The research involved in the field of ophthalmology, especially when focused in testing and in the development of medical devices or procedures that may have an impact on the corneal endothelium health, necessitates a great number of investigations, which sometimes have to face some technical difficulties.

As an example, the use of donor corneas for research purpose is unethical, as they are needed for transplantation. Fortunately, some animal models, such as the porcine eye, have anatomical and physiological properties quite similar to the human eye and therefore are useful alternatives to donor corneal tissues for performing valuable research.

Another important matter is related to the method used for collecting and analysing all qualitative and quantitative image data. In Europe, the most common method to assess the viability of the corneal endothelium is based on the trypan blue staining and visual evaluation of the mortality zones using optical microscopy. However, this method is performed manually and, therefore, the operator-dependent variability of data analysis may be a problem, especially when considering the ever-increasing number of analyses to be performed.

To overcome this problem, the Alchimia R&D team developed a standardized method for the quantitative analysis of the endothelial cell mortality with a semi-automated microscopy technology through the Fiji ImageJ software, specifically designed to help researchers in the analyses of the biological pictures.

The first step of this method is the preparation of the corneal button from the porcine eye globe and the staining of the corneal endothelium with the trypan blue. Afterwards, the stained tissue is held under the optical microscope and analysed using the most appropriate magnification. At this point, a camera connected with the microscope can take a series of pictures of the corneal endothelium. These pictures are then “transferred” to the Fiji ImageJ software that, by using special techniques to optimise the corneal images (e.g., the corneal endothelium is not a homogeneous and flat tissue), can release clear images of the central 8-mm central area (which corresponds to the usually transplanted corneal button). Within this area, the software detects the presence of the blue pixel, corresponding to the dead cells, and calculates the percentage of endothelial cells mortality in comparison to the total amount of endothelial cells.

This method was validated using negative (health corneal tissues) and positive (corneal tissues that were heavily damaged by toxic substances) controls.

In conclusion, this semi-automated quantitative system for the measurement of the corneal EC mortality resulted effective, standardized, and reproducible.

For us, this means a straightforward tool to perform a first screening of corneal tissues to compare different corneal storage conditions and experimental research on corneal tissues. In addition, when used on already commercially available products, this method will also serve as a post-market safety evaluation.

References:

• Giurgola L. et al. Development of a quantitative method to determine corneal endothelial cell mortality. Presentation at the European Associations of Tissue Banks (EATB), 2018
• Wenzel DA et al. A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons. J Vis Exp 2019;152.
• Guduric-Fuchs J et al. Immunohistochemical study of pig retinal development. Mol Vis 2009; 15:1915:1928

It is well-known that cornea transplantation is still associated with a certain – though quite low –risk of post keratoplasty infection that can pose serious threats to the transplanted patient. Since one of the possible causes includes the grafting of contaminated donor tissue, the eye banks have the responsibility to ascertain that only corneas that are free of contamination are distributed for the transplantation.

With this aim, the Alchilife R&D laboratories have an ongoing collaboration with the Eye Bank of Monza to select and validate a system to confirm the sterility of corneas by testing all the media that are used to preserve and transport the donor tissues.

As we know, most of the European eye banks store the donor corneas at 31°C in organ culture media allowing to preserve donor tissues for up to 4 weeks. This longer preservation time gives the eye banks operators the ease of performing all accurate tests on donated tissues.

The corneal storage media available in the market contain a mixture of antimicrobial agents. On one side these agents help to minimize the contamination in the preserved corneas, but on the other, they may prevent the growth of some microorganisms whose presence might not be revealed during the microbiological analysis and, as a consequence, transferred to the transplanted patient.

This is the reason why the European Pharmacopeia (EP) “Method suitability test” recommends to remove all factors that may interfere with the microbial growth before the microbiological testing.

The same Eye Bank of Monza usually performed the microbiological tests using the automatic BACTEC™ (Becton Dickinson) system, through which the preservation media samples are tested using specific BACTEC™ bottles containing resins that should maintain the antimicrobial residues. Despite the use of an automatic system, it has been shown that sometimes the results obtained can be misleading.

To avoid false-negative results, the new studied method includes an additional step in which all media to be tested are treated with a patented syringe-like CE-marked medical device, RESEP (Alchimia Srl), which also contains a special resin mixture for total elimination of the antimicrobials from the corneal storage media.

This method was validated using both samples of organ culture medium (Tissue-C, Alchimia Srl) and deswelling/transport medium (Carry-C, Alchimia Srl) by inoculum with various EP reference microbial strains before the transfer into the BACTEC™ bottles for the automatic analysis. Of course, negative controls (i.e. samples without microbial strains) and positive controls (i.e. samples with microbial strains) were also used.

The obtained results showed that the use of RESEP increased the sensitivity of the sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements.

References:

• Vignola R. et al. Validation of the BD BACTEC™ method for sterility testing of corneal preservation media according to the European Pharmacopoeia (chapter 2.6.1.). Presentations at the European Association of Tissue Banks EATB 2017 and 2018
• Vignola et al. A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia. Cell Tissue Bank 2019 https://doi.org/10.1007/s10561-019-09766-7
• Council of Europe (2013a) Sterility test. In: European Directorate For The Quality of Medicines and Healthcare (ed) European Pharmacopoeia, Eight edn, Vol I, Strasbourg Cedex, France, pp 175–178
• Schroeter J. et al. Validation of the microbiological testing of tissue preparations using the BACTECTM blood culture system. Transfus Med Hemother 2012;39(6):387–390
• Mistò R. et al. Method for sterility testing of corneal storage and transport media after removal of interfering antimicrobials: prospective validation study in compliance with the European Pharmacopoeia. BMJ Open Ophthalmol 2017;2(1): e000093.

When the visualization of the anterior capsule is compromised due to absent or insufficient red reflex, as in case of mature cataract, dense cortical cataract, dense posterior sub capsular cataract, or eyes with dense asteroid hyalosis, there is a certain risk for anterior capsular tear extending to the periphery, with a subsequent predisposition to posterior capsule rupture, vitreous loss, and posterior dislocation of nucleus.

Staining the anterior capsule with trypan blue (TB) dyes, performed for the first time by Melles et al. in 1999, helps surgeons to complete capsulorhexis in these challenging situations.

A small concentration of TB dye such as 0.05% can provide good anterior capsule staining, and endothelial toxicity is commonly believed as negligible when the TB dye is used in low concentration.

Nevertheless, for ensuring the maximum patient safety, the biocompatibility must always be ascertained, and the determination of cytotoxicity is part of the initial evaluation process according to the ISO standard.

The search for the truly reliable biocompatibility and cytotoxicity test methods has always been one of the main missions of Alchilife. This is the reason why in vitro cytotoxicity test by direct contact method according to ISO 10993-5, which is highly sensitive and able to detect even weak cytotoxicity, has always been the method of choice used in our research laboratories.

Using this approach, the Alchimia R&D department validated quantitatively and qualitatively the cytotoxicity test of vital dyes of the anterior segment according to the ISO 10993-5. By using only 30 µl of the dye applied on ARPE19 and BALB 3T3 cell lines for 24 hours, the absence or the presence of some cytotoxicity in all kinds of TB dyes can be easily and reliably ascertained.

One aspect of the cytotoxicity test methods adopted by our R&D department deserves particular attention, as it allows to “ultra-guarantee” the safety of the TB dyes to be used during anterior segment surgeries: it was chosen to perform the cytotoxicity assay not only on BALB 3T3 murine fibroblast, but also on the human retinal pigment epithelial ARPE19 cell line.

This is our way to ensure that the TB dyes are safe even in a case of inadvertent vitreous staining during cataract surgery – an event that can, although not frequently, occur during anterior segment surgery.

References:

• Parkash et al. Modified 30 G needle trypan blue staining technique under air for a uniform and consistent anterior capsule staining. Clin Ophthalmol 2017; 11:1651-1659
• Melles GRI et al. Trypan blue capsule staining of visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7-9
• ISO 10993-5, 2009. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity. https://www.iso.org/obp/ui/#iso:std:iso:10993:-5:en
• Coelho RP. Inadvertent vitreous staining by trypan blue during phacoemulsification. Vis Pan-Am 2016; 15:26-27.
• Gaur A. Inadvertent vitreous staining. J Cataract Refract Surg 2005; 31:649