(278b) Establishing Mechanically Active Synthetic Mucosal Interface in a Multi-Well Plate

Introduction: Human gastro-intestinal tract (GIT) accounts for more than 75% of the body surface that comes directly in contact with the external environment. This makes GIT the prime location for host-microbiome interactions, since 70-80% of immune cells are present along the tract. GIT epithelium consists of a monolayer of polarized cells arranged into microscopic features called villi and crypts, which increases the GIT surface area tremendously. Recent studies suggest that peristaltic motion is important for epithelial cell differentiation, polarization and structural arrangement into villi and crypts. Some progress has been made in recent times, there is still a growing need to develop model systems to integrate microbiome, epithelial cells, and immune cells in a single platform along with providing sufficient level of biological and mechanical complexity. To this end, we developed a mechanically actuated 3D tissue culture model system that can achieve the aforementioned integration of different cell types with multiple layer complexity in an easily adaptable well plate platform. Pneumatically powered mechanical actuator applies magnetic force that cause cyclic stretching of PDMS membrane supporting cell culture and can simulate peristaltic motion. Actuation frequency is easily controllable using Arduino software. In this study we demonstrate how this platform was used to mechanically stimulate colon carcinoma cells (HT-29, ATCC® HTB-38�) leading to polarization, vertical growth and 3D morphogenesis of these cells.

Materials and Methods: The in-vitro platform consists of an â??insertâ? made of three parts, a PDMS membrane (100 μm thick, 100 μm pore size, and 400 μm pore separation), a medical grade stainless steel ball embedded in a PDMS peg, and a PDMS ring. The PDMS peg was bound to the PDMS membrane and the PDMS ring bound on the outer periphery of the membrane using plasma treatment (Harrick Plasma). The PDMS membrane was coated with collagen on the apical side and HT-29 cells were seeded. The 24 well plate was then placed on the mechanical actuator (at 37^oC, and 5% CO₂) with mechanical actuation rate of 1 cycle/minute and cell culture medium was changed every day.

Results and Discussion: Cyclic stretching of HT-29 cells grown on top of a stretchable PDMS membrane cause vertical growth and formation of 3D morphological structures in HT-29 cells. Confocal fluorescence image confirms 3D growth of HT-29 cells. Commercial transwell inserts were used previously that allows for 2D culture of epithelial cells that were then exposed to pathogenic bacteria on the apical side and human peripheral blood mononuclear cells (PBMC) on the basal side. Preliminary results showed immune-modulatory effects of epithelial cells in terms of cytokine responses from PBMCs measured using ELISA (R&D systems). However, 2D cultures are not good representatives of the in-vivo physiology. Therefore, we replaced the 2D transwell system with aforementioned in-vitro platform. Currently, this system is being used to study bacterial interactions with functionally active epithelial barrier and immune cells.

Conclusions: A high throughput in-vitro platform was developed that allows for mechanical stimulation and polarization of epithelial cells via magnetic field. The system allows for compartmentalized co-culture of bacterial and mammalian cells to study the role of microbial invasion, epithelium, and cytokines in pathophysiology of human GIT.