Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation.

Near infra-red labelling and tracking of corneal endothelial cells in-vivo.

Following corneal transplantation, there is an initial, rapid decline in corneal endothelial cells (CECs) following surgery. Direct imaging of post-transplantation endothelial cells is only possible weeks after surgery and with a limited field of view. We have developed a labelling approach using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DIR) dye solution, that enables tracking of labelled CECs in vivo for at least 1 month.

Translational and Regulatory Challenges of Corneal Endothelial Cell Therapy: A Global Perspective.

Cell therapies are emerging as a unique class of clinical therapeutics in medicine. In 2015, Holoclar ( expanded autologous human corneal epithelial cells containing stem cells) gained the regulatory approval for treating limbal stem cell deficiency after chemical eye burn. This has set a precedent in ophthalmology and in medicine, reinforcing the therapeutic promise of cell therapy.

A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy.

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet's membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion.

Optimisation of Storage and Transportation Conditions of Cultured Corneal Endothelial Cells for Cell Replacement Therapy.

As the cornea is one of the most transplanted tissues in the body it has placed a burden on the provision of corneas from cadaveric donors. Corneal endothelial dysfunction is the leading indication for cornea transplant. Therefore, tissue engineering is emerging as an alternative approach to overcome the global shortage of transplant-grade corneas.

Peroxiredoxin-1 regulates lipid peroxidation in corneal endothelial cells.

Corneal transparency is maintained by a monolayer of corneal endothelial cells. Defects in corneal endothelial cells (CEnCs) can be rectified surgically through transplantation. Fuchs' endothelial corneal dystrophy (FECD) is the foremost cause of endothelial dysfunction and the leading indication for transplantation.

Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium.

Objective: We aimed to investigate the functionality of human decellularized stromal laminas seeded with cultured human corneal endothelial cells as a tissue engineered endothelial graft (TEEK) construct to perform endothelial keratoplasty in an animal model of corneal endothelial damage.

Methods: Engineered corneal endothelial grafts were constructed by seeding cultured human corneal endothelial cell (hCEC) suspensions onto decellularized human corneal stromal laminas with various coatings. The functionality and survival of these grafts with cultured hCECs was examined in a rabbit model of corneal endothelial damage after central descemetorhexis.

Functional Evaluation of Two Corneal Endothelial Cell-Based Therapies: Tissue-Engineered Construct and Cell Injection.

Restoration of vision due to corneal blindness from corneal endothelial dysfunction can be achieved via a corneal transplantation. However, global shortage of donor tissues has driven the development cell-based therapeutics. With the capacity to propagate regulatory compliant human corneal endothelial cells (CEnCs), this study evaluated the functionality of propagated CEnCs delivered via tissue-engineered endothelial keratoplasty (TE-EK) or corneal endothelial cell injection (CE-CI) within a rabbit model of bullous keratopathy.

Regulation of Oxidative Stress in Corneal Endothelial Cells by Prdx6.

The inner layer of the cornea, the corneal endothelium, is post-mitotic and unable to regenerate if damaged. The corneal endothelium is one of the most transplanted tissues in the body. Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal endothelial transplantation.

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.

Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.

Purpose: To establish a method for assessing graft viability, in-vivo, following corneal transplantation.

Methods: Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model.

Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications.

Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with diverse tissue growth applications.

In Vitro Topographical Model of Fuchs Dystrophy for Evaluation of Corneal Endothelial Cell Monolayer Formation.

A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs' endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients.

Predicative Factors for Corneal Endothelial Cell Migration.

Purpose: To characterize the effects of Descemet's 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 Descemet's 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.

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.

Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells.

One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers.

Endothelial approach ultrathin corneal grafts prepared by femtosecond laser for descemet stripping endothelial keratoplasty.

Purpose: To investigate the quality of the ultrathin corneal grafts prepared by femtosecond laser from the endothelial side for Descemet stripping endothelial keratoplasty.

Methods: Thirty human corneoscleral buttons were cut from the endothelial side by laser Doppler velocimetry (LDV) with or without viscoelastic materials coating. Two cutting depths were selected: 70 and 90 μm.

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.