Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

Summary

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

Abstract

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G', G", G^*, and tan δ) and gel point characteristics (γ_c and σ_c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is <10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more "liquid-like" behavior overall (e.g., higher tan δ). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

Introduction

Muco-obstructive airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and other respiratory conditions, such as viral and bacterial pneumonia, are prevalent health concerns worldwide. While the pathophysiology varies greatly between each condition, a common key feature is abnormal mucociliary clearance. In healthy lungs, mucus lines the airway epithelium to trap inhaled particles and provide a physical barrier against pathogens. Once secreted, airway mucus, composed of ~97.5% water, 0.9% salt, ~1.1% globular proteins, and ~0.5% mucins, is gradually transported toward the glottis by the coordinated beating of cilia^1,2. Mucins are large O-linked glycoproteins that interact via non-covalent and covalent bonds to provide the distinct viscoelastic properties of mucus, which is required for efficient transport³. Changes in the ultrastructure of the mucin network caused by altered ion transport, mucin unfolding, electrostatic interactions, cross-linking, or changes in composition can significantly affect mucus viscoelasticity and impair mucociliary clearance^4,5. Hence, identifying changes in the biophysical properties of airway mucus is essential to understanding disease pathogenesis and testing novel mucoactive compounds⁶.

Various factors can lead to the production of aberrant mucus in the lungs. In COPD, chronic inhalation of cigarette smoke triggers mucus hypersecretion as a result of goblet cell metaplasia, as well as airway dehydration via the downregulation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, causing mucus hyperconcentration and small airway obstruction^7,8. Similarly, CF, a genetic disorder associated with mutations in the CFTR gene, is characterized by the production of viscous, adherent mucus that is inadequate for transport^8,9. In brief, CFTR dysfunction induces airway surface liquid depletion, polymeric mucin entanglement, and increased biochemical interactions, which result in chronic inflammation and bacterial infections. In addition, inflammatory cells trapped in static mucus further exacerbate the viscoelasticity of mucus by adding another large molecule, DNA, into the gel matrix, worsening airway obstruction⁵. One of the best examples of the importance of mucus rheology on the overall health of the lungs is provided by the example of recombinant human DNFase (rhDNase) in the treatment of cystic fibrosis patients. The effects of rhDNase were first demonstrated ex vivo on expectorated sputum, which showed a transition from viscous mucus to a flowing liquid within minutes^10,11. Clinical trials in CF patients demonstrated that reducing airway mucus viscoelasticity with rhDNase inhalation decreased the rate of pulmonary exacerbations, and improved lung function and overall patient well-being^12,13,14. As a result, rhDNase inhalation aimed to facilitate clearance became the standard of care for CF patients for more than two decades. Similar clinical benefits were observed with the use of inhaled hypertonic saline for mucus hydration in CF, which correlated with changes in rheological properties and resulted in mucociliary clearance acceleration and improved lung function^15,16. Hence, a rapid and reliable protocol to measure mucus viscoelastic properties in clinical settings is important to optimize therapeutic approaches.

The benchtop rheometer tested herein offers a fast and convenient alternative for performing comprehensive viscoelastic measurements of mucus/sputum samples. Using dynamic oscillations with controlled angular displacement, the instrument provides deformation via a pair of adjustable parallel plates (e.g., rough or smooth geometries) to measure the torque and displacement with resolutions of 15 nN^.m and 150 nm, respectively¹⁷. A default standardized calibration combined with user guidelines adapted for non-rheology specialists allows for straightforward measurements and reduces the risk of operator errors. The device produces a strain sweep curve that is processed and analyzed in real-time (within ~5 min) and automatically provides both linear viscoelastic (G', G", G^*, and tan δ) and gel point (γ_c, and σ_c) characteristics (see Table 1).The elastic or storage modulus (G') describes how a sample responds to stress (i.e., the ability to return to its original shape), while the viscous or loss modulus (G") describes the energy dissipated per cycle of sinusoidal deformation (i.e., the energy lost due to the friction of molecules). The complex or dynamic modulus (G*) is the ratio of stress to strain, which describes the amount of internal force buildup in response to a shearing displacement (i.e., the overall viscoelastic properties). The damping factor (tan δ) is the ratio of the viscous modulus to the elastic modulus, which indicates the ability of a sample to dissipate energy (i.e., a low tan δ indicates an elastic-dominant/solid-like behavior, while a high tan δ indicates a viscous-dominant/liquid-like behavior). For gel point characteristics, the crossover strain (γ_c) is the measure of the shear strain, calculated by the ratio of the deflection path to the shear gap height, at which the sample transitions from a solid-like to a liquid-like behavior and occurs, by definition, at oscillation strain where G' = G" or tan δ = 1. The crossover yield stress (σ_c) is a measure of the amount of stress applied by the device at which the elastic and viscous moduli cross. In healthy sputa, elasticity dominates the mechanical response to strain (G' > G"). In muco-obstructive diseases, both G' and G" increase as a result of pathological mucus changes^17,18,19. The operational simplicity of the device facilitates onsite measurements and circumvents the need for sample storage/transportation/shipment to an offsite facility for analysis thus avoiding the time and freeze-thaw effects on the properties of these biological samples.

In this study, 8 MDa polyethylene oxide (PEO) solutions of different concentrations (1%-3%) were used to validate the measuring range of a commercial benchtop rheometer (Table of Materials) and the obtained concentration-dependent curve was directly compared to measurements acquired with a traditional bulk rheometer (Table of Materials). The repeatability of rheological measurements was then assessed using bronchoscopically harvested mucus from an intubated patient suffering from status asthmaticus (SA), an extreme form of asthma exacerbation characterized by bronchospasm, eosinophilic inflammation, and mucus hyperproduction in response to an environmental or infectious agent^8,20. In this case, the SA patient had been intubated for severe respiratory failure and required ECMO (extracorporeal membrane oxygenation) due to the inability to support the patient effectively and safely with mechanical ventilation alone, despite aggressive standard asthma therapies. During a clinically-indicated bronchoscopy for lobar collapse, thick, clear, tenacious secretions were noted to be obstructing lobar bronchi and were aspirated using saline washings. Immediately following collection, excess saline was removed from the aspirate and the viscoelastic properties of the remaining SA sample were analyzed using the benchtop device. Additional sample aliquots were treated with a reducing agent, tris (2-carboxylethyl) phosphine hydrochloride (TCEP), to determine whether this protocol might be used to characterize therapeutic compound efficacy ex vivo.

The results showed that this protocol and the benchtop device can be used effectively in a clinical setting. The rheological properties determined from PEO concentration-dependent curves (Figure 1A) were indistinguishable between the tested benchtop device and a traditional parallel plate rheometer (Figure 1B). Triplicate measurements of the SA mucus were repeatable, with a 10% coefficient of variation for G*, G', and G" endpoints and reflected the substantial abnormalities in mucus viscoelasticity that were clinically apparent in this patient's case (Figure 1D). Finally, ex vivo treatment with TCEP resulted in a significant reduction in G' and G", and an increase in tan δ, demonstrating responsiveness to the treatment by alterations in the mucin network (Figure 2). In conclusion, this protocol using a benchtop rheometer provides a simple and effective approach to assess viscoelastic properties of mucus samples obtained from the clinic. This capability may be used to facilitate precision medicine approaches to care, as clinicians can test the efficacy of approved mucoactive drugs onsite, which can help identify alternative treatment options. In addition, this approach can be used in clinical trials to examine the effects of investigational drugs.

Protocol

In the present study, samples were collected during a clinically indicated bronchoscopy after obtaining informed consent under a protocol approved by the UNC Institutional Review Board.

1. Sputum/mucus collection and storage

1. Collect airway mucus via sputum collection or bronchoscopy aspiration.
   1. Collect sputum either via spontaneous expectoration or induce sputum by 3% hypertonic saline inhalation. Alternatively, directly aspirate mucus from the airways during a bronchoscopy procedure.
   2. Store collected airway sputum/mucus in sterile specimen cups. In the case of sputum, remove excess saliva from the sample immediately upon collection.
   3. Place the samples on ice for transport. Limit the transport time to less than 4 h.
2. Analyze the samples at the time of collection or store at -80 °C until processed.
   1. Before storage, homogenize the mucus by gently pipetting up and down three to five times with a positive displacement pipette or pipette directly into the microcentrifuge tubes.
   2. Aliquot the samples for storage in volumes ≥500 µL to ensure sufficient volume for experiments.
      NOTE: Freezing and thawing may affect the viscoelastic properties of the sample. Only compare samples that have undergone similar freeze/thaw cycles.

2. Sample preparation

1. Pipette fresh and frozen sputa/mucus directly or homogenize specimens using a positive displacement pipette by gently pipetting up and down three to five times before aliquoting.
   NOTE: Homogenization is important for samples that contain thick plugs that can affect reproducibility.
2. Aliquot 400-500 µL of the sample into separate microcentrifuge tubes. Prepare as many aliquots as needed for repeat measurements and/or treatment with pharmacological reagents (e.g., rhDNase, N-acetyl cysteine). Incubate the aliquots to be tested at 37 °C for a minimum of 5 min prior to measurement.
3. For testing pharmacological agents (optional), use high concentrations of stock solutions to prevent sample dilution.
   1. Add between 0.4% and 10% volume (to minimize specimen dilution) of the desired reagent (e.g., TCEP) directly onto the sample. Make sure no drop of the compound stays on the side of the tube.
   2. Incubate the samples at 37 °C for the desired length of time to allow a chemical reaction (<1 h to prevent the proteolytic degradation of the mucus).
   3. Mix the mucus sample and reagent by flicking the bottom of the microcentrifuge tube every 2 min to allow progressive penetration of the reagent into the mucus sample without compromising the mucin network (e.g., mimicking ciliary beating and mucociliary clearance). When comparing multiple drug reagents, ensure that the incubation time is similar.

3. Instrument initialization and calibration

1. Turn on the machine (Table of Materials) and initialize the software.
2. Select New Measurement. Enter the sample identification number under Measure ID and the name of the operator under Operator to continue. Enter additional information or comments under Comments.
3. Select a geometry set (i.e., rough, or smooth 25 mm parallel plates) and inspect large and small plates carefully to ensure that plates are clean and in perfect condition).
   NOTE: Rough plates are designed for large volumes (350-500 µL) and smooth plates are designed for smaller volumes (250-350 µL). Using a lower or higher sample volume than recommended can cause inaccurate measurements.
4. Insert the large plate firmly on the bottom pulpit.
5. Insert the small plate gently on the upper pulpit and lock the plate by slightly rotating until hearing a "click", which indicates that the plate is properly clamped. Note that free oscillation of the upper plate is normal.
6. Wait until the temperature reaches the 37 °C target value. Then, initiate automatic calibration as prompted by the software.
   ​NOTE: Do not disturb the machine or benchtop surface during this process.

4. Sample loading

1. Using a positive displacement pipette, slowly pipette between 250 and 500 µL of the sample on the center of the large bottom plate. Once deposited on the plate, viscous samples will adopt a dome shape whereas highly elastic samples may require physical severing (use dissecting scissors).
   NOTE: Avoid introducing air bubbles. If needed, remove residual bubbles by pushing away with a pipette tip.
2. Lower the measuring head carrying the small plate via the software and observe the sample. If properly loaded on the bottom plate, the sample will make contact and be centered between the two plates.
3. To ensure that the sample fills the gap (i.e., by spreading to the edges of the plates), use the Reduce Gap function until the sample is no longer in a biconcave shape or is aligned with the edge of the plates. The Reduce Gap function lowers the measuring head in 0.1 mm increments and is limited to seven increments.
   NOTE: Monitor the sample carefully and adjust the gap progressively to avoid overspill.
   1. If a gap remains after seven increments, click on Redo Installation to return to the initial position and adjust the position and/or volume of the sample.
   2. If the gap is exceedingly reduced (e.g., biconvex shape), remove the excess sample with a spatula by a circular motion along the edge of the upper plate. Make sure to trim the excess sample gently to avoid shear stress.
      ​NOTE: At the end of this step, the edge of the sample should be aligned with the edge of the upper plate as shown in the user guidelines.
4. Lower the protective cover to avoid any accidental projection of contaminated fluids during oscillation.

5. Initiate biophysical measurement

1. To initiate measurement, click on Start Analysis. A full cycle will take 4-7 min.
   1. Avoid talking loudly and touching the device or the bench during the entire length of the cycle. A quiet environment is particularly important for the first 2 min.
      NOTE: During the cycle, the instrument performs a standardized strain sweep test, which consists of successive oscillating steps. Each step is a series of 10 oscillations at constant amplitude and frequency (1 Hz), during which the corresponding torque is measured in real-time. The strain and torque signals allow computation of the complex (G*), elastic (G'), and viscous (G") moduli, as well as the damping ratio (tan δ) at each step. Oscillations gradually increase in amplitude, which intensifies the deformation imposed on the sample.

6. Sample removal

1. Once the cycle is complete, click on Next to raise the measuring head and generate the sample analysis report.
   NOTE: For the report, the software computes the recorded data and automatically graphs two curves showing the evolution of the viscous and the elastic moduli in relation to the deformation exerted to the sample and displays the linear viscoelastic regime (i.e., a plateau at low deformation) if present. If no linear regime is detected, the values of G', G", G*, and tan δ are extracted at 0.05 strain. In addition, the crossover strain and yield stress (γ_c, and σ_c) are calculated at tan δ = 1. Data are also provided in spreadsheets for each step for further analysis.
2. Once the measuring head is fully retracted, raise the protective cover, discard the sample and carefully remove the plates. Clean and disinfect the plates using warm water and soap.
   NOTE: Dry the geometry set thoroughly before repeated use.

Representative Results

Figure 1 shows the accuracy and repeatability of rheological measurements using concentration-dependent curves of viscoelastic control, i.e., polyethylene oxide (PEO) solution, and status asthmaticus (SA) mucus. Measurements of viscoelastic characteristics of 8 MDa PEO at five different concentrations (1%, 1.5%, 2%, 2.5%, and 3%) were directly compared between the evaluated benchtop rheometer and a traditional bulk rheometer (Table of Materials). In contrast with SA mucus, PEO solutions were viscous-dominated (G" > G') in the entire strain range and did not exhibit crossover and, therefore, presented a solid-like behavior. In addition, triplicate measurements performed on 1.5% PEO solution and clinical SA mucus sample confirmed that linear viscoelastic characteristics (G*, G', and G") were highly repeatable (<10% coefficient of variation) for the values obtained from the biological sample.

The observation of lobar collapse in the SA patient suggested that mucus plugging could complicate the ability to mechanically ventilate the lungs and raised the possibility that non-standard, mucolytic therapies could be considered. In Figure 2, the protocol described herein was used to measure changes in the viscoelastic properties of mucus following treatment with a mucolytic agent. While NAC has been approved for use with COPD and CF, it was shown to have slow kinetics and low potency as a reducing agent²¹. TCEP has been shown to be highly effective at modifying the biophysical properties of mucus²². The effects of TCEP on SA mucus viscoelasticity were tested in a clinical setting using the benchtop rheometer. Mucolytic treatment resulted in a more fluid-like sample with a decrease in the complex modulus (G*) by 4.6-fold, elastic modulus (G') by 5.1-fold, viscous modulus (G") by 1.9-fold, crossover strain (γ_c) by 3.3-fold, and crossover yield stress (σ_c) by 5.7-fold, and an increase in the damping ratio (tan δ) by 2.8-fold.

Zone	Parameter		Symbol	Unit	Definition		Meaning
Linear Viscoelastic Regime (LVR)	Complex Modulus		G*	Pa	Representative viscoelastic behavior in the linear regime		Overall resistance to deformation of the molecular network
					G* = σ/γ
	Elastic Modulus		G'	Pa	Elasticity of the material in the linear regime		Rigidity of the molecular structure at rest, related to molecular network stiffness
							→0 : soft →∞ : stiff
	Viscous Modulus		G"	Pa	Viscosity of the material in the linear regime		Irreversible loss of energy while the structure is moving under very low strain
							→0 : pure solid →∞ : dissipative
	Damping Factor		tan δ	Unitless	Damping factor  in the linear regime		Energy dissipation factor, related to the molecular network morphology. Any change indicates a change in molecular nature.
					tan δ= G’’/G’		→0 : pure solid  =1: soild/liquid transition →∞ : pure liquid
Gel Point	Critical or Crossover Strain		γc	Unitless	Strain when switching from gel to flow behavior		Stretchability of the gel, the total deformation needed to start a flow or break a solid
							→0 : brittle →∞ : flexible
	Critical or Crossover Yield Stress		σc	Pa	Stress when switching to flow behavior		Strength of the gel, the amount of force needed to start a flow or break a solid
							→0 : weak   →∞ : strong

Table 1: Linear viscoelastic moduli and gel point characteristics measured by the benchtop rheometer. The device performs rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic (G', G", G^*, and tan δ) moduli and gel point characteristics (γ_c and σ_c) within ~5 min. Parameters, symbols, units, and a brief description of the measurements are provided.

Figure 1: Measurements of the viscoelastic properties of PEO solutions and SA mucus. Solutions of 8 MDa PEO were prepared at concentrations 1%, 1.5%, 2%, 2.5%, and 3%. SA mucus was harvested during a bronchoscopy procedure. For measurements using the benchtop rheometer, 25 mm rough plates and 500 µL of the sample were used. For the measurements using the traditional bulk rheometer, 20 mm parallel smooth plates and 30 µL of PEO solutions were used. Both measurements were run at a frequency of 1 Hz. (A) Curves obtained from a single cycle analyzing 1%, 1.5%, 2%, 2.5%, and 3% 8 MDa PEO, showing the evolution of the elastic modulus (G') in blue (i) and viscous modulus (G") in red (ii). (B) Curves comparing elastic (i) and viscous moduli (ii) for increasing concentrations of PEO solutions, analyzed by benchtop and traditional rheometers at 5% strain. (C) Curves showing the evolution of G' and G" of SA mucus, measured by the benchtop rheometer. Arrow indicates crossover strain (γ_c), which denotes a transition from soft-solid to liquid-like behavior_. (D) Graphs showing three replicate measurements of (i) G*, (ii) G' and (iii) G" values for 1.5% PEO (black bars) and SA mucus (gray bars) in the linear viscoelastic regime (LVR) or at 5% strain, respectively. Please click here to view a larger version of this figure.

Figure 2: Effects of TCEP treatment on the viscoelasticity of SA mucus. SA mucus was analyzed before (non-treated or NT) and after TCEP treatment (TCEP). Treatment consisted of adding 2 µL of 5 mM TCEP solution into 500 µL aliquots (final TCEP concentration of 20 µM). NT and TCEP-treated samples were incubated for 20 min at 37 °C and mixed by flicking the bottom of the tube every 2 min before analysis. Measurements were performed under oscillating strain at a frequency of 1 Hz. (A) Curves from NT and TCEP-treated SA mucus showing the evolution of (i) elastic (G') and (ii) viscous (G") moduli. The horizontal black dashed line indicates the linear viscoelastic regime (LVR) and the vertical black dotted line indicates the 5% strain reference in the event that an LVR could not be established. (B) Comparison of the complex modulus (G*), elastic modulus (G'), viscous modulus (G"), damping ratio (tan δ), crossover strain (γ_c) and crossover yield stress (σ_c) of NT and TCEP-treated mucus derived from the corresponding curves. Statistical analysis was performed, and p values were acquired using paired t-tests. Values for all graphs are shown as ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Please click here to view a larger version of this figure.

Discussion

The unique viscoelastic properties of mucus are essential in maintaining healthy airways. Internal and external factors can alter airway mucus biophysical properties, causing clinical complications characteristic of muco-obstructive diseases. Hence, monitoring changes in mucus viscoelasticity might be considered during assessments of disease status and exploration of therapies that reduce mucus viscoelasticity. Empirical studies from the 1980s demonstrated a strong correlation between mucus rheology and airway clearance using magnetic bead rheometers^23,24. In recent years, rheology has evolved to take advantage of several techniques that analyze mucus at various scales. For example, microrheological assays use microscopic probes to describe local mucus properties based on the movement of magnetic or fluorescent micrometre-size particles. However, because this technique utilizes small sample volumes, it may be difficult to obtain representative data that describe heterogeneous samples such as sputum. Further, microrheology assays require high-resolution microscopes, significant computation skills, and time-consuming analyses, and thus are poorly suited to widespread lab or clinic use.

While microrheology and macrorheology are not typically comparable, similar limitations apply to long-established devices, such as cone/parallel plate bulk rheometers. Macrorheology is performed using precision instruments equipped with rotating cones, plates, cups, and/or rotors of various dimensions to measure extremely small torques and displacements down to the sub nN^.m and sub Å ranges. To reach such high precision, most commercial rheometers require a direct connection with a compressed air supply and cooling system in an environment free of oil, dust, or noise and with a controlled ambient temperature and humidity to prevent artifact formation. Additionally, while traditional bulk rheometers can measure a wide range of materials via the adjustment of specific variables, calibration of these instruments takes significant time and requires extensive training.

In contrast, the Rheomuco benchtop rheometer was specifically designed to measure the viscoelastic properties of mucus and sputum and requires a single calibration step to perform linear viscoelastic and gel-point measurements within minutes. This benchtop device utilizes a straightforward and standardized protocol to produce rapid and accurate viscoelastic measurements without the need for extensive training in instrument calibration or rheological data analysis/calculation. The device operates by measuring torque and displacement following oscillations with controlled angular displacement to produce a strain sweep curve and establish a linear viscoelastic regime or LVR (a region of uniform viscoelastic response to strain, indicated with a horizontal dashed line in Figure 2A), before reaching the point where the sample yields. In most cases, sputum samples are within the LVR over the 1%-10% strain range. When an LVR is not detected, the value at 5% strain is commonly referenced to report on the viscoelastic characteristics of the sample. The absence of a detected LVR does not invalidate the measurement, but rather reflects a sample whose properties are distinct (more plastic) from those of most samples. The sensitivity of this instrument is optimized to match the needs of viscous and elastic fluids close to mucus while providing high tolerance to mechanical noise, which makes it ideal for the study of biological fluids in clinical settings; however, it may not be suited to study other viscoelastic materials with extremely low (e.g., saliva) or extremely high (e.g., coal-tar) elastic or viscous moduli as a result of restricted software parameters and the inability to manipulate variables such as plate shape, surface, distance, and rotational frequency. The concentration-dependent rheological measurements on PEO 8 MDa (Figure 1) allowed the estimation of sensitivity (i.e., the lower limit of detection) of this device, which is between 0.3% and 0.4% of 8 MDa PEO or <0.05 Pa for G*. An upper limit could not, however, be established owing to the difficulty of solubilizing PEO concentrations higher than 3%. Nevertheless, the device was able to report G' and G" for 3% 8 MDa PEO, which is more viscoelastic than SA mucus samples (~5-fold greater G' and 25-fold greater G" as compared with SA), suggesting a relevant dynamic range for mucus biospecimens. It should be noted that to obtain accurate measurements during oscillations, an appropriate volume of the sample must be placed in the center of the plate without the presence of bubbles. During sample loading, insufficient volume, air bubbles, and/or off-center placement will create inadequate contact with the plates, resulting in lower recorded values. Conversely, sample overflow will create excessive shear stress due to additional drag force²⁵.

This study describes how to process, store, and treat thick mucus samples immediately upon collection. One of the main challenges that confront studies of sputum rheology is the heterogeneous nature of these samples and the development of standardized measurement approaches. Sputum is an expectorated substance often contaminated with saliva that contains bacteria and digestive enzymes that can rapidly alter the mucin network and affect mucus viscoelasticity. Therefore, it is critical to remove saliva from sputum samples immediately upon collection and/or before homogenization. By nature, mucus is sticky and difficult to handle, but the use of positive displacement pipettes facilitates homogenization without compromising the mucin network, enables accurate aliquot preparation, and simplifies sample loading. Depending on the experiment, sample homogenization may not be required but can minimize variability between replicates. While processing sputum immediately after collection is recommended, airway mucus maintains unique biophysical properties after freezing and thawing. However, freezing and thawing can affect the overall rheology of a sample. Therefore, only samples that have undergone similar freeze/thaw cycles should be compared to one another. When testing the effects of mucoactive agents, initial sample homogenization is important to optimize compound diffusion. Drug delivery to the lungs via inhalation limits the volumes that access the target (i.e., mucus plug), but the constant beating of the cilia combined with mucociliary transport generates some mixing of the drug and target. To simulate in vivo treatment, small volumes of a pharmacological agent can be applied directly to samples and gradually mixed by regular agitation throughout incubation time. However, other treatment methods (e.g., drug nebulization onto the sample in a Petri dish) can be investigated. Gentle agitation during incubation will ensure progressive drug penetration without compromising the mucin network due to mechanical disruption (e.g., vortexing or sonication). Currently, TCEP is not used in clinical settings, but other mucoactive reagents, such as NAC, rhDNase, P-2119, ARINA-1, and PAAG are being investigated for a wide range of muco-obstructive conditions^21,26,27,28. For concept validation, it was demonstrated that this protocol can be used to detect significant changes in asthmatic mucus in response to TCEP treatment. A more fluid-like mucus was produced by treatment with a reducing agent, which is apparent from the lower linear viscoelastic and gel point markers, suggesting an improvement in clearance capability. Although rhDNase produced tremendous clinical benefits in CF, it is not typically used for other muco-obstructive diseases, likely due to chronically lower extracellular DNA concentrations. However, during an acute viral and bacterial infection, a strong inflammatory response can temporarily cause high extracellular DNA concentration and reduce airway clearance. Hence, rapid ex vivo testing of rhDNase efficacy on a case-by-case basis could provide guidance for treating viral- and bacterial-induced pneumonia. This could be especially valuable amid the COVID-19 pandemic, which is caused by the respiratory virus, SARS-CoV-2.

In summary, the described device provides feasible, rapid, and accurate rheologic measures. These characteristics provide the potential to investigate and monitor the status of airways diseases, as well as test the effects of novel mucoactive compounds. The rapidity and simplicity of measurements allow assays to be performed without incurring complications related to freezing and/or temporal effects of prolonged storage or transport while making these assays feasible in a wide variety of settings. Ultimately, this approach could be explored for the selection of personalized therapies from a panel of options, allowing for real-time tailoring of patient treatment.

Materials

Name	Company	Catalog Number	Comments
Capillary Pistons Tips	Gilson	CP1000
Discovery Hybrid Rheometer-3	TA Instruments		DHR-3 Bulk Rheometer manufactured by TA Instruments in New Castle, DE: Used to preform rheological tests.
Graphing Software	GraphPad Prism		GraphPad Software (San Diego, CA) used for data analysis
Microcentrifuge Tube	Costar	3621
Peltier plate	TA Instruments		Temperature control system manufactured by TA Instruments in New Castle, DE
Polyethylene oxide	Sigma	372838	8 MDa polymer used as mucus simulant
Positive Displacement Pipette	Gilson	M1000	Pipette used for handling viscous solutions
Rheomuco	Rheonova		Benchtop Rheometer manufactured by Rheonova in France: Used to preform rheological tests.
Rough Lower Geometries	Rheonova	D-1811-007	25mm Diameter
Rough Upper Geometries	Rheonova	U-1811-007	25mm Diameter
Smooth Upper Parallel Plate	TA Instruments		20mm Diameter
tris(2-carboxyethyl)phosphine	Sigma	646547-10X1ML	TCEP: Potent reducing agent.