This document reviews various methods for storing donor corneas for keratoplasty (corneal transplant surgery). It categorizes storage methods based on duration as: short-term (days), intermediate-term (weeks), long-term (months), and very long-term (years). Short-term methods include moist chamber storage and M-K medium storage. Intermediate storage involves tissue culture media with additions like chondroitin sulfate to allow storage for weeks. Long-term storage uses organ culture, which has allowed storage for months. Very long-term storage research focuses on cryopreservation.

1. 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
[cited 2012 Mar 28];43:55-8. Available from: http://www.ijo.in/text.asp?1995/43/2/55/25257
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.

2. 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

3. 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

4. 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

5. 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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