Cell culture
Caco-2 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1%PS. HT-29 cells were maintained in McCoy’s 5A medium supplemented with 10% FBS and 1% PS. All cells were cultured in an incubator maintained at 37 ℃ with a 5% CO2 atmosphere. All cell culture reagents are purchased from Gibco.
To seed the cells on the PET membrane, a ratio of 9:1 was used, resulting in a total cell count of approximately 50,000 cells. The cell-seeded chips were then placed on a rocker operating at 10 rpm and ± 8°, facilitating gentle fluid perturbation within the middle hole and ensuring regular fluid flow in the underlying channels. The culture medium was refreshed every two days to maintain cell viability and provide essential nutrients for growth. In terms of media supplementation, 150 μL was introduced into the middle hole of the chip, while 600 μL was added to the lower layer. This allocation aimed to maintain fluid level equilibrium between the middle hole and the two side holes, and ensuring adequate nutrient supply and waste removal throughout the experiment.
Finite element simulation
Simulations were conducted using the ”two-phase laminar flow” and ”incompressible Navier-Stokes” application modes within the Comsol Multiphysics Finite Element Program (COMSOL 3.5, Comsol AB, Burlington) to visualize the flow characteristics along the centerline of the upper and lower surfaces of the membrane. The oscillatory motion was applied with the bottom center point as the axis, employing an angular displacement of ± 8° and a period of 6 seconds. The results revealed that the distribution of fluid shear stress on the membrane surface exhibited intermediate symmetry and temporal variation.