Cadaveric Corneal Research Articles

Epidemiological Study and Seropositivity in Viral Panel Testing Among Corneal Tissue Donors.

Corneal tissue is a critical component for vision restoration through transplantation, yet the availability of suitable corneal tissue is limited. This limitation results in long waiting lists and high demand, especially in countries with lower donation rates compared to global benchmarks. In Mexico, the corneal donation rate remains significantly behind other Latin American countries and leading nations such as Spain. Understanding the characteristics of corneal tissue donors is essential for improving donation practices and addressing the shortage of available tissues. This descriptive retrospective study analyzed electronic medical records of cadaveric corneal donors at the General Regional Hospital 1 of the Mexican Social Security Institute of Querétaro from January 2022 to December 2023. Donors were included based on criteria such as age (2-85 years) and known cause of death. Exclusion criteria included age outside the specified range, neoplastic diseases, active systemic infections, and prior ocular surgeries. Serological testing for human immunodeficiency virus (HIV), Venereal Disease Research Laboratory (VDRL), coronavirus disease 2019 (COVID-19), hepatitis B virus (HBV), and hepatitis C virus (HCV was performed to assess viral reactivity. Statistical analyses were conducted usingIBM SPSS Statistics for Windows, Version 25 (Released 2017; IBM Corp., Armonk, New York, United States), summarizing data with descriptive statistics. The study included 185 corneal donors with a mean age of 56.34 years. The majority were male (109; 58.9%), and the leading causes of death were cardiogenic shock (34; 18.4%), hypovolemic shock (31; 16.8%), and acute respiratory failure (30; 16.2%). Exclusion due to positive serological tests included seven donors (3.8%) for HIV and seven (3.8%) for SARS-CoV-2. A total of 16 donors (8.6%) were excluded due to positive results in the viral serological panel. The most common comorbidities were chronic kidney disease (36; 19.5%), type 2 diabetes mellitus (28; 15.1%), and systemic hypertension (31; 16.8%). The study highlights a predominantly male donor profile with a mean age of 56.34 years and emphasizes cardiogenic shock as a leading cause of death. The notably higher seropositivity rate of 8.6% for various viral infections compared to international reports indicates a need for improved health interventions and screening processes. The focus on cardiovascular and respiratory causes of death underscores regional health issues affecting donation patterns. To address the organ and tissue donation shortfall, it is crucial to enhance coordination within donation teams and increase public awareness, given the significant gap in donation rates compared to leading countries.

In Situ-Forming, Bioorthogonally Cross-linked, Nanocluster-Reinforced Hydrogel for the Regeneration of Corneal Defects.

Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.

Need for new corneal restoration technologies in a post‐COVID world

In most countries, the supply of corneal material do not meet the demands for corneal transplantation. This has been exacerbated by COVID‐19. Patients have not been seen during lockdown. Many are presenting late. There has been fewer operations done during the pandemic resulting in a backlog. Even in normal times, there are cases of corneal blindness not amenable to cadaveric corneal transplantation. The clinical and research community have two tasks. One is to develop artificial corneae or contructs through biotechnology to replace cadaveric transplantation. The other is to improve keratoprostheses which are used for vascularised corneae and multiple previous failed grafts (Boston KPro1 and similar) and for dry keratinised ocular surface (the OOKP and alternatives).

Preclinical Investigation of Ab Interno Goniotomy Using Three Different Techniques.

To evaluate incisional or excisional tissue-level effects of ab interno goniotomy techniques on human trabecular meshwork (TM). The TM from human cadaveric corneal rim tissue was treated using three devices: (1) Kahook Dual Blade (KDB) GLIDE, (2) iAccess, and (3) SION. Two human corneal rims were used for each of the iAccess and SION devices and one with the KDB GLIDE, with 360 degrees of TM treated in each case. Sections were then prepared for analysis and comparison between devices. Tissue samples underwent standard histologic processing with H&E stain, followed by comparative analyses. Areas treated with the KDB GLIDE device resulted in nearly complete excision of TM overlying the canal of Schlemm without injury to surrounding tissues. The iAccess device can be used as a focal trephine to create holes or dragged for TM disruption. When used to create holes, iAccess punched through the full thickness of the TM and also disrupted the anterior scleral tissue. It caused some incisional openings through the TM but with significant leaflets remaining and minimal true "hole-punch" effect. When the device tip was dragged, iAccess incised the TM and left debris behind with little, if any, excision of tissue. SION led to both incision and excision of TM with incision predominating over excision. The various methods evaluated to perform ab interno goniotomy resulted in varying degrees of TM incision or excision. Only the KDB GLIDE device resulted in reliable excision of TM, while the other devices produced incision or minimal excision of tissue with residual leaflets and debris. Use of iAccess resulted in focal disruption of the anterior scleral wall. Because incisional approaches that leave longer residual leaflets may be more prone to fibrosis and closure compared to excisional treatments, clinical correlation will be necessary to better understand the significance of these findings with respect to relative effectiveness of intraocular pressure lowering in eyes with glaucoma.

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering.

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology.

Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells.

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic. Graphical abstract.

Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions.

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.

Congenital central corneal dermoid: A rare entity.

To understand the pathogenesis of a central corneal dermoid (CD) in a 12-day-old child, a comparison of CD specimen was done with limbal dermoid (LD) and cadaveric corneal (CC) specimens by immuno-histochemical staining. The child underwent penetrating keratoplasty for visual rehabilitation. The corneal tissue was sent for histo-pathological and immunochemistry evaluation. The corneal specimen obtained was compared the origin of central CD with LD and CC based on their antigenic expression profile. Clinically over a period of 75 months post operatively the child maintained a clear graft. Hematoxylin and Eosin staining of LD had a typical morphology including stratified keratinized epithelium, hair shaft with pilo sebaceous glands, eccrine sweat glands, lymphocytes, and blood vessels. Immuno-histochemical staining showed positive stain for Cytokeratin 3 epithelial marker in the epithelium of CC, LD, and CD. Smooth muscle maker (SMA) was identified in LD and CD but not in the CC as it is devoid of blood vessels. Limbal stem cell maker (P63) was detected only in LD. Vimentin, a mesenchymal stem cell marker stained positively in all three tissues of CC, LD, and CD. Corneal dermoid showed positive staining for mesodermal tissue components compared to both ectodermal and mesodermal components in limbal dermoid suggesting possibly a different origin of corneal dermoid.

Preclinical Investigation of Goniotomy Using Four Different Techniques.

PurposeTo evaluate the tissue-level effects of goniotomy techniques on human trabecular meshwork (TM).DesignLaboratory investigation.MethodsThe TM from human cadaveric corneal rim tissue was treated using 4 techniques: (1) microvitreoretinal (MVR) blade; (2) 360° trabeculotomy with 5-0 prolene suture; (3) the Kahook Dual Blade (KDB) Glide® device; (4) TrabEx™ device; tissue samples underwent standard histologic processing with H&E stain followed by comparative analyses.ResultsThe MVR blade exhibited incision of TM extending into the scleral wall. The TrabEx device removed a small portion of TM with large leaflet tissue remnants in all treated areas. 360° suture trabeculotomy resulted in incision of the TM proximate to Schwalbe’s line with no excised tissue evident in all treated areas. Areas treated with the KDB Glide device resulted in nearly complete excision of TM without injury to surrounding tissues.ConclusionThe various methods used for performing goniotomy or trabeculotomy resulted in varying degrees of incision or excision of TM. Only the KDB Glide device resulted in reliable excision of TM with the other devices producing incision or variable excision of tissue. Clinical correlation is required to better understand the implications of the current findings when using these methods to lower intraocular pressure in eyes with glaucoma.

Corneal dystrophies.

Corneal dystrophies are broadly defined as inherited disorders that affect any layer of the cornea and are usually progressive, bilateral conditions that do not have systemic effects. The 2015 International Classification of Corneal Dystrophies classifies corneal dystrophies into four classes: epithelial and subepithelial dystrophies, epithelial-stromal TGFBI dystrophies, stromal dystrophies and endothelial dystrophies. Whereas some corneal dystrophies may result in few or mild symptoms and morbidity throughout a patient's lifetime, others may progress and eventually result in substantial visual and ocular disturbances that require medical or surgical intervention. Corneal transplantation, either with full-thickness or partial-thickness donor tissue, may be indicated for patients with advanced corneal dystrophies. Although corneal transplantation techniques have improved considerably over the past two decades, these surgeries are still associated with postoperative risks of disease recurrence, graft failure and other complications that may result in blindness. In addition, a global shortage of cadaveric corneal graft tissue critically limits accessibility to corneal transplantation in some parts of the world. Ongoing advances in gene therapy, regenerative therapy and cell augmentation therapy may eventually result in the development of alternative, novel treatments for corneal dystrophies, which may substantially improve the quality of life of patients with these disorders.

Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells.

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.

Osteo-odonto keratoprosthesis in Egypt: a case series

Purpose The aim of this study was to evaluate the preliminary results of osteo-odonto keratoprosthesis (OOKP) performed in Alexandria Main University Hospital, Alexandria, Egypt in eyes with end-stage cicatrizing conjunctivitis.Patients and methods OOKP surgery was done in two stages. Stage 1 included preparation of the globe, buccal mucous membrane harvesting, and preparation of osteo-odonto alveolar lamina with the cemented optical cylinder. The prepared lamina was then placed in the submuscular space for 2–3 months. In stage 2, the lamina–optical cylinder complex was implanted under the buccal mucous membrane and the posterior part of the optical cylinder was placed in a central corneal opening to act as an artificial cornea, transmitting image to the retina.Results Six eyes with end-stage ocular surface disease were included in this study. Anatomical success, in the form of retention of the prosthesis, was achieved in five eyes. The procedure failed in one eye due to unhealthy canines with a small amount of bone in the lamina. Functional success, defined as restoration of vision, was achieved in four eyes. Visual improvement was not attained in one eye (in addition to the eye with anatomical failure) due to optic atrophy despite good retention of the device.Conclusion OOKP is a promising procedure to regain vision in severe cases of end-stage corneal blindness, unsuitable for cadaveric corneal grafts. Proper selection of cases is crucial to achieve the best anatomical and functional results.

The future of keratoplasty: cell-based therapy, regenerative medicine, bioengineering keratoplasty, gene therapy.

To provide an update on the state of development of novel therapeutic modalities for the treatment of corneal diseases. Novel corneal therapeutics may be broadly classified as cell therapy, regenerative medicine, bioengineered corneal grafts and gene therapy. Cell therapy encompasses cultivation of cells, such as corneal endothelial cells (CECs) and keratocytes to replenish the depleted native cell population. Regenerative medicine is mainly applicable to the corneal endothelium, and is dependent on the ability of native, healthy CECs to restore the corneal endothelium following trauma or descemetorhexis; this approach may be effective for the treatment of Peter's anomaly and Fuchs endothelial corneal dystrophy (FECD). Bioengineered corneal grafts are synthetic constructs designed to replace cadaveric corneal grafts; tissue-engineered endothelial-keratoplasty grafts and bioengineered stromal grafts have been experimented in animal models with favourable results. Gene therapy with antisense oligonucleotide and CRISPR endonucleases, including deactivated Cas9, may potentially be used to treat FECD and TGFBI-related corneal dystrophies. These novel therapeutic modalities may potentially supersede keratoplasty as the standard of care in the future.

Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy

Corneal transplantation is the only treatment available to restore vision for individuals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm (p < 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.

Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells.

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies.

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.

BackgroundThe limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue.MethodsSphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue.ResultsSpheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation.ConclusionThese observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

Boston Keratoprosthesis: Outcomes and Complications: A Report by the American Academy of Ophthalmology

To review the published literature on safety and outcomes of the Boston type I keratoprosthesis (BI-KPro) for the surgical treatment of corneal opacification not amenable to human cadaveric corneal transplantation. Searches of peer-reviewed literature were conducted in PubMed and the Cochrane Library in December 2012, July 2013, and January 2014 without date restrictions. The searches were limited to studies published in English and yielded 587 citations. The abstracts of these articles were reviewed, 48 articles were selected for possible clinical relevance, and 22 were determined to be relevant for the assessment objectives. Nine studies were rated as level II evidence and 13 studies were rated as level III evidence. Excluded were level III evidence, case reports, review articles, letters, editorials, and case series with fewer than 25 eyes. In 9 articles, a best-corrected Snellen visual acuity (BCSVA) of 20/200 or better occurred in 45% to 89% of eyes. Five articles described a BCSVA of 20/50 or better in 43% to 69% of eyes, and 4 articles found a BCSVA of 20/40 or better in 11% to 39% of eyes. Retention rates of the BI-KPro ranged from 65% to 100%. Reasons for loss of vision after BI-KPro implantation most commonly included corneal melts resulting from exposure keratopathy, endophthalmitis, and infectious keratitis or corneal ulceration. The 2 most common complications after surgery were retroprosthetic membrane formation (range, 1.0%-65.0%; mean ± standard deviation [SD], 30.0±19.0%) and elevated intraocular pressure (range, 2.4%-64.0%; mean ± SD, 27.5±18.1%). The 2 most common posterior segment complications were endophthalmitis (range, 0%-12.5%; mean ± SD, 4.6±4.6%) and vitritis (range, 0%-14.5%; mean ± SD, 5.6±4.7%). The reviewed articles on BI-KPro use suggest that the device improves vision in cases of severe corneal opacification that were not amenable to corneal transplantation using human cadaveric keratoplasty techniques. A number of severe anterior and posterior segment complications can develop as follow-up continues, making ongoing close observation paramount for patients undergoing this surgery. These complications include infection, device extrusion, and permanent vision loss.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), d-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32–80 years) or duration in eye bank corneal preservation medium prior to use (3–10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

KPro Biomaterials

AbstractResearch into keratoprosthessis(KPro) is at least 200 years old. In the early days, when corneal pathology was recognised as a specific cause for blindness, its treatment involved a replacement of the diseased cornea with artificial materials. Afterwards cadaveric corneal transplantation has proven to be more successful than prosthetic corneal substitutes. However, the need for keratoprostheses is still present. Scores of KPro devices have been proposed and many biomaterials have been investigated. Only 2 devices, the osteo‐odonto‐keratoprosthesis (OOKP) and Boston type1 KPro, have proven to be viable in the longer term although complications do occur. A true artificial cornea in form and function is still a fantasy and may not be clinically feasible. Since corneal involvement can be secondary to ocular surface disease, a universal KPro that cures every type of corneal blindness may not be a pragmatic solution. Development of biomaterials may be tailored towards KPros designed specifically for different ocular pathology rather than towards a ‘one size fit for all’ device. Translation of laboratory work into clinical practice for KPro development will require better concordance between clinicians and scientists.