The authors acknowledge the assistance of Dr. Robert Dotson of the Tulane University Department of Cell and Molecular Biology for his assistance in editing the manuscript and Dr. James Bollinger of the Tulane University Department of Chemistry for his assistance with mass spectrometry protocol design. The authors further acknowledge the Tulane University Department of Cell and Molecular Biology and the Tulane University Department of Chemistry for their support and the use of space and equipment. This work was supported by a Tobacco Product Regulatory Science Research Fellowship to C. Anderson from the Tulane University School of Science and Engineering.

1. Assembly of the Device
<ol>
	<li>Secure a 100 mL Buchner flask (Figure 1, #4) to a steel ring stand and create a vacuum trap by filling it with 50 g of calcium chloride to serve as a desiccant. Seal the flask with a rubber through-hole stopper, wrap the stopper junction with paraffin film, and run a pipette through the hole.</li>
	<li>Using vinyl tubing, connect the pipette extending from the stopper to a t-intersection hose connector.</li>
	<li>Using vinyl tubing, connect the two impinger (Figure 1, #2 &#38; #3) to each other and connect the output of the second impinger to the t-intersection hose connector.</li>
	<li>Using vinyl tubing, connect the input port of the first impinger to serve as an inhalation port (Figure 1, #1).</li>
	<li>Using vinyl tubing, connect the side arm of the Buchner flask to the input port of an airflow regulator (Figure 1, #7) and the exit port of the airflow regulator to a vacuum pump.</li>
	<li>Assemble the circuit according to the schematic in Figure 2A.</li>
	<li>Upload the PBASIC program SVL.bs1 (Figure 2B, also available at https://github.com/ChastainAnderson/SVL) to the BS1 microcontroller (Figure 1, #6) by means of a serial adaptor and the manufacturer&#39;s software.</li>
	<li>Place the 510 threaded base (Figure 1 #8) in a ring stand clamp.</li>
	<li>Using vinyl tubing, connect the solenoid valve (Figure 1, #5) to the free end of the t-intersection hose connector. 
	NOTE: The device should be complete and ready for operation, check all joints to make sure that they are air tight and apply hose clamps and vacuum grease as needed.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56709/56709fig2.jpg" /> 
Figure 2: Electrical Schematic and PBASIC Code. Figure 2A displays the electrical schematic for assembling the electrical circuit necessary to activate both the normally open solenoid valve and the heating coil of button activated electronic cigarettes (through the 510 threaded electronic cigarette tank base). The electrical parameters of the heating coil (P: Power; R: Resistance; and I: Current) are projected and should be empirically verified with a multimeter post assembly. Figure 2B displays the PBASIC timing program needed to control the circuit in Figure 2A (also available at https://github.com/ChastainAnderson/SVL). The timing constants SVT &#38; IPT (#5 &#38; #6) are in units of ms and are set to provide an activation time of 2 seconds and a downtime of 28 s. <a href="//cloudfront.jove.com/files/ftp_upload/56709/56709fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Sample Storage and Preparation
<ol>
	<li>Store all unopened conventional and electronic cigarette samples in the dark in airtight plastic bags at room temperature.</li>
	<li>Once opened, store samples in airtight plastic bags at 4 &#186;C, with a paper towel to absorb excess moisture.</li>
	<li>Pre-equilibrate all samples in a room temperature humidor at ~60% humidity for at least 30 minutes prior to use.</li>
</ol>
3. General Operation of Cigarette Smoke/Electronic Cigarette Aerosol Extraction Device
<ol>
	<li>Determine the mass of each e-cigarette cartomizer/tank pre-vaping using an analytical balance. The difference in pre/post-post vaping weight will be used to determine appropriate dosing. 
	NOTE: 3R4F reference cigarettes are assumed to contain 0.7 mg of nicotine, and the nicotine content of commercial cigarette brands can be determined by conventional analytical methods13.</li>
	<li>For sample application 1, fill the reservoir of the primary impinger with 4.3 mL of endothelial cell culture medium. For sample application 2, fill the reservoir with 5 mL of acetone.</li>
	<li>Prep the electronic cigarette or conventional cigarette for extraction:
	<ol>
		<li>If using a conventional cigarette, apply a piece of clear tape around the filter and put an easily visible mark where the cigarette paper joins the filter.</li>
		<li>If using a cigarette like e-cigarette, make sure the battery is well charged and the cartomizer tightly screwed to the battery.</li>
		<li>If using an electronic cigarette tank, make sure that an appropriate volume of electronic cigarette liquid is loaded in the tank and screw the tank onto the 510 threaded base.</li>
	</ol>
	</li>
	<li>Insert the tip of the conventional or electronic cigarette into the inhalation port (Figure 1, #1) and secure with a hose clamp.</li>
	<li>Turn on the vacuum pump.</li>
	<li>Adjust the flow meter to pull 1.65 L/minute to ensure a 55 mL puff over 2 seconds.</li>
	<li>Turn on the microcontroller. If using a conventional cigarette, light the cigarette on the first puff.</li>
	<li>Run until projected desired concentration (in parts per million or % weight/volume) is achieved.</li>
	<li>Determine the mass of each e-cigarette cartomizer/tank after vaporization using an analytical balance. Compare this measurement to the measurement taken in step 3.1 to determine the total mass consumed. Calculate the concentration of the consumed mass/volume of solvent. Use the molar concentration of consumed nicotine to normalize between products.
	<ol>
		<li>If an insufficient mass was consumed, return the electronic cigarette to the device and consume further.</li>
		<li>If a sufficient or excess mass was consumed, proceed.</li>
	</ol>
	</li>
</ol>
4. Filtration and Storage
<ol>
	<li>If extract is to be used for cell culture, filter through a 0.22 &#181;m PES syringe filter.</li>
	<li>Use the extracts immediately or store at -80 &#186;C. As part of the preparation for Anderson, et al.8, electronic cigarette aerosol was demonstrated to be stable for at least two weeks, and the stability of cigarette smoke for up to two years has been established by Crooks, et al.13.</li>
</ol>
5. Cleaning the Device
<ol>
	<li>After each extraction, rinse the tubing and reservoirs of the device with 70% ethanol and deionized water to prevent carry over between samples.</li>
	<li>Following rinsing, briefly run the empty device to allow airflow to assist the drying of the lines.</li>
</ol>
6. Sample Application 1: Neutral Red Uptake Cell Viability Assay
<ol>
	<li>Perform extract as above into 4.3 mL of endothelial cell growth medium.</li>
	<li>One day prior, seed human umbilical vein endothelial cells into 96 well plates at a density of 1 x 104 cells/wellin 100 &#181;L of endothelial cell growth medium.</li>
	<li>Treat the cells by replacing the old endothelial cell culture medium with either 100 &#181;L of fresh endothelial cell culture medium to serve as a control or 75 &#181;L of endothelial cell growth medium mixed with 25 &#181;L of a 2 mM consumed nicotine concentration extract (1.4 mg consumed nicotine into 4.3 mL endothelial cell growth medium) for a final concentration of 500 &#181;M to serve as treatment.</li>
	<li>As many of the components of both cigarette smoke and electronic cigarette aerosol are volatile, use a foil seal to keep the wells airtight.</li>
	<li>Incubate the plate 18&#8211;24 h at 37 &#176;C and 5% CO2.</li>
	<li>Prepare neutral red staining solution:
	<ol>
		<li>Create a 100x neutral red stock solution by dissolving 33 mg of neutral red dye into 10 mL of buffered salt solution.</li>
		<li>Shortly before use, dilute 100x stock solution 1:100 in cell culture medium to create 1x neutral red staining solution.</li>
		<li>Incubate neutral red staining solution at 37 &#176;C for at least 30 min prior to use and use immediately. 
		NOTE: It is normal for some crystals to precipitate during incubation. Care should be taken to avoid applying these crystals to the cell culture wells. If needed, a .22 &#181;m filter can be used to filter the neutral red stock and staining solutions.</li>
	</ol>
	</li>
	<li>Remove extract and add 100 &#181;L of neutral red staining solution per well, use excess to create at least three blank wells for proper quantification.</li>
	<li>Incubate the plate at 2&#8211;4 h at 37 &#176;C and 5% CO2.</li>
	<li>Remove neutral red staining solution and wash 3x by submersion in PBS.</li>
	<li>Apply neutral red de-stain solution (50% deionized water, 49% ethanol, 1% acetic acid).</li>
	<li>Incubate at least 10 min at room temperature with shaking.</li>
	<li>Read absorbance at 540 nm.</li>
	<li>Analyze data by subtracting out the average value of the blank wells and normalizing to the average of the blank adjusted control well value.</li>
</ol>
7. Sample Application 2: Gas Chromatography Mass Spectrometry
<ol>
	<li>Perform extraction as above into 5 mL of acetone.</li>
	<li>Run the device to achieve a final concentration of ~100 parts per million (weight of e-liquid consumed/volume of acetone) of your sample.</li>
	<li>Using a precision glass syringe, inject 1 &#181;L into the injector of a GC-MS device. Inject at 250 &#176;C with a 1:20 split ratio into a coupled gas chromatograph/quadrupole spectrometer system equipped with a ZB-5 column with the following oven protocol: 1 min at 50 &#176;C; ramp 10 &#176;C/min to 140 &#176;C; ramp 20 &#176;C/min to 300 &#176;C and hold for 10 min.</li>
	<li>Match resulting mass spectra to target library to identify aerosol components.</li>
</ol>

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The Tobacco Product Regulatory Science Research Fellowship program administered by Tulane University is funded by Altria Client Services Regulatory Affairs.

The most critical elements of this protocol are ensuring the device is clean at the start and finish of each extraction, and ensuring that all seals are maintained so that air flow remains consistent. If the device is not properly cleaned, there is a risk of carry over between samples. Additionally, if the device is left unclean for an extended period of time condensed aerosol and dried solvent can block the system. Note that it is normal for there to be a pressure drop when puffing a conventional cigarette and the airfl...

Despite a concentrated effort by health organizations, tobacco product use remains the leading cause of preventable death worldwide, with the majority of these deaths attributed to cigarette smoking1. Since entering the market in 2003, electronic cigarettes have been growing in popularity among tobacco product users. Currently, electronic cigarettes are the most popular alternative to conventional cigarettes among American adults (~5%)2 and the most popular nicotine delivery system among middle (~5.3%) and high schoolers (~16%)3. If current trends continue, electronic cigarettes can be expected to replace conventional cigarettes for future generations. However, the health consequences of electronic cigarette use remain unclear.
Research on electronic cigarettes did not start in earnest until electronic cigarette popularity rapidly increased in 20133,4. Since that time, a number of different models have been employed to address the question of their toxicity. However, the results of many studies are conflicting, and while it seems that electronic cigarettes are generally less toxic than conventional cigarettes there is no current consensus on the health consequences of electronic cigarette use5,6,7. Our previous research indicates that electronic cigarettes are significantly less toxic to the vascular endothelium than conventional cigarettes, despite their ability to cause DNA damage and the induction of oxidative stress and cell death8. However, more research is necessary before we can draw firm conclusions about the health consequences of electronic cigarette use.
As conventional cigarettes are a leading cause of preventable vascular disease9, there is a growing interest in the vascular health risk of electronic cigarette use10,11,12. In order to study the effects of electronic cigarettes on the vascular system, our lab developed a microcontroller operated smoking/vaping device (Figure 1)8. This device is capable of generating liquid extracts of either conventional cigarette smoke or electronic cigarette aerosol in either aqueous or organic solvents. As airflow is controlled by the combination of an adjustable air flow regulator and a PBASIC timing program, the device can be used to generate extracts according to any number of user defined protocols. Here we detail the assembly and operation of this device as well as two potential applications: in vitro cell viability assessment and gas-chromatography mass-spectrometry.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56709/56709fig1.jpg" /> 
Figure 1: Smoking/Vaping Device. Schematic for the physical assembly of the smoking/vaping device in both the cigarette/cigarette like electronic cigarette (e-cig) configuration (A) and the tank electronic cigarette configuration (B). Component Key: 1) Inhalation port; 2) primary collection impinger; 3) overflow impinger; 4) Buchner flask vacuum trap; 5) normally open solenoid valve; 6) BS1 microcontroller; 7) air flow regulator; 8) 510 threaded electronic cigarette tank base. <a href="//cloudflare.jove.com/files/ftp_upload/56709/56709fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>12 V AC/DC Wall Mount Adaptor</td><td>Digi-Key</td><td>T1099-P5P-ND</td><td></td></tr><tr><td>2.2 Ohm Resistors</td><td>Digi-Key</td><td>A105635-ND</td><td>Used in tandem to generate the 4.4 Ohm resistance in Figure 2A</td></tr><tr><td>330 Ohm Resistors</td><td>Digi-Key</td><td>330QBK-ND</td><td></td></tr><tr><td>510 Threaded Base</td><td>NJoy</td><td>N/A</td><td>Recovered by dismantalling a second generation NJoy electronic cigarette</td></tr><tr><td>Acetic Acid, Glacial</td><td>Sigma-Aldritch</td><td>A6283</td><td></td></tr><tr><td>Acetone (Chromatography Grade)</td><td>Sigma-Aldritch</td><td>34850</td><td></td></tr><tr><td>Basic Stamp Project Board</td><td>Digi-Key</td><td>27112-ND</td><td>This board contains the BS1 Microcontroller, serial adaptor, power switch, and a barrel pin connector for the AC/DC Wall Mount Adaptor</td></tr><tr><td>Basic Stamp USB to Serial Adapter</td><td>Digi-Key</td><td>28030-ND</td><td>An optional component to allow the BS1 serial adaptor to communicate through USB</td></tr><tr><td>Buchner Flask (Vacuum Flask) 250 mL</td><td>VWR</td><td>10545-854</td><td></td></tr><tr><td>Clear Tape</td><td>3M</td><td>S-9783</td><td></td></tr><tr><td>Clear Vinyl Tubing, 3/8&quot; ID</td><td>Watts</td><td>443064</td><td></td></tr><tr><td>EGM-2 Endothelial Cell Culture Medium</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco-Aaper</td><td>111000200</td><td></td></tr><tr><td>Flow Regulator</td><td>Dwyer</td><td>VFA-23-BV</td><td></td></tr><tr><td>Gas Chromatograph</td><td>Varian</td><td>450-GC</td><td></td></tr><tr><td>Glass Syringe, 10 mL</td><td>Sigma-Aldritch</td><td>Z314552</td><td></td></tr><tr><td>Glass Syringe, 10 &amp;micro;L</td><td>Hamilton</td><td>80300</td><td></td></tr><tr><td>High Vacuum Silicon Grease</td><td>Dow Corning</td><td>146355D</td><td></td></tr><tr><td>Hose Clamp</td><td>Precision Brand</td><td>35125</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells</td><td>ATCC</td><td>PCS-100-013&amp;nbsp;</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Varian</td><td>300-MS</td><td></td></tr><tr><td>Midget Impinger</td><td>Chemglass</td><td>CG-1820-01</td><td></td></tr><tr><td>Neutral Red</td><td>Sigma-Aldritch</td><td>N4638</td><td></td></tr><tr><td>Paraffin Film</td><td>3M</td><td>PM-992</td><td></td></tr><tr><td>Plate Seal Roller</td><td>BioRad</td><td>MSR0001</td><td></td></tr><tr><td>Plate Seal; Foil</td><td>Thermo</td><td>276014</td><td></td></tr><tr><td>Ring Stand 20&quot;</td><td>American Educational Products</td><td>7-G15-A</td><td></td></tr><tr><td>Solenoid Valve (normally open)</td><td>US Solid</td><td>USS2-00081</td><td></td></tr><tr><td>Solid State Relay</td><td>Digi-Key</td><td>CLA279-ND</td><td></td></tr><tr><td>Stand Clamp</td><td>Eisco</td><td>CH0688</td><td></td></tr><tr><td>Syringe Filter, PES, 0.22 um</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>BD Syringe</td><td>309604</td><td></td></tr><tr><td>Through Hole Stopper, Size 6</td><td>VWR</td><td>59581-287</td><td></td></tr><tr><td>Vacuum Pump</td><td>KNF Neuberger</td><td>N86KTP</td><td></td></tr></tbody></table>

a microcontroller operated device for the generation of liquid extracts from conventional cigarette smoke and electronic cigarette aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Within 24 hours of the exposure of human umbilical vein endothelial cells to either conventional cigarette smoke extract (CSE) or electronic cigarette aerosol extract (EAE), there is a significant (control vs. CSE P &#60;0.001; control vs. EAE P &#60;0.01; n = 6) reduction in cell viability (Figure 3A). Extracts were generated with a puffing profile of 2, 2 second, 55 mL puffs per minute and normalized based on molar concentration of nicotin...

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A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

a-microcontroller-operated-device-for-generation-liquid-extracts-from

Research

JoVE Journal

Bioengineering

8.3K Views.  Tulane University. Hier beschrijven we een programmeerbare laboratorium-apparaat dat kan worden gebruikt voor het maken van extracten van conventionele sigaret rook en elektronische sigaret aërosol. Deze methode biedt een nuttig instrument voor het maken van directe vergelijkingen tussen conventionele sigaretten en elektronische sigaretten, en een toegankelijk toegangspoort tot elektronische sigaret onderzoek.

Video: A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Medicine

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...

Engineering

A Versatile Kit Based on Digital Microfluidics Droplet Actuation for Science Education

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a specific example. It also has fluorescent imaging capability and closed humidified enclosure based on an ultrasonic atomizer to prevent evaporation. The kit can be assembled within a short period of time and with minimal training in electronics and soldering. The kit allows both undergraduate/graduate students and enthusiasts to obtain hands-on experience in microfluidics in an intuitive way and be trained to gain familiarity with digital microfluidics.

We describe an educational kit that allows users to execute multiple experiments and gain hands-on experience on digital microfluidics.

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Immunology and Infection

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

Source: Phillips, J. E. et al., <a href="https://app.jove.com/v/55454">Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the generation of drug aerosols and the targeted nose-only delivery of these aerosols to mice using an inhalation unit equipped with an aerosol generator. The mouse is restrained in a nose-only restrainer and connected to the inhalation unit, which delivers the drug directly into the mouse&#39;s nasal cavity. Subsequently, the drug passes through the trachea and enters the lungs.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

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