Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Thrombospondin-2 is up-regulated by TGFß2 and increases fibronectin expression in human trabecular meshwork cells.

Elevated intraocular pressure (IOP) is a major risk factor for the development of primary open-angle glaucoma (POAG). This is from an increased aqueous humour (AH) outflow resistance through the trabecular meshwork (TM). The pathogenic mechanisms leading to the increase in TM outflow resistance are poorly understood but are thought to be from a dysregulation of the TM extracellular matrix (ECM) environment. ECM modification and turnover are crucial in regulating the resistance to aqueous outflow. ECM turnover is influenced by a complex interplay of growth factors such as transforming growth factors (TGFß) family and matrix metalloproteinases (MMPs). Elevated TGFß2 levels result in an increase in ECM deposition such as fibronectin leading to increased resistance. Fibronectin is a major component of TM ECM and plays a key role in its maintenance. Thrombospondins (TSP)-1 and -2 are important regulators of the ECM environment. TSP-1 has been implicated in the pathogenesis of POAG through activation of TGFß2 within the TM. TSP-2 does not contain the catalytic domain to activate latent TGFß, but is able to mediate the activities of MMP 2 and 9, thereby influencing ECM turnover. TSP-2 knock out mice show lower IOP levels compared to their wild type counterparts, suggesting the involvement of TSP-2 in the pathogenesis of POAG but its role in the pathogenesis of POAG remains unclear. The purpose of this study was to investigate the role of TSP-2 in trabecular meshwork ECM regulation and hence the pathogenesis of POAG. TSP-1 and TSP-2 expressions in immortalised glaucomatous TM cells (GTM3) and primary human non-glaucomatous (NTM) and glaucomatous cells (GTM) were determined by immunocytochemistry, immuno-blot analysis and qPCR following treatment with TGFß2 and Dexamethasone. The level of ECM protein fibronectin was determined in TM cells using immuno-blot analysis following treatment with TSP-1 or -2. TM cells secrete TSP-1 and -2 under basal conditions at the protein level and TSP-2 mRNA and protein levels were increased in response to TGFß2 three days post treatment. Exogenous treatment with TSP-2 up-regulated the expression of fibronectin protein in GTM3 cells, primary NTM and GTM cells. TSP-1 did not affect fibronectin protein levels in GTM3 cells. This suggests that the role of TSP-2 might be distinct from that of TSP-1 in the regulation of the TM cell ECM environment. TSP-2 may be involved in the pathogenesis of POAG and contribute to increased IOP levels by increasing the deposition of fibronectin within the ECM in response to TGFß2.