Gene Expression in MCF10A cells through Differentiation on Transwells

To further understand the differences occurring in MCF10A cells as they polarize and differentiate in the Transwell® model, we performed gene expression profiling with Affymetrix Human Genome U133 Plus 2.0 Arrays. Four experimental time points, were sampled: conventional cultures of MCF10A cells grown on plastic (Monolayer) and MCF10A cells plated on Transwells® sampled at three TEER values, 200-300 Ω cm2 (Base), 1400-1600 Ω cm2 (Midpoint), and 3000-3200 Ω cm2 (Plateau). Keywords: Mammary Epithelial Cell Differentiation Cells are grown in monolayer to 90-95% confluency, trypsinized and counted for seeding onto permeable supports (Transwell®, 0.4 µm pores, polyester) in normal growth medium. MCF10A cells are seeded on 12-well Transwells® (Corning) at 105 cells/cm2. Both chambers of media were changed strictly on a 24-hour schedule. Transepithelial electrical resistance (TEER) is measured daily with Epithelial Volt-Ohm Meter (EVOM; World Precision Instruments), prior to media change. Total RNA was isolated at the indicated TEER listed above. Canonical monolayer MCF10A cells served as a control comparison. Each time point was performed on triplicate arrays except for Plateau (n=4).