ALCs are ideal biological materials that have transparent, soft and smooth basement membranes with good elasticity, hardness and toughness [1, 2, 20, 21]. Recent reports have demonstrated satisfactory therapeutic effects after autologous ALCs were transplanted to corneal ulcers, scleral flaps and macular holes [8]. LECs adhering to ALCs have the ability to proliferate, migrate and undergo fibrosis, however, which can induce complications, including inflammation, Elschnig’s pearl and fibrosis [16, 17]. LECs on ALCs should therefore be removed before transplantation to prevent related complications.

In this study, four drugs and solutions that are commonly used in clinical practice were selected for the decellularization of ALCs. We found that 2% lidocaine, 10% sodium chloride, 50% glucose and sterile water could induce the removal of LECs from ALCs at 37 °C. As the treatment time increased, the decellularization effects on ALCs increased. The decellularization effect of 10% sodium chloride solution and sterile water was significantly better than that of 2% lidocaine and 50% glucose. After treatment for 10 min, 10% sodium chloride and sterile water induced absolute decellularization of the ALCs, whereas few LECs remained on the ALCs in the 2% lidocaine-treated group and the 50% glucose-treated group.

An ideal method of tissue decellularization removes organelles and cell nuclei effectively without affecting the composition, biological activity and physical properties of tissues or organs [22, 23]. Previous studies have shown that residual cells or cell components can recruit classically activated macrophages, which then induce inflammation, delay tissue reconstruction and accelerate fibroblastic proliferation [24]. Several decellularization methods, including mechanical, chemical and enzymatic methods, are in current use. Mechanical methods, which include physical erasing, ultrasound and freeze-thaw cycles, cannot completely eliminate cells alone [25,26,27,28,29]. Enzymatic methods, such as trypsin, nuclease and enzymatic-based methods, take more time, impair collagen tissues and increase the risk of enzymatic residue [26,27,28,29]. Chemical methods involve the treatment with acid base, ionic detergent, hypertonic or hypotonic solutions [26,27,28,29]. Chemical reagents, such as acids, alkalis and ionic detergents, denature protein, destroy collagen tissue and remain residual reagents, and the material needs to be sterilized before clinical application [26,27,28,29]. Hypertonic and hypotonic solutions can alter the intracellular osmotic pressure and induce cell loosening and rupture. While performing decellularization using hypertonic or hypotonic solutions is easy and does not damage the morphological structure of the tissue, this method takes a long time and has unsatisfactory effects on deep tissue [26,27,28,29]. Because LECs are located on the surface of ALCs, hypertonic or hypotonic solutions could effectively remove LECs within a short time.

In this study, we selected several sterile, safe and reliable reagents, including 2% lidocaine, 10% sodium chloride, 50% glucose and sterile water for injection, which are commonly used in the clinic. Decellularized ALCs may therefore be used directly without additional sterilization during surgery. Lidocaine, a moderately effective anaesthetic of the aminoacylamide type, could not only cause cell degeneration, but also reduce cell adhesion, inducing decellularization [30]. Our results indicated that 2% lidocaine promoted the removal of LECs from ALCs; however, few LECs remained on the surface of ALCs after treatment for 10 min. 10% Sodium chloride (osmotic pressure: 3.42 mol/L) and 50% glucose (osmotic pressure: 2.78 mol/L) are hypertonic agents that can destroy DNA-protein connections, make cytomembranes and induce degeneration [31]. Sterile water is a hypotonic agent that can cause cell to swell and lyse [31]. Because LECs are a single layer of epithelial cells covering the surface of ALCs, hypertonic and hypotonic agents show great advantages in the decellularization of ALCs. Indeed, we found that both hypertonic and hypotonic agents could effectively induce decellularization of ALCs. The decellularization effect of 10% sodium chloride and sterile water was better than that of 50% glucose. The reason is likely related to the rapid movement of glucose through cytomembranes into the cytoplasm.

Previous studies have shown that moderate temperatures at 43 °C can induce a heat shock response in lenses [32]. We explored the decellularization effects of 10% sodium chloride and sterile water at 43 °C. We found that moderate temperature at 43 °C induced degeneration and death of LECs, which partly adhered to ALCs. The margins of the ALCs at 43 °C were curled and difficult to unfold.

In addition, the results of the hydroxyproline assay indicated that 10% sodium chloride preserved a greater level of collagen than did the sterile water. The findings indicated that 10% sodium chloride and sterile water could maintain the integrity of the lens capsular structure and support cell attachment.

In conclusion, this study identified two optimal decellularization methods for acellular ALCs. The results showed that 10% sodium chloride and sterile water are currently suitable for the decellularization of human ALCs. The decellularization process is rapid, safe and effective. The cells can adhere well to the surface of the prepared ALCs in vitro. Thus, the findings suggest that the acellular ALCs can be promising scaffolds for ocular cells. Notably, acellular ALCs do not require resterilization and may be directly used for autologous lens capsule transplantation in clinical applications.