An extensive literature review was performed, and previous reported techniques
17 18 were modified as follows. In this study, a human LEC line was used. The cell line had been immortalized with the large T-antigen of SV40 and the epithelial nature of the lens cells was confirmed in our laboratory with a pancytokeratin antibody (clone C-11; Sigma-Aldrich, Poole, UK;
Fig. 1 ).
The cells were grown to confluence in T75 flasks containing DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen, Paisley, Scotland, UK) supplemented with 10% fetal calf serum and 5 mg/mL Primocin (InvivoGen, San Diego, CA). At confluence, the cells were washed in phosphate-buffered saline (PBS), trypsinized, centrifuged at 1600 rpm, and resuspended in medium to give a cell concentration of 6 × 104/mL. For each experiment, three IOLs of the three materials were used. The IOLs chosen were silicone (SI40NB Phacoflex; Advanced Medical Optics [AMO], Santa Ana, CA), PMMA (Optical Innovations International [OII], Inc., Ontario, CA), and acrylic (Sensar AR40e; AMO). The dioptric power was matched for each IOL type, and the lenses were placed in a 12-well multiplate. A cell suspension (20 μL) was placed on the surface of each IOL and left to adhere for 2 hours at 37°C. After this time, 1 mL of DMEM with fetal calf serum was gently added to the well and the cells incubated at 37°C for 24 hours. After incubation, the lenses were examined with a phase-contrast inverted microscope (Nikon, Tokyo, Japan). With the aid of an eyepiece graticule, the total number of attached LECs per IOL was recorded. This procedure was performed by one of the authors (GM) who was blinded to the lens type. The counts were then repeated to ensure reproducibility by another of the authors (CAC). The cell counts were comparable between authors. Each experiment was duplicated (18 IOLs) and then repeated with different cell concentrations. In total, cell concentrations of 4 × 104/mL, 6 × 104/mL, and 7.5 × 104/mL were used (18 IOLs at each concentration), to test reproducibility at each dilution.
A second experiment was performed with IOLs coated with fibronectin (from bovine plasma, Sigma-Aldrich). Six lenses of each material were coated with fibronectin (total number, 18) and these coated IOLs were then seeded and incubated as before with LECs at the chosen cell concentration of 6 × 104/mL. The total number of attached LECs per IOL was again recorded and the results compared with an equal number of matched uncoated lenses (n = 18). The technique for coating the lenses was as follows; Fibronectin (from bovine plasma; Sigma-Aldrich) was diluted with sterile distilled water to give a working concentration of 2 μg/cm2. Fibronectin solution (300 μL) was added to each well of a 12-well multiplate, and an IOL was then placed in each well. The lens was allowed to incubate for 1 hour and then removed and placed in a fresh multiplate to dry at room temperature. Using the cell concentration of 6 × 104/mL the lenses were seeded and incubated as before. Statistical analysis was performed on computer (SPSS ver. 12.0.1; SPSS, Chicago, IL). Before analysis, all cell count data was log transformed. Initially, groups were analyzed with analysis of variance. Subsequent post hoc analysis was performed with the Student-Newman-Keuls test. P < 0.05 indicated statistical significance.