A translational cellular model to study the impact of high-frequency oscillatory ventilation on human epithelial cell function

Abstract

High-frequency oscillatory ventilation (HFOV) has been proposed as gentle ventilation strategy to prevent lung injury in the preterm infant. High-frequency jet ventilation leads to dimensional and mechanical airway deformation in animal airway models, which is consistent with translational studies demonstrating the impact of oxygen and biophysical stresses on normal airway cellular function. There is an overall paucity of clinical and cellular data on the impact of HFOV on the conducting airway. We developed an innovative method to test the impact of the clinical HFO Ventilator (SensorMedics 3100A) on human epithelial cell function. In this translational model, we were able to study the differential effects of biophysical stress due to HFOV independently and in combination with hyperoxia on a direct cellular level of the conducting airway system. Additionally, we could demonstrate that hyperoxia and pressure by HFOV independently resulted in significant cell dysfunction and inflammation, while the combination of HFOV and hyperoxia had a synergistic effect, resulting in greater cell death.

New & noteworthy: Traditionally, large-animal models are used to analyze the impact of clinical ventilators on lung cellular function. In our dual-chamber model, we interface high-frequency oscillatory ventilation (HFOV) directly with airway cells to study the effects of HFOV independently and combined with hyperoxia. Therefore, it is possible to study the preclinical impact of interventional factors without the high cost of animal models, thus reducing staff, time, as well as animal sparing.

Keywords: HFOV; human airway cells; modified chamber.

Fig. 1.

Schematic of experimental modulator incubator chamber.

Fig. 2.

Transepithelial electrical resistance (TER) was less than control in all treated groups. GR4 had a significant decrease of TER compared with the other experimental groups. Values are means ± 95% CI; n = 6 for each condition. *Different from control group (P < 0.001). #GR4 different from GR2 and GR3 (P < 0.05).

Fig. 3.

Cell viability of Calu-3 monolayers decreased for all experimental conditions compared with control. GR4 had a significant decrease of cell viability compared with GR2 and GR3. Values are means ± 95% CI; n = 6 for each condition. *Different from control group (P < 0.001). #GR4 different from GR2 and GR3 (P < 0.05).

Fig. 4.

Total protein concentration in apical surface wash fluid from Calu-3 monolayers increased in GR2, GR3, and GR4 compared with GR1 (P < 0.001). There was no difference in the total protein secretion among the three experimental conditions. Values are means ± 95% CI; n = 6 for each condition. *Different from control group (P < 0.001).

Fig. 5.

Level of the inflammatory mediator IL-6 corrected for viability was increased in GR2 and GR3 compared with control. Level of IL-6 was decreased in GR4 compared with GR1 and decreased compared with GR2 and GR3. Values are means ± 95% CI; n = 6 for each condition. *Different from control group (P < 0.05). #GR4 different from GR2 and GR3 (P < 0.05).

Fig. 6.

Level of the inflammatory mediator IL-8 corrected for viability was increased in GR2 and GR3 compared with control. Level of IL-8 was decreased in GR4 compared with GR1 and decreased compared with GR2 and GR3. Values are means ± 95% CI; n = 6 for each condition. *Different from control group (P < 0.05). #GR4 different from GR2 and GR3 (P < 0.05).

Fig. 7.

Semiquantitative assessment of cell morphology as the ratio of abnormal cells to total cells showed a higher ratio in GR2, GR3, and GR4 compared with GR1 (P < 0.001). Among the experimental conditions, GR4 showed a higher ratio compared with GR2 and GR3 (P < 0.001). Values are means ± 95% CI; n = 16 for each condition. *Different from control group (P < 0.001). #GR4 different from GR2 and GR3 (P < 0.05).

Fig. 8.

Representative histology. Cytospin preparation of Calu-3 cells after exposure to normoxia or hyperoxia with or without pressure by HFOV is shown. Control showed normal cellular histology. Exposure to hyperoxia (GR2) and pressure by HFOV (GR3) exhibited swollen nuclei and intracellular vacuoles as signs of cell damage. Exposure to hyperoxia and HFOV pressure combined (GR4) demonstrated the highest level of cell damage compared with other experimental conditions. All slides were examined by light microscopy at ×40 magnification (scale bar = 20 µm).