Quantifying mechanobiology using precision cut mouse lung slices
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Abstract
RATIONALE: Acute respiratory distress syndrome (ARDS) is a multi-factorial respiratory syndrome that eventually leads to an acute inflammation disorder called acute lung injury (ALI). To investigate upstream stimuli and downstream mechanistic changes, precision cut lung slices (PCLS) have emerged as a valuable pre-clinical model. We focused on the ALI subgroup known as ventilator-induced lung injury (VILI). Using a custom culture platform, we worked to establish a PCLS-based model system of VILI. We hypothesized that this model system could cyclically stretch murine PCLS (mPCLS) over a 24-hour period, while preserving sterility and viability. We hypothesized further that the experiments can be achieved in a 12-well format.
OBJECTIVES: To optimize a developed and validated mechanotransduction bioassay system for high-throughput use. Additionally, by optimizing the in vitro system, adherence and metabolic signal scopes can be enhanced and preserved in the setting of cyclical stretch.
METHODS: 1) mPCLS adherence was tested on two membrane compositions (NuSil® Gel-8100 vs. bare) prepared on a stretchable silicone membrane, and on variable air blowing times which reinforced adhesion. 2) mPCLS were plated on a custom 6-well “FlexFrame” plate, incubated for 24 hours in a sterile incubator on an established 12-well stretching device with programmed cyclical stretch parameters. 3) mPCLS were quantified for sterility with visual inspection of spores and viability using the commercially available MTT assay.
RESULTS: mPCLS successfully remained adhered to the NuSil®-coated membrane with air blowing of 20 seconds (20 psi). NuSil® adhesive did not release toxic byproducts that decrease viability. Adhered mPCLS were cyclically stretched for 24 hours at 37C to model in vivo mechanical ventilation and maintained viability. Cyclical stretching did not affect mPCLS sterility and viability.
CONCLUSIONS: By generating novel insights on PCLS adherence and viability with a new adhesive, air blowing conditions, and establishing metabolic signal scopes, we optimized an in vitro high-throughput mechanotransduction bioassay system. This foundational work enhances our ability to study VILI-related symptoms and pursue future studies on cellular mechanics, inflammatory cytokines, and airway contractility in murine and human PCLS with different genetic profiles and disease conditions.
Description
2025