Donor corneas are stored in serum-free tissue culture medium at a temperature of 2–6 °C. At this temperature the metabolic activity of endothelial cells is minimal and pumping function is lost. Corneal swelling may be prevented by the addition of water retentive compounds to the preservation medium. One of the most commonly used is the deturgescent compound dextran either alone or in association with the glucosaminoglycan chondroitin sulfate. Storage liquids also contain antibiotics (gentamicin alone or with streptomycin) that, together with the low temperature, prevent or limit the bacterial growth.

During hypothermia, the cornea shows progressive degeneration of the epithelium and the endothelium, intercellular disruption, decreased adhesion, and, eventually, cell death. Both apoptosis and necrosis occur in cells during hypothermic storage, with apoptosis appearing to predominate. The extent of endothelial loss seems to be related to the biological quality of the tissue, rather than the composition of the medium. Therefore, most corneas are transplanted after 3–5 days of storage, without displaying major alterations.

The hypothermic storage method does not allow time for obtaining preoperative microbiology controls before distribution of the tissue for transplant.

Overall, hypothermic storage seems to offer donor tissues of good quality comparable to that obtained by organ culture, provided that the storage time is kept short. Indeed, according to the literature, the risk of primary graft failure increases significantly after storage longer than 7 days. Furthermore, corneas stored longer than 7 days display epithelial alterations that may hinder the surgical procedure or delay the full recovery of the graft [8].

The organ culture storage method consists of two phases – a storage period in culture medium at 30–37 °C and a deswelling and transportation phase at 30–37 °C and room temperature in the same medium supplemented with 4–8 % dextran. Organ culture solutions are based on cell culture media. They generally consist of a base of Eagle’s MEM or its variant Dulbecco’s MEM supplemented by penicillin, streptomycin, and fungicide (amphotericin B or nystatin) to counteract the growth of microbiological contaminants and by 2–10 % fetal calf serum as a source of growth factors. A storage period of 30 days can be achieved without significant loss of endothelial cells. The evaluation of endothelium, which can show reparative phenomena during storage, is usually performed before and after storage.

Cultured corneas have preservation folds caused by swelling of the stroma in the absence of osmotic agents. These folds do not affect the quality of the tissue, provided that they remain covered by viable endothelium. Before transport and surgery, the swelling is reversed by the dextran present in the transport medium. The final thickness is reached after about 24 h and is dependent on the dextran concentration. The dextran also protects the cornea against the lower ambient temperature during transport.

Organ culture offers a longer storage time, corneal endothelium with a better defined quality, and a preoperative sterility control. Organ-cultured corneas always display an epithelium made up of 2–3 layers of viable cells. The 30-day storage period allows an efficient use of valuable donor tissue: planning of operations is easier, allowing sufficient time for the allocation of HLA-matched corneas. The disadvantages of this method are the relative technical complexity and the need for qualified staff to perform tissue culture and selection of the corneas [9].

Samples of the storage medium of cultured corneas are routinely tested for microbiology after 3–7 days in the first phase and after 1 day in the second phase. A gradual change in color of the medium is expected, but any cloudiness or significant color change of the medium is indicative of bacterial or fungal contamination. A contaminated cornea is discarded regardless of whether the microbe is pathogenic or not [10, 11].

It is still a point of debate whether the clinical outcome after grafting corneas stored by hypothermic or organ culture techniques is the same, although few studies comparing the effect of the storage methods on outcome demonstrate similar graft survival and postoperative decline in endothelial cell density [12–15]. Irrespective of the storage method used, inspection of the endothelium after a prolonged storage is essential to prevent transplantation of poor quality corneas.

Donor selection criteria for corneas used in lamellar keratoplasty are the same as for penetrating keratoplasty with a few exceptions. Corneas with prior laser photoablation surgery or noninfectious anterior stromal scars may be suitable for posterior keratoplasty, but corneas with previous intraocular surgery scars are not recommended for use since the cornea may rupture under infusion pressure while on the artificial anterior chamber [16–18].

A 3–4 mm scleral rim is needed for corneas used in lamellar keratoplasty procedures to ensure an adequate seal on the artificial chamber of the automated microkeratome.

An automated microkeratome system consists of a control unit, an artificial chamber, microkeratome turbine, and heads. The control unit should be set up in close proximity to the laminar flow cabinet. The cornea is placed using tissue forceps centrally onto the artificial anterior chamber which has been moistened by activating the irrigation system, and the chamber is locked into place. The cornea is pressurized by infusing PBS through the irrigation system. A tonometer lens is placed on the corneal surface to confirm that a minimum of 65 mmHg has been established inside the artificial chamber through the infusion of PBS. In case of anterior lenticules, the graft desired thickness is obtained by the correspondent microkeratome head. For posterior lenticules, a pachymetry reading is obtained after the removal of the epithelium, to determine which microkeratome head to use to obtain a final graft.

The corneal epithelium must be gently removed before preparation, or left in place. In the former case, the subsequent swelling of the stroma during preservation can be limited. Two points are marked on the midperiphery of the cornea using a sterile gentian violet or trypan blue marker to assist with re-aligning the cap back onto the remaining stromal bed after the cut has been made.

The microkeratome head is rotated manually across the cornea. Once the sectioning is completed, the free cap is removed from the microkeratome head and repositioned onto the corneal bed, taking care of re-aligning the marks. A wexel sponge spear is used to smooth out any bubbles between the cap and the graft bed.

Once lamellar keratectomy has been completed, the cornea should be re-evaluated by slit lamp biomicroscopy and specular/light microscopy to confirm that the tissue is suitable for the intended use [19].

Anterior corneal lenticules can be either dehydrated or freeze-dried and stored at 2–6 °C according to the eye bank’s validation protocol.

Alternatively, anterior/posterior lenticules can be placed in a cornea viewing chamber filled with preservation media (hypothermic storage) or in the transport medium (organ culture).

The donor sclera is used in allografts for a variety of procedures, most commonly to enclose orbital implants for reconstruction of anophthalmic cavities, reconstruct eyelids, cover tubes used in glaucoma surgery, repair scleral thinning, and correct lid retraction and cicatricial entropion and tumor excision. Selection criteria are the same as cited for penetrating keratoplasty, except that tissue with local eye disease affecting the corneal endothelium is acceptable for use. Being a vascularized tissue, malignancies are applied as additional contraindication. Postmortem interval may be extended.

Donor sclera is prepared from remaining ocular tissue following excision of the corneoscleral button or from donor globes which have been disqualified before corneoscleral rim excision. Since conjunctival tissue is an excellent carrier for microbes, remnants of muscles and conjunctiva must be removed.

The intraocular material is removed by using forceps, iris scissors, sterile gauze, or cotton-tipped applicators. The sclera is finally rinsed in PBS, reshaped to its original spherical form, preserved dehydrated in ethanol (70 % or higher concentration) or glycerol, fixed in formalin, freeze-dried, or frozen.

The storage and the final transport medium contain serum of animal origin in it. Apart from serum, other nutrients of animal origin have also been investigated for prolongation of the endothelial metabolic activities, such as chicken feather, ovalbumin, and pig bone amino acids, usually used in combination with other sources of nutrient supplements. Animal viruses, especially retroviruses, could integrate into the human genome and activate human oncogenes or oncosuppressor genes, while prions could lead to human forms of bovine spongiform encephalopathy (BSE). This is why synthetic media have been developed. The potential transmission of BSE primarily comes from donors who have donated their corneas and were at risk of having BSE (e.g., UK donors at the time of mad cow disease). Theoretically, there could be a transmission of animal-derived viruses that could integrate in the genome and activate oncogenes; therefore, technically it would be safer to develop and integrate a totally synthetic-/animal-free media in the routine eye banking procedures [20, 21].