During vitreoretinal procedures, the visualization of transparent structures such as the vitreous cortex, ERM, and ILM can be a challenge; however, proper staining of these tissues can increase the efficiency of a non-traumatic removal.

Vitreoretinal surgery has rapidly evolved over the past 10 years by the development of new techniques and improvement in vitrectomy machines. Moreover, a big effort has been dedicated to vital dyes formulation to enhance visualization of tissues and facilitate membranes peeling: this leads to a great reduction of complications such as iatrogenic breaks, retinal damages and, eventually, minimal postoperative discomfort for patients.

To guarantee all of this, the dye for the posterior segment of the eye must fulfil some important requirements. First of all, it has to be dense enough to easily lie down and spread onto the membrane without causing any damage because of the excessive weight. It follows that it has to selectively adhere to and stain the posterior segment eye membranes while being soluble in water to be easily washable from the tissues at the end of surgery. Finally, and most importantly, the vital dye must not be toxic for the ocular tissues.

In recent decades has strongly emerged the theme of the safety of vital dyes.

For instance, the indocyanine green has been abandoned, owing to the in vitro and in-vivo toxicity concerns. Instead, the trypan blue-based formulations tailored for use in posterior segment eye surgery are deemed to be safe and are at present unanimously considered the gold standard for ERM surgery.

In our research laboratories, the method of choice to test the safety of the vital dyes for the posterior eye segment surgery has always been a quantitatively and qualitatively validated in vitro cytotoxicity test by direct contact method according to ISO 10993-5.

This testing method has the following advantages:

• it is highly sensitive and able to detect even weak cytotoxicity;
• it is performed not only on the mouse fibroblast BALB 3T3 but also the human retina-derived ARPE-19 cell line: in this way, it is possible to meet the recommendations of the ISO standards stating that the in vitro cytotoxicity test method of a product for human use should be selected to represent, as much as possible, the situation in which the product is to be used;
• although during surgery the dye must remain in contact with the ocular tissues for a very short time (usually few seconds), the cytotoxicity test of the testing product is performed for a contact time as long as 24 hours.

References:

• Mariotti C. et al. Negative staining of the vitreous with the use of vital dyes. Eur J Pharmacol 2018;28(1):117-118
• Iuliano L. et al. Idiopathic epiretinal membrane surgery: safety, efficacy and patient-related outcomes. Clin Ophthalmol 2019; 13:1253:1265
• 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

In the United States of America, corneal tissues from deceased donors are stored in cold storage fluids that do not contain antimycotics, and undergo tissue processing for the new lamellar techniques as soon as possible after tissue procurement; therefore, in case of donor-derived infections in recipients, the only option is the treatment of the transplanted patient.

Whether the increased trend of post-keratoplasty fungal infection during the recent years in the USA might be due to the absence of antimycotic agents in the cold media or to an easier infiltration of infectious organisms in the lamellar interspaces during tissue processing, the possibility of supplementing cold storage media with antimycotic agents is attracting increasing attention as a possible solution to increase recipients’ safety.

A recent in vitro time-kill study conducted by the Alchilife R&D team on a new medium containing Amphotericin B (2.5 µg/ml) and intended for cold storage of explanted corneas demonstrated the antimycotic efficacy of the medium – despite the low temperature – against fungal strains commonly associated with keratitis and endophthalmitis, including C. albicans, C. glabrata, and C. tropicalis.

In parallel with the time-kill study, the Amphotericin B Minimum Inhibitory Concentration (MIC) for these Candida strains was also determined. By using the Etest strips containing a concentration of Amphotericin B varying from 0.002 to 32 µg/ml that was placed over each yeast strains, individually cultured on agar plates, for 24 and 48 hours, the MICs were found to vary between 0.25 and > 1 µg/ml.

These results suggested that the differences in the killing efficacy among strains may correspond to the different susceptibility to Amphotericin B according to the MICs; not less importantly, they also confirmed the good antifungal efficacy of Amphotericin B in the new product for cold storage, since the 2.5 µg/ml concentration is well above the so-determined MIC values.

	MIC (1 µg/ml)
C. Parapsilosis ATCC 90018	0,25-0,38
C. Albicans UCL	≥ 1
C. Albicans ATCC 10231	0,19-0,25
C. Glabrata BPEI	0,5
C. Albicans ATCC MYA 2876	0,25
C. Tropicalis ATCC 750	0,38
C. Albicans ATCC 90028	0,25

References:

• Aldave AJ et al. Report of the Eye Bank Association of America medical advisory board subcommittee on fungal infection after corneal transplantation. Cornea 2013; 32:149–154
• Giurgola L. et al. Antimycotic efficacy and safety of a new cold corneal storage medium by time-kill and toxicity studies. Cornea 2019; 38(19):1413-1321.
• Fontana L. et al. Interface infectious keratitis after anterior and posterior lamellar keratoplasty. Clinical features and treatment strategies. A review. Br J Ophthalmol 2018; 0:1–8

The new lamellar techniques for selective replacement of diseased layers of the cornea, which are now largely preferred over penetrating keratoplasty, have been related, at least to some extent, to an increased trend of post keratoplasty fungal infection in the USA. Two factors have been suggested as risk factors: first, these techniques might enhance infiltration of infectious organisms in the lamellar interspace and second, but no less importantly, the cold storage media commonly used in the US do not contain antimycotic agents.

Recently, our R&D team has concluded the studies to assess the in vitro killing efficacy of Kerasave against various Candida species commonly associated with keratitis and endophthalmitis, including C. albicans, C. glabrata, and C. tropicalis.

Kerasave, a new storage medium developed by Alchimia Srl (Italy), which is supplied with an Amphotericin B tablet to be added to the medium before use (thus giving a final concentration 2.5 µg/ml), is intended for the storage of donor corneas at 4°C for up to 14 days.

Kerasave showed high antifungal efficacy against susceptible fungal strains at 4°C, in particular against C. albicans and C. tropicalis. Already within 5 days of incubation, four of the six strains contaminated with 10 CFU/ml demonstrated a significant ≥ 3 Log10 reduction on average as compared to controls (p < 0.01).

The usefulness of adding an antimycotic agent to cold storage media is still a matter of debate in the USA, mainly because of some concerns about efficacy and safety issues. The results of this study help to dispel all these doubts, as:

• to obtain truly reliable results on the antimycotic efficacy, all liquid samples before plating were treated with RESEP (Alchimia srl), a syringe-like, patented, CE marked device, which contains a resin mixture for the removal of antimicrobials from samples to avoid false-negative results during the microbiology assay;
• the absence of cytotoxicity, irritation, or sensitization of Kerasave media were assessed by cytotoxicity, topical ocular irritation, and delayed hypersensitivity tests according to the ISO 10993-5 and 10993-10.

Besides, it should be reminded that transplanting the donor graft soon after tissue recovery (as commonly performed in the USA) does not leave the time for microbiological testing before surgery; in case of post kerapoplasty infection, the only option is the recipient treatment.

Therefore, the results of the time-kill studies add further data in favour of the antimycotic safety and efficacy and safety of supplementation of cold corneal storage media with Amphotericin B.

References:

• Giurgola l. et al. Antimycotic efficacy and safety of a new cold corneal storage medium by time-kill and toxicity studies. Cornea 2019, July 22, publish ahead of print.Skenderi Z. et al.
• Increased sensitivity of microbiological testing of cornea organ culture medium by additional resin treatment.BMJ Open Ophthalmol. 2018;3:e000173
• Fontana L. et al. Interface infectious keratitis after anterior and posterior lamellar keratoplasty. Clinical features and treatment strategies. A review. Br J Ophthalmol. 2018; 0:1–8.
• ISO 10993-5:2009 biological evaluation of medical devices part 5: tests for in vitro cytotoxicity. Available at: https://www.iso.org/obp/ui/ #iso:std:iso:10993:-5:en. Accessed July 6, 2019.
• ISO 10993-10:2010 biological evaluation of medical devices part 10: tests for irritation and skin sensitization. August 1, 2018. Available at: https://www.iso.org/obp/ui/”\l”iso:std:iso:10993:-10:ed-3:v1:en. Ac- cessed July 6, 2019

The Alchilife R& team, in collaboration with Mario Romano, MD, is one of the first laboratories that validated a direct contact method for the cytotoxicity assay performed according to the ISO 10993-5 standard on perfluorocarbons (PFCLs) for ophthalmic surgery.

Selecting a truly reliable method for the cytotoxicity evaluation of PFLCs is the key strategy to avoid serious concerns about patients’ safety. The unfortunate past has proved that the “extract dilution” method, despite being mentioned among the acceptable tests by the ISO 10993-5 standard, is not a reliable assay for medical devices containing volatile substances. Conversely, the direct contact test – which has always been the method of choice used in the Alchilife laboratories, is highly sensitive, able to detect even low cytotoxicity, and is gaining increased interest in the field of PFCL safety.

By exploiting the high-density properties of liquid PFCLs, the team of Alchilife researchers could ensure the effective direct contact between the tested perfluorocarbon and the cell substrate (i.e. the ARPE-19 and BALB 3T3 cell layer) for the whole duration of the test by careful injecting the perfluorocarbon liquid with the tip of the pipette immersed in the medium right above the cell lines: this creates a single dense bubble that gently lays on the cells due to the force of gravity while avoiding excessive pressure on the cell layer because of the sample heaviness. This step simulates what happens in the eye during the clinical use. The so-formed dense bubble avoids any sample evaporation or loss of contact of PFCL with the cells. This method allows to overcome the problem of PFCL insolubility in water, which prevents the sample from diluting into the medium – the latter may be one of the factors that impairs the reliability of the extraction method for testing the cytotoxicity of PFCL.

With the approach by Alchilife various amount of contact areas and contact times (up to 24 hours) can be successfully tested. As a result of this study, whose preliminary results were presented by Dr Romano at the 18^th Euretina Congress in Vienna, the direct contact cytotoxicity test of PFCLs, which warrants covering of 59% of the cell surface and contact time of 24 h according to ISO 10993-5, was validated quantitatively and qualitatively on ARPE19 and BALB 3T3 cell lines.

References:

• 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 (accessed 20 July 2019)
• Li et al. Study on the in vitro cytotoxicity testing of medical devices(Review). Biomed Rep 2015;3(5):617-620.
• Srivastava GK et al. Comparison between direct contact and extract exposure methods for PFO cytotoxity evaluation. Sci Rep 2018;8(1):1425.
• Romano M. Perfluorocarbons for intraocular use: cytotoxicity test validation according to the ISO 10993-5. Presentation at Euretina 2018
• Romano MR et al. Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo. Transl Vis Sci Technol 2019; 8(5):24.