Validation of A 3D Bioprinted Model of Airway Smooth Muscle – A Novel Tool to Study the Effects of Airway Stiffening in Asthma

Background Asthma is a chronic obstructive airway disease characterized by airway wall remodeling and aberrant contraction of airway smooth muscle (ASM), which each contribute to clinical symptoms by restricting airflow. However, the precise link between airway mechanics and ASM contractility remains elusive, because ASM cell biology is routinely studied in 2D models that inadequately replicate the complex in‐vivo 3D microenvironment of healthy or diseased airways. We used a technique called 3D bioprinting to develop an experimental model of ASM in which we could characterize the response of ASM to altered mechanical loads in a realistic 3D structure. Methods Human ASM were resuspended at 2.5×10 7 cells/mL in a bio‐ink consisting of RGD‐coupled‐alginate (0.375% w/v), fibrinogen (5 mg/mL) and collagen‐I (1 mg/mL). Cells were then bioprinted using an Aspect Biosystems RX‐1 Bioprinter, as a ring‐shaped bundle of muscle constrained within a stiffness‐modifiable acellular alginate support (0.75–1.25% w/v). After bioprinting, constructs were thrombin treated (1.25 U/mL, 30 min) to initiate fibrin polymerization and maintained in standard ASM culture conditions. Cell health, viability and morphology were assessed with LDH assays, Hoechst/propidium‐iodide and filamentous‐actin staining respectively. Expression of ASM relevant genes was measured by qPCR. Reduction in lumen area representing contractile tone was tracked by live cell microscopy. Results ASM bioprinted without acellular supports shortened excessively and rapidly lost structural integrity. Comparatively, including supports provided a mechanical load that controlled baseline shortening; cells supported by 0.75%, 1% and 1.25% alginate exhibited >45%, >25% and <15% lumen reduction respectively. Constructs bioprinted in 1% supports exhibited high cell viability (>80%) for up to 14 days in culture. Histology 4 days after printing revealed development of a highly organized tissue with evidence of cell‐cell contacts and alignment of actin fibres. Nuclei were evenly distributed in all axes indicating formation of a true 3D structure. Compared with 2D, cells in 3D had a higher mRNA abundance of myosin heavy chain, but vimentin and matrix metalloproteinase‐3 were lower (all p<0.05). Importantly, administration of acetylcholine resulted in muscle contraction, while cytochalasin D caused a dramatic decrease in tone for all tissues with structural supports. Conclusion We have validated a novel tool for studying ASM biomechanics. ASM displayed varying levels of baseline shortening to different mechanical loads, supporting that mechanical cues may have profound effects on cellular function. Further, higher abundance of contractile genes in 3D demonstrates adoption of a physiologically relevant phenotype. Moving forward, contraction, relaxation and calcium dynamics will be quantified across a range of stiffnesses. These data will help unmask mechanisms by which airway mechanics regulate ASM contractility, with important clinical relevance to asthma. Support or Funding Information Funding: NSERC Discovery Grant (ARW), Research Manitoba Studentship (JO, SS)

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