Routine donor tomography in the eye bank as a sterile screening method for improved graft selection in corneal transplantation

Purpose: To evaluate the efficiency of using anterior segment optical coherence tomography (AS-OCT) (CASIA2) as a non-invasive and sterile screening method in the eye bank to detect corneal grafts with curvature and/or thickness abnormalities, thus improving the graft selection for corneal transplantation. Methods: Two hundred donor corneal tissues mounted in sterile tissue cultivation flasks were imaged using AS-OCT (CASIA 2). The corneal tissues were preserved at least 24 hours in medium 2 (6% dextran T-500) before the measurement. All images were grabbed through the posterior surface of the corneal tissues within a 7 mm central zone to create 3D volume data. The volume data set was imported to MATLAB and, after preprocessing the data and defining the region of interest (ROI), the edge of the front and back surfaces of the corneal tissues was detected. Subsequently, the adaptation of a sphero-cylindrical surface model with raytracing was carried out. The radii of curvature for the front and back surfaces and the central corneal thickness were determined according to the method proposed by Mäurer S., Eppig T., and Langenbucher A. at the Institute of Experimental Ophthalmology, Homburg/Saar. Results: The mean steep/flat front surface radius was 7.5 ± 0.24 (6.6-7.9) / 7.7 ± 0.22 (7.1-8.8) mm, the corresponding values for the back surface being 6.6 ± 0.22 (5.9-7.1) / 6.8 ± 0.21 (6.2-7.5) mm and the mean central thickness was 582 ± 45.1 (453-693) μm. Abnormalities (beyond ± 2 SD) were found in 13 corneas (6.5%) for anterior surface curvature, 15 for corneas (7.5%) for the posterior surface, and five corneas (2.5%) for thickness. Conclusions: The AS-OCT provides an objective and sterile. In addition, it may allow, in the future, a semi-automated screening method to identify corneal morphological and refractive alterations (e.g., keratoconus, status post PRK/LASIK) to further optimize corneal donor selection in eye banks.Ziel: Bewertung der Effizienz der optischen Kohärenztomographie des vorderen Augenabschnitts (AS-OCT) (CASIA2) als nicht-invasive und sterile Screeningsmethode in der Hornhautbank zur Erkennung von Hornhauttransplantaten mit Krümmungs- und/oder Dickenanomalien, wodurch die Transplantatauswahl für die Hornhauttransplantation verbessert werden sollte. Methoden: 200 Spenderhornhautgewebe wurden im sterilen Gewebekultivierungsbehälter vermessen. Die Hornhautgewebe wurden mindestens 24 Stunden vor der Messung in Medium 2 (mit 6% Dextran T-500) konserviert. Alle Bilder wurden durch die hintere Oberfläche der Hornhautgewebe innerhalb einer zentralen Zone von 7 mm aufgenommen, um 3D-Volumendaten zu erstellen. Der Volumendatensatz wurde in MATLAB importiert, und nach der Vorverarbeitung der Daten und der Definition der “Region of interest“ (ROI) wurde die Kante der Vorder- und Rückseite der Hornhautgewebe detektiert. Anschließend erfolgte die Anpassung eines sphärozylinderförmigen Oberflächenmodells mittels Raytracing. Die Krümmungsradien für Vorder- und Rückseite sowie die Dicke der zentralen Hornhaut wurden nach der von Mäurer S, Eppig T und Langenbucher A (Institut für Experimentelle Ophthalmologie, Homburg/Saar) vorgeschlagenen Methode bestimmt. Ergebnisse: Der mittlere steile / flache Radius der Vorderfläche betrug 7,5 ± 0,24 (6,6-7,9) / 7,7 ± 0,22 (7,1-8,8) mm, die entsprechenden Werte für die Rückfläche betrugen 6,6 ± 0,22 (5,9-7,1) / 6,8 ± 0,21 (6,2-7,5) mm und die mittlere zentrale Dicke betrug 582 ± 45,1 (453-693) μm. Anomalien (außerhalb ± 2 SD) wurden bei 13 Hornhäuten (6,5%) für die Krümmung der Vorderfläche, bei 15 Hornhäuten (7,5%) für die Krümmung der hinteren Oberfläche und bei 5 Hornhäuten (2,5%) für die zentrale Dicke gefunden. Schlussfolgerungen: Das Vorderabschnittes-OCT bietet eine objektive, sterile und zukünftig halbautomatisches Screening-Methode zur Identifizierung morphologischer und refraktiver Veränderungen der Hornhaut (z. B. Keratokonus, Status nach PRK/LASIK), um die Hornhautspenderauswahl in der Hornhautbank weiter zu optimieren

Characterisations of Pre-Descemet’s (Dua’s) layer for its clinical application in keratoplasty

There exists a newly discovered, well defined, acellular, strong layer, termed pre-Descemets layer or Dua’s layer (PDL), in the cornea just anterior to the Descemets membrane. This, with the Descemets membrane, separates along the last row of keratocytes in most cases of deep anterior lamellar keratoplasty with the big bubble technique. Recognition of this layer has considerable impact on lamellar corneal surgery, understanding of posterior corneal biomechanics and posterior corneal pathology, such as descemetocele, acute hydrops and pre-Descemets dystrophies. The aim of this work was to understand the dynamics of big bubble formation in the context of the known architecture of the cornea stroma, ascertain how type 1 (air between deep stroma and PDL), type 2 (air between PDL and Descemets membrane) and mixed bubbles (combination of type 1 and type 2) form and measure the pressure and volume of air required to produce big bubbles in vitro, including the intra-bubble pressure and volume for the different types of big bubbles. We also aimed to characterise the optical coherence tomography characteristics of the different layers in the wall of the big bubbles to help surgeons identify bubbles and understand the structures seen by intra-operative OCT. Finally we evaluated the endothelial cell density and viability in tissue samples obtained for Descemets membrane endothelial keratoplasty (DMEK) and pre-Descemets endothelial keratoplasty (PDEK) by the pneumodissection technique. Air was injected in 145 corneo-scleral samples, which were unsuitable for transplantation. Samples were obtained in organ culture medium from the UK eye banks and transferred to balanced salt solution ready for injection. Different types of big bubble formed were ascertained. Air pressure and volume required to create the big bubble in simulated deep anterior lamellar keratoplasty were measured. It was found that PDL could withstand a high pressure before bursting at around 700 mm of Hg. Accurate measurements of type-2 big bubble proved challenging. The volume of the type-1 BB was fairly consistent at 0.1ml. The movement of air injected in the corneal stroma was studied from the point of exit from the needle tip to complete aeration of the stroma and formation of a BB. This was video recorded and analysed. A very consistent pattern of air movement was observed. The initial movement was predominantly radial from the needle tip to the limbus, then circular in a clock-wise and counter clock-wise direction circumferentially along the limbus, then centripetally to fill the stroma. All type 1 BB started in the centre as multiple small bubbles which coalesced to form a BB. Almost all type 2 BB started at the periphery near the limbus. Ultrastructural examination of the point of commencement of type 2 BB revealed the presence of clusters of fenestrations, which most likely allow air to escape from the otherwise impervious PDL to access the plane between PDL and DM. This was a novel discovery and explained how type 2 BB formed and why they almost always start at the periphery. The consistent pattern of passage of air was in concordance with the known microarchitecture of the central and peripheral corneal stroma. Optical coherence tomography (OCT) characteristics of different types of big bubbles were studied. Samples obtained from the UK eye banks were scanned with Fourier-domain (FD-OCT), while that obtained from Canada eye bank were scanned with Time-domain (TD-OCT). A special clamp was used to affix the corneo-scleral sample on the OCT table with its posterior surface face the machine and mounted on artificial anterior chamber. It was found that FD-OCT could demonstrate type 1 BB wall as two parallel, double contour, hyper-reflective lines with hypo-reflective space in between. It also revealed that in type-2 BB, the posterior wall showed a parallel, double-contour curved hyper-reflective line with a dark space in between. This probably corresponds to the banded and non-banded zones of DM. Dua’s layer presents as a single hyper-reflective line. In TD-OCT, the posterior wall of type-1 and type-2 BB showed a single hyper-reflective curved line rather than the double-contour line. This finding will help cornea surgeons to identify and interpret different layers of big bubble intra-operatively with high resolution OCT devices. Endothelial cell density of PDEK and DMEK tissue were calculated. Endothelial cells were counted using light microscope before pneumodissection. Air was then injected to ascertain the creation of type-1 and type-2 BB. Tissue was then harvested by trephination and endothelial cell density of both types were calculated again. It was found that the corneal endothelial cell count in PEDK tissue preparation is no worse, if not slightly better than, in DMEK tissue prepared by pneumodissection. Therefore, PDEK preparation represents a viable graft preparation technique

Characterisations of Pre-Descemet’s (Dua’s) layer for its clinical application in keratoplasty

There exists a newly discovered, well defined, acellular, strong layer, termed pre-Descemets layer or Dua’s layer (PDL), in the cornea just anterior to the Descemets membrane. This, with the Descemets membrane, separates along the last row of keratocytes in most cases of deep anterior lamellar keratoplasty with the big bubble technique. Recognition of this layer has considerable impact on lamellar corneal surgery, understanding of posterior corneal biomechanics and posterior corneal pathology, such as descemetocele, acute hydrops and pre-Descemets dystrophies. The aim of this work was to understand the dynamics of big bubble formation in the context of the known architecture of the cornea stroma, ascertain how type 1 (air between deep stroma and PDL), type 2 (air between PDL and Descemets membrane) and mixed bubbles (combination of type 1 and type 2) form and measure the pressure and volume of air required to produce big bubbles in vitro, including the intra-bubble pressure and volume for the different types of big bubbles. We also aimed to characterise the optical coherence tomography characteristics of the different layers in the wall of the big bubbles to help surgeons identify bubbles and understand the structures seen by intra-operative OCT. Finally we evaluated the endothelial cell density and viability in tissue samples obtained for Descemets membrane endothelial keratoplasty (DMEK) and pre-Descemets endothelial keratoplasty (PDEK) by the pneumodissection technique. Air was injected in 145 corneo-scleral samples, which were unsuitable for transplantation. Samples were obtained in organ culture medium from the UK eye banks and transferred to balanced salt solution ready for injection. Different types of big bubble formed were ascertained. Air pressure and volume required to create the big bubble in simulated deep anterior lamellar keratoplasty were measured. It was found that PDL could withstand a high pressure before bursting at around 700 mm of Hg. Accurate measurements of type-2 big bubble proved challenging. The volume of the type-1 BB was fairly consistent at 0.1ml. The movement of air injected in the corneal stroma was studied from the point of exit from the needle tip to complete aeration of the stroma and formation of a BB. This was video recorded and analysed. A very consistent pattern of air movement was observed. The initial movement was predominantly radial from the needle tip to the limbus, then circular in a clock-wise and counter clock-wise direction circumferentially along the limbus, then centripetally to fill the stroma. All type 1 BB started in the centre as multiple small bubbles which coalesced to form a BB. Almost all type 2 BB started at the periphery near the limbus. Ultrastructural examination of the point of commencement of type 2 BB revealed the presence of clusters of fenestrations, which most likely allow air to escape from the otherwise impervious PDL to access the plane between PDL and DM. This was a novel discovery and explained how type 2 BB formed and why they almost always start at the periphery. The consistent pattern of passage of air was in concordance with the known microarchitecture of the central and peripheral corneal stroma. Optical coherence tomography (OCT) characteristics of different types of big bubbles were studied. Samples obtained from the UK eye banks were scanned with Fourier-domain (FD-OCT), while that obtained from Canada eye bank were scanned with Time-domain (TD-OCT). A special clamp was used to affix the corneo-scleral sample on the OCT table with its posterior surface face the machine and mounted on artificial anterior chamber. It was found that FD-OCT could demonstrate type 1 BB wall as two parallel, double contour, hyper-reflective lines with hypo-reflective space in between. It also revealed that in type-2 BB, the posterior wall showed a parallel, double-contour curved hyper-reflective line with a dark space in between. This probably corresponds to the banded and non-banded zones of DM. Dua’s layer presents as a single hyper-reflective line. In TD-OCT, the posterior wall of type-1 and type-2 BB showed a single hyper-reflective curved line rather than the double-contour line. This finding will help cornea surgeons to identify and interpret different layers of big bubble intra-operatively with high resolution OCT devices. Endothelial cell density of PDEK and DMEK tissue were calculated. Endothelial cells were counted using light microscope before pneumodissection. Air was then injected to ascertain the creation of type-1 and type-2 BB. Tissue was then harvested by trephination and endothelial cell density of both types were calculated again. It was found that the corneal endothelial cell count in PEDK tissue preparation is no worse, if not slightly better than, in DMEK tissue prepared by pneumodissection. Therefore, PDEK preparation represents a viable graft preparation technique

Étude comparative de l'anatomie des plaies de greffe de cornée par tomographie de cohérence optique (OCT)

Cette thèse porte sur l’étude de l’anatomie de la cornée après 3 techniques de greffe soient, la greffe totale traditionnelle (GTT) et des techniques de greffe lamellaire postérieur (GLP) telles que la greffe lamellaire endothéliale profonde (DLEK) et la greffe endothélium/membrane de Descemet (EDMG) pour le traitement des maladies de l’endothélium, telles que la dystrophie de Fuchs et de la kératopathie de l’aphaque et du pseudophaque. Dans ce contexte, cette thèse contribue également à démontrer l’utilité de la tomographie de cohérence optique (OCT) pour l’étude de l’anatomie des plaies chirurgicales la cornée post transplantation. Au cours de ce travail nous avons étudié l'anatomie de la DLEK, avant et 1, 6, 12 et 24 mois après la chirurgie. Nous avons utilisé le Stratus OCT (Version 3, Carl Zeiss, Meditec Inc.) pour documenter l’anatomie de la plaie. L'acquisition et la manipulation des images du Stratus OCT, instrument qui à été conçu originalement pour l’étude de la rétine et du nerf optique, ont été adaptées pour l'analyse du segment antérieur de l’oeil. Des images cornéennes centrales verticales et horizontales, ainsi que 4 mesures radiaires perpendiculaires à la plaie à 12, 3, 6 et 9 heures ont été obtenues. Les paramètres suivants ont été étudiés: (1) Les espaces (gap) entre les rebords du disque donneur et ceux du receveur, (2) les dénivelés de surface postérieure (step) entre le les rebords du disque donneur et ceux du receveur, (3) la compression tissulaire, (4) le décollement du greffon, 6) les élévations de la surface antérieure de la cornée et 7) la pachymétrie centrale de la cornée. Les mesures d’épaisseur totale de la cornée ont été comparées et corrélées avec celles obtenues avec un pachymètre à ultra-sons. Des mesures d’acuité visuelle, de réfraction manifeste et de topographie ont aussi été acquises afin d’évaluer les résultats fonctionnels. Enfin, nous avons comparé les données de DLEK à celles obtenues de l’EDMG et de la GTT, afin de caractériser les plaies et de cerner les avantages et inconvénients relatifs à chaque technique chirurgicale. Nos résultats anatomiques ont montré des différences importantes entre les trois techniques chirurgicales. Certains des paramètres étudiés, comme le sep et le gap, ont été plus prononcés dans la GTT que dans la DLEK et complètement absents dans l’EDMG. D’autres, comme la compression tissulaire et le décollement du greffon n’ont été observés que dans la DLEK. Ceci laisse entrevoir que la distorsion de la plaie varie proportionnellement à la profondeur de la découpe stromale du receveur, à partir de la face postérieure de la cornée. Moins la découpe s’avance vers la face antérieure (comme dans l’EDMG), moins elle affecte l’intégrité anatomique de la cornée, le pire cas étant la découpe totale comme dans la GTT. Cependant, tous les paramètres d’apposition postérieure sous-optimale et d’élévation de la surface antérieure (ce dernier observé uniquement dans la GTT) finissent par diminuer avec le temps, évoluant à des degrés variables vers un profil topographique plus semblable à celui d’une cornée normale. Ce processus paraît plus long et plus incomplet dans les cas de GTT à cause du type de plaie, de la présence de sutures et de la durée de la cicatrisation. Les valeurs moyennes d’épaisseur centrale se sont normalisées après la chirurgie. De plus, ces valeurs moyennes obtenues par OCT étaient fortement corrélées à celles obtenues par la pachymétrie à ultra-sons et nous n’avons remarqué aucune différence significative entre les valeurs moyennes des deux techniques de mesure. L’OCT s’est avéré un outil utile pour l’étude de l’anatomie microscopique des plaies chirurgicales. Les résultats d’acuité visuelle, de réfraction et de topographie des techniques de GLP ont montré qu’il existe une récupération visuelle rapide et sans changements significatifs de l’astigmatisme, contrairement à la GTT avec et sans suture. La GLP a permis une meilleure conservation de la morphologie de la cornée, et par conséquence des meilleurs résultats fonctionnels que la greffe de pleine épaisseur. Ceci nous permet d’avancer que la GLP pourrait être la technique chirurgicale à adopter comme traitement pour les maladies de l’endothélium cornéen.This thesis aims to study the anatomy of the corneal wound following 3 techniques of corneal graft: traditional penetrating keratoplasy (PK) and two techniques of posterior lamellar keratoplasy (PLK) which are deep lamellar endothelial keratoplasy (DLEK) and Endothelial-Descemet’s Membrane Graft (EDMG) for the treatment of the endothelial corneal diseases, such as Fuch’s dystrophy and aphakic and pseudopakic bullous keratopathy. In this context, this thesis also contributes to show the utility of the time domain optical coherence tomography (TD-OCT) for studying the anatomy of surgical wounds after corneal transplantation. In this work we studied the anatomy of DLEK, before and 1,6,12 and 24 months after surgery. We used the Stratus OCT. (Version 3, Carl Zeiss, Meditec Inc.) to document the anatomy of the wound. The acquisition and the handling of the images of the Stratus OCT, an instrument originally designed for the study of the retina and the optic nerve, were adapted to analyse the anterior segment of the eye. Vertical and horizontal central images of the cornea, in addition to 4 radial measurements perpendicular to the wound at 12, 3, 6 and 9 hours were obtained. The following parameters were studied: (1) the gap between the edges of the donor disc and those of the recipient, (2) posterior surface mismatch (step) between the edges of the disc donor and those of the recipient, (3) tissue compression, (4) graft detachment, 6) elevations of the anterior corneal surface and 7) the central pachymetry of the cornea. Measurements of the total thickness were compared and correlated with those obtained with an ultrasound pachymeter. Measurements of visual acuity, manifest refraction and topography were also acquired in order to evaluate the functional results. Lastly, we compared the data of DLEK with those obtained from the EDMG and the PK, in order to characterize the wounds and to highlight the advantages and disadvantages relative to each surgical technique.Our anatomical results showed important differences between the three surgical techniques. Some of the studied parameters, like the step and the gap, were more pronounced in PK than in DLEK and completely absent in the EDMG group. Others, like tissue compression and graft detachment were observed only in the DLEK group. This let us predict that the distortion of the wound varies proportionally with the depth of recipient posterior stromal dissection. The less dissection towards the anterior surface (as in EDMG), the less it affects the anatomical integrity of the cornea, the worst case being full thickness trephination as in PK. However, all the parameters of sub-optimal posterior surface apposition and anterior surface elevation (this last only observed in PK) ended up decreasing with time, evolving with variable degrees to a topographic profile more similar to that of a normal cornea. This process appears longer and more incomplete in the cases of PK because of the type of wound, the presence of sutures and the longer healing period. The mean values of central thickness were normal after surgery. Moreover, these mean values obtained by OCT. were strongly correlated with those obtained by ultrasound pachymetry and we did not notice any significant difference between the mean values of the two measurement techniques. OCT proved to be a useful tool for the study of the microscopic anatomy of the corneal surgical wounds. The results of vision, refraction and topography of the techniques of posterior lamellar grafts showed that there was a fast visual recovery and without significant changes in astigmatism, contrary to PK with and without sutures. Posterior lamellar grafts allowed a better conservation of the morphology of the cornea, and consequently better functional results than PK. This enabled us to conclude that posterior lamellar corneal grafts could be the surgical technique of choice for the treatment of corneal endothelial diseases

Regenerative potential of corneal endothelium from patients with fuchs endothelial corneal dystrophy

La dystrophie cornéenne endothéliale de Fuchs (FECD, pour l’abréviation du terme anglais « Fuchs endothelial corneal dystrophy ») est une maladie de l'endothélium cornéen. Sa pathogenèse est mal connue. Aucun traitement médical n’est efficace. Le seul traitement existant est chirurgical et consiste dans le remplacement de l’endothélium pathologique par un endothélium sain provenant de cornées de la Banque des yeux. Le traitement chirurgical, en revanche, comporte 10% de rejet immunologique. Des modèles expérimentaux sont donc nécessaires afin de mieux comprendre cette maladie ainsi que pour le développement de traitements alternatifs. Le but général de cette thèse est de développer un modèle expérimental de la FECD en utilisant le génie tissulaire. Ceci a été réalisé en trois étapes. 1) Tout d'abord, l'endothélium cornéen a été reconstruit par génie tissulaire en utilisant des cellules endothéliales en culture, provenant de patients atteints de FECD. Ce modèle a ensuite été caractérisé in vitro. Brièvement, les cellules endothéliales cornéennes FECD ont été isolées à partir de membranes de Descemet prélevées lors de greffes de cornée. Les cellules au deuxième ou troisième passages ont ensuite été ensemencées sur une cornée humaine préalablement décellularisée. Suivant 2 semaines de culture, les endothélia cornéens reconstruits FECD (n = 6) ont été évalués à l'aide d'histologie, de microscopie électronique à transmission et d’immunomarquages de différentes protéines. Les endothélia cornéens reconstruits FECD ont formé une monocouche de cellules polygonales bien adhérées à la membrane de Descemet. Les immunomarquages ont démontré la présence des protéines importantes pour la fonctionnalité de l’endothélium cornéen telles que Na+-K+/ATPase α1 et Na+/HCO3-, ainsi qu’une expression faible et uniforme de la protéine clusterine. 2) Deux techniques chirurgicales (DSAEK ; pour « Descemet stripping automated endothelial keratoplasty » et la kératoplastie pénétrante) ont été comparées pour la transplantation cornéenne dans le modèle animal félin. Les paramètres comparés incluaient les défis chirurgicaux et les résultats cliniques. La technique « DSAEK » a été difficile à effectuer dans le modèle félin. Une formation rapide de fibrine a été observée dans tous les cas DSAEK (n = 5). 3) Finalement, la fonctionnalité in vivo des endothélia cornéens reconstruits FECD a été évaluée (n = 7). Les évaluations in vivo comprenaient la transparence, la pachymétrie et la tomographie par cohérence optique. Les évaluations post-mortem incluaient la morphométrie des cellules endothéliales, la microscopie électronique à transmission et des immunomarquage de protéines liées à la fonctionnalité. Après la transplantation, la pachymétrie a progressivement diminué et la transparence a progressivement augmenté. Sept jours après la transplantation, 6 des 7 greffes étaient claires. La microscopie électronique à transmission a montré la présence de matériel fibrillaire sous-endothélial dans toutes les greffes d’endothelia reconstruits FECD. Les endothélia reconstruits exprimaient aussi des protéines Na+-K+/ATPase et Na+/HCO3-. En résumé, cette thèse démontre que les cellules endothéliales de la cornée à un stade avancé FECD peuvent être utilisées pour reconstruire un endothélium cornéen par génie tissulaire. La kératoplastie pénétrante a été démontrée comme étant la procédure la plus appropriée pour transplanter ces tissus reconstruits dans l’œil du modèle animal félin. La restauration de l'épaisseur cornéenne et de la transparence démontrent que les greffons reconstruits FECD sont fonctionnels in vivo. Ces nouveaux modèles FECD démontrent une réhabilitation des cellules FECD, permettant d’utiliser le génie tissulaire pour reconstruire des endothelia fonctionnels à partir de cellules dystrophiques. Les applications potentielles sont nombreuses, y compris des études physiopathologiques et pharmacologiques.Fuchs endothelial corneal dystrophy (FECD) is a primary disease of the corneal endothelium. Its pathogenesis is poorly understood. No medical treatment is effective. Surgical treatment (the only available treatment) carries 10% of immunogenic rejection. Experimental models are needed in order to better understand the disease and to investigate potential autologous treatments (to prevent immunogenic rejection). The overall goal of this thesis is to develop an experimental model for FECD using tissue engineering. This was achieved in three steps. 1) An in vitro tissue-engineered FECD model was created and characterized. Briefly, Descemet’s membranes from patients with late-stage FECD undergoing Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on a previously decellularized human cornea. After 2 weeks in culture, TE-FECD corneas (n=6) were assessed using histology, transmission electron microscopy (TEM) and immunofluorescence labeling of various proteins. TE-FECD endothelium yielded a monolayer of polygonal cells well adhered to Descemet’s membrane. The TE-FECD corneal endothelium expressed the function-related proteins Na+-K+/ATPase α1 and Na+/HCO3-. Clusterin expression was faint and uniform. 2) In order to determine the best surgical procedure to transplant the TE-FECD corneas in the feline model, a DSAEK procedure was evaluated and compared to penetrating keratoplasty technique. DSAEK assessments included surgical challenges and clinical outcomes. DSAEK technique was challenging to perform in the feline model. Rapid fibrin formation was observed in all DSAEK cases (n=5). 3) The in vivo functionality of the TE-FECD corneas was assessed. TE-FECD corneas were grafted in the feline model (n=7) using penetrating keratoplasty procedure and observed for seven days. In vivo assessments included transparency, pachymetry, optical coherence tomography, endothelial cell morphometry, TEM and immunostaining of function-related proteins. After transplantation, pachymetry gradually decreased and transparency gradually increased. Seven days after transplantation, 6 out of 7 grafts were clear. Post-mortem TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE grafted endothelium expressed Na+-K+/ATPase and Na+/HCO3-. This thesis demonstrates that endothelial cells from late-stage FECD corneas can be used to engineer a corneal endothelium. Compared to DSEAK, penetrating keratoplasty is a more appropriate procedure for corneal transplantation in the feline model, since the DSAEK procedure in the feline model presently yields inconsistent clinical results. Restoration of corneal thickness and transparency demonstrates that the TE-FECD grafts are functional in vivo. This novel FECD living model suggests a potential role of tissue engineering for FECD cell rehabilitation. Potential applications are numerous, including pathophysiological and pharmacological studies