| Objective To explore a simple and effective preservation method for human corneal stromal lenticules by comparing the biological activity of them preserved in anhydrous glycerol at different temperatures and time. Methods From September 2022 to January 2023, 100 intact corneal stromal lenticules of 50 patients with moderate and high myopia were collected from the Eye Center of Suining Central Hospital from small-incision lenticule extraction(SMILE) surgery. The samples were divided into 10 groups according to random number table method and numbered, with 10 samples in each group. The lenticules were stored in anhydrous glycerol according to different preservation temperatures(4,-20, and -80℃) and time(1-month, 3-month, and 6-month). The preservation lenticules were used as the experimental group(groups 1-9) and fresh lenticules as the control group(group 10). Transmittance, hematoxylin and eosin staining(HE), terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL), immunohistochemistry(IHC), and transmission electron microscopy(TEM) were compared among all groups. Results The overall transmittance of corneal stromal lenticules in all groups was compared, and the transmittance of lenticules in all experimental groups was higher than that in control group, and the differences were statistically significant(P < 0.05). HE staining and TEM images showed that the arrangement of lenticule collagen fibers gradually became loose and disordered with the increase of preservation time and preservation temperature, the surface shape was uneven, and necrotic stromal cells were detected in all groups. The results of TUNEL staining showed a statistical difference in the apoptosis rate of corneal stromal lenticules among each group(F=8.278, P<0.001). The results of IHC showed no positive expression of HLA-DR and CD45 in both the experimental groups and the control group, and a large number of positive expression of HLA-ABC was detected only in the control group, but little or no expression was found in the experimental groups. Conclusions The preservation of human corneal stromal lenticules in anhydrous glycerol at -80℃ is simple and effective, their antigenicity is reduced, and the biological activity of them can be maintained for at least six months. |
