470786 Establishing Mechanically Active Synthetic Mucosal Interface in a Multi-Well Plate
Materials and Methods: The in-vitro platform consists of an “insert” 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 37oC, 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.
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