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 a....

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%....

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 l.......

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 insigh.......

Materials

List of materials used in this article

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