Development of Biofabrication Techniques to Engineer 3D in vitro Models of Tissue Barriers

Abstract

Tissue barriers protect the body from external pathogens and maintain homeostasis by regulating nutrient and gas transport. Dysfunction in these barriers can cause serious diseases, emphasizing the need for accurate models for therapy development. While animal models provide useful data, they have ethical, cost, and physiological limitations. Human ex vivo models are limited by ethical concerns, tissue availability, and variability. In vitro models have emerged as a solution; however, current models often oversimplify and fail to replicate critical cellular behaviors and interactions under various conditions. Modeling tissue barriers in vitro is particularly challenging due to the need for highly confluent cellular barriers within multi-compartment setups to enable realistic transport studies. This is especially complex for structures like the placenta, which features multiple layers and cell types and undergoes rapid developmental changes. The objective of this doctoral thesis was to develop a biofabrication technique using the self-assembly method to create in vitro tissue barrier models that mirror the permeability traits found in vivo. We refined the self-assembly parameters and designed a polydimethylsiloxane (PDMS) device to construct 3D tubular, membrane-like tissues suitable for advanced transport studies. Our model successfully established continuous, highly confluent endothelial barriers through self-assembly and cellular migration. Next, using BeWo cells, a choriocarcinoma cell line, we mimicked the multilayer structure of the trophoblast layers in the placenta. We then facilitated the co-culture of endothelial cells and BeWo cells within a single, heterogeneous 3D tubular construct. This setup replicated the multilayer and multi-cell type structure of the placental barrier, with appropriate cell-ECM interactions. In summary, the fabrication techniques developed in this study enable the creation of in vitro tissue barrier models that closely represent the intricate architecture of tissue barriers, offering a physiologically relevant platform for detailed transport studies and maintaining high cellular confluency and barrier integrity.