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Evaluation of MucilAir™ for Toxicity Testing In Vitro Department of In Vitro Sciences, Charles River, Tranent, Edinburgh, EH33 2NE, UK
Materials and Methods
IVTS, Liverpool
MucilAir™
www.criver.com
11/2011
MucilAir™ was obtained from Epithelix Sàrl, CH‑1228 Geneva, Switzerland. Tissues were maintained in a humidified incubator (37°C; 5% CO2 atmosphere).
Test Item
MucilAir™ was treated with the model irritant, sodium dodecyl sulphate (SDS; 0‑10 mM), for 24 h. SDS was applied in a buffered saline solution (sodium chloride, 0.9%, w/v; calcium chloride, 1.25 mM; HEPES, 10 mM).
TEER TEER was measured prior to dosing (0 h) and at 24 h and 168 h post dosing using a Millicell®-ERS meter.
Mucin Assay Surface mucus was collected at 0 h, 24 h and 168 h by lavage with buffered saline. Samples were assayed for mucin content using an enzyme-linked lectin assay (ELLA). Samples were applied to Nunc Maxisorp™ plates coated with Triticum vulgaris lectin and incubated for 30 min at 37°C. Plates were blocked with bovine serum albumin (0.1%, w/v, in PBS) for 30 min then incubated for 30 min with horseradish peroxidise (HRP) labelled soybean lectin (1 µg/mL in PBS). HRP detection was performed using BD OptEIA™ tetramethylbenzidine (TMB) substrate, incubated at ambient temperature for 15 min and stopped with 2N H2SO4. Mucin content was determined by optical absorbance (450 nm) measurement of stopped reactions.
LDH Release (%)
Culture media was collected at 0 h, 24 h and 168 h. LDH content was measured using Promega CytoTox ONE™ Homogeneous Membrane Integrity Assay. LDH release was referenced against a value for 100% release derived from MucilAir™ treated with lysis buffer (Triton-X100, 10%, w/v). R&D Quantikine® Immunoassays were used to measure Interleukin‑6 (IL‑6) and Interleukin‑8 (IL‑8) content of collected media samples. The manufacturers’ recommended protocols were followed throughout.
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Intermediate concentrations of SDS (0.6‑1.25 mM) showed reduction in TEER with little effect on mucin or LDH release. Comparison of TEER and LDH release data 24 h after dosing (Figure 4) demonstrated that 1.25 mM SDS was the lowest concentration at which toxicity was observed. SDS concentrations of 1.25‑2.5 mM produced increases in the release of IL‑6 (Figure 5) and IL‑8 (Figure 6).
Histology
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Figure 4. Comparison of TEER and LDH data at 24 h 500
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Figure 7. Representative histological appearance of untreated (0 mM SDS) and treated (0.63 to 2.5 mM SDS) MucilAir™ at 168 h post-dose
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At 24 h, MucilAir™ treated with high concentrations of SDS (2.5‑10.0 mM) demonstrated gross visible damage and loss of cilia function by light microscopy (data not shown) and suffered extensive membrane damage as measured by TEER (Figure 1), release of mucin (Figure 2) and LDH (Figure 3). 1500
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Figure 5. Effect of SDS exposure on IL-6 release by MucilAir™ 50
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Conclusions Epithelial damage was identified at ≥0.63 mM SDS using histology and ≥1.25 mM using most other measurements. Epithelial surfaces with reversible damage recovered basic cellular function, as measured by TEER and release of markers, more rapidly than they regained their pseudostratified morphology.
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Figure 6. Effect of SDS exposure on IL-8 release by MucilAir™
Figure 1. Effect of SDS exposure on TEER in MucilAir™
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The baseline, predose, values for the chosen endpoints were consistent between units for all endpoints other than IL‑6.
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Figure 3. Effect of SDS exposure on LDH Release by MucilAir™
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Following endpoint assessment of cytotoxicity or metabolic competence (data not shown), MucilAir™ was fixed using two changes of neutral buffered formalin (10%, v/v). Fixed samples were processed for histological examination and haematoxalin-eosin stained using standard methods.
Histology (Figure 7) revealed obvious morphological changes and loss of cilia at SDS concentrations of 0.63 mM and above. Samples treated with 5‑10 mM SDS were too damaged for sections to be mounted.
0h 24 h 168 h
LDH release (%)
Basic properties of MucilAir™ were evaluated as markers of chemical toxicity. Damage was evaluated by measuring transepithelial electrical resistance (TEER), cytotoxicity (using LDH release), mucin and interleukin release and cilia structure and function (microscopic observation and histological examination).
Cytotoxicity and potential irritation associated with exposure to SDS were evaluated by measuring release of lactate dehydrogenase (LDH) and interleukins into culture media.
IL-6 (pg/mL)
It is anticipated that MucilAir™ may reduce animal use in inhalation studies if it is able to identify specific liabilities that indicate a high risk of airway toxicity or predict a nontoxic starting dose for in vivo studies.
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IL-8 (ng/mL)
MucilAir™ is a mucus secreting, ciliated epithelial model of the human airway. MucilAir™ is derived from human airway cells collected from healthy donors and cultured at the air interface on permeable membranes. The culture process reconstructs a functional model of the human airway epithelium, exhibiting a pseudostratified morphology most similar in structure and function to the tracheobronchial epithelium.
TEER (Ω)
Introduction
LDH and Interleukin Release Assays
Mucin Release (% vehicle)
J Welch, A Dickie and C Roper
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Figure 2. Effect of SDS exposure on Mucin Release by MucilAir™
At 168 h, MucilAir™ treated with 5‑10 mM SDS remained severely damaged, exhibiting low TEER (Figure 1) and a reduction in LDH release (indicating cytotoxicity rather than membrane integrity, Figure 3). Mucin release in these samples remained high (Figure 2) which, as the main growth areas of these cultures were effectively devoid of cells, indicated that the viable cells remaining around the periphery of the well produced extremely high levels of mucin. Some recovery was observed in moderately damaged units (1.25‑2.5 mM) with TEER and release of LDH, IL‑6 and IL‑8 returning towards baseline levels (Figures 1, 3, 5 and 6).
In the MucilAir™ model, TEER and histological examination are the most sensitive measurements of epithelial damage.
Acknowledgements The authors gratefully acknowledge the assistance of S Constant and S Huang (Epithelix Sàrl) for technical advice and discussion.
