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

Summary

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).

Abstract

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.

Introduction

Inhalation of toxic airborne particles is a public health concern, leading to a multitude of health risks worldwide and many millions of deaths annually^1,2. Climate change, the ongoing industrial development and the rising demand for energy, agricultural and consumer products have contributed to the increase of pulmonary diseases over the last years^3,4,5,6. Knowledge and evaluation of inhalable substances regarding their acute inhalation toxicity provide the basis for hazard assessme....

Protocol

NOTE: The protocol of one exposure experiment covers a period of three days.

Day 1

1. General preparations and cultivation of cells

NOTE: The human lung adenocarcinoma epithelial cell line A549 was used for exposure experiments. Cells must be handled under sterile conditions. Other cell lines that are suitable for cultivation at the ALI can be used.

1. Prepare the growth medium (Dulbecco's Minimum Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 5 µg/mL gentamycin) and the exposure medium (DMEM, supplemented with 5 µg/mL gentamycin and ....

Results

The CULTEX RFS is a specially designed modular in vitro exposure system that enables the direct and homogenous exposure of cells at the ALI. Within a former pre-validation study, the general applicability of this exposure system and its transferability, stability and reproducibility were successfully demonstrated. In a recent research project funded by the German Federal Ministry of Education and Research, the exposure system was successfully validated and established as a prediction mode.......

Discussion

Many non-animal inhalation toxicity testing models have been developed in recent years in order to gain information about the acute inhalation hazard of inhalable particles and to reduce and replace animal experiments according to the 3R principle²⁵.

In terms of cell culture models, exposure of cells can be done under submerged conditions or at the ALI. Exposing cells under submerged conditions may affect the physico-chemical properties and thus, the toxic properties of.......

Materials

Name	Company	Catalog Number	Comments
Cells
A549	ATCC	CCL-185
Cell culture medium and supplies
DMEM	Biochrom, Berlin, Germany	FG 0415	used as growth medium
DMEM	Gibco-Invitrogen, Darmstadt, Germany	22320	used as exposure medium
FBS superior	Biochrom, Berlin, Germany	S 0615
Gentamycin (10mg/mL)	Biochrom, Berlin, Germany	A 2710
HEPES 1M	Th. Geyer, Renningen, Germany	L 0180
PBS	Biochrom, Berlin, Germany	L 1825
Trypsin/EDTA (0.05%/0.02%)	Biochrom, Berlin, Germany	L 2143
Cell culture material
CASY Cups	Roche Diagnostic GmbH, Mannheim, Germany	REF 05651794
Cell culture plates	Corning, Wiesbaden, Germany	3516	6­-well plates
Corning Transwell cell culture inserts	Corning, Wiesbaden, Germany	3450	24mm inserts; 6-­well plates; 0.4 µm
Chemicals
CASYton	Roche Diagnostic GmbH, Mannheim, Germany	REF 05651808001
Compressed Air (DIN EN 12021)	Linde Gas Therapeutics GmbH, Oberschleißheim, Germany	2290152
WST-1	Abcam, Cambridge, United Kingdom	ab155902
Instruments + equipment
CASY Cell Counter	Schärfe System GmbH, Reutlingen, Germany
Circulation thermostat	LAUDA, Lauda-Königshofen, Germany	Ecoline RE 100
CULTEX HyP - Hydraulic Press	Cultex® Technology GmbH, Hannover, Gemany
CULTEX insert sleeve	Cultex® Technology GmbH, Hannover, Gemany
CULTEX RFS - Radial Flow System Type 2 (module for particle exposure)	Cultex® Technology GmbH, Hannover, Gemany
CULTEX RFS - Radial Flow System Type 2 (module for clean air exposure)	Cultex® Technology GmbH, Hannover, Gemany
CULTEX supply
Flow controller 0-30 ml/min (IQ-Flow)	Bronkhorst Deutschland Nord GmbH
Flow controller 0-1,5 l/min (EL-Flow)	Bronkhorst Deutschland Nord GmbH
Filters (large)	Munktell & Filtrak GmbH, Sachsen, Germany	LP-050	Munktell Sterile Filter; Particle retention efficiency > 99,999%
Filters (small)	Parker Hannifin Corporation, Mainz, Germany	9933-05-DQ	Balston disposable filter
Medium pump	Cole-Parmer GmbH, Wertheim, Germany	Ismatec IPC High Precision Multichannel Dispenser	digital peristaltic pump
Microplate Reader Infinite M200 Pro	Tecan Deutschland GmbH, Crailsheim, Germany
Vakuum pump	KNF, Freiburg, Germany	N86 KT.18
Vögtlin mass flow controller 0,2-10 l/min	TrigasFI GmbH	Vögtlin red-y compact regulator, Typ-Nr.: GCR-C3SA-BA20
Water Bath	LAUDA, Lauda-Königshofen, Germany	Ecoline Staredition RE 104