Corneal Preservation Research Articles

Corneal graft survival in the cat with prolonged preservation in McCarey-Kaufman and K-Sol media.

The effects of extended preservation on transplanted corneas were investigated using McCarey-Kaufman (M-K) and K-Sol media. Cat corneas were preserved in either M-K or K-Sol at 4 degrees C for 5, 10, 15, or 20 days and were subsequently transplanted. Postoperatively, transplants were observed via slit-lamp biomicroscopy and pachymetry and at postmortem examination by light and scanning electron microscopy. In this study, we found no difference in the corneal graft clarity, thickness, endothelial cell count, or morphologic features of corneal transplants preserved in either M-K or K-Sol media. Storage for more than ten days in either M-K or K-Sol media led to primary graft failure. This study demonstrated equivalent results in corneal preservation using either M-K or K-Sol media. The development of better preservation media may require changes in essential nutrients rather than changes in osmotic constituents.

K-Sol corneal preservation at room temperature.

K-Sol, a recently developed corneal storage medium that contains purified chondroitin sulphate in tissue culture medium (TC 199), is capable of preserving corneal tissue for 14 days at 4 degrees C. To study the effect of tissue storage in K-Sol at room temperature we preserved rabbit corneas in K-Sol and M-K medium for three or seven days at 25 degrees C. All of the corneal endothelial sheets were intact after three days. At seven days the change in pH of the K-Sol medium was less than that of M-K medium. Corneas preserved in M-K medium showed swelling of mitochondria and a decrease in the number of cytoplasmic organelles. Corneas preserved in K-Sol had organised cytoplasmic organelles and nuclei. Scanning electron micrographs revealed well preserved endothelial sheets. Corneas stored in the two media showed no significant difference in thickness. A pair of human corneas preserved in K-Sol at room temperature for six days maintained about 95% of the endothelial sheet in good condition. Small separations were observed between some of the endothelial cells. However, even in these areas, the cytoplasmic organelles were well preserved. It appeared that K-Sol is more stable than M-K medium at room temperature, and that both rabbit and human corneas can be preserved in good condition in K-Sol for at least six days at 25 degrees C.

ASSESSMENT OF THE DUTCH ORGAN‐CULTURE SYSTEM OF CORNEAL PRESERVATION WITHIN THE EYE BANK OF SOUTH AUSTRALIA

Thirteen per cent of all corneas harvested by the Eye Bank of South Australia during 1986 were discarded because storage time in McCarey-Kaufman medium exceeded four days. We have therefore examined the suitability of the Dutch method of long-term corneal storage for our purposes. Twenty-two human corneas that had been discarded from the Eye Bank were assessed using the trypan blue-sucrose staining technique, and then placed into long-term storage for 15 to 17 days. They were then reassessed by vital dye staining before permanent flat-mounts were prepared for silver staining of the endothelium. A good correlation (albeit subjective) was found between the non-destructive and destructive techniques of endothelial cell assessment. Those corneas that failed to survive organ culture storage were easily detected. The Dutch system of corneal preservation and post-storage assessment seems well-suited to Australian eye-banking.

Storage of corneas at 0 °C in CPTES, a hyperkalaemic solution for corneal preservation

Storage of corneas at 0 °C in CPTES, a hyperkalaemic solution for corneal preservation

Langerhans cells in organ-cultured corneas.

We examined the effect of corneal preservation in organ culture at 34 degrees C on Langerhans cells in murine and human corneas. The presence of Langerhans cells was demonstrated by an adenosine triphosphatase stain. Langerhans cells were identified on fresh corneas and on corneas that had been in organ culture for one week or less. However, Langerhans cells were not found on most corneas after the second to third week in culture. The absence of Langerhans cells was found to be directly related to the condition of the corneal epithelium. During the second and occasionally as late as the third week in culture, the superficial layers of epithelium are sloughed. Examination of the corneas lacking Langerhans cells disclosed Langerhans cells in the sloughed epithelium. The results demonstrate that most corneas preserved in organ culture at 34 degrees C do not contain Langerhans cells at the time of transplantation.

Corneal Preservation and Eye Banking

A comparison was made of the accuracy of the Tono-Pen, Pneumatonometer, and Perkins hand-held tonometers by measuring the hydrostatically controlled intracular pressure from 10 to 50 mm Hg in human eye bank eyes. The open stopcock method was used in which the cannulated eye, the hydrostatic pressure controlling column, and a manometer were continuously open to each other. The Pneumatonometer gave accurate results at 50 mm Hg but overestimated the lower intraocular pressures; whereas, the Perkins gave satifactory results at 10 and 20 mm Hg but underestimated higher intraocular pressures. The Tono-Pen displayed the least deviation from the controlled pressure over the entire range studied. Another aspect of this study involved determining that a therapeutic soft contact lens did not have any noticeably adverse effect on the accuracy of the Pneumatonometer and Tono-Pen tonometers.

Current Advances in Corneal Preservation at 4??C with Chondroitin Sulfate-Containing Medium and the Introduction of a New Corneal Holder and Trephination System

Current Advances in Corneal Preservation at 4??C with Chondroitin Sulfate-Containing Medium and the Introduction of a New Corneal Holder and Trephination System

Chondroitin sulfate for corneal preservation at 4 degrees C. Evaluation by electron microscopy.

We tested three new corneal preservation media in which the dextran in McCarey-Kaufman (M-K) medium was replaced by three preparations of chondroitin sulfate (CS): 1.5% CS, 5.0% CS, and 2.0% modified CS (K-Sol). One cornea from each of 36 pairs of corneas was placed in M-K medium and the opposite cornea was placed in one of the CS media. Endothelial preservation was evaluated by electron microscopy after storage at 4 degrees C for 1, 3, 5, 7, 10, and 14 days. We judged cellular preservation in a masked manner from the electron micrographs according to a morphologic grading system. Preservation in 1.5% CS solution was similar to that in M-K medium. Both 5.0% CS and K-Sol gave better endothelial preservation than M-K medium. The best preservation appeared to be in K-Sol, in which only 3% of the endothelial cells were disrupted after 14 days of storage at 4 degrees C.

Replication of fungi in K-Sol corneal preservation medium at 4 degrees C.

We examined the growth of six fungi during 18 days at 4 degrees C in a corneal preservation medium containing 2.5% chondroitin sulfate (K-Sol). The number of viable organisms decreased with time for all six fungi, the average decrease being 65%. The risk of transplanting fungal organisms with a donor cornea stored for up to 18 days at 4 degrees C in preservation media containing chondroitin sulfate is no greater than that with the same cornea stored for only one day.

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 °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 °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. Current research aims to develop improved methods of corneal cryopreservation, and there is good reason to anticipate that improved methods will be available for clinical trial in the next several years.

Minnesota system corneal preservation.

The clinical and laboratory results with a modified Minnesota system of organ culture corneal preservation are presented. A refinement of our preservation technique using a closed system, as well as the addition of chondroitin sulphate to the medium is presented. Laboratory results show preservation of corneal endothelial integrity for at least 21 days with maintenance of normal corneal thickness. In addition, a 10-day quarantine system reduces the risk of donor contamination and secondary endophthalmitis. Preliminary results of the 34 degrees C and 4 degrees C closed Minnesota corneal preservation system using chondroitin sulphate show that it is safe and efficacious and allows intermediate to long-term maintenance of sterile thin tissue prior to corneal transplantation.

Decreased Endothelial Cell Survival After Transplantation of Corneas Preserved by Three Modifications of Corneal Organ Culture Technique

We placed human donor corneas in M-K medium at 4° C for 24 hours, cultured them in minimal essential medium at 34° C for two to five weeks, and then either (1) placed the corneas in M-K medium at 4° C for 48 hours before transplantation (Group 1, 47 eyes); (2) placed the corneas in M-K medium at 4° C for 16 hours before transplantation (Group 2,17 eyes); or (3) transplanted the corneas without postculture cooling to 4° C (Group 3, 11 eyes). We compared the corneas preserved by organ culture with an equal number of corneas transplanted during the same period, but preserved only in M-K medium at 4° C for one to four days. The central endothelial cell losses noted two months after keratoplasty were significantly greater in the organ-cultured corneas than in the M-K-preserved corneas in each of the three groups. The mean endothelial cell loss in the 11 organ-cultured corneas in Group 3 was significantly less than that in the 64 organ-cultured corneas in Groups 1 and 2. The corneas in Group 1 were also examined one year after keratoplasty, and the cell losses in the organ-cultured grafts remained significantly greater than those in the M-K-preserved grafts. These results indicate that the culture of donor corneas in minimal essential medium at 34° C for several weeks (after storage in M-K medium at 4° C for 24 hours) decreases the ability of the endothelial cells to survive the trauma of keratoplasty, and that postculture storage at 4° C in M-K medium decreases this ability further. Photographic assay of central endothelial cell survival was a more sensitive indicator of the efficacy of the method of corneal preservation than was the clinical success rate, which was the same (96%) in both the organ-cultured and M-K-preserved grafts. New methods of corneal preservation should be tested by the endothelial cell assay technique.

A new preservation solution for storage of corneas at low temperatures

Cryopreservation methods are only rarely used today for storing donor material for use in penetrating keratoplasty. There remains, however, a continuing need for a reliable technique for the long-term preservation of corneas. As a first step in developing an improved method of corneal cryopreservation we have studied the composition of the preservation medium. On the basis of experiments with other tissues a new solution (designated CPTES) has been formulated, containing concentrations of potassium, sodium and chloride similar to normal intracellular levels, in order to restrict the ionic imbalances that occur when temperature is reduced and ionic pumps switched off. An impermeant anion was included to reduce cellular swelling during low temperature storage. Integrity of the corneal endothelium was demonstrated following exposure to CPTES medium at 0 degree C: control of corneal hydration and the presence of an active bicarbonate - dependent pump were demonstrated by specular microscopy; normal ultrastructure of the endothelial layer was revealed by electron microscopy. The incorporation and removal of 1M dimethyl sulphoxide (Me2SO) is tolerated in corneas bathed in CPTES medium at 0 degree C, again with retention of endothelial structural and functional integrity. CPTES is proposed as a medium which will help to control osmotic and ionic disturbances during experimental procedures designed to improve methods of corneal preservation both above and below 0 degree C.

Possibilities and biological properties of variously applied methods of corneal preservation

As the demand for donor corneas increases, not only fresh donor material but also increasing numbers of preserved corneas are being transplanted. The simple eye-banking procedures such as the moist chamber and the McCarey-Kaufman medium provide sufficient endothelial cell viability for short periods. The methods of organ culture of corneoscleral disks extend storage periods to up to four weeks. Cryopreservation as a method of long-term storage has not come to be widely used owing to the complicated procedure involved and the considerable damage thus caused to endothelial cells. Thus, the organ culture method may represent a compromise between extended storage periods and good endothelial cell viability.

Penetrating Keratoplasty Using McCarey-Kaufman Preserved Corneal Tissue

Two hundred consecutive penetrating keratoplasties, using suitable donor corneas stored in modified tissue culture medium (M-K medium), were performed by a single surgeon over a 21-month period. Two hundred consecutive cultures of the donor corneosclearl rims for aerobic and anaerobic bacteria and fungi, done immediately after surgery, yielded 25 positive cultures for aerobic and anerobic bacteria and three for fungi. No clinical infections nor primary donor failures were encountered with patients after one to 21 months of follow-up. Preservation of excised corneas by trained technicians, under rigidly sterile, laminar-flow, tissue culture conditions appears to be a convenient, safe, and effective method of corneal preservation which permits elective keratoplasty.

Corneal preservation at ?4�C in a modified tissue culture medium

Addition of 6% PVP (polyvinylpyrrolidone, mol wt 40,000) to tissue culture medium No. 199 prevents it from freezing at -4 degrees C. Forty donor corneas stored for five and ten days in this medium had a mean thickness of 0.61 and 0.67 mm, respectively. By means of specular microscopy, intact endothelium was found after these storage periods. Epithelial cell layers were histologically present. Electronmicroscopic studies revealed only slight changes in the subcellular endothelial structures. During perfusion with modified Ringer's solution at +34 degrees C, the thickness of stored corneas decreased.

Organ Culture Corneal Storage at Ambient Room Temperature

• A simple Inexpensive method of organ culture corneal storage at ambient room temperature in which the media is never changed (closed system) has been developed. Human and cat corneas preserved in sealed heat-resistant culture tubes containing 50 ml of a modified Eagles minimum essential medium at ambient room temperature (22 to 24 C) retained active metabolism with adequate medium substrate and pH buffering for 7 to 12 days. Scanning and transmission electron microscopy revealed maintenance of an ultrastructurally intact endothelial layer for 6 to 7 days in human corneas and up to 14 days in cat corneas. Human endothelial function as measured by in vitro physiologic deturgescence capability was retained after up to 13 days storage. Good control of tissue sterility was achieved and observation of media clarity and pH (phenol red indicator) provided a check for microbial contamination. Closed system organ culture corneal storage at ambient room temperature provides several advantages as a method of corneal preservation. ( Arch Ophthalmol 95:869-878, 1977)

Experiments on Corneal Preservation (Short-Time Storage)

56 human eyes were stored in a moist chamber from 0 to 4 days at +4°C. 20 human corneal buttons with a scleral rim were stored in the M-K medium for 5 and 7 days at +4°C. After this procedure in both series the temperature reversal effect was measured and the living endothelial cells were counted after being stained with trypan blue and rose bengal. All eyes showed a typical swelling-shrinking phenomena but after 4 days in a moist chamber this effect was definitely less characteristic than in all other periods of storage time. In respect to the endothelial cell counts 5 day M-K medium storage corresponds to 2 day moist chamber storage, and 7 day M-K storage is comparable to 3 day moist chamber storage. The total survival time of the endothelial cells in both series is almost equal.

Corneal Preservation, Clinical and Laboratory Evaluation of Current Methods

Corneal Preservation, Clinical and Laboratory Evaluation of Current Methods

Corneal Preservation: (Clinical and Laboratory Evaluation of Current Methods).

Have we really entered the era when corneal surgeons can routinely schedule and operate on patients who require penetrating keratoplasty? Have the ingredients of the TV drama, the collection of donor material from remote places, the rush to transfer the donor to the operating surgeon, the long wait by the telephone for the call summoning the patient to immediate surgery—have all of these become a thing of the past? The answers, of course, depend on whether preserved corneal tissue is as good as fresh donor material for penetrating keratoplasty. The book Corneal Preservation is the report of a symposium held in Gainesville, Fla, in December 1970, bringing together ophthalmic surgeons, basic scientists, and Eye Bank personnel to discuss banking of donor material, preservation techniques, the changes that occur in eyes that have undergone preservation, and the application of these methods to the actual transplantation of tissue into human eyes.