Regulating temperature and relative humidity in air–liquid interface in vitro systems eliminates cytotoxicity resulting from control air exposures

May 22, 2017

DOI: 10.1039/c7tx00109f
Jose Zavala,a Rebecca Greenan,b Q. Todd Krantz,a David M. DeMarini,a Mark Higuchi,a M. Ian Gilmour a and Paul A. Whiteb

aNHEERL, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA.

bMechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario K1A 0K9, Canada.

The aim of this study was to test whether temperature and relative humidity has an influence on the cytotoxicity to BEAS-2B cells on a VITROCELL® 6 air-liquid interface system by measuring the viability (WST-1 cell proliferation assay) and lactate dehydrogenase (LDH) release.
Note from VITROCELL: We inform our customers about the possible need for humidification during installation and training. Among the need for humidification, possible other reasons for low clean air viability are clean air quality and cell handling procedures.


VITROCELL® systems permit cell exposures at the air–liquid interface (ALI); however, there are inconsistent methodologies in the literature for their operation. Some studies find that exposure to air (vehicle control) induced cytotoxicity relative to incubator controls; others do not mention if any cytotoxicity was encountered. We sought to test whether temperature and relative humidity (temp/RH) influence cytotoxicity with an unmodified (conditions A & B) and modified (condition C) VITROCELL® 6 CF with temp/ RH controls to permit conditioning of the sampled air-flow. We exposed BEAS-2B cells for 1 h to air and measured viability (WST-1 cell proliferation assay) and lactate dehydrogenase (LDH) release 6 h postexposure. Relative to controls, cells exposed to air at (A) 22 °C and 18% RH had a 47.9% ± 3.2% (p < 0.0001) reduction in cell viability and 10.7% ± 2.0% (p < 0.0001) increase in LDH release (B) 22 °C and 55% RH had a 40.3% ± 5.8% (p < 0.0001) reduction in cell viability and 2.6% ± 2.0% (p = 0.2056) increase in LDH release, or (C) 37 °C and >75% RH showed no changes in cell viability and no increase in LDH release. Furthermore, cells exposed to air at 37 °C and >75% RH 24 h post-exposure showed no changes in viability or LDH release relative to incubator controls. Thus, reductions in cell viability were induced under conditions used typically in the literature (conditions A & B). However, our modifications (condition C) overcome this shortcoming by preventing cell desiccation; regulating temp/RH is essential for conducting adequate ALI exposures.

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