This paper is supported by grants from Vertex Pharmaceuticals (Ehre RIA Award) and CFF-supported Research EHRE20XX0.

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
<ol>
	<li>Collect airway mucus via sputum collection or bronchoscopy aspiration.
	<ol>
		<li>Collect sputum either via spontaneous expectoration or induce sputum by 3% hypertonic saline inhalation. Alternatively, directly aspirate mucus...

<ol>
	<li>Button, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+periciliary+brush+promotes+the+lung+health+by+separating+the+mucus+layer+from+airway+epithelia.">A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia.</a> Science. 337 (6097), 937-941 (2012).</li><li>Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muco-obstructive+lung+diseases.">Muco-obstructive lung diseases.</a> New England Journal of Medicine. 380 (20), 1941-1953 (2019).</li><li>Rose, M. C., Voynow, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aerosolized+recombinant+human+DNase+on+exacerbations+of+respiratory+symptoms+and+on+pulmonary+function+in+patients+with+cystic+fibrosis.+The+Pulmozyme+Study+Group.">Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group.</a> The New England Journal of Medicine. 331 (10), 637-642 (1994).</li><li>Hubbard, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+of+aerosolized+recombinant+human+deoxyribonuclease+I+in+the+treatment+of+cystic+fibrosis.">A preliminary study of aerosolized recombinant human deoxyribonuclease I in the treatment of cystic fibrosis.</a> The New England Journal of Medicine. 326 (12), 812-815 (1992).</li><li>Shak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosolized+recombinant+human+DNase+I+for+the+treatment+of+cystic+fibrosis.">Aerosolized recombinant human DNase I for the treatment of cystic fibrosis.</a> Chest. 107, 65-70 (1995).</li><li>Ma, J. T., Tang, C., Kang, L., Voynow, J. A., Rubin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+sputum+rheology+correlates+with+both+acute+and+longitudinal+changes+in+lung+function.">Cystic fibrosis sputum rheology correlates with both acute and longitudinal changes in lung function.</a> Chest. 154 (2), 370-377 (2018).</li><li>Donaldson, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+clearance+and+lung+function+in+cystic+fibrosis+with+hypertonic+saline.">Mucus clearance and lung function in cystic fibrosis with hypertonic saline.</a> The New England Journal of Medicine. 354 (3), 241-250 (2006).</li><li>Patarin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+analysis+of+sputum+from+patients+with+chronic+bronchial+diseases.">Rheological analysis of sputum from patients with chronic bronchial diseases.</a> Scientific Reports. 10 (1), 15685 (2020).</li><li>Markovetz, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotracheal+tube+mucus+as+a+source+of+airway+mucus+for+rheological+study.">Endotracheal tube mucus as a source of airway mucus for rheological study.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 317 (4), 498-509 (2019).</li><li>Ramsey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+mucus+hyperconcentration+in+non-cystic+fibrosis+bronchiectasis.">Airway mucus hyperconcentration in non-cystic fibrosis bronchiectasis.</a> American Journal of Respiratory and Critical Care Medicine. 201 (6), 661-670 (2020).</li><li>Dunican, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+plugs+in+patients+with+asthma+linked+to+eosinophilia+and+airflow+obstruction.">Mucus plugs in patients with asthma linked to eosinophilia and airflow obstruction.</a> The Journal of Clinical Investigation. 128 (3), 997-1009 (2018).</li><li>Ehre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+inhaled+mucolytic+to+treat+airway+muco-obstructive+diseases.">An improved inhaled mucolytic to treat airway muco-obstructive diseases.</a> American Journal of Respiratory and Critical Care Medicine. 199 (2), 171-180 (2019).</li><li>Morrison, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+cystic+fibrosis+airway+cells+with+CFTR+modulators+reverses+aberrant+mucus+properties+via+hydration.">Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration.</a> The European Respiratory Journal. 59 (2), 2100185 (2021).</li><li>Puchelle, E., Jacquot, J., Beck, G., Zahm, J. M., Galabert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+and+transport+properties+of+airway+secretions+in+cystic+fibrosis-relationships+with+the+degree+of+infection+and+severity+of+the+disease.">Rheological and transport properties of airway secretions in cystic fibrosis-relationships with the degree of infection and severity of the disease.</a> European Journal of Clinical Investigation. 15 (6), 389-394 (1985).</li><li>Puchelle, E., Zahm, J. M., Quemada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+properties+controlling+mucociliary+frequency+and+respiratory+mucus+transport.">Rheological properties controlling mucociliary frequency and respiratory mucus transport.</a> Biorheology. 24 (6), 557-563 (1987).</li><li>Cardinaels, R., Reddy, N. K., Clasen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+errors+due+to+overfilling+for+Newtonian+fluids+in+rotational+rheometry.">Quantifying the errors due to overfilling for Newtonian fluids in rotational rheometry.</a> Rheologica Acta. 58 (8), 525-538 (2019).</li><li>Hancock, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muc5b+overexpression+causes+mucociliary+dysfunction+and+enhances+lung+fibrosis+in+mice.">Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice.</a> Nature Communications. 9 (1), 5363 (2018).</li><li>Adewale, A. 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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 ...

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 cilia1,2. 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 clearance4,5. 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 obstruction7,8. 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 transport8,9. 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 minutes10,11. 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-being12,13,14. 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 function15,16. 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&#39;, G&#34;, G*, and tan &#948;) and gel point (&#947;c, and &#963;c) characteristics (see Table 1).The elastic or storage modulus (G&#39;) describes how a sample responds to stress (i.e., the ability to return to its original shape), while the viscous or loss modulus (G&#34;) 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 &#948;) 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 &#948; indicates an elastic-dominant/solid-like behavior, while a high tan &#948; indicates a viscous-dominant/liquid-like behavior). For gel point characteristics, the crossover strain (&#947;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&#39; = G&#34; or tan &#948; = 1. The crossover yield stress (&#963;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&#39; &#62; G&#34;). In muco-obstructive diseases, both G&#39; and G&#34; increase as a result of pathological mucus changes17,18,19. 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 agent8,20. 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)&#160;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&#39;, and G&#34; endpoints and reflected the substantial abnormalities in mucus viscoelasticity that were clinically apparent in this patient&#39;s case (Figure 1D). Finally, ex vivo treatment with TCEP resulted in a significant reduction in G&#39; and G&#34;, and an increase in tan &#948;, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Capillary Pistons Tips</td>
			<td>Gilson</td>
			<td>CP1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Discovery Hybrid Rheometer-3</td>
			<td>TA Instruments</td>
			<td></td>
			<td>DHR-3 Bulk Rheometer manufactured 
			by TA Instruments in New Castle, DE: Used to preform rheological tests.</td>
		</tr>
		<tr>
			<td>Graphing Software</td>
			<td>GraphPad Prism</td>
			<td></td>
			<td>GraphPad Software (San Diego, CA) used for data analysis</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube</td>
			<td>Costar</td>
			<td>3621</td>
			<td></td>
		</tr>
		<tr>
			<td>Peltier plate</td>
			<td>TA Instruments</td>
			<td></td>
			<td>Temperature control system manufactured 
			by TA Instruments in New Castle, DE</td>
		</tr>
		<tr>
			<td>Polyethylene oxide</td>
			<td>Sigma</td>
			<td>372838</td>
			<td>8 MDa polymer used as mucus simulant</td>
		</tr>
		<tr>
			<td>Positive Displacement Pipette</td>
			<td>Gilson</td>
			<td>M1000</td>
			<td>Pipette used for handling viscous solutions</td>
		</tr>
		<tr>
			<td>Rheomuco</td>
			<td>Rheonova</td>
			<td></td>
			<td>Benchtop Rheometer manufactured by Rheonova in France: Used to preform rheological tests.</td>
		</tr>
		<tr>
			<td>Rough Lower Geometries</td>
			<td>Rheonova</td>
			<td>D-1811-007</td>
			<td>25mm Diameter</td>
		</tr>
		<tr>
			<td>Rough Upper Geometries</td>
			<td>Rheonova</td>
			<td>U-1811-007</td>
			<td>25mm Diameter</td>
		</tr>
		<tr>
			<td>Smooth Upper Parallel Plate</td>
			<td>TA Instruments</td>
			<td></td>
			<td>20mm Diameter</td>
		</tr>
		<tr>
			<td>tris(2-carboxyethyl)phosphine</td>
			<td>Sigma</td>
			<td>646547-10X1ML</td>
			<td>TCEP: Potent reducing agent.</td>
		</tr>
	</tbody>
</table>

rapid viscoelastic characterization of airway mucus using a benchtop rheometer

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&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;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 &#60;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 &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). 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.

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, P...

Watch this Scientific Journal Video about Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer at JoVE.com

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

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.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Muco-obstructive diseases are characterized by abnormal viscosity of the airway mucus. The protocol shows how to use rheology to quantify mucus viscosity and analyze data to test mucus-targeting treatments. Mucus rheology provides specific quantitative biomarkers.Using a benchtop device greatly simplifies the logistics of measuring these biomarkers in clinical specimens like human sputum samples. Using mucus rheology as a diagnosis tool for obstructive diseases is still challenge. An increasing body of evidence shows a strong correlation between rheology properties, inflammation, and bacterial infection.Mucus is always present at the body interface where it lubricates and plays a protective role. Mucus rheology can be applied to other systems like the gastrointestinal and genital tracks. To acquire accurate measurements, correct handling and placement of the sample is critical.A visual demonstration can enhance the precision of this assay. Demonstrating this procedure will be Kendall Shaffer, a research technician from the air laboratory. Begin by sorting the collected airway sputum or mucus in sterile specimen cups.In the case of sputum, remove excess saliva from the sample immediately upon collection. Place the samples on ice for transport. Limit the transport time to less than four hours.Analyze the samples at the time of collection. 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.Store the samples at 80 degrees Celsius until processed. Aliquot the samples for storage in volumes greater than or equal to 500 microliters to ensure sufficient volume for experiments. Before aliquoting, pipette fresh and frozen sputum or mucus directly or homogenize specimens.Repair the required aliquots for repeat measurements and treatments. Incubate the aliquots to be tested at 37 degrees Celsius for at least five minutes prior to measurement. For testing pharmacological agents, use high concentrations of stock solutions to prevent sample dilution.Add between 1%to 10%volume of the desired reagent directly onto the sample. Make sure no drop of the compound stays on the side of the tube. Incubate the samples at 37 degrees Celsius for the required length of time, to allow a chemical reaction.Mix the mucus sample and reagent by flicking the bottom of the microcentrifuge tube every two minutes to allow progressive penetration of the reagent into the mucus sample without compromising the mucin network. When comparing multiple drug reagents, ensure that the incubation time is similar. Turn on the machine and initialize the software.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.Select a geometry set and inspect large and small plates carefully, to ensure that plates are clean and in perfect condition. Insert the large plate firmly on the bottom pulpit. 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. Wait until the temperature reaches 37 degrees Celsius target value. Then initiate automatic calibration, as prompted by the software.Using a positive displacement pipette, slowly pipette between 250 and 500 microliters 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. 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. To ensure that the sample fills the gap, use the reduced gap function until the sample is no longer in the biconcave shape or aligned with the plate's edge. The reduced gap function lowers the measuring edge in 0.1 millimeter increments and is limited to 7 increments.If a gap remains after the 7 increments, click on Redo Installation to return to the initial position and adjust the sample's position and volume. If the gap is exceedingly reduced, remove the excess sample with a spatula by a circular motion along the edge of the upper plate. Trim the excess sample gently to avoid shear stress.Lower the protective cover to avoid any accidental projection of contaminated fluids during oscillation. To initiate measurement, click on Start Analysis. An entire cycle will take four to seven minutes.Avoid talking loudly and touching the device or the bench during the entire length of the cycle. A quiet environment is crucial for the first two minutes. Once the cycle is complete, click next to raise the measuring head and generate the sample analysis report.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.The viscoelastic characteristics of eight megadalton PEO were measured at five concentrations and directly compared between the evaluated benchtop rheometer and a traditional bulk rheometer. In contrast with PEO solutions, status asthmaticus mucus represents a solid-like elastic dominated behavior at low strain and features a crossover at high strain. In addition, triplicate measurements performed in 1.5%PEO solution and clinical assay mucus sample, confirmed that linear viscoelastic characteristics were highly repeatable for the values obtained from the biological sample.Changes were measured in the viscoelastic properties of mucus following treatment with a mucolytic agent. TCEP effects on assay 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 by 4.6 fold, elastic modulus by 5.1 fold, viscous modulus by 1.9 fold, crossover strain by 3.3 fold, and crossover yield stress by 5.7 fold, and an increase in the damping ratio by 2.8 fold.Measuring airway mucus viscoelasticity in a fast paced clinical setting can help tailor patients'treatment on a case-by-case basis, as well as provide a standardized protocol for testing novel pharmacological compounds. This technique is currently used to examine the effects of highly effective CFTR modulators on cystic fibrosis sputum and to test the potency of various drugs aimed at altering mucus properties.

rapid-viscoelastic-characterization-airway-mucus-using-benchtop

Research

JoVE Journal

Medicine

3.0K visualizações.  The University of North Carolina at Chapel Hill. As doenças muco-obstrutivas são caracterizadas pela viscosidade anormal do muco das vias aéreas. O protocolo mostra como usar a reologia para quantificar a viscosidade do muco e analisar dados para testar tratamentos direcionados ao muco. A reologia do muco fornece biomarcadores quantitativos específicos.O uso de um dispositivo benchtop simplifica muito a logística de medir esses biomarcadores em espécimes clínicos como amostras de escarro humano. O uso da reologia do muco como ...