Aerosol–Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Cell Culture
2.3. VITROCELL® Cloud 6 System
2.4. Fluorescent Microscopy for the Assessment of the Homogeneous Deposition of Surfactant
2.5. Pre-Coating of ALI Cells with Surfactant
2.6. NP Exposure Protocol
2.7. RNA Extraction
3. Results
3.1. A Uniform Deposition of Surfactant by Aerosolization with the Nebulizer
3.1.1. Reproducible Deposition Assessment by QCM Measurements
3.1.2. Homogeneous Distribution Assessment by Fluorescence Microscopy
3.2. Improvement of the Standard Initial Protocol
3.2.1. Standard Initial Protocol for NP Nebulization in VITROCELL® Cloud 6 System
3.2.2. Current Improved Protocol for NP Nebulization in VITROCELL® Cloud 6 System
3.3. Troubleshooting Flaws of the Initial Protocol
3.3.1. Preventing Rapid Drying of NPs on the Nebulizer Mesh by Modifying the Volume of Nebulized Liquid
3.3.2. Loss of Cells and Poor RNA Quality
3.3.3. Increasing the Reproducibility and Repeatability
4. Discussion
5. Conclusions
- (1)
- When using semi adherent cells, the number of cells seeded on the insert has to be increased (as compared to submerged culture conditions) to ensure that enough cells stay on the membrane after discarding the apical medium to reach a satisfying RNA yield.
- (2)
- To avoid any clogging of the mesh of the nebulizer and to obtain a reproducible cell-delivered dose, the manufacturer’s recommendations for cleaning of the nebulizer should be adhered to and the TiO NP concentration has to be decreased by increasing the nebulized suspension volume (from 57 L of 30 mg/mL to 114 L of 15 mg/mL in this case for TiO NM105 NP). This will prevent the NP suspension from both drying out on the nebulizer mesh and alteration of the NP deposition on the cells due to low cloud density in the VITROCELL® Cloud exposure chamber.
- (3)
- When two sequential aerosol exposures are performed back-to-back, when applicable, we suggest using a different nebulizer for each liquid nebulized. If the same nebulizer is used, we suggest nebulizing all replicas with the first type of aerosol and then with the second, and cleaning the nebulizer between each sequence of nebulization thoroughly as recommended by the manufacturer.
- (4)
- If it is necessary to proceed to several expositions, we recommend including each BR in each exposure session (divided into technical replicates (TR), one TR per exposition). By doing this and subsequent pooling of the TRs of each BR, the variability in deposited dose amongst expositions will be spread over every TR in the same manner for each BR.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
NP | Nanoparticles |
ALI | Air–liquid interface |
QCM | Quartz crystal microbalance |
BAL | Bronchoalveolar lavage |
BR | Biological replicate |
TR | Technical replicate |
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Initital Protcol (57 L, 30 mg/mL NP) | Current Protcol (114 L, 15 mg/mL NP) | |||
---|---|---|---|---|
Exposure | Mass Deposited (g/cm2) | Time (min) | Mass Deposited (g/cm2) | Time (min) |
Exp 1 | 2.1 | 6 | 4.2 | 5 |
Exp 2 | 7.4 | 24 | 6.1 | 5 |
Exp 3 | 7.4 | 6 | 5.7 | 5 |
Exp 4 | 3.5 | 6 | 5.5 | 5 |
Mean | 5.1 | 10.5 | 5.4 | 5 |
SD | 2.7 | 9 | 0.8 | 0 |
Initial Protocol (57 L, 30 mg/mL NP) | Current Protocol (114 L, 15 mg/mL NP) | ||||||
---|---|---|---|---|---|---|---|
Exposure | Biological Replicate | RNA Quantity (g/L) | OD 260/280 | OD 260/230 | RNA Quantity (g/L) | OD 260/280 | OD 260/230 |
1 | 89.04 | 1.66 | 1.84 | 32.31 | 2.09 | 2.09 | |
2 | 7.99 | 2.25 | 0.46 | 55.30 | 2.01 | 1.80 | |
HO | 3 | 121.07 | 2.12 | 1.14 | 69.60 | 2.13 | 2.18 |
4 | 206.30 | 2.04 | 2.32 | 16.09 | 2.08 | 1.43 | |
5 | / | / | / | 48.94 | 2.18 | 1.98 | |
1 | 3.30 | 1.86 | 0.26 | 3.61 | 1.83 | 3.72 | |
2 | 96.96 | 2.03 | 1.81 | 61.96 | 2.14 | 2.16 | |
TiO NP | 3 | 8.01 | 2.48 | 51.25 | 91.57 | 1.99 | 2.05 |
4 | 134.43 | 2.08 | 1.55 | 58.93 | 2.10 | 2.02 | |
5 | / | / | / | 66.30 | 2.06 | 2.17 | |
Mean | 83.61 | 2.07 | 1.33 | 50.46 | 2.06 | 2.16 | |
SD | 73.27 | 0.24 | 0.70 | 26.34 | 0.10 | 0.59 |
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Leroux, M.M.; Hocquel, R.; Bourge, K.; Kokot, B.; Kokot, H.; Koklič, T.; Štrancar, J.; Ding, Y.; Kumar, P.; Schmid, O.; Rihn, B.H.; Ferrari, L.; Joubert, O. Aerosol–Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction. Nanomaterials 2022, 12, 1362. https://doi.org/10.3390/nano12081362
Leroux MM, Hocquel R, Bourge K, Kokot B, Kokot H, Koklič T, Štrancar J, Ding Y, Kumar P, Schmid O, Rihn BH, Ferrari L, Joubert O. Aerosol–Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction. Nanomaterials. 2022; 12(8):1362. https://doi.org/10.3390/nano12081362
Chicago/Turabian StyleLeroux, Mélanie M., Romain Hocquel, Kevin Bourge, Boštjan Kokot, Hana Kokot, Tilen Koklič, Janez Štrancar, Yaobo Ding, Pramod Kumar, Otmar Schmid, Bertrand H. Rihn, Luc Ferrari, and Olivier Joubert. 2022. "Aerosol–Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction" Nanomaterials 12, no. 8: 1362. https://doi.org/10.3390/nano12081362