An overview of current techniques for ocular toxicity testing

Given the hazardous nature of many materials and substances, ocular toxicity testing is required to evaluate the dangers associated with these substances after their exposure to the eye. Historically, animal tests such as the Draize test were exclusively used to determine the level of ocular toxicity by applying a test substance to a live rabbit’s eye and evaluating the biological response. In recent years, legislation in many developed countries has been introduced to try to reduce animal testing and promote alternative techniques. These techniques include ex vivo tests on deceased animal tissue, computational models that use algorithms to apply existing data to new chemicals and in vitro assays based on two dimensional (2D) and three dimensional (3D) cell culture models. Here we provide a comprehensive overview of the latest advances in ocular toxicity testing techniques, and discuss the regulatory framework used to evaluate their suitability

The development of a novel human corneal substitute using decellularized corneas

The development of a novel human corneal substitute using decellularized cornea

Corneal decellularization: a method of recycling unsuitable donor tissue for clinical translation?

Background: There is a clinical need for biomimetic corneas that are as effective, preferably superior, to cadaveric donor tissue. Decellularized tissues are advantageous compared to synthetic or semi-synthetic engineered tissues in that the native matrix ultrastructure and intrinsic biological cues including growth factors, cytokines and glycosaminoglycans may be retained. However, there is currently no reliable, standardized human corneal decellularization protocol. Methods: Corneal eye-bank tissue unsuitable for transplantation was utilized to systematically compare commonly used decellularization protocols. Hypertonic sodium chloride; an ionic reagent, sodium dodecyl sulphate; a non-ionic detergent, tert-octylphenol polyoxyethylene (Triton-X); enzymatic disaggregation using Dispase; mechanical agitation; and the use of nucleases were investigated. Decellularization efficacy, specifically for human corneal tissue, was extensively evaluated. Removal of detectable cellular material was evidenced by histological, immunofluorescence and biochemical assays. Preservation of macroscopic tissue transparency and light transmittance was evaluated. Retention of corneal architecture, collagen and glycosaminoglycans was assessed via histological, immunofluorescence and quantitative analysis. Biocompatibility of the resulting scaffolds was assessed using cell proliferation assays. Results: None of the decellularization protocols investigated successfully removed 100% of cellular components. The techniques with the least residual cellular material were most structurally compromised. Biochemical analysis of glycosaminoglycans demonstrated the stripping effects of the decellularization procedures. Conclusion: The ability to utilize, reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. Reprocessing the tissue has the potential to have a considerable impact on addressing the problems associated with cadaveric donor shortage. Patients would directly benefit by accessing greater numbers of corneal grafts and health authorities would fulfill their responsibility for the delivery of effective corneal reconstruction to alleviate corneal blindness. However, in order to progress, we may need to take a step back to establish a “decellularization” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality

Keeping an eye on decellularized corneas: a review of methods, characterization and applications

The worldwide limited availability of suitable corneal donor tissue has led to the development of alternatives, including keratoprostheses (Kpros) and tissue engineered (TE) constructs. Despite advances in bioscaffold design, there is yet to be a corneal equivalent that effectively mimics both the native tissue ultrastructure and biomechanical properties. Human decellularized corneas (DCs) could offer a safe, sustainable source of corneal tissue, increasing the donor pool and potentially reducing the risk of immune rejection after corneal graft surgery. Appropriate, human-specific, decellularization techniques and high-resolution, non-destructive analysis systems are required to ensure reproducible outputs can be achieved. If robust treatment and characterization processes can be developed, DCs could offer a supplement to the donor corneal pool, alongside superior cell culture systems for pharmacology, toxicology and drug discovery studies

The use of innovative scaffolds in the development of corneal stroma-derived stem cell therapies for future corneal regeneration strategies [Abstract]

The use of innovative scaffolds in the development of corneal stroma-derived stem cell therapies for future corneal regeneration strategies [Abstract

Cultivation and characterisation of human peripheral cornea-derived endothelial cells [abstract]

To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two-step, peel-and-digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC-coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT-qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO-1, Na/K-ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed

Challenges in manufacturing an amniotic membrane alternative for corneal regeneration [abstract]

The current worldwide cornea shortages have led to the development of feasible, long-term substitutes to cadaveric donor tissue. Amniotic membrane is a clinically successful material used to reconstruct damaged corneal surfaces. However, its reliance upon the host tissue, availability, donor variation, infection risks and the fact that it is currently unsuitable for complete corneal regeneration have warranted the need for alternative solutions. Alternatives in current clinical or pre-clinical development include keratoprostheses, tissue engineered constructs, xenografts and the use of acellular matrices. With respect to corneal regeneration, there are many challenges, not least that the corneal structure is unique and difficult to replicate. When manufacturing corneal tissues, the choice of material is vital as the list of requirements is extensive. They must be biocompatible, (preferably) optically transparent, flexible, and strong, as to withstand manipulation in culture, potential suturing, irrigation and handling during surgery. Manufacturing process need to be simple and consistent, preferably at high speed and low cost. Two fundamental objectives of corneal regeneration are the maintenance of healthy cell phenotypes and the replication of the native tissue architecture. If both factors are not satisfied, the result is often regenerated tissue mimicking that of scarred native tissue

Profiles of secreted proteins from alginate-encapsulated and cultured LESC are distinct.

<p>Conditioned medium from alginate-encapsulated LESC (CM^ALG-LESC), LESC cultured in CnT20 (CM^LESC), alginate gels alone (CM^ALG), cultured CnT20 (CM^CULT), and non-cultured CnT20 (CM^NON-CULT) were treated with Laemmli buffer containing the reducing agent β-mercaptoethanol. Treated protein mixtures were separated using 15% acrylamide gels and proteins were stained using silver nitrate. Gel represents 3 individual experiments from 3 different corneoscleral rims.</p

Distribution of cp values of ECGs in different OS epithelial regions.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022301#pone-0022301-g001" target="_blank">Figure 1A</a>, the scatter graph of the Cp values of all the ECGs shows all the ECGs had cp value≤35. The order of samples 1–12 were as follows: Samples 1, 2, 3 were cornea 1, cornea 2, cornea 3, samples 4, 5, 6 were limbus 1, limbus 2, limbus 3, samples 7, 8, 9 were LEC 1, LEC 2, LEC 3 and samples 10, 11, 12 were conjunctiva 1, conjunctiva 2, conjunctiva 3. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022301#pone-0022301-g001" target="_blank">Figure 1B</a> is a box plot of q PCR cp values of the ECGs. Each box plot is mean Cp value of all OS epithelial regions. The median Cp value is represented as black line within the box plot, and it divides the Cp values into lower and upper quartile ranges. The whiskers represent the upper and lower data range for the samples tested. The box plot of PPIA demonstrates tight distribution of the samples around the median value. Similarly box plot of RPLP0 shows uniform distribution of Cp values around the median with tight whisker distribution.</p

Endogenous control genes analysed with geNorm software in this study.

<p>Endogenous control genes analysed with geNorm software in this study.</p