Investigating Crosstalk between Lymphatic and Articular Cells in an Inflammatory Context using 2D and 3D In Vitro Models Coupled with Image Analysis

Abstract

Osteoarthritis (OA) is a widespread degenerative joint disease, affecting 1 in 8 people worldwide. It is characterised by cartilage degradation and chronic inflammation. The altered behaviour of chondrocytes and synoviocytes plays a key role in OA development. However, the potential role of the lymphatic system and lymphatic endothelial cells (LECs) in joint homeostasis and OA pathogenesis has received limited attention, despite their increasingly recognized functions in immune regulation, tissue drainage, and inflammation resolution. Investigating the paracrine and physical interactions between these different cell types, under both homeostatic and inflammatory conditions, may provide new insights into OA progression.

The overarching goal of this master thesis was to explore the cellular crosstalk between selected articular components (synoviocytes and chondrocytes) and LECs through a series of complementary in vitro approaches designed to mimic the complex cellular microenvironment of the human knee joint. As this field is an emerging area of investigation, many experimental methodologies remain underdeveloped. Consequently, this study focused on the development and rigorous validation of robust in vitro protocols to accurately model cellular interactions across diverse experimental platforms, encompassing both functional assays and advanced 3D culture systems.

Commercial synoviocytes and chondrocytes were first characterised and validated. Protocols were then implemented to generate both control and inflammatory phenotypes for these two cell types. Inflammatory stimulations were shown to induce phenotypic changes consistent with OA-associated inflammation. Indirect interactions between LECs and (inflamed) articular cells were explored using concentrated conditioned media. To further refine the modelling of cellular communication, intermediate systems using Boyden chambers (Transwell assays) were developed to bridge the gap between indirect and direct co-cultures. These assays consistently revealed that LECs were more strongly influenced by inflamed synoviocytes, both in terms of proliferation and migration, whereas inflamed chondrocytes and their concentrated conditioned media had only a limited effect. In parallel, culture media were optimized to support direct co-culture experiments, which offer a more pathophysiologically relevant model for capturing cell–cell interactions. Initial efforts to model direct interactions between LECs and articular cells in 3D were conducted using two approaches: micromass system and AggreWell™-based spheroid formation. The micromasse system, however, yielded limited results for studying these interactions, whereas the TM AggreWell™ platform successfully enabled the generation of structured spheroids. Homospheroids composed of chondrocytes displayed morphological consistency with the lab’s reference micromasse system. Preliminary observations of heterospheroids suggested potential LEC integration, as indicated by podoplanin (PDPN) staining.

Together, this preliminary work provides a solid methodological foundation for future studies investigating articular–lymphatic interactions in OA. The protocols and exploratory data generated here will enable more robust experiments, with increased replication and future integration of pharmacological testing. This work therefore lays the groundwork for a more comprehensive analysis of lymphatic involvement in joint disease.