目的：探究用全飞秒激光小切口微透镜摘除术(Smile)来源的角膜基质透镜构建组织工程角膜基质支架的可行性及最佳保存条件。

方法：从Smile来源的角膜基质透镜中分离培养人角膜基质细胞(HCFs)，采用MTT法检测人纤维蛋白粘合剂(FS)对细胞的毒性作用； 将FS粘贴双层角膜基质透镜构建的双层透镜支架分别置于不同介质(无水甘油、透明质酸钠、胎牛血清、模拟湿房环境)及不同温度(常温、4℃、-20℃)中保存，对比支架透明度及硬度。

结果：MTT检测结果显示，FS作用0～72h，HCFs与正常培养细胞具有相似的增殖趋势，细胞毒性评级为0～1级，相对生存率均超过90%。FS粘贴的双层基质透镜支架表面平整、贴合紧密、透明度良好且硬度适宜。4℃保存14d后，无水甘油中9枚支架复水后均未出现开裂情况，透明度良好； 透明质酸钠中9枚支架中3枚出现开裂，剩余6枚完整，透明度尚可； 模拟湿房环境中9枚支架均无开裂现象，但皱缩严重； 胎牛血清中9枚支架全部开裂，水肿严重。保存14d后，常温无水甘油中的15枚支架其中2枚保持无色透明，5枚轻微变黄但透明度尚可，8枚严重变黄且透明度明显降低； 4℃无水甘油中的15枚支架其中5枚无色透明，10枚轻微变黄且透明度良好； -20℃无水甘油中的15枚支架均保持无色透明状态，未出现变黄情况。

结论：FS是一种安全无毒的生物胶，可利用FS粘贴Smile来源的角膜基质透镜构建稳定性好、透明度高且硬度适宜的角膜基质支架，且-20℃无水甘油是角膜基质透镜支架的较佳保存条件。

METHODS: Constructing a bilayer lens by adhering together two corneal stromal lenses with human fibrin sealant(FS). Human corneal fibroblasts were isolated and cultured from Smile derived corneal stromal lenses in vitro, and the toxicity of FS on human corneal fibroblasts was detected by MTT method. The bilayer lenses were then placed in anhydrous glycerin, sodium hyaluronate eye drops, a simulated wet room environment and fetal bovine serum groups respectively, and stored at 4℃ for 14d. The transparency, hardness and stability of the scaffolds were then compared. Afterwards, the bilayer lens scaffolds were stored in anhydrous glycerin at room temperature, 4℃ and -20℃. After 14d of preservation, the diverse effects of temperature on the transparency and hardness of the scaffolds were compared.

RESULTS: MTT results showed that the cells of the experimental group and the control group had similar proliferation trend within 0-72h. The cytotoxicity rating of the experimental group was 0 at 36-48h and 1 at 24h and 60-72h. The relative survival rate of the cells within 0-72h was over 90%. FS-bonded bilayer lens scaffold had a smooth surface, close bonding, good transparency and suitable hardness. After 14d of storage at 4℃, none of the nine bilayer lens scaffolds in the anhydrous glycerol group showed signs of cracking cracking after rehydration, and their transparency was good. In the sodium hyaluronate group, three of the nine scaffolds cracked and the remaining six were still intact. In the simulated wet room environment group, none of the 9 scaffolds cracked, but there were different degrees of shrinkage, their surface was rough and transparency was lower. In the fetal bovine serum group, all the 9 stents were cracked, and the single corneal stromal lens was soft and edema was serious. Out of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at room temperature, 2 remained colourless and transparent, 5 slightly yellowed but still remained transparent, 8 yellowed substantially with a significant reduction in transparency. Out of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at 4℃, 5 remained colourless and transparent, and 10 slightly yellowed while remaining transparent. Of the 15 bilayer lens scaffolds preserved in anhydrous glycerol at -20℃, none of the scaffolds yellowed, therefore, remaining colourless and transparent.

CONCLUSION: FS is a safe and non-toxic bio-gel. It can be used to glue Smile-derived corneal stromal lenses to construct corneal stromal scaffolds with good stability, high transparency and suitable hardness. Anhydrous glycerol at -20℃ is the best preservation condition for corneal stromal lens scaffolds.