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.