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

Abstract

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 post-exposure. 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.

Fig. 1. Experimental setup of conditions A, B, and C. (A) For condition A, the VITROCELL^® 6 CF module was mounted on a cart as specified by the manufacturer with no added temp/RH of the air-flow. (B) For condition B, we used a 3-neck flask filled with 80 mL of MilliQ water as a bubbler to increase the RH of the air-flow. (C) For condition C, we modified the VITROCELL^® 6 CF system by adding a Plexiglass enclosure, a heating pad, a heater, a fan, a diffusion humidifier, and temp/RH sensors.

Fig. 2. Viability of BEAS-2B cells exposed to vehicle air at ALI (n = 4) measured 6 h post-exposure. (A) Cells exposed to air experienced a significant reduction in viability (p < 0.001) compared to incubator controls. (B) Cells exposed to air experienced a significant reduction in viability (p < 0.001) compared to incubator controls. (C) No change in viability was observed (p = 0.14) between condition C and incubator controls. Bars represent average percent viability ± SEM (two-tailed unpaired t-test compared with incubator controls; * denotes significant difference).

Fig. 3. LDH release by BEAS-2B cells exposed to vehicle air at ALI (n = 3) measured 6 h post-exposure. (A) Cells exposed to air experienced a significant increase in LDH release (p < 0.001) compared to incubator controls. (B) Cells exposed to air experienced a non-significant increase in LDH release (p = 0.21) compared to incubator controls. (C) Cells exposed to air experienced a small decrease in LDH release (p = 0.02) compared to incubator controls. Bars represent average percent LDH release ± SEM (two-tailed unpaired t-test compared with incubator controls; * denotes significant difference).

Fig. 4. Viability of BEAS-2B cells exposed to vehicle air by ALI (n = 6) measured 24 h post-exposure for condition C. No reduction in viability was observed (p = 0.1098) compared to incubator controls. Bars represent average percent viability ± SEM (two-tailed unpaired t-test compared with incubator controls).

Fig. 5. LDH release by BEAS-2B cells exposed to vehicle air at ALI (n = 6) measured 24 h post-exposure for condition C. Cells exposed to air experienced no change in LDH release (p = 0.61) compared to incubator controls. Bars represent average percent LDH release ± SEM (two-tailed unpaired t-test compared with incubator controls).