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| CURRENT OPHTHALMOLOGY |
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| Year : 1995 | Volume
: 43
| Issue : 2 | Page : 55-58 |
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A review of methods for storage of corneas for keratoplasty
Prasanta Kumar Basu
Department of Ophthalmology, University of Toronto, Canada, USA
Correspondence Address:
Prasanta Kumar Basu
37 Chambery Crescent, Unionville, Ontario, L3R 6L6, Canada
USA
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 8818310 
This article reviews the various methods used for the storage of the donor cornea for keratoplasty. The methods have been classified in terms of the duration of storage as (a) short-term, (b) intermediate term (c) long-term and (d) very long-term.
The practical importance of the moist-chamber method of short-term storage has been discussed. A short report on a new intermediate-term corneal storage medium using steroid as a lysosome membrane stabilizer has also been included. Organ culture of corneas for long-term storage is not popular in the developing countries due to lack of appropriate storage facilities. Cryopreservation as a long-term storage technique still attracts researchers' attention. Keywords: Corneal blindness - Keratoplasty - Corneal storage methods.
How to cite this article:
Basu PK. A review of methods for storage of corneas for keratoplasty. Indian J Ophthalmol 1995;43:55-8 |
A corneal storage system for penetrating keratoplasty has two main general objectives: (1) to maintain the endothelial viability and integrity so that an optional number of viable endothelial cells remain attached to the Descemet's membrane, thereby keeping the endothelial water-pump mechanism intact; and (2) to increase the duration of storage so that an efficient use of the donor corneas can be made.
The sudden arrest of the aqueous humour formation after death, and the depletion of nutrients and oxygen supply to the eye for variable lengths of time, especially at room temperature, result in the initial damage of the corneal cells by autolysis.[1] The time during which the cadaver is exposed to the room temperature should, therefore, be as short as possible.
In terms of the duration of storage of the donor material, the period of preservation has been arbitrarily classified as (a) short-term storage (b) intermediate-term storage (c) long-term storage and (d) very long-term storage.
| SHORT-TERM STORAGE | |  |
For storing corneal donor materials for a few days, two techniques are used: Moist-chamber storage and M-K medium storage.
| Moist-Chamber Storage | | |
The whole donor eye is kept in a sterile jar filled with saturated moist atmosphere at 4°C. If the cadaver time (time between the death of the donor and the enucleation of the donor eye) is short (4 to 6 hours), the donor eyes can be stored for two days in the moist chamber. The shorter the time of storage, the better is its effects on the cornea.
The intact donor eye enables the surgeon to modify the excision technique of the transplant. The most important criticism against the moist-chamber storage method is that in an intact eye, the endothelium is exposed to postmortem changes in the aqueous humour. However, our knowledge about these changes is still rudimentary.[2]
The moist-chamber storage technique is the simplest and the least expensive of all the storage techniques. It is advantageous in many situations, particularly in the developing countries where the need for donor corneas is the greatest and the benefits of tissue culture laboratories and bacteriologically controlled areas are not generally available to the eye banks. In these situations, efforts should, therefore, be concentrated on collecting as many donor eyes as possible within a short time after donors' death. For this reason, keratoplasty surgeons should consider corneal grafting as an emergency surgery rather than an elective one.
| M-K Medium Storage | | |
Historically, this is the first successful method for storing excised cornea (corneoscleral button) in a chemically defined tissue culture medium at 4°C. This method appears to be better than the moist-chamber method as the donor corneas are not exposed to the stagnant aqueous humour of questionable composition,[3]
M-K medium, described by McCarey and Kaufman,[4] is a mixture of tissue culture medium (T-C 199)[5] and dextran (5%, 40,000 molecular weight). As a colloidal osmotic agent, dextran prevents excessive stromal swelling of the excised corneas in the liquid medium. Besides dextran, the medium includes other additives, such as, HEPES (N[1][2]hydroxyethylpiperazine-N-ethane-sulphonic acid) as buffer, penicillin and a combination of gentamicin and polymyxin as antibiotics. Since the medium uses HEPES, phenol red (phenol-sulfonphthalein) as pH indicator is not included. According to McCarey and Kaufman, the M-K medium has been found to increase the corneal storage time to approximately 96 hours.
| INTERMEDIATE-TERM CORNEAL STORAGE | | |
The development of the intermediate-term corneal storage medium has facilitated a better maintenance of the donor cornea for periods, for which, the moist chamber and the M-K medium methods are both inadequate. A longer period of corneal storage has allowed a flexible surgical scheduling which is often required for the donor material evaluation, blood testing and transportation. In the intermediate-term corneal storage, as in the M-K medium storage, excised corneoscleral buttons are kept in biochemically defined tissue culture medium and incubated at 4°C.[6]
The addition of chondroitin sulfate has been a key development in the genesis of intermediate-term corneal storage media. Unlike in the western countries, in Japan, chondroitin sulfate has been used in storage media for more than 20 years. Mizukawa and Manabe[7] reported successful preservation of whole donor globes immersed in a solution of chondroitin sulfate at 4°C for upto three days. The exact mechanism by which chondroitin sulfate protects the donor cornea has not yet been conclusively established. It probably acts as an antioxidant and free-radical scavenger to protect cell membranes. It may also act as a cation-exchange resin regulating cation fluxes across cell membranes through the formation of chelation complexes.[8]
Several corneal storage media containing chondroitin sulfate have been developed for clinical use in the United States of America and Europe. They are K-Sol (Cilco, Huntington, West Virginia), Chondroitin Sulfate Storage Medium (CSM), Dexsol, Optisol (Chiron Ophthalmics Inc. Irvine, California), and Likorol (Opsia Pharma, France). However, K-Sol and CSM are no longer commercially available. It has been demonstrated that Optisol as compared to Dexsol, K-Sol and MK medium, can preserve the corneal endothelium better yielding a thinner cornea upto two weeks.[6] A thinner cornea not only permits a better evaluation and easier manipulation of the donor tissue at the time of the surgical procedure, but also helps an earlier visual rehabilitation.
A brief account on the biochemical composition of Dexsol and Optisol are given in the Table.[8]
It appears that the development of the intermediate-term corneal storage has taken a new direction. A brief account of this is given below.
| Steroid Containing Corneal Storage Medium | | |
Basu and Hasany[1] suspected that cellular autolysis caused by the release of hydrolytic enzymes from lysosomes could be an important factor in the degradation of the corneal donor material during storage, and speculated that steroids could be used to reduce the autolytic damage of the corneal cells.
To test this hypothesis, Basu et al[9][10][11][12][13][14][15][16] made a series of studies to determine the effect of steroids on stored corneas. Their studies showed that media containing steroids were more beneficial with regard to. the viability of the corneal cells and preservation of the ultrastructure. Their work was confirmed by Hull et al.[17],[18]
In view of the above encouraging reports, recently at the Eye Bank Laboratory of the University of Toronto, a new medium for intermediate-term corneal storage, containing hydrocortisone has been developed for clinical use. In preliminary studies, in almost all the parameters so far tested, this new medium compared well with Optisol. As it is easily prepared in the Eye Bank Laboratory, its cost of production has been a fraction of that of the commercially prepared media. (Personal communication, Dr. D. Rootman, Scientific Director, Eye Bank Laboratory, University of Toronto).
| LONG-TERM CORNEAL STORAGE | | |
| Organ Culture of the Cornea | | |
The organ culture method of corneal storage has evoked interest in researchers of several places in countries including Denmark,[19] the Netherlands,[20]France,[21] UK[22] and Australia.[23]
In 1972, Doughman[19] and his team (Minnesota, USA) began research on the corneal organ culture of donor cornea for long-term corneal storage. Their extensive investigations demonstrated adequate endothelial cell function of human corneas stored at 34°C for at least five weeks. Clinically, when the average storage time was 25 days, 80 percent of the transplanted corneas remained clear.
In the Minnesota method, the cornea following organ culture is deswelled in M-K medium at 4°C; while in the Dutch procedure, the culture medium is supplemented with dextran to dehydrate the cornea at 31°C.
Though different incubation temperatures (e.g. 37°C, 34°C, 31°C, room temperatures, 4°C) have been reported in the literature, it does not appear that the level of temperature is a very important factor unless it is too high or too low.
Organ storage seems to specifically reduce the HLA-DR antigen load of the donor corneas without affecting HLA-A, -B, -C antigens.[24]
| VERY LONG-TERM CORNEAL STORAGE | | |
| Cryopreservation of the Cornea | | |
In 1954, Eastcott et al[25] were the first to store full-thickness corneas by freezing after pretreating them with 15 percent glycerol. One of their grafts was highly successful and the rest were partially successful. Kaufman and Capella[26] reported successful preservation of donor corneas for periods up to one year by cryopreservation. Although corneal cryopreservation is not a common procedure, yet continual interest in this field has been shown in various countries.[27-29] It is possible that in future an ideal method for corneal cryopreservation will be developed using different cooling rates, transfer temperatures and cryoprotectants.
In conclusion, the functional status of the endothelium and a sustained state of corneal detergescence are of great clinical importance in the development of corneal preservation. As our knowledge of the human corneal endothelium increases, so would increase our anticipation of developing an optimum medium. Addition of antioxidants, additional energy sources and other nutritive substrates,[30]as well as substances like dextran, chondroitin sulfate and steroids provides an exciting prospect for corneal preservation and may help us develop an ideal medium.
| Acknowledgement | | |
I am grateful to the Lion's Club, District A-16, Ontario, Canada, and to the Department of Ophthalmology, University of Toronto, for their financial support. I thank Dr. David Rootman and Mr. S.M. Hasany of the Eye Bank of Canada (Ontario Division) for their assistance; and Mrs. Rajni Lola, Research Secretary, Department of Ophthalmology, University of Toronto, for her help in preparing the manuscript.
| References | | |
| 1. | Basu PK, Hasany SM. Autolysis of the cornea of stored human donor eyes. Can J Ophthalmol 9:229-235, 1974.  |
| 2. | Schimmelfennig BH. Tissue storage and tissue typing, short-term - state of the art In: Corneal Surgery, 2nd Ed, Brightbill FS (ed). St. Louis, Mosby, 1993, pp. 597-609. |
| 3. | Bito LZ, Salvador EV. Intraocular fluid dynamics II. Postmortem changes in solute concentration. Exp Eye Res 10:273, 1970. |
| 4. | McCarey BE, Kaufman HE. Improved corneal storage. Investigative Ophthalmol 13:165-173, 1974. |
| 5. | Morgan JF, Morton HJ, Parker RC. Nutrition of animal cells in tissue culture. I Initial studies on synthetic medium. Proc Soc Exp Biol Med 73:1, 1950. |
| 6. | Lindstrom RL, Kaufman HE, Skelnik BS, et al. Optisol corneal storage medium. Am J Ophthalmol 114:345-356, 1992. |
| 7. | Mizukawa T, Manabe R. Recent advances in keratoplasty with special reference to the advantage of liquid preservation. Folia Ophthalmol Jpn 19:1310-1318, 1968. |
| 8. | Steinemann TL, Kaufman HE, Lindstrom RL, et al. Intermediate-term storage media (K-Sol, Dexsol, Optisol). In: Corneal Surgery, 2nd Ed, Brightbill FS (ed). St. Louis, Mosby, 1993, pp. 609-613. |
| 9. | Chin Fook TJ, Ranadive NS, Basu PK. The prevention of autolysis of stored cornea using steroid as a lysosome membrane stabilizer. Can J Ophthalmol 10:482-486, 1975. |
| 10. | Spencer JA, Dixon WS, Ranadive NS, et al. Factors in the survival of stored corneas. Can J Ophthalmol 12:123-127, 1977. |
| 11. | Basu PK, Hasany SM. The prevention of autolysis in stored corneas by lysosome stabilization. A histochemical study. Can J Ophthalmol 12:48-52, 1977. |
| 12. | Basu PK, Hasany SM, Doane FW, et al. Can steroid reduce endothelial damage in stored corneas? Effect on cell viability and ultrastructure. Can J Ophthalmol 13:31-38, 1978. |
| 13. | Liao HR, Hasany SM, Lin BJ, et al. Biochemical analysis of the cornea stored in steroid medium. Can J Ophthalmol 14:274-280, 1979. |
| 14. | Basu PK, Hasany SM, Ranadive NS, et al. Damage to the corneal endothelial cells by lysosomal enzymes in stored human eyes. Can J Ophthalmol 15:137-140, 1980. |
| 15. | Basu PK, Hasany SM. Preventing corneal autolysis after a donor's death. Can J Ophthalmol 17:70-73, 1982. |
| 16. | Hasany SM, Basu PK. Changes of MK medium during storage of human cornea. Br J Ophthalmol 71:477-483, 1987. |
| 17. | Hull DS, Green K, Bowman K, et al. Corneal endothelial cell function after storage in MK medium and hydrocortisone. Can J Ophthalmol 14:114-116, 1979. |
| 18. | Hull DS, Green K, Buyer J. Cornea endothelial bicarbonate fluxes following storage in moist chamber, MK medium, and MK medium with added hydrocortisone. Invest Ophthalmol Vis Sci 18:484-489, 1979. |
| 19. | Doughman DJ, Lindstrom RL, Skelnick DL, et al. Long-term organ culture corneal storage: Minnesota system. In: Corneal Surgery, 2nd Ed, Brightbill FS (ed). St. Louis, Mosby, 1993 pp. 614-622. |
| 20. | Sperling S, Olsen T, Ehlers N. Fresh and cultured corneal grafts compared by post-operative thickness and endothelial cell density. Acta Ophthalmol 59:566-575, 1981. |
| 21. | Piquot X, Delbosc B, Herve P, Royer J. Preservation of human corneas in organ culture: Results of a feasibility clinical protocol. Bull Soc Ophthalmol Fr 90:429-432, 1990. |
| 22. | Redmond RM, Armitage WJ, Whittle J, et al. Long-term survival of endothelium following transplantation of corneas stored by organ culture. Br J Ophthalmol 76:479-481, 1992. |
| 23. | Williams KA, Noack LM, Alfrich SJ, et al. Assessment of the Dutch organ-culture system of corneal preservation within the Eye Bank of South Australia. Aust NZ J Ophthalmol 16:21-25, 1988. |
| 24. | Pels E, van der Gaag R. HLA-A, B, C and HLA-DR antigens and dendritic cells in fresh and in organ culture preserved corneas. Cornea 3:231-239, 1984-1985. |
| 25. | Eastcott HHG, Gross AG, Leigh AG, et al. Preservation of corneal grafts by freezing. Lancet 1:237-239, 1954. |
| 26. | Kaufman HE, Capella JA. Preserved corneal tissue for transplantation. J Cryosurg 1:125-129, 1968. |
| 27. | Ehlers N, Sperling S. Ultrastructure of cryopreserved, functioning human corneal endothelium. Acta Ophthalmol 61:245-253, 1983. |
| 28. | Delbosc B, Harve P, Carbillet JP, et al. Corneal cryopreservation in man: A proposal for an original technique. J Francais D'Ophthalmologic 7:321-331, 1984. |
| 29. | Ruusuvaara P. Long-term follow-up of cryopreserved corneal endothelium. Acta Ophthalmol (Copenh) 66:687-691, 1988. |
| 30. | Lindstrom RL. Advances in corneal preservation. Trans Am Ophthalmol Soc 88:555-648, 1990. |
[Table - 1]
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