High-throughput Mucus Microrheology for Phenotyping and Disease Modeling.

Mucus plays an integral role for the barrier function of many epithelial tissues. In the human airways, mucus is constantly secreted to capture inhaled microbes and pollutants and cleared away through concerted ciliary motion. Many important respiratory diseases exhibit altered mucus flowability and impaired clearance, contributing to respiratory distress and increased risk of infections. Understanding how mucus rheology changes during disease progression and in response to treatments is thus of great interest for subtyping patients and tailoring treatments, probing disease mechanisms, and tailoring therapies; however, basic research of mucus rheology is greatly hampered by the lack of scalable and user-friendly rheometry assays for the small volumes of mucus typically produced by in vitro respiratory models and in clinical ex vivo settings. To address this challenge, we developed a streamlined, high-throughput protocol leveraging Differential Dynamic Microscopy (DDM) to reliably measure the frequency-dependent microrheology of minuscule (3-10 L) mucus samples using standard epifluorescence microscopy. Our method does not require time-consuming user-interventions common in particle tracking routines and measures microrheology at the time scale of mucus relaxation (1-20s), hence greatly reducing assay time. We demonstrate the successful application of our method in mucus samples harvested from state-of-art air-liquid-interface (ALI) human respiratory cultures to assess mucus rheology in airway disease models and different culture conditions. To show that our approach equally applies to other types and sources of human mucus, we also validated our method in clinical samples of cervical mucus. We envision that our method can be seamlessly adopted by non-expert users, without the need for specialized equipment or extensive training, to study diseases and their treatments in the respiratory, intestinal, reproductive and other mucosal organ systems. This advancement opens up new avenues for large-scale studies, providing new insights into the role of mucus rheology previously limited by data accessibility and resource constraints.