MucilAir: a Novel Human 3D Airway Epithelium Model for Long Term Toxicity Testing
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1 MucilAir: a Novel Human 3D Airway Epithelium Model for Long Term Toxicity Testing Samuel Constant, Ph.D., COO samuel.constant@epithelix.com Sàrl 14, Chemin des Aulx CH-1228 Plan les Ouates Genève - Switzerland +41 (0) / Fax: +41 (0) epithelix@epithelix.com 1
2 MucilAir Isolation, amplification & seeding Primary Human Cells Ready-to-use Fully differentiated Epithelium Mucus Ciliated Cells Mucus Cells Basal Cells Patients Normal Asthmatic Smokers COPD CF Air-interface, differentiation 2
3 Morphology and Histology Similar, if not identical, ultra-structures as in the native tissues
4 In vitro, de novo Ciliogenesis on MucilAir A time course of cilia formation monitored by anti-β-tubilin antibody D 21 D 30 D 45 D 60 4
5 Immunohistochemistry Various ions channels and cell markers are detected in our in vitro model of airway epithelium Na-K ATPase Occludin CFTR AQP3 5
6 The main characteristics of MucilAir It closely mimics the morphology and functions of the normal human airway epithelium. It has a unique shelf-life of one year. It is easy to handle and maintain. Adapted to high-throughput screening and testing. Serum free. 6
7 Applications of MucilAir 7
8 Functional In Vitro Assays using MucilAir Toxicity Testing (cell viability and tissue integrity) Effect on ciliogenesis Effect on cilia beating (mucociliary clearance) Effect on Mucin secretion Measurement of pro-inflammatory mediators Simulation of chronic inflammation Electrophysiological activity (TEER, ion channels) Drug transport, etc. 8
9 Toxicity Testing MucilAir is a powerful tool to quantify Acute, Chronic and Long Term effect of molecules Cell viability (EC 50 & ET 50 ) Resazurin test (Quantitative) Tissue Integrity Trans Epithelial Electrical Resistance (Quantitative) Cilia beating Monitoring Visual Inspection (Qualitative) Time points Acute = 1 hour exposure Chronic = Multiple 1 hour exposures Long Term exposure (e.g. 24h, 48h, etc.) Type of Compounds Solid, liquid Gas, nanoparticles, smoke: Cultex Chambers CULTEX 9
10 Acute Toxicity (1 hour Exposure) N N Cell viability 150% Nicotine (N=9) 100% Viability [%] 50% 0% Concentration [mm] Tissue Integrity Nicotine (N=3) TEER [ohm.cm2] Patient 1 Patient 2 Patient Concentration [mm] 10
![Chronic Toxicity (Multiple 1 hour Exposures) HCl HN Acute Toxicity on Propranolol (N=9) O OH 100% EC 50 = 8 mm Viability [%] 50% 0% 0 1 2 3 4 5 6 7 8 9 10 Concentration](/docs-images/87/97338881/images/11-0.jpg "[mm] Concentrations 0 mm 1 mm 2.5 mm 4 mm 5.5 mm 7 mm 8.")
11 Chronic Toxicity (Multiple 1 hour Exposures) HCl HN Acute Toxicity on Propranolol (N=9) O OH 100% EC 50 = 8 mm Viability [%] 50% 0% Concentration [mm] Concentrations 0 mm 1 mm 2.5 mm 4 mm 5.5 mm 7 mm 8.5 mm 10 mm Expo 1 (Day 0) EC 25 X Expo 2 (Day 7) Expo 3 (Day 14) Expo 4 (Day 21) Expo 5 (Day 28) Expo 6 (Day 35) 11
12 Inflammatory reaction on MucilAir 2 exposure modes Apical exposure Air Liquid Ideal for gas, particles. Typical reaction with liquids : 24 hours apical exposure Secretion of IL-8 (ng/ml) Basal NaCl (0.9%) NaCl (0.9%) + TNF-a 12
13 Inflammatory reaction on MucilAir 2 exposure modes Apical exposure Air Basolateral exposure Liquid Ideal for gas, particles. Typical reaction with liquids : Ideal for liquids No inflammation induced by vehicle 24 hours apical exposure Secretion of IL-8 (ng/ml) Basal NaCl (0.9%) NaCl (0.9%) + TNF-a 13
14 Inflammatory reaction on MucilAir Our primary in vitro epithelium responds to pro-inflammatory mediators (TNF-α) in a physiological manner 80 Secretion of IL-8 (ng/ml) TNF-α N = 10 0 Day 0 Day 2 Day 5 Day 7 Day 9 A time course of IL-8 secretion (an inflammatory marker) after 30 minutes of TNF-α stimulation on a 3 months old epithelium. Note that this same epithelium can be reused to simulate chronic inflammation. 14
15 Measurement of Mucin secretion Enzyme-linked lectin assay (ELLA) The test is based on the affinity of lectins for mucins. It allows the quantitative measurement of the secreted mucins. Acute, long term or chronic exposition Positive control : ATP stimulation 24 hours apical exposure Secretion of mucin (ng/ml) Basal NaCl (0.9%) stimulated 15
16 Molecule effect on Mucociliary clearance Phase contrast or using fluorescent particles Direct measurement of particles velocity 16
17 Electrophysiology on MucilAir The cells from our in vitro epithelium show typical electrophysiological responses to ion channel inhibitors and activators Current measurement in modified Ussing chamber Amiloride Isoproterenol Bumetanide R(Ω.cm 2 ) (resistance) 438 ± 18 (N=203) Amiloride (sodium channel inhibitor) reduces the current. Isoproterenol, a CFTR activator (chloride channel) increases current. Bumetanide, a general channel blocker, abrogates the total channel current activities. 17
18 Evaluation of drug transport across MucilAir dcr/dt (Atenolol) 2,5 2 y = 0,0217x - 0,0013 R 2 = 1 1,5 Cr (µm) 1 0,5 0-0, Molecule P app (cm/s) Atenolol 55 x 10-6 Propranolol 10 x 10-6 Ibuprofen 157 x 10-6 Insulin 2 x 10-6 t (Min) Time course of the rate of permeation of Atenolol from the apical to basal lateral side during a period of 90 min (triplicate). LC/MS detection High reproducibility 18
19 Acknowledgements Ludovic Wiszniewski, Ph.D., CEO Song Huang, Ph.D., CSO Jean-Paul Derouette, Ph.D. Mireille Caul-Futy Fondation Liechti 19
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