Medium-term preservation of human corneas in an enriched culture medium at +37 degrees Centigrade

A study of 36 penetrating keratoplasties using donor corneas stored at +37 degrees C in organ culture incubation for an average of 12 days prior to transplantation is reported. The criteria for the survival of corneal grafts were mean central corneal thickness, clarity of the cornea and mean endothelial cell density. A total of 86% of the grafts remained clear after 12 months follow-up time. The mean central corneal thickness was 0.47 mm. The mean endothelial cell density was 1452 cells/mm2. Based on the results of this study, organ culture reported here appears to be a safe and efficient method of medium to long-term corneal storage.     

Preservation of human corneas in an enriched culture medium at +37 degrees Centigrade: histological and biochemical analyses

Using the methods of Doughman, Sperling and Pels 66 human corneas were preserved up to five weeks in a modified tissue culture medium at +37 degrees C. The organ culture medium consisted of the following ingredients: Dulbecco Iscov's medium, 2% fetal calf serum, penicillin 100 IU/ml, Streptomycin 0.5 mg/ml, and fungizone 5 mg/ml. After culture the corneal endothelium was examined by light microscopy after staining with trypan blue 0.3% and alizarin red 1%. The number of dead cells was counted and the morphological alterations were described at 7, 14, 21 and 35 days. Biochemical analysis of the medium (pH, lactate, glucose) during storage has allowed the comparison of three kinds of storage: 50 ml, 20 ml, and 20 ml with 10 ml substitution weekly. After culture the number of dead cells did not exceed 1% at each period indicating that no dead cell was present at that time. Alteration of the cell shape and formation of rosette and joint meetings of 5 to 8 cells were the dominating morphological changes of the endothelial cells. The endothelial layer was intact and coherent on to 35 days culture. Endothelial cells loses during culture were not determined. The biochemical studies have shown the better quality (pH stability, anaerobic ratio) of storage in 50 ml medium or 20 ml 50% weekly substitution medium. This last kind of storage has given the best metabolic conditions for the preserved corneas. However for long period of storage up to 3-4 weeks, renewing 50% of the medium is found appropriate. At the end of this study a clinical trial is proposed.     

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.     

Human grafts preserved at +37 degrees C. Initial clinical results

This study reports the clinical use of donor corneas stored at +37 degrees C in organ culture medium. Twenty-five penetrating keratoplasties were performed. The mean storage time of organ-cultured donor corneas was 12.3 days (3-21). The follow up of the patients was 12 months. The criteria for the survival of corneal grafts was mean central corneal thickness, clarity of the cornea and mean endothelial cell density. The graft survival was 92% at 12 months. The mean central corneal thicken was 0.54 mm. The mean endothelial cell density was 1625 cells/mm2. The mean decrease in endothelial cell density at 12 months was 40.6%.     

Tolerance of corneas to multimolar dimethyl sulfoxide at 0 degrees C. Implications for cryopreservation

Attempts to improve current methods of cryopreservation of corneas, whether by conventional freezing and thawing or by vitrification in the absence of ice, will require the use of high concentrations of cryoprotectants. In this study we extend our previous investigation of the tolerance of rabbit corneas to multimolar concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) added and removed at 0 degrees C in CPTES, a hyperkalaemic preservation solution containing the impermeant anionic buffer N-Tris(hydroxymethyl)methyl-2-aminoethane sulphonate (TES). Isolated corneas were exposed to 1, 2 or 3 mol/l Me2SO at 0 degrees C to minimize any effect due to temperature-dependent chemical toxicity and attention was given to the procedure for diluting Me2SO from the tissue in order to minimize osmotic stress to the endothelium. Endothelial integrity following these procedures was assessed both by the ability to control stromal hydration during perfusion on the specular microscope and by the structural integrity when examined by light and electron microscopy. The presence of an active endothelial pump and good morphology were demonstrated in corneas exposed to 1 and 2 mol/l Me2SO; serial dilution of the cryoprotectant was more beneficial than a single-step direct dilution. Corneas immersed directly into 3 mol/l Me2SO were irreparably damaged irrespective of the method of dilution. Sequential addition of 1 M, then 2 M and finally 3 M cryoprotectant followed by serial dilution was, however, tolerated by the endothelium and minor alterations to the structural integrity of the endothelial layer were rapidly repaired. The osmotic nature of these observations are analyzed and discussed.     

The prevention of autolysis in stored corneas by lysosome stabilization. A histochemical study.

We wished to determine if dexamethasome, acting as a lysosome stabilizer, could reduce the release and activation of the lysosomal acid hydrolases, and thus retard autolysis of stored corneas. One cornea (experimental) of a rabbit was soaked in a 2 per cent steroid solution and the other cornea (control) in physiological saline for 3 hours at 23 degrees C. The experimental and control corneas were then processed histochemically to show the localization of the lysosomal marker enzymes beta-glucuronidase and acid phosphatase. Compared to the controls the steroid treated corneas showed reduced enzyme activity suggesting that autolysis during storage had been retarded.     

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells

BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.     

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.     

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.     

Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n?=?12) and fibroblastic-like (thin and very elongated cells, n?=?6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in?vitro studies of this disease.     

Optical microscopy study of human sclera stored in different media

PURPOSE: To describe microscopic changes in the structure of human sclera immediately after enucleation (negative control group) and to compare them after being stored for three months in four different media: pure glycerin, absolute alcohol, benzalkonium chloride diluted in absolute alcohol (1:5000), and benzalkonium chloride diluted in balanced salt solution (1:5000). The comparison took into consideration their final state of conservation. DESIGN: Experimental study, laboratory investigation. METHODS: Optical microscopy was used to study the specimens after they had been in storage for one, two, and three months. The scleral fragments were prepared in thin plates, dyed, and then submitted to histologic analysis by two masked specialists. Unpreserved scleral fragments obtained right after enucleation were assessed and served as negative controls. RESULTS: The collagen fibers of scleras stored in glycerin presented with a more regular pattern, closer in appearance to the negative control group. Scleras stored in the other three media presented contorted collagen fibers. CONCLUSIONS: Our findings suggest that glycerin is close to being the ideal storage medium for sclera, because it maintains the structural characteristics of the collagen fibers. Research is ongoing to determine how to increase the bactericidal and antiviral properties of glycerin storage.     

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium

PURPOSE: To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. METHODS: Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. RESULTS: When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. CONCLUSIONS: FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.     

Influence of temperature on corneas stored in culture medium. A comparative study using functional and morphological methods

PURPOSE: To investigate the influence of storage temperature on corneal swelling and on endothelial morphology in cultured corneas. MATERIAL AND METHODS: Forty-eight rabbit corneas were separated into four groups of 12. The corneas were stored in culture medium at 37 degrees (group 37), 34 degrees (group 34), 31 degrees (group 31) and 23 degrees (room temperature) (group 23), respectively. All the corneas were monitored by weight recordings on days 0, 1, 2, 3, 4 and 7. On day 7, corneas were prepared for scanning electron microscopy and endothelial cell counts were performed. RESULTS: Lowering the temperature of the culture medium resulted in less swelling. Both temperature and storage time had significant effects on corneal swelling (p < 0.001). On day 7, the observed mean weight increase was 131.2%, 143.0%, 172.5% and 199.7% in groups 23, 31, 34 and 37, respectively. The estimated mean daily weight increase for the corneas were 2.6%, 4.0%, 9.1% and 16.0% in groups 23, 31, 34 and 37, respectively. Scanning electron microscopy showed an intact endothelial layer in all groups after 7 days and there were no statistically significant differences in endothelial counts between groups 23, 31 and 34. In group 37, the cell borders were difficult to distinguish after 7 days and no meaningful count could be performed. CONCLUSIONS: The swelling rate of cultured corneas is significantly less at 23 degrees and 31 degrees than it is at 34 degrees and 37 degrees during the first week. This is most likely the result of a greatly increased barrier effect at lower temperatures. Whereas weight recording revealed profound differences between the groups, scanning electron microscopy and endothelial cell counting did not. The results support the hypothesis that storage at 37 degrees is not optimal in culturing corneas. Lowering the temperature below body temperature, and even lower than 31 degrees, results in less corneal swelling.     

Can steroid reduce endothelial damage in stored corneas? Effect on cell viability and ultrastructure

We studied the effect of steroid on the viability and integrity of the endothelium of variously stored corneas of different species of animals and man. Paired corneas of rodents and humans were used,--one cornea (experimental) being treated with steroid and the other (control) not. Rat and guinea pig corneas were kept at 37 degrees C for one hour in phosphate buffered saline with or without hydrocortisone 21-sodium succinate (10-6M). Human corneas were kept at 4 degrees C for 4 days in M-K medium with or without hydrocortisone 21-sodium succinate (10-6M). The viability of the endothelia (rat and guinea pig) was tested with para Nitro Blue Tetrazolium and trypan blue dye exclusion tests. The ultrastructural changes in the control and experimental endothelial (guinea pig and human) were examined by scanning electron microscopy. Viable cells P less than 0.001) were more numerous and ultrastructural alteration less in the endothelia of the steroid treated corneas compared to the controls.     

Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats

PURPOSE: The feasibility of corneal reconstruction with cultured adult human corneal endothelial cells (HCEC) was examined in a nude rat model. METHODS: Endothelial cells were removed from the corneas of Lewis rats using a sterile cotton swab. Cultured adult HCEC labelled with a fluorescent marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) were seeded onto the denuded Descemet's membrane. Then the corneas were centrifuged, incubated for 2 days, and transplanted into the eyes of nude rats using the penetrating keratoplasty technique (HCEC group). Control nude received corneas denuded of endothelium and without HCEC. The operated eyes were observed for 28 days after transplantation, and then were subjected to histological and fluorescein microscopic examination. RESULTS: The mean corneal thickness was significantly smaller in the HCEC group than in the control group throughout the observation period. The corneal endothelial cell density of the grafts at 28 days postoperatively ranged from 2425 to 3250 cells mm(-2) (mean+/-sd, 2744+/-337 cells mm(-2)). Fluorescein microscopy at 28 days after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the HCEC group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet's membrane. CONCLUSIONS: Cultured adult HCEC function well and maintain corneal transparency for 1 month after transplantation in nude rats.     

Variations in endothelial morphology of normal corneas and after cataract extraction. A specular microscopic study

The central corneal endothelium of 22 normal subjects and 11 unilateral aphakic subjects was photographed with the non-contact specular microscope. The aphakic patients had undergone intracapsular cataract extraction one to four years previously. Endothelial cell densities were estimated. All cells which had been counted were then grouped according to their number of neighbours. The percentages of cells having 4, 5, 6, 7, or 8 neighbours were means +/- SD): 0.4 +/- 0.8; 18.0 +/- 3.4; 64.1 +/- 6.5; 15.9 +/- 2.6 and 0.9 +/- 0.8 per cent, respectively in the normal eyes, and 0.8 +/- 1.3; 20.9 +/- 3.7; 57.4 +/- 5.6; 19.6 +/- 3.9 and 1.4 +/- 1.2 per cent in the aphakic group, respectively. The frequency of cells having 5, 6, or 7 neighbours were significantly different in the two groups. Cells with 9 or 10 neighbours were not seen in the normal group but occurred in 2 and 1 of the aphakic eyes, respectively. In the normal group the frequency of hexagonal cells was found to correlate to the cell density (r = 0.46, 2P less than 0.01) and age (r = -0.40,2P less than 0.05).     

Electron microscopic study of the endothelium of stored and cryopreserved monkey corneas

Summary Monkey corneas were either cryopreserved at a controlled rate over liquid nitrogen in a solution containing dimethylsulfoxide, sucrose and serum albumin, or were stored in moist chambers at 4°C for 48 hours. The endothelium was examined by scanning and transmission electron microscopy and the results compared. The entire endothelial layer of cells was always intact and there was little evidence of freeze-thaw-induced structural damage in the cryopreserved corneas. Mitochondrial swelling and clumping of nuclear chromatin was observed in the stored corneas.

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Zusammenfassung Hornhäute von Rhesusaffen (Macaca arctoides) wurden entweder nach der Methode von Kaufman und Capella gefrierkonserviert oder in feuchten Kammern bei +4°C für 48 Std gelagert. Anschließend wurde das Endothel elektronenmikroskopisch untersucht. Die vergleichende Auswertung ergab, daß die gesamte endotheliale Zellschicht immer intakt war. Die gefrierkonservierten Hornhäute waren in ihrer Struktur durch den Gefrierprozeß nur geringfügig geschädigt. In den bei + 4° C gelagerten Hornhäuten waren Schwellung der Mitochondrien und Verklumpung des Kernchromatins zu beobachten.

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This investigation was conducted while Dr. Waller was a Fellow in the Departments of Ophthalmology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin U.S.A. This work was supported in part by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg (WA 298/1) (Dr. Waller); and by a grant (EY 00625-01A1) from the United States Public Health Service-National Eye Institute, Bethesda, Maryland; and by Designated Research Funds from the Veterans Administration (Dr. Van Horn).     

Effects of human epidermal growth factor on endothelial wound healing of human corneas.

Paired human donor corneas (age, 73 +/- 12 yr), preserved in organ culture medium, were used to evaluate the effect of human epidermal growth factor (hEGF) on endothelial wound closure rate (WCR), on morphometric parameters (cell size, shape, and density), and on cell division in the wound area. The endothelium of the corneas was mechanically wounded (area, 4.9 +/- 0.9 mm2). For each pair, one cornea was treated with 10 ng/ml hEGF, while the mate served as control. WCR was assessed by daily staining of the corneas with trypan blue. Morphometric data were obtained after alizarin staining. Mitotic activity was assessed using 3H-thymidine autoradiography. Addition of hEGF significantly increased the WCR compared to the control group. In the closed wound (between 4-9 d), the mean cell size in the center averaged 1940 microns2 in the control group and 1287 microns2 in the hEGF-treated group (P less than 0.01). Fifteen days after wounding, the mean cell sizes averaged 1910 microns2 and 1427 microns2 in the control and hEGF-treated group, respectively (P less than 0.01). All corneas exposed to hEGF had higher endothelial cell densities than the control corneas. In the early stages of wound closure, the cells in the transitional zone in hEGF-treated corneas had a somewhat more elongated shape. However, hEGF did not affect the final cell shape within the closed wound. Autoradiographic results revealed that hEGF accelerated DNA-synthesis, although only to a limited extent. The results indicate that, in human corneas, hEGF promotes endothelial wound healing predominantly by cell migration, at least in corneas from senior donors.     

Medium term preservation of corneas. Comparative study of 4 preservation media

A comparative study of four preservation medium was made (Corneal Storage Solution, K-Sol, RPMI Hepes buffer, RPMI bicarbonate buffer). After a five days preservation delay there is no statistical difference between the percentage of the endothelial cellular mortality amongst the three first medium. In the fourth medium the cellular mortality is statistically higher. For longer preservation delay between 5 and 15 days, only the corneas preserved in K-Sol medium have a cellular mortality under 20% and could be used for keratoplasty.