus Halberstadt, Department of Ophthalmology, Inselspital, 3010 Bern, Switzerland; markus.halberstadt{at}insel.ch


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 Methods
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