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

Summary

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.

Abstract

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.

Introduction

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 ARDS^6,7. 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—as opposed to intravenous anesthesia—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 isoflurane^7,9 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)-κB, 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 repair^13,14,15. 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 "inhaled" sevoflurane or isoflurane, we understood from previously published studies that fractions of sevoflurane have only been assessed in the "air" interface^17,18,19 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 "physiologic" 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 "air" fractions and "medium" 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.

Protocol

1. Culture of Alveolar Epithelial Cells (hAELVi)

1. Thawing
   1. 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 °C).
   2. 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.
   3. Aspirate the supernatant and resuspend the cell pellet with 5 mL of the cultivation medium. Then, transfer the cells to a T25 flask.
   4. Cultivate the cells under standard conditions (5% CO₂, 95% humidified air, 37 °C)
2. Splitting
   1. 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.
   2. Aspirate the cultivation media of the cells with a sterile pipette.
   3. Wash the cells once with 4 mL of Dulbecco’s Phosphate Buffered Saline (DPBS) and aspirate the DPBS.
   4. Add 1 mL of Trypsin/EDTA solution (TE) to the cells prior to incubating at 37 °C for 3 min until the cells start to detach; check for detachment under a microscope.
   5. 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.
   6. Use the trypan blue dye exclusion assay to determine the viability of the cells. Take 30 µl of the cell suspension and add 30 µl of a 0.4% solution of trypan blue in a tube. After that, effectively mix the solution 3-5 times using a 100 µl pipette. Take 10 µl of the solution and put it under the coverslip of the hemocytometer.
   7. 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 – (100 / total number of cells x number of dead cells)
   8. 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.
   9. Cultivate the cells in an incubator under standard conditions (5% CO₂, 95% humidified air, 37 °C)
   10. Once the cells are completely confluent, they are ready for the experiment.

2. Preparation of an Air-tight Chamber

NOTE: The construction plan for the air-tight chamber is depicted in Figure 1.

1. 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.
2. Drill a 2.5 cm diameter hole on the bottom side of the lateral wall.
3. Insert a corrugated tube with a green mark, which will serve as the gas-air mixture input pipe, and seal it with silicon.
4. Drill a second 2.5 cm diameter hole on the top side of the opposite lateral wall.
5. 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.
6. Drill a tight 4 mm diameter hole at the center of the mean wall of the box.
7. 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.
8. Place a digital thermometer/hygrometer inside the air-tight chamber.

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.

1. Work under a laboratory extractor hood.
2. Customize an anesthetic machine circuit to switch the gas line of nitrous oxide by carbon dioxide (CO₂).
3. 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 °C
4. Install the air-tight chamber on a hot plate, providing a heating plate temperature of 37°C.
5. Put the 6 well-plate containing the hAELVI cells into the air-tight chamber and seal the lid.
6. 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.
7. 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).
8. Note the composition of gas mixture and the sevoflurane or isoflurane concentration as measured by an external gas analyzer and displayed on the screen.
9. Once the target values are achieved, reduce the fresh gas flow rate to 1 L/min.
10. The air-tight chamber can be maintained with this gas flow rate as long as necessary for the experiment.

4. Measure Sevoflurane or Isoflurane by Chromatography

1. Preparation of samples
   1. 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.
   2. 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 °C if you do not use them immediately.
   3. 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 °C.
   4. 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.
2. 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.
   1. 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.
   2. 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 °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.
   3. For calibration curves and controls, prepare the samples by spiking 50 µL of IS (100 mg/L) into 1 mL of the cellular sample matrices.
   4. Prepare sample solutions with 200 µL of saturated sodium chloride water solution in a 10 mL headspace tube, screwed hermetically tight with a Teflon-sealed cap.
3. Gas chromatography and mass spectrometry
   1. For sample analyses, use headspace injections in a gas chromatography, coupled with the mass detection method.
   2. Carry headspace tubes for 10 min at 80 °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 °C with a split flow at 100 mL/min for 2 min at the start of the chromatography run.
   3. 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.
   4. Simultaneous to the injection, start with an oven temperature of 60 °C for 1 min and increase until a temperature of 140 °C is reached at a rate of 20 °C/min. Then, increase the temperature again until 250 °C is achieved. The total time of the run is 7 min.
   5. Carry out the chromatography separation using a fused-silica capillary column (30 m x 1.4 µm, 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.
   6. 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.

Results

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 per...

Discussion

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—for the first time—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.

Materials

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