Abstract
Purpose :
As a major component of human cornea, collagen type I has been heavily investigated as a biomaterial for corneal tissue engineering. Transparency and stable mechanical properties can be challenging to achieve. The aim of the study is to develop an easy-to-use method to generate a transparent collagen gel for use in corneal research.
Methods :
Lyophilised bovine collagen type I (Sigma) was dissolved in 0.1M acetic acid, neutralised firstly with 5M NaOH to reach pH7.3-7.6, followed by addition of common ions (Na+, K+ ions were tested). The final 5.4 mg/mL solution was examined for precipitation at 5min, 15min, 1 day and up to 6 days. It was then mixed with 0.1% riboflavin, transferred to various types of petri dishes for UV treatment. The final product was subject to manual handling using tweezers. Human corneal epithelial cell line (HCE-T), primary human corneal keratocytes (passage 2 which showed a fibroblast-like morphology) and primary endothelial cells are cultured on the generated collagen gel and cell growth was examined by Olympus light microscope.
Results :
The salt solution test showed that Na+ can maintain the clarity of neutralised collagen solution for up to 6 days at room temperature, but not K+. Combining Na+ and K+ also maintains the clarity of collagen solution for up to 6 days. The collagen solution maintained physiological pH, and formed a solid gel with 15min UV treatment inside a biohazardous hood. Depending on the volume and size of petri dish used, a thickness from 100µm to 2mm can be readily achieved. All generated products showed mechanical strength strong enough to be manipulated by tweezers. HCE-T cells were able to reach confluence on the collagen gel. Both primary human keratocytes (P2) and endothelial cells expanded on the collagen gel. The collagen gel appeared to degrade over time in keratocytes but not in other cell culture systems.
Conclusions :
This method can be used in all standard biological laboratories to generate a clear type I collagen gel that is easy to handle, may support corneal cell growth and allow imaging. It is degradable by keratocytes, although longer culturing time for epithelial and endothelial cells may be needed to examine the interaction between the cells and the gel. Thickness can be adjusted while maintaining mechanical properties implying it can be potentially used as a customisable carrier for corneal cells in cell therapy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.