Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Development of Human In Vitro Bronchial and
Alveolar Models to Evaluate Molecular Mechanisms of
Pulmonary Diseases
Yuksel Cetin
yuksel.cetin@jrc.it
European Center of Validation of Alternative Methods
Institute for Health and Consumer Protection
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Outline
Pulmo-Net introduction
Human in vitro bronchial model
Human in vitro alveolar model
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
PULMO-NET
6th Framework Programme for Research and Technological
Development (FP6)
Marie Curie Research Training Network in the field of pulmonary
research
Consortium (universities, research institution, industrial partner) of
teams located in different countries that propose a common research
project
RTN is primarily based on providing a vehicle to train young
researchers and to facilitate the transfer-of-knowledge
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Coordinator
Universite Paris-Sud
UPS, France
Universitat Konstanz,
UKON, Germany
Medizinische Universitad
Innsbruck, MUI
Austria
Across Barriers G.m.b.H
Germany
Universidad
Complutense de
Madrid, UMC, Spain
Universitatsklinikum Ulm
UKU, Germany
Joint Research Center
IHCP-ECVAM
Italy
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Overall AIM of PULMO-NET
To create a network of experts in the area of pulmonary biology
To develop in vitro systems for lung toxicity and pathogenesis
To establish and pre-validate the most promising in vitro
pulmonary tests
To train early-stage and experienced researchers in various fields
http://www.elegantweb.at/d103-b1375d38/
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
ECVAM Contribution to the Pulmo-Net Tasks
To set up in vitro model(s) to study induced damage to
pulmonary barrier after exposure to volatile chemicals,
airborne pathogens and particles
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Bronchial Barrier
Calu-3
In Vitro Human
Pulmonary Barrier
Alveolar Barrier
NCI-H441 & HPMECST1.6R
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Airway Epithelium
(bronchial tract)
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Calu-3 as a Suitable In Vitro Model of the
Human Bronchial Barrier
Human bronchial adenocarcinoma that form confluent
mixed cultures of ciliated and secretory cells
Air
Medium
Form Tight junctions (high resistance, low permeability)
Produce mucous
Functional P-glycoprotein activity
Pro-inflammatory response
Under Air-Interfaced culture closely mimic the in vivo situation
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Calu-3 under Air-Liquid conditions produce mucous
Calu-3 20X AL
Alcian blue staining for mucous detection
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Endpoints for Assessing Pulmonary Toxicity
Cytotoxicity: Neutral Red Uptake Assay (NRU)
Barrier function: - Transepithelial Resistance (TEER)
- Permeability (FITC-Inulin)
- Tight Junction immunostaining
Bronchial function: Mucous production (QPCR / ELISA)
Pro-inflammatory response: Cytokine release (ELISA for IL-6,
IL-8, RANTES, etc.)
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Mucous
Viscoelastic gel containing water, carbohydrates, proteins,
lipids and Mucous glycoproteins (Mucins)
Two layers: upper gel, lower fluid (where cilia beat)
Function: protective barrier against pathogens and toxins,
contribution to the innate mucosal defense system
Major components of mucous- MUC5AC and MUC5B
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Toxicity testing -Experimental design
Toxicant range finding- 96 well plate
NRU
Toxicant range finding- Transwell
Air interfaced culture
Exposure to
Toxicants
TEER
NRU
Calu-3
Defining concentrations + Time points
For further experiments
Apical Treatment in PBS+
Or
Calu-3
Basolateral Treatment in 0.1% FBS
24 hours
1.
2.
3.
TEER and Permeability
Collect samples for ELISA (mucous, cytokines)
RNA isolation- Real Time PCR for MUC5 mRNA
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Cadmium Chloride (CdCl2)
CdCl2 is an important industrial and environmental pollutant which is
highly toxic for the respiratory system.
Wide variety of toxic effects such as: interfering with the normal
action of Zn2+ and other essential metals, oxidative stress,
mimicking/ antagonizing Ca2+ physiological actions.
Cd2+ can selectively disrupt E-cadherin-dependent cell-cell
junction in various type of epithelial cells.
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Assessment of Cytotoxicity After 24h Exposure to
Cadmium Chloride by Neutral Red Uptake
IC 50= 72mM
Cell Viability (%)
125%
100%
75%
50%
25%
0%
10
100
Cadmium Chloride (µM)
1000
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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TEER is more sensitive and specific end point of the barrier damage
Apical / TEER
% Control
Apical / NRU
180
160
140
120
100
80
60
40
20
0
Basolateral / TEER
IC 50 values
Basolateral / NRU
0
10
20
30
40
50
100
200
Exposure
End point
IC 50
(mM)
Apical
TEER
NRU
94
> 100
Basolateral
TEER
NRU
15
48
96 well plate
NRU
72
300
Cadmium chloride (mM)
Calu-3 cells were 14 days in culture before exposure to CdCl2 for 24 hours
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Significant difference in barrier damage kinetics measured by TEER
(apical vs. basolateral treatment)
Basolateral Treatment
time 0
1 Hour
4 Hours
6 Hours
9 Hours
24 Hours
time 0
1 Hour
4 Hours
6 Hours
9 Hours
24 Hours
140
180
160
TEER (% control)
TEER (% control)
Apical Treatment
140
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
1
10
20
30
Cadmium chlorid (mM)
40
50
100
0
1
10
20
30
40
Cadmium chlorid (mM)
50
100
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Significant Up-regulation of MUC5 expression levels
after 24h exposure to CdCl2
Calu-3 cells were 14 days in culture before exposure to CdCl2 for 24 hours
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Bronchial Barrier
Calu-3
In Vitro Human
Pulmonary Barrier
Alveolar Barrier
NCI-H441 & HPMECST1.6R
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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In vivo Alveolar System
 Alveolar Type I
 Alveolar Type II
 Produce surfactants
Lipids; 80-90% phosphaditylcholine
Proteins; SP-A, SP-B, SP-C, SP-D
 Lowers surface tension
 Host defense
Structure of alveolus (Hawgood & Clements, 1990)
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Setting up of an in vitro human alveolar model
Monoculture of epithelial cell line
Epithelial Cells
COL-Coated Membrane
Endothelial Cells
Co-culture of epithelial and endothelial cell lines
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Co-culture of Human Alveolar Barrier
In vitro cultures of alveolar epithelial cells,
NCI-H441
alone or in conjunction with endothelial cells
Functional pulmonary barrier
High degree of differentiation
Multicellular bodies
Polycarbonate Membrane
Secretion of surfactant proteins
Pro-inflammatory response
Ref: Kirkpatrick et al, 2004. Lab. Invest., 84:736-752
HPMEC
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Co-culture of Alveolar System
Human Lung Epithelial Cell Line
NCI-H441
Human Pulmonary Microvascular
Endothelial Cell Line
HPMEC-ST1.6R
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Endpoints
To assess toxicity on pulmonary barrier
- TEER
- Permeability
To assess specific pulmonary function
- Surfactant secretion
To assess pro-inflammatory response
- Cytokine release
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Optimization of In vitro Co-culture of Alveolar Barrier
Seeding cell densities
Incubation time of HPMEC-ST1-6R
Serum effect
Dexamethasone effect
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Barrier Formation of Mono- and Co-cultures
300
Monoculture;+Dex
Monoculture; -Dex
250
TEER, ohm.cm2
Coculture; +Dex
200
Coculture; -Dex
150
100
50
0
0
5
10
Days in cultures
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Permeability of Sodium Fluorescein Across
the Mono- and Co-cultures
5.2x10-6
4.8x10-6
Coculture - DEX
7.8x10-6
Monoculture - DEX
5.3x10-6
5.9x10-6
1.2x10-9
Coculture +DEX
Monoculture +DEX
3.6x10-6
2.3x10-7
Basolateral to Apical
Apical to Basolateral
0,E+0 1,E-06 2,E-06 3,E-06 4,E-06 5,E-06 6,E-06 7,E-06 8,E-06 9,E-06
0
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Tight Junction Proteins of NCI-H441
Occludin
ZO-1 (red)
Phalloidin (green)
E-Cadherin
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
Other endpoints
Surfactant
Lamelar bodies and exocitosis by flourescent method
Immunostaining & westernblotting
SP-A, SP-B, SP-C, SP-D
Inflamation
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Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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Future experiments
Testing of selected nanoparticles
Comparison of results obtained from bronchial and
alveolar models
Hacettepe Congress Centre, Ankara, Turkey on 12-13, November 2007 – JRC Enlargement & Integration Action 2007, Workshop on Alternative Methods
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