Speaker
Description
Introduction
In-vitro models advanced significantly with the introduction of 3D culture technology providing a physiologically relevant microenvironment. While tissue morphology and functionality have greatly improved, bioengineered tissue models often lack long-term stability with a decrease in cell viability and tissue integrity, preventing their use for chronic exposure and long-term studies in wound healing. Traditional 3D methods lack precise control of culture parameters, therefore we have utilised a novel perfusion platform that allows for enhanced control of the in-vitro microenvironment to promote improved replication of native physiological conditions.
Methodology
We aim at improving the longevity of our in-vitro full-thickness skin model by fine-tuning the culture microenvironment, considering the nutrient-metabolite-signalling factor balance, gaseous environmental conditions and simulation of in-vivo perfusion. Skin models are generated by culturing human primary fibroblasts in the porous Alvetex® scaffold, providing an ideal 3D environment for cell infiltration and ECM deposition, and subsequent seeding of primary human keratinocytes onto the robust dermis to form a multi-layered and stratified epidermis at the air-liquid interface. Dynamic culture conditions were introduced using in-house built and commercially available perfusion systems with adjustable flow velocities at different culture stages also allowing for variation of the flow pattern. Using computational fluid dynamics, fluid flow can be characterised to predict shear stress and mass transfer.
Results
Analysis of co-factors during skin model maturation demonstrated their stability under common culture conditions and revealed the superior significance of nutrient-metabolite balance for functional tissue maturation in models generated from fibroblasts obtained from varied age groups. The integration of dynamic culture phases through perfusion enhanced scaffold infiltration and build-up of multiple cell layers. Analysis of extracellular matrix proteins such as Collagen I demonstrated increased deposition and improved distribution uniformity throughout the model. In low-serum media, perfusion was found to shorten the tissue maturation period which allowed for the formation of a robust overlying epidermis at an earlier time point, reducing overall model maturation time. Improved maturation seems to be achieved through advanced cell differentiation mediated by signalling factors such as TGF-β1. This result was found to be transferable across different perfusion platforms investigated in these studies.
Conclusion
Perfusion is a valuable tool to bridge the gap between in-vitro and in-vivo conditions. Understanding how different perfusion patterns affect flow characteristics and in turn gene expression, endogenous protein production and the build-up or disruption of signalling factor gradients, will support the development of long-term skin model cultures with maintained tissue functionality. Ultimately, this will allow for prolonged studies to be carried out to increase the significance of in-vitro research.
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