An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations.

Descemet Membrane Endothelial Keratoplasty Learning Curve for Graft Preparation in an Eye Bank Using 645 Donor Corneas.

PURPOSE: To investigate the learning curve of Descemet membrane endothelial keratoplasty (DMEK) graft preparation in an eye bank. METHODS: Four operators prepared 645 DMEK grafts using the stripping technique between 2014 and 2017 at the Veneto Eye Bank Foundation, Italy. Endothelial cell loss (ECL) and tissue wastage were recorded retrospectively after DMEK preparation and correlated with the number of tissues prepared each year by each operator. On average, our operators performed 1 donor preparation a week over the course of this study. Only donors older than 60 years were used in this study, and approximately 10% of donors had diabetes. The Wilcoxon test for paired data and 1-way ANOVA were used for checking statistical significance with the Tukey test as post hoc analysis. P < 0.05 was considered statistically significant. RESULTS: ECL did not change significantly over time from operator 1. Significant ECL drop was noted from operator 2 between years 2014-2016 (P = 0.0049) and 2017 (P = 0.0094); from operator 3 between years 2015-2016 (P = 0.0288) and 2017 (P = 0.0097); and from operator 4 between 2015-2016 (P = 0.0469) and 2017 (P = 0.0331). Operators 1 and 3 did not show a significant difference, considering every 50 grafts prepared by each operator. Operator 2 showed significant ECL drop between 1 to 50 and 51 to 100 (P = 0.0002) and 1 to 50 and 101 to 150 (P = 0.0001) grafts. Operator 4 showed significant ECL drop between 1 to 50 and 101 to 150 (P = 0.002) and 51 to 100 and 101 to 141 (P = 0.0207) grafts. No intraoperator difference was observed per 50 grafts (P > 0.05). CONCLUSIONS: There is a learning curve for DMEK graft preparation. ECL and tissue wastage can be reduced with practice and skills. However, each operator may be limited to his or her own learning capability.

Recombinant human serum albumin for corneal preservation.

PURPOSE: To study the performance of a completely synthetic organ culture (OC) preservation system containing recombinant human serum albumin (rHSA) for preservation of human donor corneas. METHODS: Twenty-four paired donor corneas were randomly collected, and one cornea from each donor was preserved in synthetic (experimental) and serum-based media (control). The tissues were assessed at day 0; after 6 days of preservation at room temperature (RT) in Cornea Trans® and Cornea Prep II® ; after 28 days at 31°C in Cornea Syn® [with rHSA] and Cornea Max® [with foetal calf serum (FCS)] and; 4-day post deswelling in Cornea Trans® and Cornea Jet® . Thickness was determined with optical coherence tomography (OCT) and transparency with a validated, custom device. Morphology, endothelial cell density (ECD) and mortality were observed after treating the tissues with Trypan blue and sucrose. Glucose uptake by the cells was analysed. Data were compared using non-parametric paired Wilcoxon tests with p < 0.05 deemed significant. Histology using periodic acid-schiff (PAS), expressions of p63, CK12, αSMA and ZO-1 were analysed, and cell apoptosis postpreservation was studied. RESULTS: Corneas stored in synthetic media showed a higher and statistically significant value as compared to serum-based media in terms of viable endothelial cell density (VECD), mortality, morphology and glucose uptake postpreservation. Histology showed presence of all the layers, all the markers were expressed, and no apoptosis was observed in either media. CONCLUSION: The new synthetic preservation system containing rHSA (and other confidential constituents) showed better preservation effects than traditional media containing serum.

Polarization of human donor corneas.

To investigate the de-orientation effect of DSAEK grafts by observing the cross patterns and polarization power of human donor corneas using a polarizing device (Lumaxis(®)). Forty human donor corneas were placed in small petri-plates with epithelial side facing up. Polarizing power (arbitrary unit) and crosses were monitored and recorded by the software. The tissue was marked at 'Superior' position to ensure that the base and the polarizer are in alignment with each other after the cut. The anterior lamellar cut was performed using microkeratome. The lenticule was placed back in the same position as marked to mimic the alignment. The tissue was further rotated by 45° ensuring that the base of the cornea and the polarizer were in alignment. The polarization power and 'crosses' were identified at each step. The average of forty corneas from pre-cut to post-45° angular change showed statistically significant difference (p < 0.05) in terms of polarizing power. The cross-shaped pattern deformed and lost the sharpness towards 45° angle. However, multiple variances in terms of 'cross-patterns' were observed throughout the study. Lumaxis(®) was able to determine the worst quality tissue in terms of polarization (no black zone and crosses). Despite the quality of cross pattern which can be used as an additional objective parameter to evaluate the optical properties of the corneal tissue, this preliminary study needs to be further justified in terms of clinical relevance whether polarization changes with oriented or de-oriented grafts have any effects and consequences on the visual acuity.

Development of a new superficial punch for Descemet's Membrane Endothelial Keratoplasty donor tissue preparation.

Endothelial Keratoplasty (EK) is a corneal surgical procedure that allows a selective transplantation of the posterior layer of the cornea. Descemet's Membrane Endothelial Keratoplasty (DMEK) is one of the EK procedures in which the diseased Descemet's Membrane and the endothelium are replaced with a healthy donor tissue. To achieve this, the donor cornea is cut superficially from the endothelial side and the tissue can be separated using specific instruments like Pierse Notched, Acute or Fogla forceps. However, the pressure required to punch the superficial layer has always been a challenge and therefore a calibrated device to punch and excise the required superficial layer has been designed. This new model of punch will help to identify the peripheral edge of the DMEK lenticule which in turn helps to excise the tissue exclusively, further reducing the donor tissue wastage, as seen with the current tissue preparation methods.

Transplantation failure due to inadvertent reversal of human donor corneas.

PURPOSE: To emphasize and create awareness of the possibility of inadvertently grafting an inverted corneal button and describe 4 cases that showed this phenomenon on human donor corneas during preservation and transportation from an eye bank. METHODS: Three out of 4 tissues showed inadvertent inversion during transportation and 1 was identified as inverted during preservation in the eye bank. Out of the 3 tissues that were shipped, 2 tissues were transplanted and 1 was identified as inverted prior to transplantation and shipped back to the eye bank. RESULTS: Four tissues showed inversion at different time points. The anatomy was clearly visible with a naked eye that showed the presence of trabecular meshwork on its outside along with some residual choroid. The endothelial cell count was in the range of transplantation without any mortality even after inversion. The inverted corneas showed a mountain-like shape with grooves as compared to the normal cornea. CONCLUSIONS: Previous reports have identified tissue inversion post-transplantation. We describe inadvertent inversion of donor tissues during preservation and transportation from an eye bank. It is recommended to check the presence of corneo-scleral rim, trabecular meshwork, grooves, and residues of choroid if present inside or outside before transplantation to ensure accurate grafting and avoid transplantation failures.

A superfusion apparatus for ex vivo human eye irritation investigations.

A superfusion apparatus (SA) was developed to maintain isolated human corneas ex vivo under conditions which mimic the natural eye environment in vivo, including controlled temperature, tear flow and intraocular pressure. The SA was designed, developed and tested for use in ophthalmic pre-clinical research and to test new pharmaceutical formulations. Corneas undergo an equilibration process in the new physiological environment for one day. The test was then initiated by the application of the test substance, incubation, and temporal assessment of corneal damage using various parameters. The effects of mild and severe irritant concentrations of NaOH (2% and 8%, respectively) on corneal opacity, swelling and epithelial integrity were studied, and the inflammatory status assessed using F4/80 and MPO as macrophages and neutrophils markers, respectively. The SA was then used to test new artificial tear formulations supplemented with silver ions as an active constituent, showing different degrees of inflammatory responses as indicated by the migration of MPO and F4/80 positive cells towards the epithelium. The human cornea superfusion apparatus was proposed as a model for acute eye irritation research.

Preloaded donor corneal lenticules in a new validated 3D printed smart storage glide for Descemet stripping automated endothelial keratoplasty.

PURPOSE: To design and validate the efficacy of three-dimensional (3D) printed smart storage glide (SSG) which is capable of preserving and delivering posterior lenticules for Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Laboratory investigation (A) was followed by clinical validation (B). Unsuitable corneas for transplantation (n=20) were used for study A. These tissues were cut using a standard microkeratome, punched and loaded into the SSG and preserved for 7 days in transport media. Endothelial cell density (ECD), Trypan blue and Alizarin red staining for endothelial morphology, thickness measurements and glucose uptake, cell apoptosis and immunostaining post preservation were analysed. For study B, clinical grade corneas (n=14) were preloaded in SSG and grafted in patients with indications of Fuchs' dystrophy (n=8), pseudophakic bullous keratopathy (n=3), posterior polymorphous dystrophy (n=2), and previous DSAEK failure (n=1). Standard DSAEK included descemetorhexis under air and bimanual delivery of the tissue under infusion of buffered saline solution through an anterior chamber maintainer placed at the 12 o'clock position. Main outcome measures for study B were less surgical time, best spectacle-corrected visual acuity (BSCVA), speed of visual recovery, and ECD. RESULTS: For study A, an average ECD loss was 2.30±3.21%, thickness increased by 30.80±20.85% and one-third of glucose was utilised during the preservation phase. Alizarin red showed hexagonality of the cells. Cell apoptosis was not observed and expression of ZO-1 was noted on the preserved tissues. In study B, 25% ECD loss was observed after 6 months. BSCVA improved to 20/25 or better within 3 months after DSAEK. Mean surgical time recorded was 21 min. CONCLUSIONS: This paper describes the development, design, laboratory and clinical validation of a 3D printed SSG which helps to store and deliver posterior lenticules, therefore allowing transportation of quality-controlled precut tissues.

Synthetic versus serum-based medium for corneal preservation in organ culture: a comparative study between 2 different media.

PURPOSE: To study the effect of a synthetic medium and compare it with a serum-based medium for corneal preservation in organ culture using an overall quality assessment system. METHODS: A randomized study with blinded observers was performed comparing parameters such as thickness, transparency, viable endothelial cell density (VECD), morphology, and overall quality (OQ) of the corneal tissues preserved in synthetic and a serum-based medium, respectively. Seven human paired corneas were randomly selected and assessed at day 0 (initial), day 2 (before organ culture), day 30 (before deturgescence/deswelling storage), and 48 hours post deswelling. Thickness was determined with optical coherence tomography and transparency with a validated, custom device. The morphology and VECD were observed after treating the tissues with trypan blue and sucrose. Data were compared using paired t tests with p<0.05 deemed significant. RESULTS: Parameters were similar at the initial stage between the groups with no statistically significant difference. However, after preservation in the deturgescent medium, the corneas stored in a serum-based medium showed a higher and statistically significant OQ value (p = 0.0317). CONCLUSIONS: The OQ of a serum-based medium was higher than that of the synthetic medium. A higher rate of transparency and reduction in thickness was observed in the serum-based medium at the end of the storage. Although complete synthetic media may have distinct advantages of being serum/animal-free, the quality of the cornea is of a reasonable concern when it is deemed for transplantation.

Bubble technique for Descemet membrane endothelial keratoplasty tissue preparation in an eye bank: air or liquid?

PURPOSE: To compare the big-bubble method using air and liquid as medium of separation for Descemet membrane endothelial keratoplasty (DMEK) lenticule preparation in an eye bank. METHODS: Donor corneas (n=20) were immersed in liquid [tissue culture medium (TCM)]. Air and liquid was injected using a 25-gauge needle in the posterior stroma or as near to the stroma-Descemet membrane (DM) phase as possible to create a complete bubble of larger diameter. The endothelial cell density and mortality were checked pre- and postbubble after deflating the tissue. Four pairs of tissues were used to analyse the intracellular tight junctions and three pairs for histological examination and DNA integrity studies, respectively. RESULTS: The yield obtained using air was 80%, whereas that with liquid was 100%. Single injection was required in six cases; twice in two cases; three and four times in one case each with air bubble, whereas seven cases required single injection; twice in two cases; and thrice in just one case with liquid bubble. The average diameter of the final lenticule was 9.12 (±1.71) mm for air bubble and 9.78 (±1.75) mm for liquid bubble with p=0.4362 (no statistical significance). Endothelial cell mortality postbubble preparation was 8.9 (±12.38)% for air and 6.25 (±9.57)% for liquid (p=0.6268). CONCLUSIONS: DM and endothelium could be separated exclusively using air or liquid bubble. However, liquid bubble seems to have certain advantages over air such as the generation of yield, larger diameter and higher maintenance of endothelial cell density and integrity.

A validated biorepository of retina and choroid tissues for gene expression studies.

Research studies on pathologies affecting the posterior segment of the eye are usually carried out either in animal models or cell lines of human origin that mimic the molecular patterns occurring in the human retina-pigment epithelium-choroid (RPC) complex in vivo. As this is not always the case, we were prompted to validate a biorepository of RPC tissues for research purposes. A PubMed literature search on "retina," "choroid," "bio-bank," or "biorepository" as keywords did not lead to any publication describing the collection and banking of samples from the RPC complex for research purposes. The possibility to obtain access to a validated collection of high quality human RPC tissues as starting material is likely to lead to more appropriate findings and treatments, which eventually may improve human ocular health. Here we show that when tissues are harvested (T <25 hours from donor death) and stored appropriately, RNAs are not degraded (RNA Integrity Number Values >8.0) and express specific genes and molecular/biochemical pathways occurring in the RPC complex. These quality controlled tissues/RNAs comprising the biorepository could therefore be used for gene expression studies by research scientists and clinicians interested in testing their hypotheses in a more appropriate setting, thus replacing studies performed on less relevant animal models and cells in vitro, and directly extrapolating the findings to human pathophysiology.

DMEK lenticule preparation from donor corneas using a novel 'SubHyS' technique followed by anterior corneal dissection.

PURPOSE: To describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas. METHODS: 30 human donor corneas were immersed in liquid (organ culture (OC) storage medium). Using a 25-gauge needle, approximately 0.3 mL of OC was injected (SubHyS) in the posterior stroma to create a liquid bubble. The bubbled cornea was mounted onto a modified artificial chamber with the epithelial side facing the air. The endothelium was protected with a viscoelastic solution. The anterior cornea was excised with a Barron radial vacuum trephine and the residual peripheral stroma was removed manually using micro-scissors. The DEG was dismounted and washed. The endothelial cell density (ECD) and mortality of the prepared DEG was determined. All the DEGs were preserved in deturgescent medium for 7 days using a cornea claw which was fixed to the sclera. ECD and mortality were checked post preservation. RESULTS: Complete detachment of Descemet's membrane and stroma was achieved in all 30 cases. Bubble burst was observed in five cases (excluded from the study) due to overfilling of the liquid. The average diameter of the excised DEG was 10.96 mm. The average endothelial cell loss post preservation was 27.69%. Histological analysis confirmed elimination of the residual stroma (n=13). CONCLUSIONS: The DEGs can be preserved in a deturgescent medium for up to 7 days. The procedure provides a standardised, pre-validated (quality assured), pre-separated, no-touch, ready-to-use tissue and also reduces the preparation time. Further, the tissues can be trephined as per the surgeon's convenience and can either be rolled or a contact lens could be used for final delivery of the DEG using a surgical glide.

Descemet membrane endothelial keratoplasty tissue preparation from donor corneas using a standardized submerged hydro-separation method.

PURPOSE: To standardize a novel submerged hydro-separation technique for Descemet membrane endothelial keratoplasty (DMEK) graft preparation from donor corneal tissues. DESIGN: Experimental study, laboratory investigation. SETTING: The Veneto Eye Bank Foundation, Venice, Italy. STUDY POPULATION: Fifty-four random human donor corneal tissues unsuitable for transplantation. INTERVENTION: Donor corneas were laid in a sterile basin partially filled with tissue culture medium. A 25 gauge needle with 1 mL mounted syringe was filled with the tissue culture medium. The needle (with bevel up) was bent to 90 degrees and was inserted in the posterior cornea initiating beneath the trabecular meshwork. It was further advanced toward the midperiphery, ensuring that only the bevel was inserted, considering it as a threshold of insertion. The liquid was injected with a medium to high pressure into the posterior stroma or in the Descemet membrane-stroma interface to create the bubble. The tissues were preserved for 7 days in tissue culture medium at 31°C. Parametrical, physiological and histological analyses were carried out. MAIN OUTCOME MEASURES: Larger-diameter tissue, no tissue wastage, reproducibility, and preshipment evaluation. RESULTS: Complete detachment was achieved in all the cases without any tissue wastage. Average diameter of the excised graft was 10.80 (±0.28) mm and endothelial cell loss post preservation was 11.48%. Expression of tight junction protein and regular morphology was observed post preservation. No signs of cell apoptosis were seen. Histological analysis showed elimination of residual stroma in most of the cases. CONCLUSIONS: The submerged hydro-separation method reduces tissue wastage. It allows preshipment evaluation, thus allowing a validated tissue to be transported from the eye banks to the surgeon. Because of the liquid interface, the peeling of the DMEK graft becomes easy for transplantation.

A quantitative method to evaluate the donor corneal tissue quality used in a comparative study between two hypothermic preservation media.

To standardize a new evaluation technique for calculating the overall quality (OQ) of the donor cornea and validate it using a comparative study of corneas preserved in Optisol-GS and Cornea Cold®. Thirty pairs of donor corneas were selected for a 4 week in vitro comparative study using masked observers. Physiological parameters like thickness, transparency, viable endothelial cell density (VECD) and morphology were transformed to numerical range (0-4) to obtain the OQ. Microbiological examination was performed using Bactec instrument. Students t test showed statistically better results (p < 0.05) from week 3 for thickness, week 2 for transparency and week 1 for morphology and VECD; statistical significance (p < 0.05) was found for OQ from week 2 for the corneas preserved in Cornea Cold® compared to Optisol-GS. Epithelial quality was similar regardless of the medium. Microbiological examination showed absence of aerobic and anaerobic microorganisms in both media. OQ method is efficient, consistent and easy, now validated for comparative studies. Further refinement is necessary for its use at eye-banks, bio-banks and research or transplantation purposes. Cornea Cold® is a promising hypothermic corneal storage medium with preservation time ≤21 days. This permits higher flexibility, evaluation accuracy, longer duration for surgical preparation and ease of transportation.

A portable device for measuring donor corneal transparency in eye banks.

To develop a portable device for measuring the donor corneal transparency and validate its efficacy for corneal evaluation in the eye-banks and for research. The transparency device (TD) has a light source, a detachable system for corneal insertion and a base for light transmission. The probe detects the transmitted light which is measured by a lux-meter. A contact lens was set as 'control' to reduce the light scattering concern, an empty petri-plate as 'blank' and the cornea as 'sample'. Two experts and non-experts (masked) observed the corneas for subjective analysis which was then compared using the TD. The parameters observed were scars, foreign-body, stromal-deformities, folds, thickness and opacity which were then converted to a relative overall percentage by the observer. Twenty corneas were evaluated for correlation, five tissues to obtain standard-deviation and twenty-four pairs for a comparative study. Experts mimicked the eye-banks with long-term experience while non-experts mimicked the emerging eye-banks. Subjective values by the experts closely resembled the measurements by TD. The average correlation between the experts and the non-experts to TD was 0.985 and 0.960 respectively. TD showed higher reproducibility than experts followed by the non-experts. The comparative study showed that increase in thickness reduces the transparency. TD is portable, easy, efficient, maintains sterility and less expensive hence the emerging eye-banks and researchers can use to raise their standards and evaluate the transparency for in vitro tests and comparative studies. The suitable transparency for the cornea deemed for clinical applications was found to be >75 %.

Posterior lamellar graft preparation: a prospective review from an eye bank on current and future aspects.

Descemet membrane endothelial keratoplasty (DMEK) is a corneal surgical technique which selectively replaces the damaged posterior part of the cornea with a healthy donor graft retaining the rest of the tissue intact. There is a need to validate and standardize the donor tissue before grafting due to certain issues that can lead to consequences such as graft failure due to poor endothelial cell count, higher mortality, detachment of the graft, or increased surgical expenses, time, and effort. Thus, prospective potential surgeons and eye banks should now aim at developing new improved surgical techniques in order to prepare the best suited, validated, precut, preloaded, and easy to transplant tissue to reduce pre- and postsurgical complications. This could be achieved by defining parameters like graft thickness, accepted mortality threshold of the endothelial cells, and behavior of grafts during preservation and transportation along with using more sophisticated instruments like microkeratome and femtosecond lasers for graft preparation. Thus, a rapport between the eye banks and the surgeons along with the advanced instruments can overcome this challenge to find the best possible solution for endothelial keratoplasty (EK).

Effect of postmortem interval on the graft endothelium during preservation and after transplantation for keratoconus.

PURPOSE: To investigate the effect of postmortem intervals and prognostic factors on endothelial cell density (ECD) of human donor corneas during preservation and at 1 and 3 years after transplantation in patients transplanted for keratoconus. METHODS: Two different studies were performed: (1) with 733 donor corneas selected for the preservation study and (2) 64 patients with keratoconus selected retrospectively from 2 hospital clinics. The corneas were evaluated on the basis of the ECD during preservation, study A, and at 1 and 3 years after transplantation, study B. The effect of ≥ 10 hours of postmortem interval on the percentage of corneal endothelial cell loss (ECL) was determined. RESULTS: The multiple regression showed no statistical significance (P = 0.827) of postmortem interval on ECL during preservation. However, for patients with keratoconus, the postmortem interval was statistically significant at both 1 year (P < 0.0001) and 3 years after transplantation (P < 0.0001). CONCLUSIONS: The postmortem interval has no influence on the ECD during preservation. However, it has a statistically significant effect on the ECL after transplantation for patients transplanted for keratoconus, and therefore, it becomes eligible to be one of the potential factors affecting the ECD apart from surgical trauma.

Banking of donor tissues for descemet stripping automated endothelial keratoplasty.

PURPOSE: The demands for precut lamellar grafts for Descemet stripping automated endothelial keratoplasty rose in our eye bank from 74 in 2007 to 408 in 2010. To meet this expanding requirement, we explored the possibility to preserve these preparations in organ culture. METHODS: Organ cultured corneas, stored in a medium containing 6% dextran, were mounted on a Moria artificial anterior chamber, deprived of the epithelium and then cut with a microkeratome. The posterior lamella was protected by positioning the anterior stromal cap, trephined at a diameter of 8.5 mm and stored at 31°C in the medium containing dextran. The endothelium was examined with trypan blue and alizarin staining and tested for its glycolytic activity (conversion of glucose into lactate). RESULTS: Incubation for a period of 1 week caused a small increase in the cell loss observed after trephination (from 6.2% to 10.6%). After 2 weeks, the decrease in endothelial cell density was 19.9% but the endothelial organization remained intact. The rate of glycolysis remained unchanged during the 2 weeks of preservation, with the majority of glucose uptake accounted for by lactate production. The thickness of the lenticules remained constant, ranging from 170 to 180 µm during the preservation. CONCLUSIONS: The lamellar grafts for Descemet stripping automated endothelial keratoplasty may be stored in organ culture for 2 weeks without damaging the endothelium or increasing the overall thickness.

A simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision(4,5,6). The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina(5,6). The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve(5,6). The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.