CURRENT OPHTHALMOLOGY Year : 1995 | Volume : 43 | Issue : 2 | Page : 55-58

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

PMID: 8818310

Abstract

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 facil ities.

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

How to cite this URL: Basu PK. A review of methods for storage of corneas for keratoplasty. Indian J Ophthalmol [serial online] 1995

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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 ultrastruc ture. 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 cont inual 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 .

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