Obtaining good results in corneal transplants is not possible without having safe corneal tissue and of adequate quality. For this, the selection of donors, the evaluation of the viability of the tissue and its conservation by different means is fundamental.

SELECTION OF THE DONOR

To decide if a potential donor is suitable, we follow the recommendations of the EEBA (European Eye Bank Association), available through its website (www.europeaneyebanks.org). We need to collect certain information regarding the donor using reliable information sources and carry out a serological study (tables 1 to 3). The presence of various pathologies can also contraindicate the donation (table 4).

Although there are no absolute limits in terms of age, this is a factor to be considered. Several studies indicate that an advanced age of the donor does not decrease the survival of the grafts^2,3, and some even show that the corneas cultivated by elder donors are associated with a better graft survival⁴ and that their endothelium are more stable in culture media⁵. In practice, the greater the age of the donor, the lower the percentage of corneas accepted for a transplant that includes the endothelium. The use of corneas of infants (<6 months) and, above all, of neonates is also controversial. Although their endothelial density is high, they have a higher antigenic capacity, since they also have higher stromal cell density. In addition, its great elasticity also poses problems.

OBTAINING THE OCULAR TISSUE

It is advisable to obtain the cornea or the eyeball with a time not exceeding 6-12 hours after death, as a guarantee of the quality of the graft for further conservation¹. The "corpse time" accepted in clinical studies varies from 4 to 48 hours, although the current consensus considers that it should not exceed 6 hours. It includes two periods:

- 1) Exposure of the corpse at room temperature. During this period, the cessation of aqueous humor formation causes endothelium nutrition problems, which compromises its viability.

- 2) Storage of the corpse in a cold room between 4 °C and 8 °C. The low temperature decreases the endothelial pump mechanisms and prevents metabolic stress. Under these circumstances, the corneal endothelium can survive longer.

The extraction must be carried out observing strict standards of asepsis. First of all, a washing and disinfecting of the skin of the eyelids is carried out, and a sterile field is applied to isolate the donor eye. A lid speculum is placed, carefully not to damage the epithelium. The fornixes are washed with saline or BSS^® solution and 5% povidone-iodine aqueous solution is instilled. A peritomy is performed 360° close to the limbus to include the minimum conjunctiva, since this is considered contaminated tissue (Figure 1). Then we can extract the entire eyeball or a segment or corneoscleral cap with at least 2-3 mm scleral margin. This will be of great importance in order to correctly place the tissue in the artificial anterior chamber (to perform the microkeratome cut for endothelial lamellar keratoplasty). Once properly identified and labelled, it will be kept at 4 °C (video 2.3.1).

ASSESSMENT OF THE CORNEA

Bio microscopy with slit lamp

The first inspection of the eyeball or the corneoscleral segment is performed by biomicroscopy with a slit lamp, with attention to the following details (Figures 2A and 2B):

a) Epithelium: if there is de-epithelization, exposure, opacities or foreign bodies.

b) Stroma: degree of edema or striae, senile arch or other opacities and scars from surgery.

c) Endothelium: if there are folds, some area of detachment or guttata corpuscles.

d) Limb-scleral ring: if it is well centered, its width and any remains of conjunctiva.

A study using optical coherence tomography (OCT) of the anterior pole can also be done by means of a special adapter to the OCT clinical device (Figure 3). This allows obtaining a topographic pachymetry.

Endothelial specular microscopy

Specular microscopes can be classified according to their objective contacts or not the cornea. Most eye banks use non-contact specular microscopes for evaluation of the corneal buttons within the storage vials.

Since the corneas are preserved at 4 °C, at this temperature the endothelial Na-K⁺ ATPase pump is inactivated, which induces edema and causes the endothelial surface to lose its smoothness. Therefore, the endothelium is not easily visualized in most corneas at 4 °C and should be heated to about 25 °C. To obtain a good endothelial image a variable heating time is required, from 45 minutes to more than 2 hours. The reasons for this variability are unknown. When the cornea is heated, the endothelial pump is reactivated, and the stroma recovers a more normal turgor (it is "de-edematized"). This cold-hot-cold cycle has no adverse effects on the morphological parameters of the endothelium⁶.

One of the most commonly used specular microscopes in eye banks is the Konan CellChek EB-10 Eye Bank System (Figure 4)(Video 2.3.2). It has two methods for endothelial counting (Konan Centre and Flex Centre). The "Centre Method" is usually used, which consists of marking the center of a set of contiguous cells on which the analysis will be made. It allows determining of the density, the polymegatism (size variation) and the cellular pleomorphism (variation of the shape), as well as to perform a pachymetry (deduced from the focal plane). The images thus obtained can be evaluated qualitatively and quantitatively (table 5).

Optical microscopy

It is the preferred technique, in particular for the study of corneas in hot culture medium. An inverted microscope, equipped with phase contrast equipment and a digital camera, is recommended (Figure 5). This study requires the cellular limits to be evidenced (Figure 6) by means of a hyperosmolar agent (usually sucrose) and a vital dye (trypan blue), which stains only the nuclei of the cells with the damaged wall.

Currently there are 3 methods of endothelial analysis with optical microscopy:

1. Direct estimation through a grid incorporated in one of the eyepieces.

2. Manual counting on photographs with a calibrated grid overprinted.

3. Study of a digital image by means of a computer program of image analysis.

Estimation of endothelial cell density

Regardless of the technique, the accuracy will depend on the ability to identify the individual cells according to the quality of the image⁷. The estimation of endothelial cell density can be performed by:

A) Subjective comparison method: it provides an approximate value of the cell density by comparing the image of the donor with a set of hexagonal patterns calibrated for certain cell densities. This is usually done during the direct inspection of the endothelium.

B) Framework method: the number of cells within a frame is counted, which can be of fixed or variable size. In the fixed frame analysis, the cell count is performed within a defined area; the most accepted method is to count the number of cells completely inside the frame and those that touch the edges only on two of the sides of the frame (Figure 7). This can lead to errors if the total number of cells is low or there is a significant pleomorphism. Therefore, it is recommended to analyze at least 200 μm^{2 8}, which increases the evaluation time if it is manual. The variable frame analysis is carried out with a specific computer program. The technician defines the area to be analyzed and marks each cell. The program calculates the density by dividing the number of cells by the area of ​​the frame. Some programs allow the identification of individual cells by determining the cell borders, which provides morphometric and statistical data (video 2.3.3).

C) Corner method: the area of the cells is determined by digitizing the intersections of the cell borders.

D) Centre method: the center of the contiguous cells is marked, and the density is calculated using a mathematical algorithm.

Evaluation of endothelial cell morphology

The ideal endothelium has high cell density and low polymegatism and pleomorphism. It is therefore presented as a mosaic composed mainly of small hexagons of the same size. The evaluation of the degree of visualization of the cellular borders of the endothelium, of the background noise and of the surface of the visible area of the cells, helps to determine the viability of the donor cornea. A good visualization of the endothelial cells and their response to the osmotic stimulus are signs of good vitality. The inability to see cell boundaries, together with an accelerated loss during culture, indicates poor quality of the cornea.

Recently the circularity (4π* area/perimeter²) has been proposed as a new parameter that relates the area to the cell perimeter⁹. Its values oscillate between 0 and 1, with this representing a perfect circle. Ideally the endothelial cell resembles a hexagon, but the greater the geometric regularity of a set of cells, the greater the circularity. Since not all cells are hexagonal after being subjected to hot storage, this parameter would better describe their morphology. In Figures 8 A, B and C, by means of microlithographic mosaics of different cell densities (Eurokeratotest study)¹⁰, we obtain by means of the graphical representation of the distribution of the values of cell area, perimeter and circularity, an objective assessment of polymegatism and pleomorphism.

Since 2011, our Tissue Bank uses the circularity together with the "coefficient of variation" (CV) of the area, in the assessment of donated corneas prior to its cultivation process at 31 °C and its use in transplants. We have only found the use of this parameter in a work published in 1993 in cultured corneas¹¹. High levels of pleomorphism and polymegatism are signs of poor cell viability and are more frequent in corneas with low endothelial counts. Special attention should be paid to the presence of "gutta" type corpuscles, which are seen under the microscope as round structures with smooth edges and size similar to endothelial cells. Each eye bank establishes its criteria for accepting a cornea as viable for transplants that include the endothelium. Normally they include a minimum of cellular density between 2,000 and 2,500 cells/mm², together with pleomorphism and polymegatism in a minimum degree. The percentage of nuclei stained by trypan blue should be less than 10%. Table 6 lists the reasons for excluding a donor cornea due to insufficient viability.

MEANS OF STORAGE AND CONSERVATION

Short term: cooling in a humid chamber

It consists of introducing the entire eyeball in an airtight vial, on a bed of sterile gauze soaked in saline, and refrigerate it at 4 °C. This allows maintaining an acceptable viability up to 48 hours after the extraction (Figure 9). It has the advantage of being simple and economical, but the exposure of the endothelium to aqueous cadaverous humor limits the storage time.

Medium term: cold culture media

The isolated corneoscleral segment is introduced in a liquid medium at 4 °C. The first of this type was that of McCarey-Kauman (M-K) in 1973, based on a standard culture medium (TC-199) enriched with amino acids and vitamins. In addition to the classic electrolytes (Na⁺, K⁺) it carries calcium (like the BSS), without which the endothelial cells retract, as well as 5% dextran to counteract the tendency to stromal edema due to its oncotic effect. Its use has spread especially in the American eye banks, with variants of M-K such as Optisol GS^®, which also contains chondroitin sulphate (as an antioxidant) and precursors of ATP. It has been argued that this method allows storage for up to 14 days, but many eye banks prefer not to exceed the week.

Long term: hot organic culture

The use of a hot organic culture medium (at 31°C to 37 °C) allows the corneas to be preserved up to four weeks, under similar environmental conditions to the physiological ones. This method has spread especially in Europe for logistical reasons, to increase the storage of donor corneas. It requires a medium type MEM (minimum essential medium) enriched with bovine serum and other additives, antibiotics and antifungals. Strict periodic controls must be carried out to guarantee sterility¹². Because the epithelial turnover persists during the culture, the corneas must remain suspended vertically to avoid deposition of debris on the endothelial cells (Figure 10). This medium lacks oncotic agents (dextran), potentially toxic for the endothelial cell, so that stromal edema occurs during storage. Therefore, the cornea must be transferred to a drying medium (with dextran) 36 to 48 hours before use (Figures 11A and 11B). In each change of solution microbiological controls must be carried out, and before being used, it is mandatory to check again the state of the endothelium.

Very long term: cryopreservation

Cryopreservation involves cooling the tissue to temperatures well below the freezing point, so that all metabolic activity ceases. This would theoretically allow an almost indefinite conservation. However, the complexity and cost of the process and the difficulty in avoiding damage to the endothelium have severely limited its use in corneas, except for rare occasions of urgent transplants to save the eye when fresh tissue or in culture is not available.

Freezing damages the cells in the first place by formation of ice crystals inside them that break them. To avoid this, "cryoprotectants" are used, which are essentially dehydrating agents such as dimethyl sulfoxide (DMSO), followed by a slow cooling process. However, the cryoprotectants themselves have a toxic effect. Currently cryopreservation employs a standard culture medium (TC-199 or RPMI) with DMSO in progressive concentrations, to which 20% albumin can be added. A "ramp" program is followed in the biological freezer, where the temperature drops 1 °C/min to -80 °C and then 5 °C /min to -140 °C. The corneas thus treated are stored in nitrogen vapor phase (video 2.3.4).

The recent boom in lamellar techniques, such as deep anterior lamellar keratoplasty (DALK) or the lenticular implants obtained from cutting with femtosecond laser ("SMILE" technique), have opened new horizons for cryopreservation, especially after demonstrating the viability of keratocytes together with the cryopreserved collagen structure^13-15.

Vitrification is a form of ice-free cryopreservation. It poses considerable challenges, due to the very high concentrations of solute required to achieve it at practicable cooling speeds. The use of cryoprotectants that do not penetrate the cells could lead to simpler methods of corneal cryopreservation¹⁶.