Original Article

Corneal storage at 4 ' Celsius in a chondroitin sulphate and dextran containing medium Maria Morton, BSc*

Abstract Corneas were stored at 4°C in a chondroitin sulphate and dextran containing medium from one to ten days. The viability of the corneal endothelium was assessed after storage with the vital stain, trypan blue.

Key words: Chondroitin sulphate, cornea, dextran, endothelium, storage medium.

Chondroitin sulphate (CS) containing media have been found to support endothelial viability longer than McCarey and Kaufman (M-K) medium.'-' The two most commonly used media which contain chondroitin sulphate are K-Sol and CSM and both have successhlly preserved the corneal endothelium for 10 to 14 days at 4"C.4,5 Comparative studies have shown no significant differences between these two However, a clinical observation by Bourne found that postoperative endothelial cell loss correlated with storage time in K - S O ~ . ~ He suggested that corneas should not be stored for longer than 10 days.

One disadvantage of the CS-containing media is the increase in corneal thickness during storage. The addition of dextran can overcome this problem."

The present study was undertaken to assess the viability of the corneal endothelium after various lengths of storage and to try to determine a suitable storage period under the conditions which exist .in

'Senior Scientific Officer, Eye Pathology, Sydney Eye Hospital, New South Wales.

the Sydney Eye Bank where 66% of the potential donors are over sixty years of age and death to preservation varies considerably from donor to donor (four to 24 hours).

Material and methods The eyes after enucleation were received by the Eye Bank Laboratory in moist pots. They were examined with a biomicroscope for epithelial, stromal and endothelial abnormalities. To get valid results, the corneas used for this study had an endothelium judged suitable for penetrating keratoplasty, but were otherwise rejected due to stromal abnormalities or unsatisfactory blood results.

The corneoscleral rims were removed from donor eyes using standard eye banking techniques and placed in 20 mL of chondroitin sulphate, dextran (CSD) containing medium (Table 1) and stored at 4 "C. This medium (CSD) is manufactured, tested and supplied by Cytosystems Pty Ltd NSW on a fortnightly basis.

After storage, the quality of the endothelium was assessed by staining with 0.25% trypan blue in 0.9% saline for 90 Dead and damaged cells as well as Descemet's membrane where the cells have lifted off stained blue. The percentage of cells stained and the pattern of staining were all carefully noted.

Results Corneas in which fewer than 30% of the endothelial cells stained with trypan blue were considered to have survived the storage satisfactorily (Table 2). The results indicate that endothelial cell loss increases with storage time (Figure 1). The rate at

Rtprinr requesrs: Ms M Morton, Eye Pathology, Sydney Eye Hospital, Sir John Young Crescent, Woolloomooloo, New South Wales 201 1, Australia.

Corneal storage in a chondroitin sulphate and dextran medium 21 1

Table 1. CSD medium

T C 199 with Hank’s salts Chondroitin sulphate 1.35% Dextran 1 .OO% HEPES buffer 25 mM L-glutamine 24mM Penicillin 100 IUlmL Streptomycin 100 ug/mL pH 7.4 Osmolarity 340 Phenol red as indicator

(bicarbonate free)

~~ ~~

which the cells degenerate varies and it is not possible to predict this rate either from the history of the donor or from the appearance of the eye at the time of biomicroscopy. A number of corneas showed a high percentage of cell death near the periphery. (Cornea numbers 5, 11, 13, 27, 28 and

34). The pattern and position of the dead cells suggest mechanical damage during the removal of the cornea from the donor eye.

Discussion Presumably, the physiological state of the endothelial cells of different donor corneas varies at the time of the death. Therefore, it is not surprising that their ability to withstand post- mortem stresses including storage will vary from donor to donor.

The length of time a cornea can be preserved is really determined by the quality of that particular cornea; however, all corneas should be used as soon as possible.

There is a great demand for donor corneas in NSW so there is a need to utilise the maximum

Table 2. Donor characteristics and staining results

Hours from death to

preservation

Days in Percentage cells viable medium

Cornea Donor age Cause of Number (Years) death

1 78 cv 10 1 90 2 71 cv 18 1 90 3 71 cv 18 3 95 4 69 cv 14 4 80 5* 67 cv 12 5 60 6 65 cv 8 5 90 7 73 0 13 5 80 8 64 cv 15 5 90 9 58 CA 5 5 85

10 71 cv 12 5 90 11* 65 cv 7 5 20 12 64 cv 22 6 90 13* 73 cv 15 6 75 14 73 0 9 6 85 15 48 T R 12 6 80 16 68 cv 14 6 85 17 72 CA 8 6 80 18 42 0 16 6 80 19 78 0 17 7 90 20 73 T R 6 7 95 21 62 cv 8 7 95 22 49 cv 19 7 90 23 60 0 8 7 80 24 84 cv 15 7 80 25 74 cv 7 7 90 26 76 0 14 8 90 27* 69 cv 14 8 70 28* 29 cv 19 8 40 29 77 cv 12 9 75 30 4.5 0 23 9 90 31 65 0 10 9 80 32 19 0 25 9 90 33 0.5 0 18 10 85 34* 14 cv 24 10 70

*Indicates corneas which have shown a high percentage of cell death near the periphery. The pattern and position of the dead cells suggest consider- able mechanical damage. CV = cardjovascular; CA = cancer; T R =trauma; 0 =other.

212 Australian and New Zealand Journal of Ophthalmology 1992; 20(3)

1 oo

80

- - 8 60 - P

- 0 a\

a 40

B

20

0 0 1 2

+ + +

+

+

I

3 4 5 6 7 8 9 1 0

Days in Storage

Figure 1 Approximate rate of endothelial cell deterioration. The graph shows endothelial deterioration as linear. This may not

be the case.

number of corneas possible. Because of the variation in the survival rate of the corneal endothelium from donor to donor, the Sydney Eye Bank Laboratory recommends a five-day maximum storage limit on all corneas and uses the CSD medium exclusively.

Acknowledgements The author would like to thank The Lions Eye Bank of New South Wales for supplying the tissue for this project. Thanks are also due to Dr M Filipic for his support, to Ms Barbara Bojarski for her technical assistance and to Miss Felice Hunter for her secretarial expertise.

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