The purpose of this study is to compare the in vitro cytotoxicity tests according to the ISO 10993-5 (2009) standards using direct contact and the test on liquid extracts of compounds previously identified as possible toxic impurities in perfluorocarbon liquids (PFCLs) for use in vitreoretinal surgery. Compounds including perfluorooctanoic acid (PFOA), 1H-perfluorooctane (1H-PFO), 2H-tridecafluoro-2-methylpentane, 1H,2H-octafluorocyclopentane, and 2H,3H-decafluoropentane were analyzed by ¹⁹F NMR before and after extraction using an aqueous solution and tested by both the direct contact and liquid extract tests in L929, BALB 3T3, and ARPE-12 cells. The concentration that reduced in vitro cell viability by 30%, the cytotoxicity concentration threshold (CC₃₀), was determined for each compound. ¹⁹F NMR spectroscopy confirmed the immiscibility of perfluoro-n-octane (PFO) and 1H-PFO and the solubility of PFOA with the extraction vehicle. The other samples reacted with the extraction vehicle, releasing fluoride ions. Using the direct contact test, the CC₃₀ of PFOA, 1H-PFO, 2H-tridecafluoro-2-methylpentane, 1H,2H-octafluorocyclopentane, and 2H,3H-decafluoropentane corresponded to 48 124, 50, 14, 8035, and 46 ppm, respectively. The method on liquid extracts did not detect cytotoxicity in three out of five tested compounds, and CC₃₀ could not be determined. In conclusion, the in vitro cytotoxicity test by direct contact revealed a positive correlation between cell toxicity and the concentration of the tested substance. Conversely, the test on liquid extracts hardly detected the cytotoxicity of toxic impurities in PFCLs. Thus, only the cytotoxicity test by direct contact, according to ISO 10993-5 (2009), is a sensible and reliable method to detect possible cytotoxic impurities in PFCLs to guarantee patient safety.

Purpose: To compare two commercially available staining solutions (MembraneBlue Dual® by D.O.R.C., Netherlands, and TWIN by AL.CHI.MI.A. S.R.L., Italy), in terms of intraoperative handling, staining efficacy and safety, in eyes undergoing surgery for idiopathic epiretinal membrane (ERM).

Methods: In this observational cross-sectional study, the performance of the dyes used during the procedure (cohesion, ERM and internal limiting membrane [ILM] staining efficacy) was scored by the surgeon using a customized questionnaire after 10 procedures with each of the two dyes. Best-corrected visual acuity (BCVA), central foveal thickness (CFT), blue-light fundus autofluorescence (BAF), and microperimetry-determined retinal sensitivity were reviewed preoperatively and then at 1 and 3 months after surgery.

Results: ILM staining efficacy with TWIN was scored 2.89 ± 0.33 by the surgeons, which turned out to be higher than with MembraneBlue Dual® (1.90 ± 0.31, P = 0.0002). The cohesion score was 2.70 ± 0.48 for TWIN and resulted significantly higher than with MembraneBlue Dual® (1.60 ± 0.51, P = 0.0010). BCVA, CFT and retinal sensitivity were similar in the two groups, 1 and 3 months postoperatively (P nonsignificant for all).

Conclusions: Both TWIN and MembraneBlue Dual® dyes showed suitable staining properties and equivalent safety and efficacy profiles, both intra- and postoperatively. The TWIN dye might offer a solution for surgeons who prefer a more cohesive and stable dye.

Background/Aim: We compared the quality of human donor corneas stored in a cold storage medium containing 2.5 μg/ml of amphotericin B (Kerasave, AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy) and Optisol-GS (Bausch & Lomb Inc., Bridgewater, NJ, USA) for 14 days.

This study aimed to assess the cytotoxic effect of low molecular weight components (LMWC) and conventional silicone oils (SOs) 1000 cSt with different degree of purification (raw, intermediate, and purified) using in vitro cytotoxicity tests. Direct contact cytotoxicity tests were performed in BALB 3T3 and human retinal pigment epithelial cells (ARPE-19) using quantitative and qualitative evaluation according to the ISO 10993-5 (2009) standards. Conventional SOs 1000 cSt in form of raw, intermediate (intermediate product obtained during distillation process), and purified SO (final product after distillation) and a concentrate of LMWC (including siloxane chains with molecular weight up to 1557 g/mol) were directly applied to 100% of cell layer area for 24 h. Cell viability was quantified using 3-(4,5-dimethylthiazole-2-yl)-2,5–28 diphenyltetrazolium bromide (MTT) and neutral red uptake assays in ARPE-19 and BALB3T3, respectively. All tested samples, including the concentrate of LMWC, resulted to be not cytotoxic according to ISO 10993-5 in both qualitative and quantitative evaluations. However, the cellular viability was significantly higher in the intermediate and purified SO compared with the raw SO in ARPE-19 cells. No reduction in cell viability was detected by LMWC.

The absence of cytotoxicity was observed for all tested samples in both BALB3T3 and ARPE-19 after 24 h of application. A direct cytotoxic effect is not likely to be involved in the potential complications related to SO and LMWC. Long-term potential adverse effects of SO could be related to the raw material and to different concentrations of LMWC.

Purpose: To compare the performance of Kerasave (AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy) containing 2.5 μg/mL of amphotericin B and Optisol-GS (Bausch & Lomb, Bridgewater, NJ) cold corneal storage media on donor corneas during routine eye bank procedures.

Methods: Forty-four paired donor corneas were preserved after swab sample collection and povidone-iodine decontamination. Right and left corneas were immersed in Kerasave and Optisol-GS, respectively, and stored at 4°C before the initial evaluation. Paired corneas were assigned to processing subgroups for penetrating keratoplasty (n = 20), Descemet stripping automated endothelial keratoplasty (n = 14), or Descemet membrane endothelial keratoplasty (n = 10). Endothelial cell density, central corneal thickness, slit-lamp examination, and endothelial cell damage were assessed at different intervals. Sterility testing was performed on media samples.

Results: At the initial evaluation, after 25.6 ± 3.2 hours of storage, the mean central corneal thickness of all corneas in Kerasave (n = 22) was greater than those in Optisol-GS (n = 22) (571 ± 12 μm vs. 526 ± 10 μm, respectively; P = 0.006). All other metrics were comparable between Kerasave and Optisol-GS in processing subgroups at all time intervals. Corneal swabs were positive in 90% of corneas before decontamination with povidone-iodine. At the initial evaluation, fungal contamination was detected in 24% and 19% of Kerasave and Optisol-GS, respectively. At the final evaluation, no fungi was detected in Kerasave and 1 Optisol-GS sample was positive (P = 0.999).

Conclusions: Metrics of corneas stored in Kerasave and Optisol-GS were comparable. Kerasave might be considered an antifungal-possessing alternative to Optisol-GS.

Abstract: The eye lens is mainly composed of the highly ordered water-soluble (WS) proteins named crystallins. The aggregation and insolubilization of these proteins lead to progressive lens opacification until cataract onset. Although this is a well-known disease, the mechanism of eye lens protein aggregation is not well understood; however, one of the recognized causes of proteins modification is related to the exposure to UV light. For this reason, the spectroscopic properties of WS lens proteins and their stability to UV irradiation have been evaluated by different biophysical methods including synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies. Moreover, dynamic light scattering, gel electrophoresis, transmission electron microscopy, and protein digestion followed by tandem LC–MS/MS analysis were used to study the morphological and structural changes in protein aggregates induced by exposure to UV light. Our results clearly indicated that the exposure to UV radiation modified the protein conformation, inducing a loss of ordered structure and aggregation. Furthermore, we confirmed that these changes were attributable to the generation of reactive oxygen species due to the irradiation of the protein sample. This approach, involving the photodenaturation of proteins, provides a benchmark in high-throughput screening of small molecules suitable to prevent protein denaturation and aggregation.

This study aimed to evaluate the possibility to extend the storage of unused organ-cultured donor corneas. After 28 days of corneal culture in TISSUE-C (AL.CHI.MI.A. S.R.L., Italy) and 5-day storage in transport/deswelling medium CARRY-C (AL.CHI.MI.A. S.R.L., Italy), 25 corneas that were deemed suitable for transplantation were transferred in fresh TISSUE-C at 31 °C for additional 7 days and then in fresh CARRY-C at room temperature for 24 h. Tissues were assessed for endothelial cell density (ECD), endothelial mortality and morphology after the standard and the extended corneal storage.
In addition, the effect of donor age < 85 years and ≥ 85 years on corneal characteristics was assessed. After the extended storage, 6 out of 25 tested corneas (24%) showed ECD values below the acceptance limit (< 2000 cells/mm2). 19 corneas (76%) were still suitable for transplantation and showed a 5.9% loss in ECD, which was not statistically significant (P = 0.0949) compared to standard storage period. The two donor age groups did not show statistically significant differences in any tested parameter, although a trend for lower ECD and higher mortality in Descemet's folds after standard storage was observed in the ≥ 85 age donor group. Thus, the attempt of the current study to provide new sight-restorative options for unused tissues and increasing the availability of corneas in case of shortage gave encouraging results. Although a higher vulnerability of corneas from very old donors could not be statistically demonstrated in the present study, higher sample size could be required for prolonging the shelf life of these tissues.