Development of Novel Microfluidics System for In vitro Modelling of Pulsatile Glucocorticoids Rhythms

Abstract

Glucocorticoids (GCs) are among the most commonly used therapeutics due to their anti-inflammatory and immunosuppressive properties. Despite their benefits, they are associated with significant side effects, particularly with long-term use. Current therapeutic regimens typically use constant GC administration, whereas physiological GCs are secreted in daily (circadian) and hourly (ultradian) rhythms. This mismatch may contribute to GC-associated side effects. This research investigated how the pattern of GC administration influences glucocorticoid receptor (GR) expression. I hypothesised that pulsatile GC administration, mimicking natural ultradian rhythms, could preserve GR expression and responsiveness, thereby reducing ligand-induced downregulation and its associated side effects. To test this, in-vitro downregulation and recovery studies were performed using the GR-HiBiT-tagged A549 human lung epithelial cell line. Cells were treated with physiological doses of hydrocortisone (HCT) under constant exposure conditions. GRα expression was quantified using the HiBiT Lytic Detection System. Results showed that a 1 hour exposure to 50nM hydrocortisone (HCT) caused significant downregulation of GRα, which was reversed after 24 hours of hormone withdrawal. To model physiological pulsatile GC rhythms and study their effect on GR expression, a novel microfluidics system was successfully developed and optimised, providing to the best of my knowledge, the first platform specifically designed to study pulsatile GC exposure under controlled and physiologically relevant conditions. Preliminary data using this newly developed pulsatile microfluidics system together with physiological GC exposure suggested that a hormone withdrawal period (inter-pulse interval) of only a few hours is sufficient to maintain GR expression, on which cellular sensitivity to GCs depends