A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Summary

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

Abstract

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

Introduction

Personal sampling using in vitro techniques could provide comprehensive information regarding the biological effects of aerosols in the workplace.¹ Exposures to contaminants in the air include exposures to the chemical itself, to the collected air samples, under submerged conditions where the gas is introduced to the cell suspension, intermittent exposures using a device such as a rocker, or direct exposures at the air-liquid interface (ALI).² Many of these techniques are performed with cells grown in suspension or the collection of samples prior to the exposure, each of which can affect the toxicological study ....

Protocol

Operators must wear personal protective equipment (e.g. lab coat, gloves, goggles) when performing steps 1, 2, 3, 5, and 6.

1. Preparation of Materials

1. Prepare materials for system assembly and exposure to ensure repeatability.
   1. Make sure to use new or 70% ethanol cleaned ¼” inner diameter conductive tubing and ¼” outer diameter connectors for the system assembly.
   2. Store test materials including filters, PIVEC components, tweezers, and particle powders in a well-controlled environment, with respect to the temperature and humidity, for at least 24 h prior to the experiment.
      NOTE: The te....

Results

Occupational in vitro toxicology involves maintaining cellular viability while performing aerosol exposure. The PIVEC system is shown in Figure 2, including the temperature and humidity control and the worn PIVEC. The temperature was maintained using a battery-powered resistive heater and the aerosol humidified using increased natural humidification through a porous, wetted tube. In a controlled aerosol setting inside a laboratory, the PIVEC can be s.......

Discussion

Filter cassettes provide a simple, inexpensive method of collecting aerosols in the breathing zone; however, aerosol samples extracted from filters do not represent the entire aerosol (i.e. gases, volatiles, and particulates) and consequently limit the assessment of related biological effects. Using the initial design of the 37 mm filter cassette, the PIVEC is designed to maintain portability and mimic the in vivo deposition of particles from inhalation. The PIVEC is significantly smaller than current ALI exposu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Scanning mobility particle sizer (SMPS)	TSI, Inc.	3910	NanoSMPS
Optical particle sizer (OPS)	TSI, Inc.	3330
Stainless Steel Pipe, 4" Long	McMaster-Carr	4830K116	Standard-Wall 304/304L, Threaded on Both Ends, 1/8 Pipe Size
Brass Ball Valve with Lever Handle	McMaster-Carr	4112T12	Compact High-Pressure Rating, 1/8 NPT Female
Steel Pipe, 2" Long	McMaster-Carr	7753K121	Standard Wall, Threaded on One End, 1/8 Pipe Size
HEPA filter	GE Healthcare	09-744-12	HEPA-Cap Disposable Air Filtration Capsule
Vacuum Generator	PISCO USA	VCH10-018C
PIVEC	VCU		For design please contact authors
Resistive heater
1/4" barbed connectors	Zefon International, Inc.	459743
Porous tubing	Scientific Commodities, Inc.	BB2062-1814A	Hydrophilic 10 um pores
Battery power bank
Cell culture insert	Fisherbrand	353095	24 well plate insert
Filter Forceps	Fisherbrand	09-753-50
Transfer Pipette	ThermoScientific	13-711-27
Glass Fiber Filters	SKC	225-7	Binder-Free Type AE Filter 37 MM 1.00 um pore
Ultra Micro Balance	A&D	BM-22	Housed in environmental chamber
37 mm filter cassette	SKC	225-3250	Filter Cassette Blank, 37 mm, Clear Styrene
Variable flow vacuum pump	SKC	220-5000TC	AirChek TOUCH, 5 to 5000 mL/min
Copper Particles	U.S. Research Materials, Inc.	US1090	40 nm
Copper Particles	U.S. Research Materials, Inc.	US1088	100 nm
Copper Particles	U.S. Research Materials, Inc.	US1117M	800 nm