The EYECULTURE consortium, funded by the German Federal Ministry of Education and Research (BMBF) under grant numbers 161A574C and 031A574C, carried out a seven‑year project (01 August 2015 – 31 July 2022) to develop an organotypic long‑term culture system for adult retinal tissue on titanium dioxide nanotube scaffolds (NTS). The project’s third sub‑task, led by Prof. Dr. Stefan G. Mayr of the University of Leipzig, focused on optimizing scaffold production, establishing a robust retinal culture, and characterizing mechanical and pathological changes in the tissue.

Electrochemical anodization was used to tailor the diameter and wall thickness of the TiO₂ nanotubes, producing highly superhydrophilic surfaces that immediately wet with culture medium. The scaffolds were cleaned by a combination of chemical and physical methods, allowing repeated sterilization and reuse. Production capacity was increased by a factor of five, enabling the generation of large batches of uniform scaffolds for parallel experiments. Mechanical loss modules were measured for the scaffolds at loading forces of 0.05 N (samples A & B) and 0.1 N (samples C & D), providing baseline data for subsequent tissue‑scaffold interactions.

Porcine retinal explants, obtained fresh from a local slaughterhouse after euthanasia and exsanguination, were seeded onto the NTS and maintained in organotypic culture for extended periods. Laser scanning microscopy and a custom tissue stretcher were employed to monitor the stretching behavior of retinal nerve fibers and the endfeet of Müller glial cells. This approach allowed the first detailed observation of retinal delamination under mechanical load. Environmental scanning electron microscopy (ESEM) revealed that the retinal tissue retained its native morphology, cellular viability, and functional markers over the culture period, outperforming conventional culture methods that lack a defined nanotubular architecture.

A bioreactor was designed to accommodate eye cups, providing controlled perfusion and mechanical stimulation that mimics physiological conditions. Within this system, the team simulated pathological states such as retinal stretching and biomechanical stress, correlating observed tissue damage with measured mechanical properties. The correlation study (AP7) linked specific pathological changes to alterations in the mechanical response of the retina, offering insights into disease mechanisms and potential therapeutic interventions.

The project also addressed the 3R principle by replacing in‑vivo animal experiments with ex‑vivo tissue cultures derived from slaughterhouse waste, thereby reducing animal use and improving ethical compliance. Collaboration with the veterinary department at Leipzig, the Leibniz Institute for Surface Modification, and the slaughterhouse Emil Färber GmbH ensured a multidisciplinary approach, combining expertise in surface physics, tissue engineering, and animal science.

Overall, the EYECULTURE sub‑project demonstrated that TiO₂ nanotube scaffolds can support long‑term, organotypic retinal cultures that preserve physiological structure and function. The mechanical characterization and pathological simulations provide a platform for studying neurodegenerative diseases and testing pharmaceuticals in a human‑relevant, ethically responsible in‑vitro system.