Development of methods to study diffusion in hydrogels for cornea-on-a-chip

Details

Supervisors

Dupont-Gillain, Christine C.

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

According to the World Health Organisation, 2.2 billion people worldwide suffer from visual impairment, 1 billion of which could have been prevented or treated. The ageing of the population is exacerbating this problem, increasing the need to develop effective ocular treatments. Currently, the models used in preclinical diffusion and toxicity tests for ocular drugs raise ethical concerns relating to animal experimentation and reliability issues due to the complexity of the structure of the human cornea, that cannot be fully recapitulated in models. This master thesis is carried out in the context of an interdisciplinary project which aims to create a cornea-on-a-chip model that faithfully reproduces the structure of the human cornea (epithelium, stroma and endothelium) and its environment in vivo. Inside this chip, the stromal layer is simulated using a gelatin-derived hydrogel, such as gelatin-methacrylate (GelMA) or gelatin-norbornene (GelNB). To design this model, an understanding of the diffusion behaviour in hydrogels is essential. The aim of this master thesis is therefore to develop methods for the characterization of diffusion in GelMA and GelNB hydrogels. The adopted strategy was to develop two methods for determining the diffusion coefficient of selected molecules (fluorescein sodium salt – FSS; rhodamine B – RhoB): the diaphragm tank method (DTM) and fluorescence recovery after photobleaching (FRAP). DTM and FRAP were compared for measuring FSS diffusion coefficients in GelMA and GelNB. DTM, though more time-consuming and challenging to manipulate, provided higher diffusion coefficients with significant inter-sample variation. FRAP, being quicker and easier, yielded lower measurements. However, the results from FRAP are highly dependent on data treatment, which can introduce greater variability than the inter-sample variation seen with DTM. This study also evaluates the partition coefficient, which determines the distribution of solutes between the surrounding solution and the gel, in order to understand the influence of solute-polymer interactions on diffusion. This study also evaluates the partition coefficient, which determines the distribution of solutes between the surrounding solution and the gel, to understand the influence of solute-polymer interactions on diffusion. The partition coefficients measured for each gel-solute combination (GelMA-FSS, GelMA-RhoB, GelNB-FSS, and GelNB-RhoB) are all greater than 1,indicating a retention effect on the polymer network. Factors such as the nature of the solute, the nature of the polymer, tortuosity, and mesh size directly affect this retention. These results contribute to the advancement of the cornea-on-a-chip model, providing a solid basis for comparing the diffusion values obtained in the gel alone with those measured when the gel is integrated into the chip. The development of these techniques for assessing the diffusion coefficient also makes it possible to advance our scientific understanding of diffusion in hydrogels and to determine how the polymer network of the hydrogel may influence the diffusion of a solute. Determination of the partition coefficient has shown that solute-polymer interactions can influence diffusion, underlining the importance of considering this parameter when designing ocular treatments. This research paves the way for future developments in the characterisation and optimisation of hydrogels for ocular medical applications, improving the effectiveness of treatments and reducing the ethical problems associated with preclinical testing.