Corneal lenticule storage before reimplantation

Abstract

Purpose: To explore the optimal lenticule storage conditions that maintain lenticule integrity and clarity.

Methods: A total of 99 lenticules obtained from myopic patients undergoing small incision lenticule extraction (SMILE) were divided into four combinations for short-term storage conditions: PBS, Dulbecco's Modified Eagle's Medium (DMEM), Optisol GS, or anhydrous glycerol. Two thirds of the lenticules were further stored for 4 weeks under eight different conditions. Clarity evaluation with transmittance measurements, cell-death assays with terminal deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL), collagen fibril spacing and necrotic response assessed with transmission electron microscopy (TEM), and immunohistochemistry analysis for human leukocyte antigens (HLAs) and CD45 for immunogenicity, and matrix metalloproteinase (MMP)-2 for keratocyte response, were undertaken at baseline, 48 h (short term), and 4 weeks (long term).

Results: The TUNEL and immunogenicity results were comparable among the groups. The mean percentage of TUNEL-positive cells across all groups was 24.3% ± 11.8% and 62.9% ± 20.7% at the 48 h and 4 week time points, respectively. HLA-ABC+, HLA-DR+, and CD45+ cells were extremely rare, and MMP-2 expression ranged from non-detectable to minimal, under all conditions at all time points. Transmittance at 4 weeks was significantly different among groups with the greatest maintenance of clarity seen in the lenticules stored initially in DMEM at 4 °C for 48 h followed by cryopreservation in serum-free medium or glycerol at 4 °C followed by storage at room temperature. At TEM analysis at 4 weeks, the lenticules cryopreserved in liquid nitrogen, regardless of storage solutions, had significantly narrower inter-fibrillar distance than controls, while glycerol-preserved lenticules, at either room temperature or -80 °C, maintained the inter-fibrillar distance.

Conclusions: Clarity, structural integrity, and low immunogenicity under various conditions, at 4 °C or room temperature for short-term storage, offer encouragement for lenticule storage. It can be undertaken without access to s specialized and potentially expensive laboratory setup at least within the first 48 h before transportation to larger facilities for long-term storage.

Figure 1

Flow diagram showing the experimental design. IHC: immunohistochemistry, PBS: phosphate buffered solution; DMEM: Dulbecco’s Modified Eagle’s Medium; SFM: serum-free medium; FBS: fetal bovine serum; CS: chondroitin sulfate; DMSO: dimethyl sulfoxide; RT: room temperature; LN₂: liquid nitrogen; TEM: transmission electron microscopy.

Figure 2

Spectrum-wide transmittance trends and mean transmittance over time in different groups. A: Representative color photographs of lenticules at different time points. B: Mean transmittance over the entire spectrum of visible light for the eight groups across all time points.

Figure 3

TUNEL cell-death detection. A: Representative images of lenticules stained with terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) after 48 h storage for different groups. B: Representative images of lenticules stained with TUNEL after 4 weeks of storage for different groups. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Original magnification: 200X; scale bar = 100 μm. C: Mean percentage of TUNEL-positive cells in all groups after 48 h. D: Mean percentage of TUNEL-positive in all groups after 4 weeks of storage. No statistically significant inter-group differences were found at each time point.

Figure 4

Transmission electron microscopy of the stromal lenticule showing transverse section of collagen fibrils following 4 weeks of storage (A–H: groups 1–8; I: control). Scale bar = 0.2 μm.

Figure 5

Transmission electron micrographs of necrotic keratocytes in the experimental and control groups following 4 weeks of storage. In the control lenticule, the chromatin in the cell nucleus was condensed, whereas in the lenticules in groups 1–8, there was irregular clumping of chromatin, cytoplasmic vacuoles, and swelling nuclei. These necrotic keratocytes were less observed in groups 7 and 8. Scale bar = 2 μm.

Figure 6

Expression of MMP-2 at the central part of lenticules at 48 h and 4 weeks. There was minimal expression of matrix metalloproteinase (MMP)-2 in groups 4 and 6–8, and the expression was not detectable in the other groups. Negative control ((-) C): fresh lenticules; positive control ((+) C): primary human stromal fibroblasts. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Original magnification: 200X; scale bar = 50 μm.