Name: in vitro method to control concentrations of halogenated gases in cultured alveolar epithelial cells
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Description: Watch this Scientific Journal Video about In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells at JoVE.com

The authors acknowledge the Auvergne Regional Council (&#34;Programme Nouveau Chercheur de la R&#233;gion Auvergne&#34; 2013) and the French Agence Nationale de la Recherche and the Direction G&#233;n&#233;rale de L&#39;Offre de Soins (&#34;Programme de Recherche Translationnelle en Sant&#233;&#34; ANR-13-PRTS-0010) for the grants. The funders had no influence in the study design, conduct, and analysis or in the preparation of this article.

1. Culture of Alveolar Epithelial Cells (hAELVi)
<ol>
	<li>Thawing
	<ol>
		<li>Pipette 4 mL of cultivation ready-to-use human alveolar epithelial (huAEC) medium in a 15 mL plastic tube and quickly thaw the vial in a preheated water bath (37 &#176;C).</li>
		<li>Transfer the thawed cell suspension to a 15 mL plastic tube containing 4 mL of the medium before centrifuging the tube at 200 x g for 5 min.</li>
		<li>Aspirate the supernatant and resuspend the cell pellet with 5 mL of the cultivation medium. Then, transfer the cells to a T25 flask.</li>
		<li>Cultivate the cells under standard conditions (5% CO2, 95% humidified air, 37 &#176;C)</li>
	</ol>
	</li>
	<li>Splitting
	<ol>
		<li>Check the status of the cells microscopically. When the cells are 80-90% confluent, split the cells, following steps 1.2.2 through 1.2.10.</li>
		<li>Aspirate the cultivation media of the cells with a sterile pipette.</li>
		<li>Wash the cells once with 4 mL of Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) and aspirate the DPBS.</li>
		<li>Add 1 mL of Trypsin/EDTA solution (TE) to the cells prior to incubating at 37 &#176;C for 3 min until the cells start to detach; check for detachment under a microscope.</li>
		<li>Resuspend the cells with 2 mL of the culture medium and centrifuge the cells at 200 x g for 5 min. Then, aspirate the supernatant and resuspend the cell pellet again with 3 mL of the cultivation medium.</li>
		<li>Use the trypan blue dye exclusion assay to determine the viability of the cells. Take 30 &#181;l of the cell suspension and add 30 &#181;l of a 0.4% solution of trypan blue in a tube. After that, effectively mix the solution 3-5 times using a 100 &#181;l pipette. Take 10 &#181;l of the solution and put it under the coverslip of the hemocytometer.</li>
		<li>Count both the total number of viable cells and the number of dead cells (blue) in the areas of the hemocytometer. Then, calculate the cell viability [%] using the equation: cell viability = 100 &#8211; (100 / total number of cells x number of dead cells)</li>
		<li>Transfer the aliquot with the resuspended cell pellet to a new cell culture T25 flask or a 6 well-plate. Add the cultivation medium to the cells to achieve a total of 5 mL of cultivation medium in the T25 flask, or 1 mL for each well of the 6 well-plate.</li>
		<li>Cultivate the cells in an incubator under standard conditions (5% CO2, 95% humidified air, 37 &#176;C)</li>
		<li>Once the cells are completely confluent, they are ready for the experiment.</li>
	</ol>
	</li>
</ol>
2. Preparation of an Air-tight Chamber
NOTE: The construction plan for the air-tight chamber is depicted in Figure 1.
<ol>
	<li>Use a hermetic polypropylene box with a capacity of 6.5 L. The length, width, and height are 30 x 20 x 15.5 cm, respectively. Please note, the volume of the box is lower than the theoretical volume because of the rounded corners.</li>
	<li>Drill a 2.5 cm diameter hole on the bottom side of the lateral wall.</li>
	<li>Insert a corrugated tube with a green mark, which will serve as the gas-air mixture input pipe, and seal it with silicon.</li>
	<li>Drill a second 2.5 cm diameter hole on the top side of the opposite lateral wall.</li>
	<li>Insert another corrugated tube with a red mark and connect it with a charcoal filter, which will serve as the gas-air mixture output pipe, and seal it with silicon.</li>
	<li>Drill a tight 4 mm diameter hole at the center of the mean wall of the box.</li>
	<li>Insert short infusion tubing that is connected at a manifold with a rotating male luer-lock, which will be plugged into a gas analyzer, and seal it with silicon.</li>
	<li>Place a digital thermometer/hygrometer inside the air-tight chamber.</li>
</ol>
3. Expose Alveolar Epithelial Cells to Halogenated Agents (Sevoflurane and Isoflurane)
NOTE: A schematic drawing of the device is depicted in Figure 2.
CAUTION: Although animal studies have revealed no evidence of fetal harm or impaired fertility, and a very small study during cesarean sections did not show any untoward effects on the mother or fetus, the safety of using halogenated agents (e.g., sevoflurane or isoflurane) during labor and delivery has not been demonstrated to date. Furthermore, no controlled data has been collected during human pregnancies. Therefore, performing experiments using sevoflurane or isoflurane while pregnant should be strongly discouraged.
<ol>
	<li>Work under a laboratory extractor hood.</li>
	<li>Customize an anesthetic machine circuit to switch the gas line of nitrous oxide by carbon dioxide (CO2).</li>
	<li>Plug the air-tight chamber with the green-marked, corrugated tube into the customized anesthetic machine circuit. Insert a heated humidifier (such as those used on ICU ventilators) into the pipe between the anesthetic machine and the air-tight chamber to warm the gas flow mixture to approximately 37 &#176;C</li>
	<li>Install the air-tight chamber on a hot plate, providing a heating plate temperature of 37&#176;C.</li>
	<li>Put the 6 well-plate containing the hAELVI cells into the air-tight chamber and seal the lid.</li>
	<li>Regulate the gas flow rates (i.e., the mixture of air and CO2) to quickly obtain the standard conditions, defined as 5% of CO2 and 95% of humidified air.</li>
	<li>Open the halogenated agent evaporator and choose the percentage desired (in the present study, the tested concentrations of sevoflurane and isoflurane were 4% and 1%, respectively).</li>
	<li>Note the composition of gas mixture and the sevoflurane or isoflurane concentration as measured by an external gas analyzer and displayed on the screen.</li>
	<li>Once the target values are achieved, reduce the fresh gas flow rate to 1 L/min.</li>
	<li>The air-tight chamber can be maintained with this gas flow rate as long as necessary for the experiment.</li>
</ol>
4. Measure Sevoflurane or Isoflurane by Chromatography
<ol>
	<li>Preparation of samples
	<ol>
		<li>At different time points, very briefly open the chamber to take out the studied samples (in the present study, we used a 6 well-plate) and close the lid. Keep the other samples in the box. Then, aspirate 1 mL of the medium contained in each sample with a multi-volume adjustable micropipette.</li>
		<li>Put the medium into 10 mL headspace chromatography vials, which should be screwed hermetically tight with a Teflon-sealed cap. Freeze the chromatography vials at -20 &#176;C if you do not use them immediately.</li>
		<li>Prepare a stock solution of sevoflurane and another stock solution of isoflurane, both at 50 g/L in methanol. Simultaneously, prepare a stock solution of chloroform (internal standard, IS) at 2 g/L in methanol. Store all standard solutions at -20 &#176;C.</li>
		<li>Prepare working solutions of sevoflurane and isoflurane at 50, 500, and 5000 mg/L in ultrapure water/dimethyl sulfoxyde (50/50; v/v). For internal standardization, the working solution is fixed at 100 mg/L in methanol.</li>
	</ol>
	</li>
	<li>Analysis of cellular samples 
	NOTE: The extraction procedure is based on the previously validated method of gas chromatography and mass spectrometry from Bourdeaux et al.1 and uses the same parameters of sensibility and specificity. Sevoflurane and chloroform (IS) were used in this protocol, and isoflurane was associated with the multiparametric analytical method. Briefly, for mass spectrometry acquisition, the method was developed after pure solution injection. Then, m/z was confirmed with reference standards and literature data. Three m/z were selected for each analyte (except IS): one m/z for quantification (the most abundant and the higher), for which abundance was calculated by integrating the area under the curve for quantification, and two m/z for confirmation. Using three m/z, analytes could be specifically identified because all m/z had the same retention time, as well as because all m/z amounts (relative of ion confirmation vs. quantification) were the same in the pure standard and in all samples. With this acquisition mode, analytes could be identified and quantified with good specificity.
	<ol>
		<li>Construct a calibration 8-point curves with the concentration ranges of 0.5-400 mg/L and multiple quality controls (0.5, 1, 5, 10, 20, 75, 200, and 400 mg/L). 
		NOTE: To validate each calibration, four quality controls were used: the lower limit of quantification (C1 = 0.5 mg/L), two intermediate levels (C2 = 20 mg/L and C3 = 75 mg/L) and the final level (C4 at 400 mg/L; upper level of quantification). All standards and controls were analyzed in cultured cell matrices to avoid the matrix effect. For each calibration curve, a blank matrix was analyzed to validate that there was no interference with cultured cellular and internal standards.</li>
		<li>Prepare a stock solution of sevoflurane and another stock solution of isoflurane, both at 50 g/L in methanol. Simultaneously, prepare a stock solution of chloroform (internal standard, IS) at 2 g/L in methanol. Store all standard solutions at -20 &#176;C. Then, prepare working solutions of mixed sevoflurane/isoflurane at 50, 500, and 5000 mg/L in ultrapure water / dimethyl sulfoxyde (50/50; v/v). For IS, the working solution is diluted at 100 mg/L in methanol.</li>
		<li>For calibration curves and controls, prepare the samples by spiking 50 &#181;L of IS (100 mg/L) into 1 mL of the cellular sample matrices.</li>
		<li>Prepare sample solutions with 200 &#181;L of saturated sodium chloride water solution in a 10 mL headspace tube, screwed hermetically tight with a Teflon-sealed cap.</li>
	</ol>
	</li>
	<li>Gas chromatography and mass spectrometry
	<ol>
		<li>For sample analyses, use headspace injections in a gas chromatography, coupled with the mass detection method.</li>
		<li>Carry headspace tubes for 10 min at 80 &#176;C with a heater shaker. Then, withdraw and inject 1.5 mL of the gas sample into the gas chromatograph. Set the parameter of the injector at 260 &#176;C with a split flow at 100 mL/min for 2 min at the start of the chromatography run.</li>
		<li>Use Split/splitless injector with a carrier mode-programmed pressure. First, keep the gas pressure at 40 kPa for 0.15 min. Then, increase the rate program pressure to 150 kPa at 125 kPa/min before setting a rate of 16 kPa/min to 300 kPa pressure for 5 min.</li>
		<li>Simultaneous to the injection, start with an oven temperature of 60 &#176;C for 1 min and increase until a temperature of 140 &#176;C is reached at a rate of 20 &#176;C/min. Then, increase the temperature again until 250 &#176;C is achieved. The total time of the run is 7 min.</li>
		<li>Carry out the chromatography separation using a fused-silica capillary column (30 m x 1.4 &#181;m, 0.25 mm ID). Perform mass experiments with a single ion monitoring (SIM) condition, and monitor ion quantification m/z 181, and ion qualifications m/z 151 and 51 simultaneously at a retention time (RT) of 2.30 min for sevoflurane, m/z 149 (ion quantification), m/z 115 and 87 (ion qualifications) for isoflurane at RT 2.8 min, and m/z 83 (ion quantification) for chloroform at RT 3.70 min.</li>
		<li>Determine the concentrations of sevoflurane and isoflurane in the cell culture by their area ratios to that of the IS using a weight quadratic fit. The lower limit of quantification (LLOQ) for sevoflurane and isoflurane was at 0.5 mg/L and the upper limit of quantification (ULOQ) was 400 mg/L.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Our protocol describes an easy method to expose cells to a precise fraction of a halogenated anesthetic agent, such as sevoflurane or isoflurane. Furthermore, we report here&#8212;for the first time&#8212;a rigorous correlation between both the gas fraction and the concentration of sevoflurane and isoflurane inside the culture medium itself. This fundamental step now allows us to safely use our air-tight chamber to study the effects of these halogenated agents in a cultured monolayer of human alveolar epithelial cells.
Currently, most research teams studying the effects of sevoflurane in alveolar cells use a jar that is first saturated with halogenated gas and then sealed. In this case, sevoflurane may be metabolized, and it could be speculated that the fraction of volatile agent may decrease linearly over time, leading to an unstable gas concentration. However, the correlation between the gas fraction of sevoflurane and its concentration in the culture medium is not clearly reported in the literature. Usually, the concentration of sevoflurane used in these experiments is chosen based on a simple relationship between the gas fraction and the MAC. MAC was introduced in 1965 and is the concentration of a vapor in the lungs that is needed to prevent a motor response (movement) in 50% of subjects in response to a surgical stimulus (pain)22. MAC is used to compare the strength, or potency, of anesthetic vapors. In ICU patients, MAC is correlated to FeSevo and the clinical Richmond Assessment Agitation-Sedation Scale (RASS)23. Although it is a useful indicator in daily clinical practice, the relevance of this parameter has never been investigated in the setting of experimental in vitro research. In our protocol, using chromatography analyses of the medium, we determined the precise correlation between the sevoflurane contained in the gas fraction and the sevoflurane dissolved into the medium. With this method, the specific effect of a volatile agent is expressed according to the real concentration in the medium rather than based on the approximation of a clinical effect. This important element allows the study of the specific effect of a precise concentration of a halogenated agent on cells growing in a medium, in order to compare the effects of different concentrations of inhaled agents. Furthermore, as the air-tight chamber is very easy to use, this method allows researchers to replicate the experiment with precision.
Another important point that may preclude the use of the correlation between gas fraction and MAC in experimental research is that a halogenated agent has low solubility in blood (blood/gas partition coefficient at 37&#176;C = 0.63 to 0.69 for sevoflurane). A minimal quantity of sevoflurane is mandated to dissolve in the blood before the pressure in the alveoli achieves equilibrium with the pressure in the arterial. Thus, during the induction of anesthesia, the alveolar (end-tidal) concentration (AF, alveolar fraction) of sevoflurane rapidly increases around the inspired concentration (FI, inspired fraction). However, in vitro culture conditions do not allow such mechanisms, and usual cell media mainly consist of aqueous solutions. Furthermore, the solubility coefficient between water/gas (partition coefficient at 37&#176;C = 0.36 for sevoflurane) is lower than between blood and gas, underlying the critical importance of performing chromatography analyses.
Additionally, when a sealed jar is used, the atmospheric oxygen in the jar is absorbed by the cells with the simultaneous generation of carbon dioxide. This effect is probably insignificant in short experimental procedures, but for longer experimental durations, cells that are deprived of oxygen would switch to an anaerobic metabolism; this change in metabolism may induce a certain degree of bias in experimental analyses. In contrast to the sealed jar, when using our air-tight chamber, both oxygen and halogenated agent flows are adjustable over time to maintain the targeted level. This major characteristic of our protocol therefore allows the design of in vitro experiments for long time periods (e.g., more than one day), making it an interesting tool to study the cellular mechanisms involved in lung epithelial injury and repair over time, especially when halogenated agents are used. Indeed, the effects of inhaled anesthetic agents on lung cells or tissue during alveolar injury remain poorly investigated to date while this alternative therapy seems to show very encouraging results12.
However, there are limitations to this technique. First, an anesthetic machine circuit is needed to provide oxygen, carbon dioxide, and halogenated agent gas flows. Using such a device is mandatory to set the flow rate and maintain stable concentrations over time. Second, to sample the medium prior to chromatography analyses, the air-tight chamber is briefly opened, which induces a transient decrease in the gas concentrations. As we use an anesthetic machine circuit, gas flows are thereafter increased until expected concentrations are achieved again on the gas analyzer. Third, we have measured the concentration in the medium for only one fraction of each halogenated agent, chosen a priori based on previous study. Fourth, to stabilize the cell medium to the growth of alveolar epithelial cells, we need to use carbon dioxide at a concentration of 5%. Indeed, no anesthetic machine circuit provides such a concentration of carbon dioxide. Therefore, the anesthetic machine circuit needs to be customized to allow the connection of carbon dioxide gas flow in place of nitrous oxide. Such a connection should be used exclusively in the setting of experimental research and should cautiously be unplugged after each experiment. Furthermore, to avoid any risk for humans, we invite researchers to use a devoted anesthetic machine circuit to perform this protocol and not to use a machine dedicated to clinical anesthesia.
The main advantages of this technique are that it is relatively inexpensive and very easy to adopt, even when researchers have never manipulated an airtight chamber before. Moreover, with our protocol, the results of dissolved sevoflurane and isoflurane concentrations are reproducible, which represents a major quality criterion for experimental research. In addition, our system could allow for the study of other volatile halogenated agents, such as desflurane. Indeed, a simple change of the type of gas evaporator device would be sufficient in this case. Similarly, our system could provide a means to study the concentrations of sevoflurane or isoflurane dissolved in any type of medium with different solubilities, such as water, blood, or oil.
Our experiment represents a fundamental step that is part of a larger project designed to test the hypothesis that sevoflurane and isoflurane may exert beneficial effects on lung injury, inflammation, and AFC through RAGE-mediated pathways. A primary culture of human alveolar epithelial cells will be used for mechanistic investigations of transepithelial fluid transport, channel-specific fluid transport (e.g., using pharmacological antagonism), epithelial paracellular permeability, wound repair, cell migration and proliferation, with or without a halogenated anesthetic agent (sevoflurane or isoflurane), alone or combined with cytomix (in vitro model of alveolar injury)24.
In conclusion, this protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for this frequent and life-threatening syndrome.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar injury, lung edema, and hypoxemic respiratory failure. Although ARDS represents more than 10% of intensive care unit (ICU) admissions and nearly 25% of ICU patients requiring mechanical ventilation, it is still an under-recognized challenge for clinicians, with a hospital mortality rate of 35-45%1. Despite intense research, the identification of an effective ARDS pharmacologic therapy or prevention has failed to date. Two major features contribute to mortality in ARDS: impaired alveolar fluid clearance (AFC) (i.e., the altered resorption of alveolar edema fluid from distal lung airspaces) and severe inflammation2. Since ARDS mortality remains high, current initiatives should also include primary prevention; however, a key challenge is to identify at-risk patients in whom ARDS is likely to develop and who would benefit if ARDS were prevented.
Volatile halogenated anesthetics, such as sevoflurane and isoflurane, are widely used to provide general anesthesia in the operating room. Worldwide, more than 230 million patients undergoing major surgery each year require general anesthesia and mechanical ventilation3, and postoperative pulmonary complications adversely affect clinical outcomes and healthcare utilization4. The use of sevoflurane instead of propofol was associated with improved lung inflammation in patients undergoing thoracic surgery and significant decreases in adverse events, such as ARDS and postoperative pulmonary complications5. Similarly, pretreatment with isoflurane had protective effects on respiratory mechanics, oxygenation, and hemodynamics in experimental animal models of ARDS6,7. Although further studies are warranted to address the impact of inhaled agents on outcomes in noncardiac surgery, a similar decrease in pulmonary complications has been recently observed in a meta-analysis, demonstrating that inhaled anesthetic agents&#8212;as opposed to intravenous anesthesia&#8212;are significantly associated with a reduction in mortality for cardiac surgery8.
Specific prospective data about the use of volatile agents for the sedation of ICU patients to prevent or treat lung damage is lacking. However, several trials now support the efficacy and safety of inhaled sevoflurane for the sedation of ICU patients, and preclinical studies have shown that inhaled sevoflurane and isoflurane7,9 improve gas exchange, reduce alveolar edema, and attenuate inflammation in experimental models of ARDS. Additionally, sevoflurane mitigates type II epithelial cell damage10, whereas isoflurane maintains the integrity of the alveolar-capillary barrier through modulation of tight junction protein11. However, further studies are needed to verify to what extent the experimental evidence of organ protection from inhaled sevoflurane and isoflurane could be translated to humans. A first single-center randomized controlled-trial (RCT) from our group found that early use of inhaled sevoflurane in patients with ARDS was associated with improved oxygenation, reduced levels of some pro-inflammatory markers, and reduced lung epithelial damage, as assessed by the levels of the soluble form of the receptor for advanced glycation end-products (sRAGE) in plasma and alveolar fluid12.
Taken together, the beneficial effects of sevoflurane and isoflurane on lung injury could point to multiple biological pathways or functional processes that are dependent on the RAGE pathway, namely alveolar fluid clearance (AFC), epithelial injury, translocation of nuclear factor (NF)-&#954;B, and macrophage activation. In addition, sevoflurane may influence the expression of the RAGE protein itself. Since previous research by our research team and others supports pivotal roles for RAGE in alveolar inflammation and lung epithelial injury/repair during ARDS, we designed an experimental model to provide a translational understanding of the mechanisms of sevoflurane in lung injury and repair13,14,15. The in vitro effects of sevoflurane and isoflurane were investigated in a novel human alveolar epithelial primary cell line specifically designed to study the air-blood barrier of the peripheral lung, hAELVi (human Alveolar Epithelial LentiVirus immortalized), with alveolar type I-like characteristics including functional tight junctions16.
While preparing the design of our in vitro investigations (e.g., cultures of alveolar epithelial cells at the air-liquid interface with exposure to &#34;inhaled&#34; sevoflurane or isoflurane, we understood from previously published studies that fractions of sevoflurane have only been assessed in the &#34;air&#34; interface17,18,19 using standard monitors (similar to those used in a clinical setting). Halogenated agent concentrations were usually chosen according to the minimum alveolar concentration (MAC) values (e.g., in humans, for sevoflurane, 0.5, 1.1, and 2.2 vol%, representing 0.25, 0.5, and 1 MAC, respectively; for isoflurane, 0.6, 0.8, and 1.3 vol% representing 0.25, 0.5, and 1 MAC, respectively)20. Indeed, sevoflurane and isoflurane concentrations have never been investigated in the culture medium itself, thus limiting the validity of previous experimental models/instruments. Furthermore, most experiments used an anaerobic jar that was sealed after the air mix containing sevoflurane had been flushed inside. As our goal was to study alveolar epithelial cells under &#34;physiologic&#34; conditions, we believed that such an anaerobic state may not be optimal and would not be compatible with long experimental durations. Therefore, we developed our own system to culture cells at the air-liquid interface and expose them to halogenated agents (sevoflurane and isoflurane) with the aim of providing precise controlled &#34;air&#34; fractions and &#34;medium&#34; concentrations for these agents. In our opinion, this experimental step, which has not been reported to date in the literature, is mandatory prior to any further in vitro investigations of sevoflurane and isoflurane.

<table><tbody><tr><td>Sevoflurane</td><td>Baxter</td><td></td><td>Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged</td></tr><tr><td>Isoflurane</td><td>Virbac</td><td></td><td>Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged</td></tr><tr><td>Human Alveolar Epithelial cells</td><td>InScreenex</td><td>INS-CI-1015</td><td></td></tr><tr><td>huAEC Medium (ready-to-use)</td><td>InScreenex</td><td>INS-ME-1013-500ml</td><td></td></tr><tr><td>Anesthetic machine circuit</td><td>Drager</td><td>Fabius</td><td></td></tr><tr><td>Gas analyzer</td><td>Drageer</td><td>Vamos Plus</td><td></td></tr><tr><td>Anesthetic gas filter</td><td>SedanaMedical</td><td>FlurAbsord</td><td></td></tr><tr><td>Heated Humifier</td><td>Fisher&amp;amp;Paykel</td><td>MR850</td><td></td></tr><tr><td>Chamber</td><td>Curver</td><td>00012-416-00</td><td></td></tr><tr><td>Gas chromatography coupled with mass detection</td><td>Thermo Fisher Scientific, San Jose, CA, USA</td><td>Trace 1310 with TSQ 8000evo</td><td></td></tr><tr><td>Fused-silica column (30 m x 1.4 &amp;#956;m, 0.25 mm ID)</td><td>Restek, Lisses, France</td><td>Rxi-624Sil MS</td><td></td></tr></tbody></table>

in vitro method to control concentrations of halogenated gases in cultured alveolar epithelial cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

The concentrations of the sevoflurane and isoflurane, which dissolved in the medium over time, are reported in Table 1 and Table 2, respectively.
The courses of the sevoflurane and isoflurane concentrations in the medium were similar over time. Immediately after the required concentration of halogenated agent was set, concentrations rose over the first hour. A plateau was then reached, which persisted until the administration of the halogenated agent was stopped. After administration interruption, concentrations decreased within one hour (Figure 3).
After the first hour, the median concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. No significant difference was found between the different experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58554/58554fig1.jpg" /> 
Figure 1: Construction plan of the air-tight chamber <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58554/58554fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58554/58554fig2.jpg" /> 
Figure 2: Schematic drawing of the device <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58554/58554fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58554/58554fig3.jpg" /> 
Figure 3: Concentration of sevoflurane (n = 5) and isoflurane (n = 5) over time. A) Concentration of halogenated agent over time. Values are expressed in mg/L. Values are expressed in mean and SEM. B) Concentration of halogenated agent over time for each experiment. Value are expressed in mg/L. C) Fraction of halogenated agent over time in the air-tight chamber measured by the gas analyzer. Values are expressed in percentages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58554/58554fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="3">Time</td>
			<td colspan="2">Concentration of sevoflurane in the medium</td>
			<td>Fraction of sevoflurane</td>
		</tr>
		<tr>
			<td colspan="2">(mg/L)</td>
			<td>in the air-tight chamber</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>IC</td>
			<td>(%)</td>
		</tr>
		<tr>
			<td>5 min</td>
			<td>27</td>
			<td>[19 - 31]</td>
			<td>4.6</td>
		</tr>
		<tr>
			<td>30 min</td>
			<td>152</td>
			<td>[142 - 152]</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>1 h</td>
			<td>251</td>
			<td>[243 - 332]</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>4 h</td>
			<td>259</td>
			<td>[256 - 271]</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>8h</td>
			<td>265</td>
			<td>[237 - 280]</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>24 h (stop)</td>
			<td>218</td>
			<td>[196 - 247]</td>
			<td>4.3</td>
		</tr>
		<tr>
			<td>Stop + 5 min</td>
			<td>237</td>
			<td>[214 - 241]</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Stop + 30 min</td>
			<td>92</td>
			<td>[91 - 104]</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Stop + 1 h</td>
			<td>57</td>
			<td>[42 - 58]</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations of sevoflurane dissolved in the medium over time. Numerical data are expressed as a median value with interquartile range for the concentration and as percentage for the fraction. IQR (for interquartile range)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="3">Time</td>
			<td colspan="2">Concentration of isoflurane in the medium</td>
			<td>Fraction of isoflurane</td>
		</tr>
		<tr>
			<td colspan="2">(mg/L)</td>
			<td>in the air-tight chamber</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>IC</td>
			<td>(%)</td>
		</tr>
		<tr>
			<td>5min</td>
			<td>2</td>
			<td>[2 - 2.5]</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>30min</td>
			<td>16</td>
			<td>[4 - 18]</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>1h</td>
			<td>22</td>
			<td>[18 - 27]</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>4h</td>
			<td>30</td>
			<td>[25 - 31]</td>
			<td>1.4</td>
		</tr>
		<tr>
			<td>8h</td>
			<td>22</td>
			<td>[15 - 26]</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>24h (stop)</td>
			<td>26</td>
			<td>[23 - 27]</td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>Stop + 5min</td>
			<td>19</td>
			<td>[12 - 25]</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Stop + 30min</td>
			<td>4</td>
			<td>[4 - 4]</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Stop + 1h</td>
			<td>1</td>
			<td>[0.8 - 1]</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 2: Concentrations of isoflurane dissolved in the medium over time. Numerical data are expressed as a median value with interquartile range for the concentration and as percentage for the fraction. IQR (for interquartile range)

Watch this Scientific Journal Video about In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells at JoVE.com

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...

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Nanyang Technological University

Research

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Medicine

6.5K Views.  CHU Clermont-Ferrand. Deze methode kan helpen bij het beantwoorden van de belangrijkste vraag op het gebied van longletsel en reparatie, zoals het testen van gehalogeneerde aanvulling als potentiële nieuwe therapieën voor acute respiratoire noodsyndroom. Het belangrijkste voordeel van deze techniek is dat het afhankelijk is van een zeer eenvoudige methode om de cellen te kweken en bloot te stellen aan gehalogeneerde stoffen om nauwkeurige en gecontroleerde concentraties te bereiken. Gebruik voor de bouw van de l...

Video: In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

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 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Immunology and Infection

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Bioengineering

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

Developmental Biology

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Biology

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

In Vitro Model of Murine Airway Epithelial Cells and Chronic Smoke Exposure

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Author Spotlight: Development and Characterization of an In Vitro Model to Study Chronic Cigarette Smoke Exposure and Its Impact on Airway Epithelial Cells in COPD Research

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

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