Improving Endothelial Explant Tissue Culture by Novel Thermoresponsive Cell Culture System

ABSTRACT

Aim

Studying cell migration of corneal endothelial cells in vitro is challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, we report a thermoresponsive culture technique designed to maintain cellular viability, and to reduce tissue handling in order to analyze in vitro endothelial cell migration from corneal grafts.

Materials and Methods

As a test tissue, fifteen Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts were used that were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2–3 weeks in a humidified atmosphere at 37°C and 5% CO₂.

Results

All grafts could be successfully cultured inside the thermoresponsive polymer solution for periods of up to 21 days. Using this system, cell migration could be assessed by light microscopy at fixed time intervals. At the end of the culture period, the gel could be removed from all grafts and immunohistochemistry analysis showed that endothelial cells were able to maintain confluence, viability, and junctional integrity. Some problems were encountered when using the thermoresponsive cell culture system. These were mostly structural inconsistencies during the sol-to-gel transition phase that resulted in the formation of tiny bubbles in the matrix. Additionally, areas with different viscosity resulted in optical distortions showing up as folds throughout the matrix which can persist even after several cycles of culture medium exchange. These effects had impact on the imaging quality but did not affect the viability of the explant tissue.

Conclusion

This study proves that temperature-reversible hydrogel is a very useful matrix for studying in vitro corneal endothelial cell migration from explant grafts and allows for subsequent biological investigation after gel removal.

KEYWORDS:

• Polymer matrix
• gel matrix
• corneal transplantation
• endothelial cell culture

Disclosure of interest

Dr. Melles is a consultant for DORC International and SurgiCube International. Alina Miron, Daniele Spinozzi, Jessica Lie, Silke Oellerich and Sorcha Ni Dhubhghaill declare that they have no conflict of interest.

Ethical statement

The study was carried out following the tenets of the declaration of Helsinki and the Barcelona Principles.

Financial support

None

Additional information

Funding

None