Organ Culture (Normothermic Storage) of the Corneoscleral Button

Technical Aspects

The corneosclereal buttons are stored in an incubator at 30–37°C. The procedure was developed by Doughman (1976), modified by Sperling(1979) and further developed and refined in Europe [16]. The technique provides storage of the corneoscleral buttons up to 4–6 weeks or even longer.

The technique is more complex compared to hypothermic storage. It requires microbiological surveillance during storage and mandatory evaluation of the endothelium at the end of the storage period. In addition, the storage solution, consisting of tissue culture medium, foetal bovine serum as source for energy, proteins, growth factors and antibiotics, does not contain macromolecules to maintain corneal hydration in vitro [16]. This swelling of the cornea is reversed before surgery by transfer of the tissue to a dextran-supplemented storage medium (referred to as the Transport or Thinning medium) [16]. This later medium is also used to transport the cornea (conveniently) at room temperature. Due to the number of steps involved in organ culture storage, relative to hypothermic storage, the cornea is not directly available for surgery.

Storage Period

The storage time in the organ culture medium may be up to 4–7 weeks after excision. Renewal of the medium during storage (at 10–14 days) may promote an extended storage time [16].

Depending on the eye bank, reported maximum times for the cornea placed in transport medium vary from 1 to 7 days and the dextran concentration varies from 4% to 8%. This largely determines the extent of the reversal of corneal swelling (usually to a level dependent on the preference of the recipient surgeons). Time spent in transport medium is limited by the toxic effects of dextran over time, which is ingested in cellular vacuoles [16].

Microbiological Safety

The antibiotics present in the storage solution are effective during organ culture. In the absence of antibiotics and despite extensive corneal decontamination before organ culture, the corneas themselves have been responsible for greater than 30% microbial contamination of cultures [16]. Even in the presence of antibiotics, the organ culture mediuma remains a highly nutrient medium for the continued growth of any contaminating organisms. Thus, microbiological testing and surveillance of the storage solution is mandatory and a quarantine period for the corneas is required until the results of microbiological testing of the culture solution are available. Any contaminated organ cultures are discarded. When these procedures are followed, the reported incidence of endophthalmitis from corneas transplanted after organ culture is 0–0.1% (EEBA Directory).

The rate of contaminated cultures varies between eye banks. It appears to be dependent on several variables: the antibiotic cocktail in the medium (narrowto wide-spectrum antibiotics), in situ excision or enucleation, and the post-mortem

delay between death of the donor and collection and storage (EEBA Directory). Most importantly, organ culture may become the method of choice when corneas are suspected to be contaminated, for example, from a donor with bacteraemia [17]. The storage itself acts as an in-built microbiological surveillance system, since any culture containing microorganisms not controlled by decontamination and antibiotics will become contaminated, and the cornea not used for transplant.

Tissue Evaluation

Although mandatory only after storage, inspection of the endothelium is often performed both before and after storage (EEBA Directory). Differences in endothelial cell loss in individual corneas may occur as a result of wound healing processes and impaired endothelial viability due to post mortem time, cause and circumstances of death, etc. In this way, organ culture might be considered as a stress test or an inbuilt viability test for the cornea. Corneas showing significant cell loss will be discarded before surgery [16]. Interestingly, 100% cell loss has been found associated with a herpes infection [18].

The specular microscope is not suitable for the inspection of organ-cultured corneas as it requires an un-swollen cornea. Therefore, the endothelium of organcultured corneas is generally assessed by light microscopy together with vital dye staining. These methods were developed along with the introduction of the organ culture procedure in Europe [19]. It allows the inspection of the entire endothelial surface irrespective of the corneal thickness and transparency. In addition, the use of the vital dye trypan blue allows the detection of non-viable cells and denuded Descemet’s membrane. The method is invasive and requires the use of hypotonic solutions to temporarily swell the intracellular spaces so that the endothelial cells can be visualised. This must be done using aseptic conditions. It is variously performed (depending on the eye bank) as a bright field microscope or a phase contrast image using inverted or non-inverted microscopes. The induction and the swelling pattern are dependent on storage time and medium and therefore, interpretation of the images requires experience and standardised working conditions.

For parameters such as cell counts, some type of image analysis is required. The light microscope set up needs to be well-calibrated for these purposes [20]. Modern image software aims for automated cell analysis that is independent of the observer and experience. However, so far, reliable results can only be obtained after much time-consuming interactive manipulation which requires experienced observers. Confocal microscopy is a promising development for future analysis of eye bank corneas but it awaits the development of a non-contact version for eye banking for it to gain widespread acceptance.

Other Aspects

Organ-cultured corneas are evaluated at the end of the storage period and therefore eye banks using this technique have less limitations in regard to post-mortem to enucleation and excision times (EEBA Directory). This also serves to increase the potential donor pool.