Ex VIVO eye globe preservation

Abstract

A device for storing and maintaining viability of an ex vivo enucleated eye globe, the device comprising: a platform comprising an eye bed, wherein the eye bed is part-spherical and is radiused to substantially correspond to the dimensions of an eye globe to be received within the device, and wherein the eye bed comprises at least one drainage aperture, and wherein the platform further comprises an attachment means configured to hold a cannula in place. The device may form part of a system also including: an enucleated eye globe resting on the eye bed; a cannula retained in place by the attachment means, the cannula connected at a first end to the eye globe and at a second end to a reservoir of perfusion fluid; and a pump to enable transfer of the perfusion fluid into the eye globe via the cannula.

Description

[0001] EX VIVO EYE GLOBE PRESERVATION

[0002] TECHNICAL FIELD

[0003] The invention relates to a device and a system for maintaining viability of an enucleated ex vivo eye globe, and to a method of maintaining the viability of such an eye globe.

[0004] BACKGROUND

[0005] According to the World Health Organization, in 2023, vision loss affected at least 2.2 billion people worldwide. Retinal degenerative diseases, glaucoma and traumatic vision loss are major causes of vision loss worldwide, and so far, there are very few therapies which reduce symptoms, and none which restore vision.

[0006] The retina is a complex neural tissue in the posterior chamber of the eye, responsible for converting light signals into neural signals and for their transmission to the brain. It has a high metabolic demand and avascular zones, whose function relies heavily on maintenance of structural integrity.

[0007] Although many animal species, such as fish and amphibians, have evolved mechanisms to readily regenerate their retinas upon injury, human retinas do not regenerate. Restoration of vision therefore remains a significant challenge, as current therapeutic interventions are limited to slowing the progression of vision loss, as for example anti- VEGF, the gold-standard treatment for age-related macular degeneration (AMD).

[0008] As the retina does not readily regenerate following damage, various therapies aiming at regenerating or restoring vision, are currently being tested in clinical trials or remain in the experimental phase. These therapies include cell replacement therapies, retinal prostheses, optogenetics and gene therapy patients. Despite some very interesting advances in vision restoration in specific populations, there remains many diseases where these treatments have limited or no efficacy. Whole eye transplantation (WET) is not currently available, although it could be a potentially viable option for restoring vision. Achieving WET with vision from the transplanted eye will challenge several paradigms and require the advancement of several techniques which are currently extremely difficult to perform. Preservation of the eye outside the body, is an important challenge to be overcome in order to enable its use as an experimental “model” for pre-clinical assessment of therapies, which can expedite the approval process and the development of new ocular therapeutics for the clinical use. However, the complexity of the eye’s composition; its anterior and posterior compartments, lens, musculature, its complex retinal layers and its intrinsic connection to the brain via the optic nerve, make the eye an inherently challenging organ to maintain ex vivo.

[0009] Currently, pre-clinical evaluation of potential therapies for retinal degeneration is largely carried out in rodents, which have a vastly different ocular anatomy to humans. Namely, the mouse eye has a large lens occupying almost the entire eye globe, while the human lens occupies less than one quarter of the eye globe. Moreover, the human retina contains critical areas which confer precise central vision, namely the macula and fovea, which are absent in mouse retinas. The macula is a major sight of degeneration in human visionthreatening diseases such as AMD, Diabetic Maculopathy and Diabetic Macular Edema (DME). Therefore, therapies validated in species without a macular area is of limited relevance to human retinal degenerative diseases.

[0010] The retina from various species (including rats, pigs, non-human primates and humans) can show electrical activity outside the body, when isolated and maintained in close-to physiological in oxygenated medium. However, a rapid decay of the electroretinogram (ERG) signal was observed within 35 min of enucleation at 37°C. Maintaining the retina inside the intact eye in physiological conditions is a challenge, and it is known that the human retina rapidly ceases its role in light detection and response following circulatory cessation. To date, it remains unknown whether retinal function can be preserved in intact eye globes ex vivo, given the challenges of delivery of nutrients and oxygen to the retina inside the post-mortem eye. This challenge is essential to overcome for WET, as the eyes would need to be maintained intact if to be transplanted.

[0011] While large organ transplantation is nowadays a successful remedy for patients suffering from conditions such as kidney failure, lung failure, heart failure and skin damage from burns, the eye contains specific anatomical features which complicate its transplantation, as alluded to above. In the case of WET, the restoration of a functional connection between the donor eye’s optic nerve to the recipient’s optic nerve and brain is of crucial importance. Peripheral nerve axonal elongation following injury (axotomy) readily occurs, however it is extremely limited or absent in the central nervous system (CNS), where ongoing research efforts are attempting to understand how to stimulate CNS regeneration.

[0012] Basic research aiming to regenerate the optic nerve is ongoing, and reconnection of retinal ganglion cell (RGC) axons to the brain (lateral geniculate body) can be enhanced using stem cell technology. However, this approach is still immature and other strategies should be developed. Notwithstanding these hurdles, a recent breakthrough approach successfully achieved whole eye and face transplantation in one patient. The eye was transplanted with the face, showing good aesthetic results. Moreover, angiography showed successful perfusion of the retina, maintenance of eyeball volume, and more importantly, recorded electroretinogram and responses of the visual cortex to light stimulation was reported. However, the patient remained unable to see from the transplanted eye.

[0013] Another major challenge for WET is restoring perfusion to the retina, particularly within a short time after enucleation, as the retina is sensitive to ischemia. However, clinical cases have shown that even after ischemic events, where vision is temporarily impaired, normal vision can be restored after a period of time. One such example is Amaurosis fugax, a temporary vision loss condition caused by a transient obstruction to the retinal blood flow, in which vision can return once normal blood flow is re-established. Temporary vision loss, lasting approximately one day, can occur in humans due to conditions such as retinal migraine, retinal vasospasm, central retinal artery occlusion, epileptic seizures and Uhthoff phenomenon. However, in experimental studies of central retinal artery occlusion, recovery of sight is very limited if perfusion is not restored within 90 min. These previous reports suggest that the entire visual system has some degree of tolerance to ischemia. Thus, the question arose as to whether restoration of blood flow and physiological conditions in an ex vivo eye globe could be sufficient to preserve ERG signals.

[0014] It is against this background that the invention has been devised.

[0015] SUMMARY OF THE INVENTION

[0016] In a first aspect, the invention provides a device for storing and maintaining viability of an ex vivo enucleated eye globe. The device comprises a platform comprising an eye bed. The eye bed is part-spherical and is radiused to substantially correspond to the dimensions of an eye globe to be received within the device. The eye bed comprises at least one drainage aperture. The platform further comprises an attachment means configured to hold a cannula in place.

[0017] The device may further comprise a housing comprising a main housing, a lid, and a base that together define an enclosure. The platform may rest within the enclosure on the base, and the lid may comprise an opening configured to receive the cannula passing therethrough into the enclosure. The drainage aperture may be in fluid communication with a drainage channel in the base of the housing.

[0018] The drainage channel may be in fluid communication with a tube connector.

[0019] The device may further comprise a closure mechanism, configured to bring the device into a closed configuration and to retain the device in said closed configuration. The closure mechanism may comprise respective pairs of latch arms on each of the lid and the base, and corresponding ledges to each of the pairs of latch arms on side walls of main housing, each ledge defining a latching surface for the associated latch arm to bear against when the latch arm is in a closed position.

[0020] The device may further comprise a first gasket between lid and the main housing and a second gasket between the base and the main housing so that operation of the closure mechanism to bring the device into the closed configuration generates a hermetic seal around the enclosure.

[0021] The device may further comprise a window in a wall of the main housing. The window may be detachable from the wall of the main housing. This may be via a twist-off mechanism, or the window may be hinged to the wall of the main housing.

[0022] The attachment means may comprise a clip received in a recess in the platform. The device may further comprise a holding means configured to maintain an eye globe in place on the eye bed.

[0023] The device may further comprise a stand attached to the base configured to raise the base above a surface on which the device rests.

[0024] The radius of curvature of the eye bed may be between 1 cm and 4 cm. In a second aspect, the invention provides a system. The system comprises: the device described above;; a cannula retained in place by the attachment means of the device, the cannula being connected at a first end to a reservoir of perfusion fluid and being operatively connectable at a second end to an eye globe in order to deliver the perfusion fluid thereto; and a pump to enable transfer of the perfusion fluid along the cannula.

[0025] The perfusion fluid may comprise oxygenated Ringer’s solution, a physiological buffer solution, blood serum, blood or a blood substitute.

[0026] The system may comprise at least one electrode, the electrode being configured to measure an electroretinogram (ERG) signal from the retina of an eye globe. The or each electrode may be a wireless electrode.

[0027] The system may further comprise a drainage tube in fluid communication with the drainage channel at a first end and with the reservoir of perfusion fluid at a second end so as to enable recirculation of used perfusion fluid.

[0028] The device may further comprise a second cannula configured to deliver a hydration fluid configured to maintain hydration of a cornea of an eye globe. The device may comprise a second attachment means, wherein second cannula may be retained in place by the second attachment means. The second cannula may be a non-contact cannula.

[0029] The device may further comprise a third cannula configured to deliver a medication or diagnostic agent to an eye globe. The device may comprise a third attachment means, wherein the third cannula is retained in place by the third attachment means.

[0030] The second attachment means and / or the third attachment means may be carried on a flexible arm mounted to the platform of the device.

[0031] The system may further comprise a temperature control system to enable delivery of a temperature control fluid into the device. The platform of the device may comprise a bore extending therethrough from an inlet port to an outlet port to define a fluid conduit for the temperature control fluid passing underneath the eye bed. The temperature control fluid may be the perfusion fluid. The system may further comprise a coaxial heat exchanger extending around tubing connecting the cannula to the reservoir of perfusion fluid.

[0032] The system may further comprise an eye globe resting on the eye bed, wherein the cannula is operatively connected at its first end to the eye globe so as to enable delivery of the perfusion fluid thereto.

[0033] The cannula may be operatively connected to the eye globe via an artery thereof, wherein the artery is preferably one of the ophthalmic artery, the internal carotid artery, the central retinal artery, or one of the ciliary arteries thereof.

[0034] The pump may be configured to enable delivery of the perfusion fluid into the eye globe at a rate of between 0.005 mL / minute and 5 mL / minute.

[0035] The system may be configured to ensure that the eye globe maintains viability for 1 hour post-enucleation when perfused with the perfusion fluid via the cannula, preferably for 3 hours post-enucleation, more preferably for 6 hours post-enucleation, more preferably for 12 hours post-enucleation, more preferably for 24 hours post-enucleation, more preferably for 48 hours post-enucleation.

[0036] The eye globe may demonstrate viability through the generation of an ERG signal greater than 1.5 standard deviations over the background noise level, preferably greater than 2 standard deviations over the background noise level, more preferably greater than 3 standard deviations over the background noise level, and more preferably greater than 4 standard deviations over the background noise level.

[0037] In a third aspect, the invention provides a method of maintaining the viability of an ex vivo enucleated eye globe. The method comprises: placing the eye globe on a part-spherical eye bed of a platform, the eye bed being radiused to substantially correspond with the dimensions of the eye globe; operatively connecting a cannula to the eye globe and retaining it in place with an attachment means received on the platform; delivering a perfusion fluid to the eye globe via the cannula; and allowing outflowing perfusate to drain through at least one aperture in the eye bed.

[0038] The method may comprise operatively connecting the cannula to the eye globe via an artery thereof. The method may comprise delivering the perfusion fluid at a rate of between 0.005 mL / minute and 5 mL / minute. In some embodiments, the method may comprise delivering the perfusion fluid at an initial rate of between 0.05 mL / minute and 5 mL / minute and then subsequently delivering the perfusion fluid at a rate of between 0.005 mL / minute and 5 mL / minute. The method may comprise determining an optimal delivery rate for the perfusion fluid using a machine learning algorithm.

[0039] The method may comprise recirculating the used perfusion fluid draining out of the at least one aperture into a reservoir of the perfusion fluid connected to the cannula, wherein the platform and the eye globe are contained within a hermetically sealed enclosure.

[0040] The method may comprise mapping the vasculature of the eye globe using a machine learning algorithm.

[0041] The method may further comprise cooling the eye globe before delivering the perfusion fluid thereto and / or warming the eye globe during delivery of the perfusion fluid thereto by delivering a temperature control fluid into a device within which the eye globe is received.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] So that it may be more readily understood, the invention will now be described with reference to the following figures, in which:

[0044] Fig. 1A shows representative images of the central, mid and peripheral retina after 3-, 10- and 24-hours post-mortem at room temperature;

[0045] Fig. 1 B shows quantification of the maximum number of nuclear rows in inner nuclear layer (INL) and outer nuclear layer (ONL) of the retina;

[0046] Fig. 1C shows representative images of retinae stored for 3-, 10- and 24-hours postmortem;

[0047] Fig. 1 D shows retinal degeneration severity grading of Haematoxylin and Eosin stained retinal sections obtained from eyes stored for 2, 3, 4, 5, 6, 10 and 24 hours post-mortem and the distribution of degradation severities at 3, 10 and 24 hours post-mortem, with retinae examined in the central, mid and peripheral zones;

[0048] Fig. 2A shows representative images of retinal pigment epithelium cell morphologies of retinas stored for 3-, 10- and 24-hours post-mortem;

[0049] Fig. 2B shows the distribution of retinal pigment epithelium cell size according to number of sides, comparing 3-, 10-, and 24-hours post-mortem;

[0050] Fig. 2C shows the number of 9-, 10-, 11- and 12-sided retinal pigment epithelium cells as percentage of entire population at 3-, 10-, and 24-hours post-mortem;

[0051] Figs. 3A and 3B show representative Haematoxylin and Eosin staining of eyes stored for 3, 10 and 24 hours at 24°C and 4°C, respectively;

[0052] Fig. 3C shows a violin plot comparing the severity of degeneration of the retinal samples from eyes stored for 3, 10 and 24 hours post mortem at 24°C (leftmost three violins) and 4°C (rightmost three violins), each point on the violin plot indicating the average result from the central retina in one eye;

[0053] Fig. 3D shows a violin plot of the viability of cells stored for 3 and 24 hours post mortem at 4°C, having been examined using the Countess cell counter;

[0054] Fig. 3E shows a violin plot of number of live cells taken from retinal samples stored for 3 and 24 hours post mortem at 4°C, those cells having been examined using flow cytometry with calcein marking live ells and DAPI marking dead cells;

[0055] Fig. 3F shows a violin plot of the size of live cells taken from retinal samples stored for 3 and 24 hours post mortem at 4°C;

[0056] Figs. 4A, 4B and 4C show proteomic analyses of cell death markers, retinal neuronal and glial markers, and photoreceptor markers, respectively, from samples taken from the porcine retina and stored at 4°C at 3, 10 and 24 hours post mortem for each marker; Figs. 5A and 5B show perspective and exploded views of a device for storing and maintaining viability of an enucleated eye globe, respectively;

[0057] Fig. 5C shows a cross-sectional view of an embodiment of the device of Figs.5A and 5B;

[0058] Fig. 6A shows a perspective view of an embodiment of a platform of the device of Figs. 5A and 5B;

[0059] Fig. 6B shows a perspective view of an alternative embodiment of a platform of the device of Figs. 5A and 5B;

[0060] Fig. 7 shows a top view of an embodiment of a platform of the device of Figs. 5A and 5B;

[0061] Fig. 8 shows a schematic diagram of one configuration of the device of Figs. 5A and 5B, together with associated fluid circuits for the delivery of perfusion fluid and hydration fluid to an eye globe inside the device;

[0062] Fig. 9 shows a coaxial heat exchanger that may be used to heat perfusion fluid travelling to an eye inside the device of Figs. 5A and 5B;

[0063] Fig. 10A shows graphs detailing how the perfusion pressure (top panel) and intraocular pressure bottom panel) change over the first hour of perfusion of 8 different porcine eye globes;

[0064] Fig. 10B shows OCT scans showing the opening of retinal vessels (marked by arrows) and choroidal vessels (marked by a box) after 5 minutes of perfusion with oxygenated Ringer’s solution;

[0065] Fig. 10C shows OCT scans of ten porcine retinas and a violin plot showing how the overall retinal thickness changes over one hour of perfusion;

[0066] Fig. 11 A shows retinal fundus fluorescein angiography (FFA) images of progressive vessel perfusion in an ex vivo porcine eye following cannulation; Fig. 11 B shows a confocal maximum intensity projection of the whole retinal vasculature of a casted, flat mounted retinal specimen, showing successful microvascular perfusion to the capillary level after vascular casting;

[0067] Fig 11C shows a micro-CT image of retinal microvascular perfusion in an ex vivo porcine eye after vascular casting;

[0068] Fig. 11 D shows a single vessel resolution of an ex vivo porcine eye following perfusion and vascular casting;

[0069] Figs. 11 E and 11 F show a porcine eye with vascular casting performed at 5 and 54 hours post-mortem, respectively, showing perfusion of the choroidal and retinal vascular system;

[0070] Fig. 11G shows a magnified view within the boxed region highlighted in Fig. 11 B;

[0071] Fig. 11 H shows a segmented and reconstructed image of the region shown in Fig. 11G, showing disconnections in the vasculature;

[0072] Fig. 12A shows a reconstructed vasculature of a perfused ex vivo porcine eye generated by a machine learning algorithm following a micro-CT scan;

[0073] Fig. 12B shows the partition of the eye globe shown in Fig. 8A into octants and bullseye representation;

[0074] Figs. 12C and 12D show total segmental vessel lengths and volumes, respectively, in vascular networks casted at 3, 5, 32 and 54 hours post-mortem, with outliers due to leaking of the casting material indicated (*);

[0075] Fig. 13A shows the proportion of viable cells disaggregated from retinas in non-perfused eyes over time post-mortem at 24°C compared to the proportion of viable cells disaggregated from retinas in eyes perfused for 24 hours with Ringers solution at 0.5 mL / minute;

[0076] Fig. 13B shows the comparison of cell sizes of live retinal cells disaggregated from nonperfused eyes at 3 hours and 24 hours post-mortem at 24°C compared to the cell sizes of live retinal cells disaggregated from eyes perfused with Ringer’s solution for 24 hours postmortem at 24°C;

[0077] Fig. 13C shows flow cytometry analysis of live and dead cells using calcein as a live cell marker and DAPI as a dead cell marker for retinal cells disaggregated from non-perfused eyes at 3 hours and 24 hours post-mortem at 24°C compared to retinal cells disaggregated from eyes perfused with Ringer’s solution for 24 hours post-mortem at 24°C;

[0078] Fig. 13D corresponds to Fig. 3A, but includes an additional panel showing a representative image of Haematoxylin and Eosin staining of a porcine retina having undergone perfusion with oxygenated Ringer’s solution for 24 hours post mortem at 0.5 mL / min at 24°C;

[0079] Fig. 13E corresponds largely to Fig. 3C but includes an additional violin showing the severity of degradation of a retinal sample from a porcine eye having undergone perfusion for 24 hours post mortem at 0.5 mL / min at 24°C;

[0080] Fig. 14A shows the effect of systemic oxygen saturation levels on ERG responsiveness;

[0081] Fig. 14B shows the correlation between systemic oxygen saturation and ERG amplitude in vivo,

[0082] Fig. 14C shows the dependence of ERG response in in vivo porcine eyes from varying intensities of illumination by light;

[0083] Fig. 15A shows a representative recording from an ERG-positive perfused ex vivo porcine eye, stimulated at various time points over a three-hour period following enucleation, showing a characteristic signal with stable amplitude (peak-to-peak);

[0084] Fig. 15B shows a representative waveform from an ex vivo perfused eye where no ERG response is present, the eye having been stimulated at various time points following enucleation;

[0085] Fig. 15C shows overall ex vivo ERG responses of perfused porcine eyes to light for up to 12 hours of perfusion with oxygenated Ringer’s solution;

[0086] Fig. 15D shows the ex vivo ERG response of an individual porcine eye over time, where periods marked with an asterisk (*) indicate that perfusion was switched off; Fig. 16A shows the average estimated decay time of the ERG amplitude for ex vivo perfused porcine eyes and ex vivo non-perfused porcine eyes;

[0087] Fig. 16B shows a linear extrapolation of the normalised averaged ERG amplitude of all ex vivo perfused porcine eyes (solid circles) and non-perfused porcine eyes (empty diamonds), along with an exponential decay fit for both data sets;

[0088] Fig. 17A shows a violin plot of the length of ischemia time post-enucleation against the ex vivo ERG responsiveness of perfused porcine eyes;

[0089] Fig. 17B shows a linear regression analysis of in vivo ERG amplitude against ex vivo ERG response duration showing an R-squared value of 0.08865;

[0090] Fig 17C shows the effect of in vivo interocular pressure on ex vivo ERG responsiveness;

[0091] Fig. 17D shows a violin plot of the in vivo baseline retinal function against the ex vivo ERG response;

[0092] Fig. 18A shows a retinal fundus image showing perfusion of BriteVu casting material in an ex vivo human donor eye;

[0093] Fig. 18B shows an OCT scan of a human donor retina after perfusion with BriteVu casting material; and

[0094] Figs. 19A and 19F show comparisons of cell viability in contralateral human donor eyes between eyes perfused for 3 hours with Ringer’s solution and non-perfused eyes by Countess cell counting (Fig. 19A) and flow cytometry for calcein (Fig. 19B).

[0095] DETAILED DESCRIPTION

[0096] The invention aims to provide a device that enables maintenance of viability of an eye globe ex vivo, in order to allow for its use as a model for pre-clinical assessment of therapies and, eventually, development of a viable whole eye transplantation (WET) procedure. The eye globe may be sourced from a cadaver, or from heart-beating, cold- perfused donors after brain death (DBD) and circulatory death (DCD). In this description, the term ‘viability’ may refer to retinal cell viability. Retinal cell viability may be determined by detecting an electroretinogram (ERG) signal from the retina inside the intact eye globe that is distinguishable over background noise. Alternatively, retinal cell viability may be determined through analysis of a disaggregated retinal segment revealing a proportion of live cells above a predetermined threshold. This can be achieved using various methods, such as flow cytometry using calcein as a live cell marker, or the use of machine learning algorithms to identify the live cells.

[0097] To identify the best preservation strategy needed to maintain retinal viability, a number of initial experiments were carried out to characterise the degeneration of the retina in intact ex vivo eyes over time by analysing the retina structure. As room or higher temperature might be required for assessing pre-clinical therapies in ex vivo eyes, particularly where electrophysiology is used to evaluate function, the experiments were carried out at room temperature. Previous experiments showed that the retinal light-driven activity in explanted retinas was extended by 2.5 fold compared to activity at 37°C, moreover at room temperature (24°C), the metabolic demand of tissue is significantly reduced, thus preservation of function is enhanced.

[0098] The initial experiments focused mainly on pig eyes. Pig eyes were chosen due to their dimensional and anatomical similarity to human eyes: importantly they include a “visual streak”, which is a fovea-like, cone-rich zone in the central retina, which is similar to the macula of the human eye and is responsible for sharp, detailed central vision. The porcine retina also has a similar retinal ganglion cell distribution to human retinas. Furthermore, pig eyes are ethically and sustainably favourable, as they can be easily obtained as waste products of the meat industry. Consequently, the numbers of live animals needed in pre- clinical ophthalmology studies would be reduced and treatments arriving to clinical trials would undergo validation in whole eyes ex vivo before progressing to live animals and to patients.

[0099] Porcine eye obtention and preparation

[0100] Porcine eyes were enucleated from recently slaughtered pigs, a local slaughterhouse (Mafrica, Manresa, Spain). The breeds of pigs used in experiments were (Landrace x LargeWhite) x Pietrain or (Landrace x LargeWhite) x Duroc. Pigs are stunned into unconsciousness on a carbon dioxide wheel, and suspended by the hind legs, before rapid exsanguination performed by severance of the brachiocephalic trunk at the neck. Exsanguination occurs during 95 seconds, during which time, the eyes are extracted. Eyes are extracted by separating the eye globe from the ocular cavity using a paring knife, briefly washed in betadine, and placed in saline with heparin sodium 50 UI / mL, on ice, for transport and delivered to the laboratory within 2.5 hours post-mortem.

[0101] To prepare porcine eye globes for perfusion, the following steps were performed. The conjunctiva was trimmed, and the muscle tissue surrounding the optic nerve was carefully dissected to reveal a perfusable artery, such as the ophthalmic artery which lies adjacent to the optic nerve and is distinguishable due to its size and thick arterial walls compared to veins. Use of the ophthalmic artery is therefore preferred, although other arteries that can be canulated for this purpose include the internal carotid artery, the central retinal artery and ciliary arteries. When extracting the eye globe, it is important to ensure the optic nerve is extracted with the eye globe to preserve the arterial supply to the eye. Maintaining these vessels intact enables subsequent cannulation of one or more arteries for perfusion of the eye, which is critical to the method of the invention. This is because the ophthalmic artery and the central retinal artery both run along the optic nerve. If the optic nerve is cut too close to the eye globe, these vessels may be severed, although it is possible to maintain one or more of the arteries supplying the eye without inclusion of the optic nerve. Retaining a sufficient portion of the optic nerve helps to keep these vessels intact, helping to enable retinal perfusion and, consequently, maintenance of tissue viability under quasi- physiological conditions, as will be discussed below. The extracted eye may therefore preferably retain a minimum optic nerve length of about 1 cm, 1.5 cm, or 2 cm, more preferably at least 2 cm. The extracted eye preferably retains a minimum arterial length of about 0.5 cm, 1 cm, 1.5 cm. 2 cm, 2.5 cm, more preferably at least 2 cm, to facilitate cannulation and perfusion in accordance with the method of the invention. A 26G cannula (BD Neoflon Ref. 391349) was positioned in the ophthalmic artery and secured using nonabsorbable braided silk suture 6 / 0 (Fine Science Tools Item no. 18020-60). Perfusion was driven by a LongerPump BT100-1 L peristaltic, or Braun syringe pump at a rate of 0.5 mL / min. Oxygenation was performed by bubbling solutions with carboxygen (95% O2, 5% CO2) for 30 minutes prior to use.

[0102] For preparation of paraffin embedded retinal sections, the anterior chamber of the eye, including the lens and vitreous humour, were removed and the remaining posterior chamber eyecup was fixed in 6% paraformaldehyde (PFA) at 4°C overnight. The following day, retinae were dissected out of eyecups, and wax processed and embedded for paraffin section staining. Paraffin sections were cut at a 5 pm thickness and mounted on super- frost slides. For staining, the citraconic anhydride antigen retrieval method was used at 95 degrees for 30 minutes. Haematoxylin and Eosin (H&E) staining, and 4’,6-diamidino-2- phenylindole (DAPI) immunostaining were performed by adapting previously published protocols (Byrne et al. 2021). Histology images were captured using a Leica DM6000 B microscope. Retinal neurons labelled with DAPI were imaged using LSM980 Zeiss confocal microscope.

[0103] The number of nuclear rows in the inner and outer retina were quantified manually using the multi-point tool in FIJI. At least 3 areas per scaled image, and at least 3 images per retinal area (central, mid, periphery) per biological sample were used to calculate the average number of neurons per area.

[0104] Following overnight fixation of the eye cup in 6% PFA, retinal pigment epithelium (RPE)- choroids were dissected away from the sclera and retina. RPE flatmounts were washed with phosphate buffered saline (PBS) and blocked with 10% goat serum for one hour at room temperature. Alexa Fluor™ 488 Phalloidin (# A12379, Thermofisher) was applied at a dilution of 1 :1000 in 5% goat serum in PBS, for 2 hours at room temperature. Flatmounts were washed 3 times in PBS, and mounted with VECTASHIELD® Antifade Mounting Medium (H-1000-10, Vector laboratories) in 50mm FluoroDish glass bottomed dishes (FD5040-100, World Precision Instruments). Flatmounts were imaged using a Leica CTR7000 HS microscope, and RPE cell structure was analysed using Cellpose.

[0105] Phalloidin labelled images of the RPE cells of porcine retinal explants were analysed using Cellpose. Images were denoised and segmented using a diameter of 200 pixels and a flow threshold of 0.2. A mask of the segmentation outline of the Phalloidin positive areas was created, representing the borders of the RPE cells. RPE cell shape descriptors, namely cell area and cell shape (no. of sides) were obtained and both the average(most commonly occurring characteristic) and the frequencies at which each characteristic is present, were compared between eyes maintained for different durations post-mortem.

[0106] Histology sections were graded according to the severity scale outlined previously (Mouiee et al., 2021), by 2 blinded evaluators. Briefly, damage to the inner and outer nuclear layers was classified according to the following criteria. Retinas were deemed healthy when the typical retinal layers (Outer Nuclear Layer (ONL), Outer Plexiform Layer (OPL), Inner Nuclear Layer (INL), Inner Plexiform Layer (IPL), Ganglion Cell Layer (GCL)), were present, without any damage. The presence of any tears in either the ONL or INL meant an image had at least a low severity of damage. Loss of nuclear density in some areas increased the severity to moderate degeneration, and uniform loss of nuclear density was graded as severe degeneration.

[0107] For flow cytometry experiments, retinae were isolated from intact eye globes after maintenance for 3, 10 or 24 hours post-mortem at 4 degrees or at room temperature in saline with heparin sodium 50 UI / mL. For analysis of live and dead retinal cells, retinae were disassociated in 800 pL Papain containing DNAse in a shaker at 650 RPM at 37 degrees, for 30 minutes. After digestion, the cell suspension was spun down at 200 RPM at room temperature for 5 minutes to pellet the cells. The digested cells of one entire retina was resuspended in 2 mL PBS. For staining, 100 pL of the retina cell suspension was added to 1900 pL PBS and 2 pL Calcein-AM was added. Calcein-AM staining was conducted for 20 minutes on ice before cytometry. Dyes were not washed before cytometry. Immediately before cytometry, 2 pL DAPI was added to each sample. Samples were filtered using Falcon® 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap, product no. 352235. Flow cytometry analysis using LSRII cytometer and FlowJo analyses. The population of cells were identified from all events detected, using Compensated IndoViolet A versus FSC-A gates. Events below 50K FSC-A were considered debris, and the events above 50K FSC-A were considered cells. Next, taking the population of cells only, Calcein positive live cells were identified, and represented as a percentage of total cells.

[0108] Results of initial experiments on porcine eyes

[0109] Immediately after enucleation, pig eyes were maintained for 3, 10 or 24 hours at room temperature in PBS. Retinas were isolated from intact eye globes and were fixed at the different time points. Retinal neuron loss was then investigated using DAPI staining during 24 hours post-mortem in ex vivo eyes (Fig.lA). The number of rows of neurons in retinal sections from the centre, middle and peripheral retina, were quantified (Fig.l B). We did not observe a significant reduction of nuclear rows in the inner nuclear layer (INL), or outer nuclear layer (ONL) in the centre, middle and peripheral retina.

[0110] Haematoxylin and eosin (H&E) staining was then carried out to investigate the preservation of the retinal structure in the first 24 hours post-mortem. The tissues showed clear sign of structural disruption, as was evident by irregularity in the nuclear and plexiform layer, starting from 10 hours post mortem (Fig.1 C) . Retinal degeneration severity was graded, and it appeared to gradually increase with time post-mortem (Fig.1 D).

[0111] Next, investigations were carried out as to whether clinically relevant retinal pigment epithelial (RPE) alterations occurred ex-vivo. To this end, RPE structure was examined over various post-mortem times (Fig.2). RPE morphology was well maintained, and the overall cell area did not change between early (2 hours) and advanced (24 hours) postmortem times (Fig.2B). However, we observed a sign of cellular expansion, namely, very small number of RPE cells (<5%) had increased numbers of sides at 24 hours post-mortem compared to 3 or 10 hours post-mortem (Fig.2C). This is consistent with RPE dysmorphia, reported in aged and age-related macular degeneration human donor RPE cells, and cell body extension, reported in ageing mice. This suggests that a certain level of damage occurs in the RPE of ex-vivo eyes, although only in very small percentages of the overall population of cells 24h post mortem.

[0112] Although room temperature is the preferred condition for testing pre-clinical therapies in ex-vivo eyes, changes were also investigated changes in retinal cell viability in eyes stored at 4°C , which further lowers the metabolic activity of the tissue (Fig.3). 4°C is currently used to preserve large organs before their transplantation. However, it is generally thought that the electrophysiological activity of neurons is optimal at temperatures above 30°C, although there is a scarcity of published articles documenting this in mammals, and it has not been investigated in intact ex vivo eye globes. Importantly, this temperature also allows reducing bacterial proliferation, ensuring that the observed changes could be more confidently attributed to the time post-mortem rather than other factors.

[0113] Figs.3A and 3B show the results of H&E staining of eyes stored for 3, 10 and 24 hours at room temperature (24°C, Fig.3A) and 4°C (Fig.3B). Fig.3C shows a violin plot of graded severity scores for the degradation in the various samples, with the three violins on the left of the plot representing samples at 4°C at, from left to right, 3, 10 and 24 hours postmortem and the three violins on the right representing samples at 24°C, again at 3, 10 and 24 hours post-mortem. In Fig.3C, it can be seen that there is no statistically significant difference between the samples at 4°C and 24°C with regards to the severity of degradation of the cells. This is shown by the ‘ns’ labels on the figure, a convention that is adhered to throughout the figures. Where a statistically significant difference between data is seen, asterisks are used to indicate this, with a larger number of asterisks indicating a greater significance of the difference. One asterisk (*) indicates a p-value < 0.05, two asterisks (**) indicates a p-value < 0.01 , three asterisks indicates a p-value <0.001.

[0114] Single cells were also disaggregated from retinas from intact eyes that were stored at 4°C for 3 hours and 24 hours. These cells were examined using the Countess Cell counter. The percentage of viable cells at 3 hours and 24 hours is shown in Fig.3D. Fig.3E shows the percentage of live cells at 3 and 24 hours post mortem, with calcein dye marking live cells and DAPI marking dead cells. Finally, Fig.3F shows the respective sizes of live cells at 3 and 24 hours post mortem. Figs.3A to 3F show significant degradation in retinal cell viability, and cell size after 24 hours, indicating that storage at 4°C is not sufficient to preserve the retina in a post-mortem eye.

[0115] To understand whether the observed retinal cell death could be attributed to altered protein expression in the post-mortem retina, proteomics was conducted on eyes maintained for up to 24 hours post-mortem, at 4°C. No changes were observed in cell death related proteins at 3, 10 and 24 hours post-mortem (Fig.4A), with the darker bars indicating longer time intervals. Overall, very few proteins were found to change over time. Relevant retinal neuronal and glial markers and photoreceptor markers, such as Vimentin, GFAP, Rhodopsin and others, with known roles in retinal function (Fig.4B, 4C), were examined over time, and no significant differences between timepoints were found. Contrary to the assumptions of rapid protein degradation in post-mortem tissues, we did not find changes, which suggests that proteins are rather stable at 4°C between 3- and 24-hours postmortem. However, since retinal cell viability is impaired at both 4°C and room temperature 24 hours post-mortem, further intervention is required to avoid damage and allow for possible resuscitation of the eye.

[0116] Despite some expected alterations, the retina and RPE sheet overall structure were preserved for at least 10 hours post-mortem even at room temperature. It was therefore reasoned that it may be possible to detect retinal light responses in ex vivo eyes and that delivering oxygen to the retinal tissue via the physiological circulation of the eyes could restore function and thus resuscitate the eye. Additionally, it was reasoned that to preserve the eye’s functionality, the haemodynamic function in both retina and choroidal vascular beds must be maintained.

[0117] Device for enabling maintenance of viability of a cadaveric eye globe ex vivo Following these initial experiments, a device to enable delivery of oxygen to an enucleated ex vivo eye globe was developed.

[0118] Fig. 5a shows a perspective view of the device 10 in the form of a box, while Fig.5b shows the box 10 in an exploded view. The box 10 is generally cuboidal in shape and comprises a main housing 12, a base 14 and a lid 16 that together define an internal enclosure 18. A platform 20 rests on the base 14 inside the enclosure 18. The platform 20 comprises a part-spherical eye bed 22 at a first end thereof to support an enucleated eye globe (not shown). The eye bed 22 is radiused to substantially correspond to the dimensions of the eye globe to be received thereon. For example, for human and porcine eyes, the radius of curvature of the eye bed 22 may be between 1 cm and 4 cm.

[0119] The lid 16 comprises an opening 24 through which a cannula 23 can be inserted. The cannula 23 is used to deliver a perfusion fluid, or perfusate, to the eye globe when the eye globe is stored inside the box 10. The cannula 23 is supported and held in position by an attachment means such as a clip 25 (shown in Fig. 6A) that is received in a recess 26 in the platform 20. This enables the cannula 23 to remain in the correct position with respect to the eye globe when the eye globe is in place on the eye bed 22. A first end of the cannula 23 is inserted into a perfusable artery of the eye globe, such as the ophthalmic artery, the internal carotid artery, the central retinal artery, and the ciliary arteries and subsequently sutured to enable delivery of perfusate to the entire vasculature of the eye, including the retina, via the cannula 23. The perfusate supplies oxygen to the eye globe, helping to maintain the function of the retina and thus maintain the eye globe in a viably functional state.

[0120] At a second end, the cannula 23 is connected to a reservoir of a perfusion fluid, or perfusate (not shown in Fig. 6A). The perfusate may be oxygenated Ringer’s solution. The skilled person will be aware that the term ‘Ringer’s solution’ encompasses a wide compositional range and will appreciate that the exact composition used may not be of central importance to the invention. The key purpose of the use of Ringer’s solution is allow the delivery of oxygen to the vasculature of the eye globe, in particular to the retina. As such, a number of other fluids may be suitable for use as the perfusion fluid, such as a physiological buffer solution, blood serum, blood or a blood substitute. A pump may operate on the perfusate in the reservoir to control the delivery of the perfusate to the eye globe via the cannula 23. The pump may mediate delivery of the perfusate at a rate of 30 mL / hour (0.5 mL / minute), for example. Initial perfusion rates may range from 0.05 to 5 mL / minute, and once the ocular vasculature has received an initial round of perfusate, maintenance perfusion can be achieved using the same flow rate, or even lower perfusion rates such as 0.005 to 5 mL / minute.

[0121] The pressure of perfusion fluid, which is detected upstream of the eye globe, may lie between 0.01 and 5000 mbar, although a range of 25 to 130 mbar is more typical. Since the aim is to achieve an intraocular pressure within a physiological range (e.g. 20-25 mmHg), having this flexibility in the pressure of the fluid column allows for use in eyes in poorer conditions which might require different pressures, or perfusates of different viscosities. If the flow rate is kept fixed, the fluid pressure will vary with the diameter of the tubing or vessel the fluid flows in. A machine learning algorithm may be used to simulate perfusion of the eye globe and so determine an optimal perfusion rate and pressure.

[0122] The vasculature of the eye is complex and so, rather than connecting a second cannula to a vein to collect outflowing perfusate, the eye bed 22 comprises a number of drainage apertures 28 that allow outflowing perfusate to drain through the eye bed 22. The drainage apertures 28 are in fluid communication with a drainage channel 28a, seen best in the cross-sectional view of Fig. 5C in the base that leads to a tube connector 30. A drainage tube 28b can be connected to the tube connector 30 to collect the perfusate. In some embodiments, the drainage tube 28b may be in fluid connection with the reservoir of perfusion fluid to allow for recirculation of the perfusate, although the outflowing perfusate would need to be reoxygenated, and potentially may need replenishment of nutrients / electrolytes, depending on the precise composition of the perfusion fluid. In other embodiments, the drainage tube 28b simply lead to a waste collection container.

[0123] Since the drainage channel is located in the base 14, the tube connector 30 projects downwardly out of the box 10. As such, in order to provide sufficient space underneath the box 10 for the tube connector 30 and the drainage tube, the base 14 may be mounted on, or otherwise connected to, a stand 32 that raises the box 10 above the surface on which it rests. The stand 32 may comprise openings to allow the delivery tube to pass outside the footprint of the stand 32 without the stand 32 being forced to rest unevenly on the surface.

[0124] The main housing 12 comprises a first end wall 34a, a second end wall 34b and two side walls 36. The side walls 36 each comprise two oppositely oriented protruding ledges 38. The two ledges 38 on each side wall 36 may be considered to form a ledge pair, with one upper ledge 38a and one lower ledge 38b per side wall 36. Each ledge 38 defines a latching surface 40, which defines the widest point of the ledge 38, with the body of the ledge 38 tapering back to the side wall 36 away from the latching surface 40. The latching surface 40 of the upper ledges 38a faces downwards when the box 10 is in an upright orientation, with the latching surface 40 of the lower ledges 38b facing upwards.

[0125] Each ledge 38 is associated with respective arms 42: two of these arms 42 are located on the lid 16 with another two arms 42 on the base 14. Each arm 42 is rotatably attached to a side of the respective component of which they are a part (i.e. , either the lid 16 or the base 14) via respective hinges 44. The arms 42 are able to rotate around the hinges 44, with a distal portion 42a of each arm 42 being configured to engage the latching surface 40 of the associated ledge 38 when rotated to lie against the side wall 36 of the main housing 12.

[0126] When an arm 42 is engaged with the associated latching surface 40 of one of the ledges 36, it may be considered to be in a closed position. When all of the arms 42 are in the closed position, the box 10 may be considered to be in a closed configuration, where the lid 16 and base 14 are each retained against the main housing 12 in order to prevent an eye globe stored in the enclosure 18 from falling out. In this way, the arms 42 and ledges 38 together form a closure mechanism for the box 10.

[0127] The skilled person will appreciate that, while the latch arms 42 and ledges 38 shown in Figs.5A and 5B are especially convenient for the generally cuboidal box 10 shown in those figures, the exact shape and nature of the closure mechanism is not of central importance to the invention and that other closure mechanisms can be employed for the same purpose of closing the box 10 and may be more appropriate for doing so for certain box morphologies.

[0128] The box 10 additionally comprises a first, upper gasket 46 and a second, lower gasket 48, each gasket being made of a silicone elastomer. The upper gasket 46 is located in between the main housing 12 and the lid 16, while the lower gasket 48 is located in between the main housing 12 and the base 14. The gaskets 46, 48 enable the creation of a hermetic seal around the enclosure 18 when the action of the closure mechanism brings the box 10 into the closed configuration. For example, in the embodiment of Figs.5A and 5B, the engagement of the arms 42 with the latching surfaces 40 of the ledges 38 urges the lid 16 and base 14 against the upper and lower gaskets 44, 46, and in turn, the main housing 12 to create the hermetic seal. Creation of a hermetically sealed enclosure 18 is useful as it minimizes contact with the external environment, facilitating recirculation of outflowing perfusate. A hermetically sealed enclosure also enables use of perfusion fluids that need to be isolated from the air.

[0129] The first end wall 34a is provided with a window 50 to enable observation of the eye globe inside the enclosure 18 while the box 10 is closed. The window 50 may be made of any suitable transparent material, such as glass or methacrylate. To ensure maintenance of the hermetic seal when the box 10 is in the closed configuration, the window 50 may additionally comprise a window gasket 52. In certain configurations, the window 50 may be detachable from the first end wall 34a. This can facilitate manipulation of the cornea and sclera while the eye globe is in place on the eye bed 22, or the application of electrodes or trochars for electrophysiological assessments or administration of therapies, respectively. The detachment of the window 50 from the first end wall 34a may be enabled by a twist-off mechanism.

[0130] The box 10 may further include an access mechanism that is openable and closable to allow selective access to the internal enclosure 18 without requiring movement of the eye globe, interruption of perfusion or removal of the lid 16. The access mechanism may also be sealed against the rest of the main housing 20 to enable maintenance of the hermetic seal when the access mechanism is closed. In certain embodiments, the window 50 may form the access mechanism.

[0131] With the eye globe in place on the eye bed 22, one or more electrodes may be attached to the eye globe in electrical communication with the retina. The electrodes may be used to stimulate the retina and measure the electrical response thereof to generate an electroretinogram (ERG). As alluded to earlier, the ERG signal so generated may be used to determine the viability of the eye globe for transplantation purposes. The ERG signal can also be used to assess the efficacy of certain treatments applied to the eye globe, as part of pre-clinical tests.

[0132] In some embodiments, the electrodes may be wireless electrodes that communicate with an external processor via a wireless communication protocol. Various wireless communication protocols, such as Bluetooth, WiFi, etc. will be known to the skilled person. The use of wireless electrodes is beneficial as it avoids any complications with maintenance of the hermetic seal around the enclosure 18 while still enabling the transfer of ERG signals by the electrode to the external processor. In other embodiments the electrodes may be connected to wires that run through the housing, either through the main housing 12, or the lid 16 or the base 14.

[0133] Fig. 6B shows an embodiment of the platform 20. In this embodiment, a second clip 25a is received in the recess 26 in the platform 20, or in a second recess, distinct from the recess 26. As shown in Fig. 6B, the second clip 25a may support a second cannula 23a used to deliver medicament, drugs, etc to the eye globe. The second cannula 23a may enter the enclosure 18 through a second opening in the box 10 (not shown in Fig. 6B). A third clip 25b may also be in place on the platform 20 to support a third cannula 23b used for corneal hydration purposes. The configuration of the additional cannulae 23a, 23b may vary. For example, in Fig. 6B, both the clip 25 and the second clip 25a are located on the platform 20, while the third clip 25 is carried on an arcuate flexible arm 27. The arm 27 may extend between two attachment points 29 on the platform 20. However, as shown in Fig. 6C, each of the second and third clips 25a, 25b may be carried on flexible arms 27. It should also be appreciated that, while each cannula 23, 23a, 23b is shown in Figs. 6A and 6B to be needles that would contact the eye globe, this need not be the case. For example, non-contact cannulae may be used for corneal hydration or for delivery of medication via eye drops.

[0134] The eye box 10 may also include a temperature control system to enable the temperature of the eye globe to held at a desired level. Fig.7 shows an exemplary configuration of the platform 20 for this purpose, seen from a top view. The platform 20 in this case comprises a fluid heating circuit that incudes an inlet port 54, an outlet port 56 and a fluid conduit extending 58 therebetween, which takes the form of a bore through the body of the platform 20 and extends underneath or in close proximity to the eye bed 22.

[0135] The inlet and outlets ports 54, 56 are connected to a reservoir of temperature-controlled fluid, which therefore allows for heating or cooling of the eye globe via conduction through the platform 20 when circulated through the fluid conduit 58, depending on the temperature of fluid. Use of a fluid for temperature control avoids the introduction of electrical noise associated with electrical heating or cooling elements and thereby prevents interference with electrophysiology measurements. As an alternative to the use of a separate fluid heating circuit, the eye box 10 may instead utilise the perfusion fluid itself to control the temperature of the eye globe. In such cases, the temperature of the perfusion fluid may itself be controlled in order to control the temperature of the eye globe, heating or cooling the eye globe from within the vasculature of the eye. Regardless of the exact configuration of the temperature control system, a temperature sensor must be embedded within the system to enable monitoring of the fluid and the eye globe. For example, a Lauda thermal conditioning unit, or other similar system, may be used in order to control the temperature of the reservoir of the fluid.

[0136] The eye globe may be heated to approximately 37°C, or cooled to approximately 4°C, or controlled at an intermediate temperature by virtue of the temperature control system. For example, the eye globe may be cooled for general tissue preservation before the start of perfusion and heating during perfusion to enable restoration of electrophysiology and measurements thereof.

[0137] Fig. 8 shows a schematic diagram of one way in which the eye box 10 and associated systems may be configured. Fig. 8 shows the eye globe in place in the box 10, resting on the eye bed 22. There are two fluid circuits created, one for the perfusion fluid and one for a hydration fluid. The fluid circuit for the perfusion fluid functions in a similar way to that described above, with perfusion fluid stored in a reservoir 60 and being delivered to the eye globe via a cannula (not shown) inserted in an artery, such as the ophthalmic artery or the central retinal artery. The perfusion fluid is transported away from the eye globe via a second cannula (also not shown) connected to a suitable vein at one end and back to the reservoir 60 at the other end. Pumps 62 are used in each part of the perfusion fluid circuit to drive the flow of fluid. Each part of the perfusion fluid circuit is also fitted with a pressure sensor 64 to monitor the pressure of the perfusion fluid at that location. An oxygenation module 65 may also be used for acellular perfusates to enable medical oxygen to be bubbled into the reservoir.

[0138] The hydration fluid is also stored in a reservoir 66, and is delivered to the eye globe via a pump 68 and a third cannula (not shown), which may be a contacting or non-contacting cannula, as described above. The hydration fluid is collected by the eye bed 22 and flows out of the drainage apertures and the drainage channel (both not shown), aided by a drain pump 70.

[0139] In the configuration of Fig. 8, both the perfusion fluid and the hydration fluid return to the respective reservoirs 60, 66 for re-use. However, this is not necessary and each fluid may simply be transported to a waste collection after use. The skilled person will appreciate that the perfusion fluid may also be passively drained via the drainage apertures 28 in the eye bed 22 together with the hydration fluid. Both reservoirs are temperature controlled via temperature conditioning units 72, such as a Lauda thermal conditioning unit. The various pumps 62, 68, 70, the pressure sensors 64 and the temperature conditioning units 72 are controlled by a controller 74 which can control the flow rate and pressure for each fluid, and the temperature thereof. The control of the perfusion fluid circuit may implement closed-loop control in order to keep the flow rate and pressure of the perfusion fluid within a desired range, for example via use of a PI D algorithm.

[0140] In order to mitigate any temperature variation of the perfusion fluid (either warming of a cooled fluid or cooling of a heated fluid) as it travels out of the reservoir 60 to the eye globe, the tubing of the circuit for inflowing perfusate may be enclosed within a coaxial heat exchanger 76, shown in Fig. 9. The perfusion fluid flows along a central channel 78 of the heat exchanger 76, surrounded by conditioned water from the temperature conditioning unit 72.

[0141] Finally, the box 10 may comprise a further opening to allow the collection of samples, either from the eye globe or a sample of outflowing perfusate from the eye globe. In addition, the eye bed 22 or platform 20 may comprise an integrated clip, or other holding means to maintain the eye in place on the eye bed, especially when any sort of intervention to the eye globe is carried out.

[0142] In vivo ERG on porcine eyes

[0143] For ERG experiments, in vivo ERG was performed on anaesthetised, intubated pigs, of both sexes, 35-52 kg in weight, in a veterinary surgery at Universitat Autonoma de Barcelona. Prior to in vivo ERG, pupils were dilated using compound tropicamide eye drops (5 mg / ml) and eyes were hydrated Electrode contact Gel ( by Parker laboratories) used to avoid corneal dehydration and improve the signal transmission.

[0144] Animals were dark-adapted for at least 30 minutes before recordings. All recordings were performed using a custom-made ERG system, consisting of an electrooculogram amplifier (BioPac, Ref. EGG100C), an isolated power supply module (BioPac, Ref.lPS100C), a multifunctional data acquisition device(National instruments Ref. USB-6001), a disposable corneal electrode ERG-Jet (Fabrinal Ref. F-06), a disposable subdermal needle electrode (by Technomed Ref. TE / S43-438) and a blue LED used as flash stimulus controlled by a purpose-built Software interface, built in MatLab. Eyes were stimulated with light power of 5 nW / mm2(on the cornea), with 20 ms pulse duration, one second rest and a total of 30 pulses. The recorded data was then analysed. Firstly, the 50 Hz noise was filtered using readily available MATLAB functions, followed by the bandpass filtering of the data (1-100 Hz) to extract the ERG signal. The average of the 30 repetitions was calculated and then the ERG amplitudes were then defined as the difference between the maximal and minimal values of the average signal. Noise level was defined as two standard deviations of the average signal amplitude of the time window where no response is expected (400-900 ms from stimulus onset).

[0145] Ex vivo experiments on perfused porcine eyes

[0146] Once normal retinal function was confirmed by in vivo ERG recordings in both eyes in the surgical room, eyes were enucleated from end-point anaesthetized, intubated pigs, and placed in saline. Eyes’ ophthalmic arteries were immediately cannulated and stabilised in the box 10, where they were kept for the duration of the ex vivo experiments. Once cannulated, the eyes were perfused with freshly prepared carboxygenated Ringer’s solution 110 mM NaCI, 22 mM NaHCO3, 2.5mM KOI, 1.6mM MgCI, 1mM CaCI2, NaH2PO4, and 10mM) at a flow rate of 30mL / hour and kept at room temperature, around 24°C. The cannulation and perfusion strategy aimed to optimise a method previously reported by Rousou et al in 2019.

[0147] Eyes were perfused at a constant flow rate of 0.5 mL / min. As can be seen in the lower panel of Fig.10A, this led to a gradual increase in intraocular pressure (IOP), followed by a plateau at a level close to normal IOP in pigs, which is around 20-25 mmHg. This was accompanied by the perfusion pressure remaining stable for at least 60 minutes, as seen in the upper panel of Fig.10. OCT imaging showed the retinal vessels and choroidal vessels opening after 5 minutes of perfusion. This is shown in Fig.10B: the top panel shows the baseline before perfusion, with the bottom panel showing the retinal vessels, marked by arrows, and the choroidal vessels, marked by the box on the right hand side, having opened. OCT imaging also showed no change in retinal thickness after an hour of perfusion (Fig.10C), indicating an absence of retinal edema. Fig.100 also includes a violin plot showing the retinal thickness of the various samples at the start of perfusion, and after one hour of perfusion.

[0148] Retinal vessel re-perfusion was assessed by retinal fundus imaging and fluorescein angiography performed at room temperature (Fig.11 A). The vessels were filled with the fluorescein-containing medium in approximately 90% of >10 well cannulated eyes analysed (Fig.11 A). Having developed the cannulation strategy, vascular casting was conducted to map the vessels within the entire ex vivo eye globes. The eyes were perfused with BriteVu casting reagent, fixed and imaged by Micro-CT scanning. The retina was examined using confocal microscopy to confirm whether smaller capillaries difficult to see using Micro-CT were also perfused (Fig.11 B). BriteVu casting material was clearly visible in retinal micro-vessels, evidencing their efficient perfusion (Fig.11 B). The entire ocular vasculature, including the retina, choroid and iris was efficiently filled with the casting material (Fig.11C), and individual perfused vessels were resolvable (Fig.11 D). Remarkably, vascular casting was possible even 5 (Fig.11 E) and 54 hours (Fig.11 F) postmortem, suggesting preservation of the overall vascular system a long time post-mortem.

[0149] Analysis of retinal structure (Fig.1), perfusion (Fig.11), and vascular mapping (Fig.12) suggested that the retina was sufficiently preserved for restoration of perfusion, even multiple hours post mortem. Retinal cellular viability was then examined in post-mortem eyes, to understand if restoration of perfusion with oxygenated Ringer’s solution could be sufficient to maintain the retina alive in the post-mortem eye. Cell viability was assessed by performing cell counts and flow cytometry on disaggregated retinas after 3 or 24 hours post-mortem. Two groups of eyes maintained for 24 hours were analysed, one without perfusion and the other after perfusion with Ringer’s solution and the retinal cell survival was compared with eyes analysed 3 hours post-mortem without perfusion (Fig.13). While retinal cell viability was significantly reduced 24 hours post-mortem compared to 3 hours post-mortem, preservation of retinal cell viability was observed in eyes perfused with Ringer’s solution compared to un-perfused eyes, when evaluated with automatic cell counting (Fig.13A). Live cell size was also significantly larger in 3 hour post-mortem and 24 hour-post-mortem perfused eyes, compared to 24 hour post-mortem non-perfused eyes (Fig.13B). The protection of cell viability offered by perfusion with Ringer’s solution was confirmed by flow cytometry analyses of Calcein labelled live cells in disaggregated retinas (Fig.13C).

[0150] Perfusion with Ringer’s solution also indicated some beneficial effects for the slowing of retinal structure degradation, as seen in Figs.13D and 13E. Fig.13D corresponds to Fig.3A, but includes an additional panel showing H&E staining of a retina having undergone 24 hours of perfusion with Ringer’s solution at room temperature. Fig.13E corresponds to the right hand side of Fig.3C, but includes an additional violin for samples that have undergone 24 hours of perfusion. Following these encouraging results, retinal resuscitation in ex vivo perfused eyes was also tested by evaluating whether the retina could respond to stimulation with light flashes entering the cornea.

[0151] Prior to ex vivo ERG testing, the effect of certain vital parameters (namely oxygenation and IOP) on electrophysiology was investigated in vivo, to understand how pivotal they were to detection of ERG in the ex vivo eye (Fig.14). In intubated pigs, reducing blood oxygen saturation levels led to lower ERG amplitudes, suggesting that oxygen levels are an important although not fully linear parameter, with R2= 0.7026 (Fig.14A,14B). This suggests that blood oxygen is closely related to ERG response, and there are some windows of oxygenation levels during which changes in oxygen levels do not alter ERG response, probably due to the extremes of the oxygen saturation curve being associated with either the maximal ERG response (upper oxygen saturation), or no ERG response (below lower threshold of oxygen saturation). Importantly, ERG amplitude increased linearly with light intensity, reaching saturation at the higher intensities, as shown in Fig.14C.

[0152] In assessing the ERG response, noise levels were determined for each eye after electrode placement, by conducting a stimulus with the light fully isolated in aluminium foil, preventing stimulation of the eye. ERG recordings were then performed using the same method described above for the in vivo ERG recordings. ERG signals were recorded approximately every 5 minutes, until the signal was indistinguishable from the noise. Eyes are dark adapted within 5 minutes post-mortem, and ERG measurements commence as soon as the perfusion cannula is secured (usually 15-20 minutes post-mortem).

[0153] Robust ERG signals were detected during perfusion of the eyes with oxygenated ringer. Positive response was defined by the presence of a characteristic and repeated ERG- waveform above the noise level as a signal with amplitude at least two standard deviations higher than the noise level (Fig.15A). Fig.15B, on the other hand, shows the ERG amplitude for non-responsive perfused eyes. ERG signals were detected during perfusion of the eyes with oxygenated Ringer’s solution in 15 out of 36 eyes (41.4% responsive). During perfusion, ERG amplitude gradually decreased until reaching plateau, which was maintained for up to 12 hours (Fig.15C). In some cases, recording was stopped due to logistical reasons rather than the eye ceasing to respond. Fig.15D shows the ERG amplitude for an individual eye over 12 hours. ERG response decays during periods marked with an asterisk when perfusion was switched off; the signal is revived when perfusion is restored.

[0154] The decay of signal upon hypoxia was much faster in non-perfused eyes than in perfused eyes, as shown in Fig.16A. Fig.16B shows the normalised averaged ERG amplitude of all perfused eyes, which shows a gradual decrease over 12 hours, as well as the normalised average ERG amplitude of all non-perfused eyes, which decreases to zero almost instantly. These results and those shown in Fig.15D highlight the ability of the invention to maintain eye functionality for up to 12 hours after death through perfusion, as well as the dependence of the resuscitation of retinal activity on perfusion. Optimization of the entire perfusion system (including perfusate) may yield in a longer persistence of the ERG signal.

[0155] Possible causes of the variability of the ERG response detected in ex vivo eyes were also investigated. There was no significant difference in ischemia time, i.e., the time from enucleation to reperfusion, in ERG responsive and unresponsive eyes (Fig.17A). The baseline in vivo ERG amplitude of responsive eyes was compared to the duration of ex vivo response, and a very weak positive or no correlation was observed, with an R-squared value of 0.08865 (Fig.17B). Moreover, intraocular pressure (IOP) values obtained in the initial in vivo testing were compared with subsequent ex vivo responsive and non- responsive eyes. Here, was no significant difference was found, indicating that pre-existing IOP is not the cause of the difference in ERG response (Fig.17C). ERGs were recorded for all eyes in vivo before ex vivo recording were performed. There were no significant differences in baseline ERG amplitudes between ERG-responsive and unresponsive eyes (Fig.17D). These results suggest that additional factors determine ex vivo ERG responses.

[0156] Tests on ex vivo human eyes

[0157] Experiments were also carried out on enucleated human eye globes in order to assess the applicability of the methods described above for human eyes.

[0158] The enucleated human eyes were placed in the device 10 for perfusion in a similar way to that described above for the porcine eyes. After perfusion, fluorescein angiography was carried out in the retina of the perfused eyes. Visualisation of the casting material by Micro- CT scanning suggested efficient perfusion of the eyes, indicating that the techniques developed for the porcine eyes has good applicability and transferability to human eyes. This is very promising for the development of whole eye transplantation in humans. Retinal fundus imaging of human eyes perfused with BriteVu casting reagent was also performed. The result is shown in Fig.18A, where perfusion of the white casting material can be seen. The white casting material was also imaged via OCT (Fig.18B), suggesting efficient perfusion of the human retina. Human eyes from four donors have also been perfused with Ringer’s solution. Cell viability was compared after 3 hours perfusion against unperfused eyes by countess cell counting (Fig.19A) and flow cytometry for calcein (Fig.19B). In both cases, perfusion with Ringer’s solution led to a greater percentage of viable retinal cells, which is another promising result for the transferability of the method to human eyes.

[0159] Al modelling of porcine eye vasculature

[0160] Although perfusion was achieved effectively, among multiple samples and at different time points post-mortem, our observations revealed differences in the extent of the penetration in the distal branches. To quantitatively compare these variations a detailed reconstruction of the vascular anatomy of the perfused porcine eyes was performed using an Al segmentation method (Fig.12).

[0161] The vascular segmentation pipeline used a supervised deep learning approach. Ground truth data were sourced from 4 micro-CT datasets, which were divided into sub-blocks and 310 were randomly picked to be manually annotated. Training, validation and test sets were assigned at 75:15:10 ratio. 3D ll-Net ( igek et al., 2016), V-Net (Milletari, Navab and Ahmadi, 2016), Deep Residual ll-Net (Zhang, Liu and Wang, 2018) and Attention ll-Net (Gitonga, 2023; Oktay et al., 2018) were trained 5 times each and top F1 scores were compared. As shown in table 1 , Attention ll-Net was most performant and was used for the subsequent processing.

[0162] Table 1 : Outcome of training segmentation Al architectures.

[0163] Table 2. Global anatomical statistics of whole eye samples.

[0164] The segmented vascular mask image was skeletonized and converted to a sparse graph format yielding an effective data compression from around 70 GB to a few kilobytes. Vessel diameters were detected using a modified version of Rayburst sampling (Rodriguez et al., 2006) applied in a predictor-corrector fashion, which initially used a local thickness filter as a first pass to tune a truncated, oriented sampling core that is adaptively scaled by the thickness. Full width half maximum criterion was used for ray termination.

[0165] Connected components were identified from the full graph, which often revealed smaller subtrees isolated from the main network. The detachments were assumed to have been introduced post-casting (since occlusions would not have allowed Britevu to penetrate distally), and thus subtrees were included in the quantification.

[0166] In order to enable a comparison between different eye globes, all reconstructed vascular network meshes were aligned to a standard orientation using the lens plane (from which anterior-posterior axis was identified), and the optic disc (which was selected to be the 0 angular position). The equator of the globe, which was assumed to bisect the posterior and anterior hemispheres, was then defined, finally resulting in the division of the globe into 8 parts (Fig.12B).

[0167] The vascular segments contained within each octant were identified, and the total vascular length and volume, and vascular density were calculated.

[0168] Although perfusion was achieved effectively, among multiple samples and at different time points post-mortem, observations revealed differences in the extent of the penetration in the distal branches. To quantitatively compare these variations a detailed reconstruction of the vascular anatomy was performed using an Al segmentation method (Fig.12A). Global anatomical measurements are shown in Table 2. The samples casted at 32 and 54 hours post-mortem suffered from localised vessel rupture and leakages which inflated the true total vessel volume (expressed as a percentage of globe volume), though the sample at 54 hours showed a clearly reduced level of vessel filling regardless relative to other samples. The total length and volume densities of the vascular segments are shown for each region in Figs.12C and 12D, respectively. Across the samples casted at varying time points (3, 5, 32 and 54 hours PM), some broadly consistent patterns can be seen including a higher level of vascularisation in the posterior segment, and variation between octants (characterised by coefficient of variation ranging between 0.22 to 0.46 for total length density). On the other hand, the samples casted at later time points (32 hours and up) appear to exhibit a greater level of variation across the regions, associated by increased instances of perfusate leakage. Though further samples need to be tested in future, these results may be indicative of loss of integrity in the vascular walls to withstand pressure by 32 hours post-mortem.

[0169] In addition, the segmentation pipeline was also applied to retinal vascular images (Fig.11 B) to reconstruct distal networks including capillaries which are orders of magnitude smaller. A region measuring 34 mm2in cross-section (Fig. 11G) was fully reconstructed (Fig.11 H), showing that the methodology is capable of handling a differing imaging modality as well as imaging resolution. Direct overlay of the images confirmed that every segment within the imaging volume was faithfully reconstructed, albeit disconnections among the segments were found (indicated by arrows in Fig.11 H). It is likely they were introduced during the mounting of the retina on a glass slide, as they are also present in the images while the filling in the vessel trees distal to the disconnections was complete.

[0170] Conclusions

[0171] The experiments carried out show that room temperature perfusion with Ringer’s solution without biological material, which may add risks of immune reactions, is sufficient for resuscitation of the retinal function in the intact eye globe, during the first hours postmortem. Our data also indicate that cold storage of the eyes during the first hours postretrieval might preserve the retinal structure. Thus, future development of a protocol in which the eyes are warmed up after cold storage might also lead to improved results. These findings defy common beliefs that neural tissues die and their functions are instantly lost following death, or after cessation of circulation. Instead, the data presented here corroborate the idea that organs, even neural organs, might be revived post-mortem, as in case of eyes to be used for whole eye transplantation (WET) in the future. The experiments carried out provide novel data showing that neuronal responses can be elicited from the retina in intact eyes for multiple hours ex vivo, in agreement with previous studies from isolated retinas, as well as from retinal organoids. This is also in agreement with the recent finding in which an ERG signal was detected in one patient after face allograft and eye reperfusion. Interestingly, during the surgical procedure the explant from the donor was carried out side-by-side with the face transplant in the recipient. The novelty of the findings presented herein is that resuscitation and preservation of intact eye globes can be observed for up to 12 hours out of the body with a simple intervention. This timeframe may be sufficient for WET within the same hospital, in a side by side donor and recipient situation.

[0172] The invention may be more easily understood with reference to the following numbered clauses:

[0173] 1. A device for storing and maintaining viability of an ex vivo enucleated eye globe, the device comprising: a platform comprising an eye bed, wherein the eye bed is part-spherical and is radiused to substantially correspond to the dimensions of an eye globe to be received within the device, and wherein the eye bed comprises at least one drainage aperture, and wherein the platform further comprises an attachment means configured to hold a cannula in place.

[0174] 2. The device of Clause 1 , further comprising a housing comprising a main housing, a lid, and a base that together define an enclosure, wherein the platform rests within the enclosure on the base, and wherein the lid comprises an opening configured to receive the cannula passing therethrough into the enclosure, and wherein the drainage aperture is in fluid communication with a drainage channel in the base of the housing.

[0175] 3. The device of Clause 1 or Clause 2, wherein the drainage channel is in fluid communication with a tube connector.

[0176] 4. The device of Clause 2 or Clause 3, further comprising a closure mechanism, configured to bring the device into a closed configuration and to retain the device in said closed configuration. 5. The device of Clause 4, wherein the closure mechanism comprises respective pairs of latch arms on each of the lid and the base, and corresponding ledges to each of the pairs of latch arms on side walls of main housing, each ledge defining a latching surface for the associated latch arm to bear against when the latch arm is in a closed position.

[0177] 6. The device of Clause 4 or Clause 5, further comprising a first gasket between lid and the main housing and a second gasket between the base and the main housing so that operation of the closure mechanism to bring the device into the closed configuration generates a hermetic seal around the enclosure.

[0178] 7. The device of any of Clauses 2 to 6, further comprising a window in a wall of the main housing.

[0179] 8. The device of Clause 7, wherein the window is detachable from the wall of the main housing.

[0180] 9. The device of Clause 8, wherein the window is detachable from the wall via a twist- off mechanism.

[0181] 10. The device of Clause 8, wherein the window is hinged to the wall of the main housing.

[0182] 11 . The device of any preceding clause, wherein the attachment means comprises a clip received in a recess in the platform.

[0183] 12. The device of any preceding clause, further comprising a holding means configured to maintain an eye globe in place on the eye bed.

[0184] 13. The device of any of Clauses 2 to 12, further comprising a stand attached to the base configured to raise the base above a surface on which the device rests.

[0185] 14. The device of any preceding clause, wherein the radius of curvature of the eye bed is between 1 cm and 4 cm.

[0186] 15. A system comprising: the device of any preceding clause; a cannula retained in place by the attachment means, the cannula being connected at a first end to a reservoir of perfusion fluid and being operatively connectable at a second end to an eye globe in order to deliver the perfusion fluid thereto; and a pump to enable transfer of the perfusion fluid along the cannula.

[0187] 16. The system of Clause 15, wherein the perfusion fluid comprises oxygenated Ringer’s solution, a physiological buffer solution, blood serum, blood or a blood substitute.

[0188] 17. The system of Clause 15 or Clause 16, comprising at least one electrode configured to measure an electroretinogram (ERG) signal from the retina of an eye globe.

[0189] 18. The system of Clause 17, wherein the or each electrode is a wireless electrode.

[0190] 19. The system of any of Clauses 15 to 18, further comprising a drainage tube in fluid communication with the drainage channel at a first end and with the reservoir of perfusion fluid at a second end so as to enable recirculation of used perfusion fluid.

[0191] 20. The system of any of Clauses 15 to 19, wherein the device comprises a second cannula configured to deliver a hydration fluid configured to maintain hydration of a cornea of an eye globe.

[0192] 21. The system of Clause 20, wherein the device comprises a second attachment means, and wherein the second cannula is retained in place by the second attachment means.

[0193] 22. The system of Clause 20 or Clause 21 , wherein the second cannula is a noncontact cannula.

[0194] 23. The system of any of Clauses 15 to 22, wherein the device comprises a third cannula configured to deliver a medication or diagnostic agent to an eye globe. 24. The system of Clause 23, wherein the device comprises a third attachment means, wherein the third cannula is retained in place by the third attachment means.

[0195] 25. The system of Clause 21 , Clause 22 or Clause 24, wherein the second attachment means and / or the third attachment means are carried on a flexible arm mounted to the platform of the device.

[0196] 26. The system of any of Clauses 14 to 25, further comprising a temperature control system to enable delivery of a temperature control fluid into the device.

[0197] 27. The system of Clause 26, wherein the platform of the device comprises a bore extending therethrough from an inlet port to an outlet port to define a fluid conduit for the temperature control fluid passing underneath the eye bed.

[0198] 28. The system of Clause 26, wherein the temperature control fluid is the perfusion fluid.

[0199] 29. The system of Claim 28, further comprising a coaxial heat exchanger extending around tubing connecting the cannula to the reservoir of perfusion fluid.

[0200] 30. The system of any of Clauses 14 to 29, further comprising an eye globe resting on the eye bed, wherein the cannula is operatively connected at its first end to the eye globe so as to enable delivery of the perfusion fluid thereto.

[0201] 31 . The system of Clause 30, wherein the cannula is operatively connected to the eye globe via an artery thereof, wherein the artery is preferably one of the ophthalmic artery, the internal carotid artery, the central retinal artery, or one of the ciliary arteries.

[0202] 32. The system of Clause 30 or Clause 31 , wherein the pump is configured to enable delivery of the perfusion fluid into the eye globe at a rate of between 0.005 mL / minute and 5 mL / minute.

[0203] 33. The system of any of Clauses 14 to 32, wherein the eye globe maintains viability for 1 hour post-enucleation when perfused with the perfusion fluid via the cannula, preferably wherein the eye globe maintains viability for 3 hours post-enucleation, more preferably wherein the eye globe maintains viability for 6 hours post-enucleation, more preferably wherein the eye globe maintains viability for 12 hours post-enucleation, more preferably wherein the eye globe maintains viability for 24 hours post-enucleation, more preferably wherein the eye globe maintains viability for 48 hours post-enucleation.

[0204] 34. The system of any of Clauses 14 to 33, wherein the eye globe demonstrates viability through the generation of an ERG signal greater than 1.5 standard deviations over the background noise level, preferably greater than 2 standard deviations over the background noise level, more preferably greater than 3 standard deviations over the background noise level, and more preferably greater than 4 standard deviations over the background noise level.

[0205] 35. A method of maintaining the viability of an ex vivo enucleated eye globe, the method comprising: placing the eye globe on a part-spherical eye bed of a platform, the eye bed being radiused to substantially correspond with the dimensions of the eye globe; operatively connecting a cannula to the eye globe and retaining it in place with an attachment means received on the platform; delivering a perfusion fluid to the eye globe via the cannula; and allowing outflowing perfusate to drain through at least one aperture in the eye bed.

[0206] 36. The method of Clause 35, comprising operatively connecting the cannula to the eye globe via an artery thereof.

[0207] 37. The method of Clause 35 or Clause 36, comprising delivering the perfusion fluid at a rate of between 0.005 mL / minute and 5 mL / minute.

[0208] 38. The method of Clause 37, comprising delivering the perfusion fluid at an initial rate of between 0.05 mL / minute and 5 mL / minute and then subsequently delivering the perfusion fluid at a rate of between 0.005 mL / minute and 5 mL / minute.

[0209] 39. The method of any of Clauses 35 to 38, comprising determining an optimal delivery rate for the perfusion fluid using a machine learning algorithm. 40. The method of any of Clauses 35 to 39, comprising recirculating the used perfusion fluid draining out of the at least one aperture into a reservoir of the perfusion fluid connected to the cannula, wherein the platform and the eye globe are contained within a hermetically sealed enclosure.

[0210] 41. The method of any of Clauses 35 to 40, comprising mapping the vasculature of the eye globe using a machine learning algorithm. 42. The method of any of Clauses 35 to 41, further comprising cooling the eye globe before delivering the perfusion fluid thereto and / or warming the eye globe during delivery of the perfusion fluid thereto by delivering a temperature control fluid into a device within which the eye globe is received.

Claims

CLAIMS1. A device for storing and maintaining viability of an ex vivo enucleated eye globe, the device comprising: a platform comprising an eye bed, wherein the eye bed is part-spherical and is radiused to substantially correspond to the dimensions of an eye globe to be received within the device, and wherein the eye bed comprises at least one drainage aperture, and wherein the platform further comprises an attachment means configured to hold a cannula in place.

2. The device of Claim 1, further comprising a housing comprising a main housing, a lid, and a base that together define an enclosure, wherein the platform rests within the enclosure on the base, and wherein the lid comprises an opening configured to receive the cannula passing therethrough into the enclosure, and wherein the drainage aperture is in fluid communication with a drainage channel in the base of the housing.

3. The device of Claim 1 or Claim 2, wherein the drainage channel is in fluid communication with a tube connector.

4. The device of Claim 2 or Claim 3, further comprising a closure mechanism, configured to bring the device into a closed configuration and to retain the device in said closed configuration.

5. The device of Claim 4, further comprising a first gasket between lid and the main housing and a second gasket between the base and the main housing so that operation of the closure mechanism to bring the device into the closed configuration generates a hermetic seal around the enclosure.

6. The device of any of Claims 2 to 5, further comprising a window in a wall of the main housing.

7. The device of any of Claims 2 to 6, further comprising a stand attached to the base configured to raise the base above a surface on which the device rests.

8. A system comprising:the device of any preceding claim; a cannula retained in place by the attachment means, the cannula being connected at a first end to a reservoir of perfusion fluid and being configured to be operatively connectable at a second end to an eye globe in order to deliver the perfusion fluid thereto; and a pump to enable transfer of the perfusion fluid along the cannula.

9. The system of Claim 8, wherein the perfusion fluid comprises oxygenated Ringer’s solution, a physiological buffer solution, blood serum, blood or a blood substitute.

10. The system of Claim 8 or Claim 9, comprising at least one electrode configured to measure an electroretinogram (ERG) signal from the retina of an eye globe.

11. The system of any of Claims 8 to 10, further comprising a drainage tube in fluid communication with the drainage channel at a first end and with the reservoir of perfusion fluid at a second end so as to enable recirculation of used perfusion fluid.

12. The system of any of Claims 8 to 11 , further comprising a temperature control system to enable delivery of a temperature control fluid into the device.

13. The system of any of Claims 8 to 12, further comprising an eye globe resting on the eye bed, wherein the cannula is operatively connected at its first end to the eye globe so as to enable delivery of the perfusion fluid thereto.

14. The system of Claim 13, wherein the cannula is operatively connected to the eye glove via an artery thereof.

15. The system of Claim 13 or Claim 14, wherein the pump is configured to enable delivery of the perfusion fluid into the eye globe at a rate of between 0.005 mL / minute and 5 mL / minute.

16. A method of maintaining the viability of an ex vivo enucleated eye globe, the method comprising:placing the eye globe on a part-spherical eye bed of a platform, the eye bed being radiused to substantially correspond with the dimensions of the eye globe; operatively connecting a cannula to the eye globe and retaining it in place with an attachment means received on the platform; delivering a perfusion fluid to the eye globe via the cannula; and allowing outflowing perfusate to drain through at least one aperture in the eye bed.

17. The method of Claim 16, comprising operatively connecting the cannula to the eye globe via an artery thereof,18. The method of Claim 16 or Claim 17, comprising delivering the perfusion fluid at a rate of between 0.005 mL / minute and 5 mL / minute.

19. The method of any of Claims 16 to 18, comprising recirculating the used perfusion fluid draining out of the at least one aperture into a reservoir of the perfusion fluid connected to the cannula, wherein the platform and the eye globe are contained within a hermetically sealed enclosure.

20. The method of any of Claims 16 to 19, further comprising cooling the eye globe before delivering the perfusion fluid thereto and / or warming the eye globe during delivery of the perfusion fluid thereto by delivering a temperature control fluid into a device within which the eye globe is received.

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