Effects of ranibizumab (Lucentis®) and bevacizumab (Avastin®) on human corneal endothelial cells

All corneas used in this study were taken under the legal responsibility of the Department of Ophthalmology, Lions Eye Bank (Federal authority Paul-Ehrlich-Institut: PEI.G.11601.01.1) within the facilities of the University Hospital Heidelberg. Forty-six human donor corneas were included in the study. They were not suitable for transplantation for one or more of the following reasons:

Individual exclusion criteria for each donor cornea are summarized in Table 1.

In the case of poor quality of the corneas (endothelial cell count < 500 cells per mm², endothelial cell necrosis > 50%) or contamination of the culture medium, the respective test part was stopped and repeated with a new cornea.

Written consent of relatives to use the tissue for scientific purposes was present for all corneas used in this study.

Corneas included in this study were randomly assigned to one of 6 study groups or the control group and cultured for 1, 3, 7 or 14 days. Three corneas were used per time point in each group. The corneas were cut in half (2 corneas were quartered). One half was used in the control group and the other half in one of the three study groups. All corneal segments were cultivated in 48-well plates containing 2 ml of respective liquid (BSS, medium and agent if any). Study groups contained different concentrations of ranibizumab (Lucentis®) or bevacizumab (Avastin®) (group 1: 250 μg / ml, group 2: 25 μg / ml, group 3: 2.5 μg / ml). Study agents were diluted in balanced salt solution (BSS). One milliliter of each dilution was added to 1 ml culture medium (Cornea Max®, Eurobio). In contrast, corneas of the control group were cultured in 1 ml culture medium supplemented with 1 ml of undiluted BSS solution, only.

Corneal endothelial cells were qualitatively and quantitatively evaluated for abnormalities at baseline using a specular microscope (EM-3000, Tomey Corporation, Aichi, Japan). Only corneas with a coefficient of variation (CV: defined as standard deviation of the cell size expressed as percentage divided by the mean cell size) below 40 indicating low polymegathism levels and a hexagonality over 50% were used for culturing. Before and after 1, 3, 7 or 14 days of cultivation all corneas were stained with 0.06% trypan blue (VisionBlue, D.O.R.C., Zuidland, The Netherlands) for evaluation of endothelial cell viability. In detail, two drops of 0.06% sterile isotonic trypan blue solution were applied to the corneal specimen’s endothelial cells facing up. After incubation for 60 s, corneas were rinsed with balanced salt solution. Subsequently endothelial cell counting was performed manually by light microscopy (Olympus CKX41, Olympus Europa Se & Co Kg, Hamburg, Germany). We performed cell counting in real time with the support of the NAVIS software (NIDEK Technologies Srl, Padova, Italy). Endothelial cells were counted within a central rectangle in L-configuration. The endothelial cell count of the vivid cells was calculated as endothelial cells/mm² (ECD).

In addition, morphologic parameters such as endothelial cell morphology (cell size and shape) and cell viability were evaluated at each time point. All corneas were dehydrated for 24 h in a dextran-containing culture medium (Cornea Jet®, Eurobio) before cell counting.

Endothelial cells were evaluated for potential cell damage by determination of LDH activity within the culture medium. LDH is secreted by damaged endothelial cells. The enzyme is capable of converting the pale yellow test reagent into red (tetrazolium) which is colorimetrically detectable by a photometer at a wavelength of 490 nm. The measured light intensity is directly proportional to the amount of LDH released into the supernatant of the culture medium [17]. In detail, 96-well microtiter culture plates were sensitized by incubating with 100 μLVEGF at a concentration of 0.15 mg/L in coating buffer (1 mol/L carbonate–bicarbonate buffer, pH 9.6) overnight at + 4 °C. Plates were washed 4-times with PBS containing 0.05% Tween 20. The remaining unspecific protein-binding sites were blocked by incubation with 200 μL blocking buffer (PBS containing 1% BSA) for 2 h at room temperature. Plates were subsequently washed as follows: After adding 100 μL of 1:100 diluted standards, quality controls (QCs), or test samples plates were incubated for 1 h at 37 °C in an iEMS incubator-shaker (Labsystems, Helsinki, Finland) with a subsequent rinsing. Then, 100 μL of conjugated-anti IgG antibodies diluted in 1% PBS-BSA was added to each well for 1 h at room temperature and subsequently rinsed. Finally, 100 μL o-Phenylenediamine (OPD) (prepared by dissolving tablet sets in 20 mL distilled water) was added and the reaction was allowed to develop at room temperature in the dark. The color reaction was stopped by adding 50 μL of 2 mol/L sulfuric acid to each well. Reading was performed at two wavelengths (492 and 620 nm) using an iEMS ELISA plate reader (Labsystems). The plate background absorbance at 620 nm was subtracted from the absorbance at 492 nm.

Corneas showed variable endothelial cell findings in the study and control groups. Endothelial cell counts did not shown any statistical significant differences after treatment with ranibizumab (Lucentis®) and bevacizumab (Avastin®). The overall cell loss and the corneal area were comparable within study groups (Table 2).

Figure 1 illustrates hexagonal shaped human endothelial corneal cells at a magnification of 10× and 20× obtained by light microscopy. No major differences in regard to cell morphology (size, shape) and viability were detected among the study groups and the controls over time (Fig. 1). No statistical significant endothelial cell loss occurred during incubation with ranibizumab (Lucentis®) and bevacizumab (Avastin®) compared to the control group. A significant natural loss of cells as usually seen during prolonged incubation in vitro is illustrated in the control groups (Fig. 2).