In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Summary

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid 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 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.

Abstract

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.

Introduction

Exposure to toxic inhaled chemicals (TICs)/gases such as chlorine (Cl₂) remains an ongoing health concern in accidental exposures as well as in their potential use as a chemical threat agent. Although the lungs are the primary target, organs such as heart and brain are also affected^1-3. In vivo models are generally used for testing toxicity from TICs, but in vitro assays for toxicity assessment are simpler, faster and more cost effective. In vitro models also allow for extensive investigation of agent-cell interactions that may be difficult to evaluate in vivo. Such in vitro exposure systems are rare and moreover, in some conventional models where toxic agents are added to the culture medium in which cells are submerged, the properties of the agents can change due to interactions and binding to components in the medium. In such scenarios cell culture systems such as air-liquid interface (ALI) cultures of primary human airway epithelial cells, proposed here, that can be directly exposed to gaseous agents could be promising.

Epithelial cells lining the airway are the first lines of defense against inhaled toxic chemicals. The human airway epithelium forms a physical barrier between the lumen and the underlying cells in the lung and participates in the response of the lung. It produces a number of cytokines and other pro- and anti-inflammatory agents as well as secretes mucus/airway surface liquid (ASL) covering the epithelium. One of the limitations in conventional submerged in vitro culture systems is also that the ASL and mucus that cover the epithelial surface is removed or diluted. This does not reflect the physiological condition of lung epithelial cells that are exposed to air. Thus, an ideal in vitro system for TIC toxicity testing should replicate this architecture. There is great interest in developing rapid screening methods that predict in vivo toxicity. Epithelial cells grown at the ALI differentiate and have well-differentiated structures and functions compared to cells grown submerged and serve a superior model of the airways.

In this study, we describe the use of air-liquid-interface culture of human airway (tracheobronchial) epithelial cells for testing poisonous inhaled gas toxicity and compare it with a submerged cell culture of cardiomyocyte, hence studying another important target of toxicity.

Protocol

1. Rat Cardiomyocyte Cultures

1. All experiments were performed under protocols approved by the institutional animal care and use committee, IACUC.
2. Obtain rat cardiomyocytes from the hearts (ventricles) of male rats (240-260 g) using methods described previously⁴. Briefly, anesthetize animals using an intraperitoneal injection of pentobarbital (100 mg/kg; confirm anesthesia by toe pinch method) and then remove hearts into 10.0 ml, 1 mM Ca²⁺ containing Krebs Ringer buffer, pH 7.4.
3. Rinse the hearts 5-6x to remove blood and then switch to a Ca²⁺ free Krebs Ringer buffer containing 0.02% protease and 0.06% collagenase A (5.0 ml/heart). Incubate the hearts in this solution for 10-15 min at 37 °C with occasional shaking.
4. After 10-15 min, wash out the enzymatic solution with Ca²⁺ free Krebs Ringer buffer for an additional 5 min. Release the cells from the flaccid tissue using a 25 ml pipette by pipetting the suspension up and down several times.
5. Separate the cells from tissue by filtering through a 70 μm nylon mesh and allow them to settle in Krebs Ringer buffer containing 0.1 mM Ca²⁺. Suspend the cell pellet in 1.0 ml Krebs Ringer buffer containing 0.2 mM Ca²⁺.
6. Carefully layer over 5.0 ml 60 μg/ml bovine serum albumin, BSA, in 15 ml centrifuge tubes, to separate cardiomyocytes from nonmyocytes and allow them to settle for 30 min. The cardiomyocytes are heavier and move to the bottom of the tube. Remove carefully the supernatant cells and media. Repeat this step once to further purify the cells.
7. Gradually transition by washing (in steps using 0.25 mM, 0.5 mM, 0.75 mM, and 1.0 mM Ca²⁺ containing buffer) the ventricular myocytes/cardiomyocytes to 1.0 mM Ca²⁺ containing buffer and resuspended in ACCT medium consisting of Dulbecco’s Modified Eagle Medium, DMEM containing 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, and 100 μg/ml streptomycin.
8. Plate the cardiomyocytes in ACCT medium at a density of 100 to 150 cells/mm² on 100 mm or 35 mm laminin coated plastic culture dishes or 40 x 22 mm glass coverslips precoated with laminin (1 μg/cm²). After 1 hr, wash the dishes with 2.0 ml ACCT to remove cells that are not attached. Add 10.0 ml of fresh media and incubate the cells.

2. Differentiated Air-liquid Interface (ALI) Culture of Human Airway Epithelial Basal Cells

1. Procure human tracheobronchial tissues from National Disease Research Interchange under Institutional Review Board approved protocols. Cell harvest and perform culture using a published procedure^5,6. This procedure uses cell cultures as they are a precise model for human inhalation exposures to TICs, however, if needed one can isolate and grow at ALI mouse and/or rat airway epithelial cells^7,8.
2. Briefly, make small pieces (~¼ inch) of the tissue after removing all connective tissue and lymph nodes. Wash tissue several times in Lactated Ringer solution. Add tissues to a 50 ml conical tube and add protease solution (1% Protease/0.01% DNase in minimal essential media, MEM, tissue to fluid ratio (1:10)).
3. Rock the tissue at 4 °C for overnight. End the dissociation of the tissue by adding 10% fetal bovine serum. Scrape the epithelial surface with a surgical scalpel and collect the cells by centrifugation (2,000 rpm for 10 min).
4. Plate cells at passage one on collagen-coated snapwells in special ALI medium prepared as described in detail by Fulcher et al⁹. Culture for 5 days before changing to air-liquid interface (ALI) by removing apical media.
5. Feed the cells using ALI media every alternate day and allow the cells to differentiate for additional 2-3 weeks (observe numerous beating cilia and mucus secretion) before performing exposures. Wash the apical surface with warm PBS along with media changes.

3. Chlorine Exposure

1. Contain the chlorine exposure system (CES) inside a qualified chemical hood with an operational face velocity of 100 fpm that provides the necessary secondary containment to prevent exposure of personnel in the event of accidental chlorine leaks.
2. Operate the system under slight positive pressure (0.5 inches of water). Dry air is fed at 15 L/min and an appropriate level of chlorine is fed to attain the desired final chlorine concentration. The chambers have a locking lid with 4 mini BCU locks and a low durometer silicone gasket to provide the pressure seal.
3. Deliver the Cl₂ mixture through a Mass Flow Controller. The CES uses a compressed gas cylinder containing 1.0% Cl₂ in dry nitrogen.
4. Regulate the dilution airflow using the custom designed control panel and similarly regulate the Cl₂ concentration delivered to the exposure chambers. A low volume sampling pump pulls the exhaust from the chambers into a chlorine analyzer to monitor concentrations that are then recorded on a data logger connected to the analyzer.
5. Measure the flow rates within the chambers prior to exposure using a flow meter to assure equal delivery and exhaust rates.
6. Prepare the cell cultures for exposure by removing the supernatant media and adding fresh media (basolateral media in ALI cultures). Any pretreatments with agents could be performed at this time.
7. Expose the ALI cultures of airway epithelial cells to Cl₂ gas (50, 100, or 300 ppm for 30 min) in the two sealed polysulfone biocontainment chambers. The cardiomyocytes (submerged or confluent cultures on membranes) are exposed to 50 or 100 ppm Cl₂ for 15 min.
8. After exposure flush the chambers with air until the Cl₂ level falls below 1 ppm and can be safely opened to remove cells (within 5 min).

4. Transepithelial Electrical Resistance (TER) Measurement

1. Measure the TER of air-liquid-interface, ALI, cultures using an epithelial voltohmmeter with a pair of silver chloride “chopstick” electrodes.
2. Equilibrate the chopstick electrode in the ALI media 15 min before use.
3. Add warm media to the apical (1.0 ml) and basolateral (2.0 ml) surface and measure the TER using the chopstick electrodes of the voltohmmeter.
4. Dip the shorter arm of the electrode in apical media and the longer arm in the basolateral media. Click the ‘measure’ button on the voltohmmeter to evaluate the electrical resistance.
5. The voltohmmeter has the option to measure ohms or k ohms. Subtract the resistance across a cell-free culture support from the resistance measured across each cell layer to yield the transepithelial resistance (TER).

5. Caspase Measurement

1. Add fresh ALI media (2.0 ml) to the basolateral surface post exposure and incubate the cell at room temperature. Collect supernatant media at 4 and 24 hr.
2. Measure caspase 3/7 activity in the media supernatants by using a commercial caspase 3/7 assay kit.

6. Western Blot and Immunocytochemistry

1. Perform western blots using cell lysates as previously described¹⁰. Suspend the protein lysate (20 μg) in 5x reduced sample buffer and boil for 5 min. Subject the protein lysate to SDS-PAGE (4-15%) and transfer the separated proteins to a nitrocellulose membrane by electrophoretic blotting.
2. Block non-specific binding by incubating the membrane with 5% milk in wash buffer (PBS + 0.1% detergent) and probe the membranes with primary antibodies against SERCA2 or sarcomeric actin at 1:1,000 dilution, overnight at 4 °C. Next, wash and incubate membranes with the respective peroxidase-conjugated secondary antibodies and develop for detection as described before¹⁰ using commercial peroxidase detection kit.
3. For immunocytochemistry treat live cells grown on inserts or glass coverslips in 6-well plates with 0.4% Triton-X-100 in 10 mM sodium citrate buffer for 20 min after rinsing with PBS.
4. Block nonspecific binding by treating the cells with 5% donkey serum for 20 min, and then incubate the cells with non-specific IgG or individual primary antibodies specific to sarcomeric actin or Ki-67.
5. Wash the cells with PBS and incubate with fluorescent-conjugated secondary antibodies to detect the primary antibody and a nuclear stain (1 μg/ml DAPI).
6. Wash with PBS and mount coverslips using commercial mounting media.
7. Visualize the staining using a fluorescent microscope.

Representative Results

Primary rod shaped cardiomyocytes attach on laminin matrices and spread and differentiate into confluent cultures (Figure 1A and its inset). These cells were further characterized on the basis of sarcomeric actin and SERCA2 expression (Figures 1B and 1C). Rat cardiomyocytes are highly susceptible to chlorine toxicity as 15 min exposure to 100 ppm chlorine caused extensive cell rounding and death in submerged cultures and disruption of confluent layers on cells grown on laminin coated membranes (Figure 1D). There was also enhanced apoptotic cell death as indicated by caspase 3/7 release in cardiomyocytes grown on inserts (Figure 1E).

Exposure of differentiated human airway epithelium (Figure 2, inset to panel 1 showing a cross section of cell culture inserts with columnar, ciliated and goblet cells) to chlorine caused sloughing and lifting of cell membranes at both low (100 ppm) and high (300 ppm) concentrations (Figure 2 panel A2 and A3). Damage by low chlorine concentrations was quickly reversed (Figure 2 panel A5), however, cells exposed to higher chlorine concentrations had delayed or no repair ability (Figure 2 panel A6) as shown by visual inspection as well as cell proliferation assessment by Ki-67 staining (Figure 2 panel A9). Trans epithelial electrical resistance, TER measurements and caspase activity further confirmed these results and provide evidence for loss of membrane integrity and apoptotic cell death upon chlorine exposure (Figure 2, panel B and C). Thus our studies describe the development of an in vitro Cl₂ exposure system that causes loss of membrane integrity and death of airway epithelium and cardiomyocytes. This effect may not be due to non-physiological pH changes in the cardiomyocytes as the pH of the media after exposure to chlorine was maintained at ~7.4 as measured by using a pH meter in the collected media postexposure.

Figure 1. Rat cardiomyocyte isolation and exposure to chlorine. Rat cardiomyocytes were isolated as described in the protocol and plated on laminin-coated plastic dishes (panel A, a representative light microscopic image) or laminin coated inserts. Inset to panel A shows a rod shaped cardiomyocyte spreading and differentiating. The cardiomyocytes were also characterized based on the sarcomeric actin expression (panel B showing a representative image detected by immunofluorescence and lane a panel C showing a representative western blot scan) and abundant SERCA2 expression (panel C lane b). Chlorine exposure (100 ppm 15 min) caused extensive cell death in submerged cultures as well as disruption of the cell monolayer on the inserts (panel D showing the representative photomicrographs). Caspase 3/7 release (panel E) in the supernatant media of cardiomyocytes grown on inserts, at 4 hr and 24 hr post chlorine exposure was also measured as described in the text. Values shown are mean ± SEM and * indicates significant (p < 0.05) difference from 0 ppm control.

Figure 2. Effect of chlorine exposure on differentiated human airway epithelial air-liquid interface (ALI) cultures. Human airway epithelial basal cells were cultured on collagen coated snapwells. After day 5 the apical media was removed. Differentiated cultures (consisting of basal, ciliated, columnar, and goblet cells as shown in the inset in top left panel of panel A) were exposed to chlorine (100 or 300 ppm) for 30 min. The TER was measured and media was changed and cells incubated for 24 hr. At 24 hr TER was measured again and apical media was collected for caspase release measurement and the cell membranes were fixed for immunohistochemistry. The open arrow in panel A parts 2 and 3 shows sloughed off epithelial layer and black arrows show empty spaces on the insert. The arrowhead in panel A part 5 shows regenerated epithelium. Parts 7, 8, and 9 show cellular proliferation as assessed by Ki-67 immunostaining. Values shown are mean ± SEM and * indicates significant (p < 0.05) difference from 0 ppm control.

Discussion

The most common type of acute toxic exposures occurs when one breathes a poisonous chemical into the lungs. These chemicals may also be quickly taken up in the bloodstream and may impact other organs such as brain and heart. Inhalation toxicity of various agents using animal models are studied and reported widely, however the mechanisms are less well understood. This is a major hurdle in developing effective therapies. Absence of in vitro exposure systems is a primary reason behind the lack of mechanistic insights. Here we describe cell culture models of heart muscle and airways to study impact of toxic inhaled chemicals such as chlorine exposure. Chlorine reacts rapidly with aqueous surfaces to form hydrochloric and hypochlorous acid^3,11,12. Chlorine can also combine with reactive oxygen species (ROS) to produce highly reactive compounds that may lead to oxidation of critical proteins and enzymes of the airway surfaces¹³. Studies using submerged cultures may only demonstrate effects of by products such as HOCl in case of chlorine rather than the gas itself. Exposure of differentiated ALI cultures of human airway epithelial cells described here would allow study of direct interactions of TICs such as chlorine with cell surfaces in absence of aqueous media similar to what occurs in vivo.

Using primary rat cardiomyocytes, we also describe that chlorine exposure causes rapid cell death in submerged cultures. These cells are unable to grow at ALI as they do not polarize and form tight junctions. Therefore, we also utilized confluent cultures of cardiomyocytes grown on inserts with a thin layer of media. Exposure of these cell monolayers to chlorine demonstrated membrane disruption and enhanced caspase release suggesting apoptosis may play a role.

This study demonstrates that airways (the primary target of inhalation that can be replicated by ALI cultures) as well as organs such as heart that do not grow at ALI may be studied using in vitro exposure systems. These in vitro exposure models can easily be adapted to assess effects of other toxic inhaled chemicals (TICs). Using these models we anticipate to provide detailed mechanistic understanding of the toxicities and develop novel strategies to mitigate the toxic events associated with TIC/chlorine and then evaluate them further in vivo. Although, these exposures are short in duration the exposure system needs to better replicate cell culture conditions. Exposure to gases such as chlorine limits the use of humidity as it may corrode the device. The ALI cultures could be rocked to simulate respiration as previously utilized in our ozone exposure system^14,15. We are currently working in these directions.

Materials

Name	Company	Catalog Number	Comments
Rats	Harlan Laboratories	Sprague-Dawley
Pentobarbital	Sigma-Aldrich	P3761
Chlorine	AirGas, Inc	X02NI99CP163LS1
Caspase 3/7 kit	Promega	G8091
Epithelial voltohmmeter and chopstick electrode	World Precision Instruments	EVOM and STX2
Snapwell inserts	Corning	07-200-708
70 micron nylon cell strainer	Corning	#352360
Polysulfone biocontainment chambers	BCU, Allentown Cage Equipment	BCU
DMEM	Life technologies	12491-015
Sarcomeric actin antibody	Abcam Cambridge, MA	ab28052
SERCA2 antibody	Affinity Bioreagents, Golden, CO	MA3-9191
Ki-67 antibody	Dako, Carpinteria, CA	M7248
Alexa 488-conjugated secondary antibody	Invitrogen, Grand Island, NY	A11029
BSA	Sigma-Aldrich	A9418
Carnitine	Sigma-Aldrich	C0283
Taurine	Sigma-Aldrich	T8691
Creatinine	Sigma-Aldrich	C6257
Krebs Ringer Buffer	Sigma-Aldrich	K4002
Protease	Sigma-Aldrich	P5147
Collagenase	Sigma-Aldrich	C6885
DNAase	Sigma-Aldrich	DN-25
Lactated Ringer solution	Abott Laboratories	7953
Donkey serum	Fisher Scientific	017-000-001
PBS, phosphate buffered saline	Sigma-Aldrich	D1408
4-15% SDS-PAGE gels	Bio-Rad	456-1083
Nitrocellulose membrane	Bio-Rad	162-0115
Dergent, Tween	Sigma-Aldrich	P1379
Peroxidase detection kit	Pierce	3402
DAPI	Sigma-Aldrich	D9542
Mounting media, Fluormount G	eBiosciences	00-4958-02
Sodium citrate	Sigma-Aldrich	71497
Collagen	Sigma-Aldrich	C7521
MEM	Sigma-Aldrich	M8028
Laminin	BD biosciences	354259
Penicillin/streptomycin	Life Technologies	15070063
FBS	Gibco	200-6140AJ