Precision-cut lung slices (PCLS) are ideal for measuring small airway contraction. However, these measurements are currently limited to acute exposure scenarios that typically last a few minutes to a few hours. Using an insulin-supplemented culture medium, we prolong the small airway contractility in mouse PCLS for up to two weeks. Compared to conventional culture medium, insulin-supplemented culture medium provides no additional benefit in preserving cellular viability or airway structure. However, it protects the airway smooth muscle (ASM) against a loss of smooth muscle myosin heavy chain (SMMHC) expression. We elucidate the significance of this new culture medium for chronic disease modeling of IL-13-induced airway hyper-responsiveness.