Benjamin L. George

Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach:

Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy.

The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach

The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). We have recently described a dual media approach for the propagation of human CECs. In this work, we characterize the effects of a Rho-kinase inhibitor Y-27632 on the cultivation of CECs propagated using the dual media culture system. Seventy donor corneas deemed unsuitable for transplantation were procured for this study. We assessed the use of Y-27632 for its effect at each stage of the cell culture process, specifically for cell attachment, cell proliferation, and during both regular passaging and cryopreservation. Lastly, comparison of donor-matched CEC-cultures expanded with or without Y-27632 was also performed. Our results showed that Y-27632 significantly improved the attachment and proliferation of primary CECs. A non-significant pro-survival effect was detected during regular cellular passage when CECs were pre-treated with Y-27632, an effect that became more evident during cryopreservation. Our study showed that the inclusion of Y-27632 was beneficial for the propagation of primary CECs expanded via the dual media approach, and was able to increase overall cell yield by between 1.96 to 3.36 fold.

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro

BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

A cost-minimization analysis of tissue-engineered constructs for corneal endothelial transplantation.

Corneal endothelial transplantation or endothelial keratoplasty has become the preferred choice of transplantation for patients with corneal blindness due to endothelial dysfunction. Currently, there is a worldwide shortage of transplantable tissue, and demand is expected to increase further with aging populations. Tissue-engineered alternatives are being developed, and are likely to be available soon. However, the cost of these constructs may impair their widespread use. A cost-minimization analysis comparing tissue-engineered constructs to donor tissue procured from eye banks for endothelial keratoplasty was performed. Both initial investment costs and recurring costs were considered in the analysis to arrive at a final tissue cost per transplant. The clinical outcomes of endothelial keratoplasty with tissue-engineered constructs and with donor tissue procured from eye banks were assumed to be equivalent. One-way and probabilistic sensitivity analyses were performed to simulate various possible scenarios, and to determine the robustness of the results. A tissue engineering strategy was cheaper in both investment cost and recurring cost. Tissue-engineered constructs for endothelial keratoplasty could be produced at a cost of US

Predicative Factors for Corneal Endothelial Cell Migration

880 per transplant. In contrast, utilizing donor tissue procured from eye banks for endothelial keratoplasty required US

Comparison of intra-ocular pressure changes with liquid or flat applanation interfaces in a femtosecond laser platform.

3,710 per transplant. Sensitivity analyses performed further support the results of this cost-minimization analysis across a wide range of possible scenarios. The use of tissue-engineered constructs for endothelial keratoplasty could potentially increase the supply of transplantable tissue and bring the costs of corneal endothelial transplantation down, making this intervention accessible to a larger group of patients. Tissue-engineering strategies for corneal epithelial constructs or other tissue types, such as pancreatic islet cells, should also be subject to similar pharmacoeconomic analyses.

Performing Reliable Lens Capsulotomy in the Presence of Corneal Edema With a Femtosecond Laser

PURPOSE To characterize the effects of Descemets stripping, Rho-associated protein kinase inhibitor Y-27632, and donor age on endothelial migration in human corneas maintained in ex vivo culture. METHODS Twenty-eight cadaveric human corneas underwent ex vivo culture in either standard or Y-27632-supplemented culture medium for 14 days. The posterior surface of each cornea was manipulated to create two types of wounds: scratched wound--corneal endothelial cells (CECs) were denuded from the Descemets membrane (DM) to leave behind a bare but intact DM; and peeled wound--both the DM and overlying CECs were stripped to leave behind bare corneal stroma. Endothelial migration was assessed via Trypan blue staining. Morphologic traits of CECs were assessed via Alizarin red microscopy and scanning electron microscopy. RESULTS The CECs migrated preferentially over scratched wounds compared with peeled wounds. Y-27632 supplementation accelerated endothelial migration over scratched wounds. Endothelial migration decreased with advanced donor age for both wound types, regardless of exposure to Y-27632. Y-27632 supplementation resulted in a less rapid decline in endothelial migration for donors older than 50 years of age for scratched surfaces. Greater cell density and hexagonality was observed over scratched wounds compared with peeled wounds, regardless of Y-27632 supplementation. CONCLUSIONS The presence of an intact DM, Y-27632 supplementation, and young donor age are factors that promote endothelial migration in an ex vivo human cornea culture model. The negative effect of age on endothelial migration can be mitigated by the presence of an intact DM and Y-27632 supplementation.

Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients

Cataract surgery is the most common surgical procedure and femtosecond laser assisted cataract surgery (FLACS) has gained increased popularity. FLACS requires the application of a suction device to stabilize the laser head and focus the laser beam accurately. This may cause a significant escalation in intra-ocular pressure (IOP), which poses potential risks for patients undergoing cataract surgery. In this study we aimed to assess the effect of the Ziemer LDV Z8 femtosecond cataract machine on IOP. We demonstrated through a porcine model that IOP was significantly higher with a flat interface but could be abrogated by reducing surgical compression and vacuum. Pressure was lower with a liquid interface, and further altering angulation of the laser arm could reduce the IOP to 36 mmHg. A pilot series in patients showed comparable pressure rises with the porcine model (30 mmHg). These strategies may improve the safety profile in patients vulnerable to high pressure when employing FLACS with the Ziemer LDV Z8.

Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice

Purpose To determine the effects of the Ziemer LDV Z8 liquid interface femtosecond laser platform during capsulotomy under different energy settings in the presence of corneal edema. Methods Cadaveric porcine eyes (n = 36) employed at less than 6 and greater than 24 post enucleation hours to simulate clear/edematous corneas, underwent capsulotomy with the Ziemer LDV Z8 femtosecond laser (5-mm diameter, energy 90%, 130%, or 150%). Lens capsules were removed for evaluation by scanning electron microscopy and rupture strengths determined by the single column universal testing system. Following ethical approval, 23 patients had lens capsules removed during routine cataract surgery following manual or Z8 capsulotomy and subjected to TUNEL assay. Results There was no difference in edge morphology or rupture strength (120, 113, and 118 mN at increasing energy, P = 0.42) in the clear cornea. Only 50% of capsulotomies succeeded at 90% energy in an edematous cornea, improving with increased energy (75% completion at 130%, 100% at 150%). Rupture strength in edematous corneas was not significantly different at 112, 133, and 114 mN for 90%, 130%, and 150%, respectively (P = 0.3). In human samples, increased TUNEL-positive cells were seen at 130% energy, but not at 150% (0.0 manual vs. 0.2 [90%] vs. 2.1 [130%] vs. 0.6 [150%], P < 0.05). Conclusions Because of the low energy delivered by a femtosecond nanojoule platform, even incremental increases in energy appeared to have minimal effect on lens capsule morphology and strength and negligible influence on cell death. Furthermore, increasing energy appeared to enhance consistency and the ability to complete a capsulotomy in an edematous cornea.

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

Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor β-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor β-induced protein (amino acid 611–642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.