Presented at the 2018 Society of Toxicology Annual Meeting.

1. Understanding Air-Liquid Interface
Cell Exposure Systems:
A Comprehensive Assessment of Various
Systems Under Identical Conditions
José Zavala, PhD
ORISE Postdoctoral Fellow
NHEERL, EPHD, Inhalation Toxicology Facilities Branch
Mentor: Mark Higuchi
Email: zavala-mendez.jose@epa.gov
SOT Annual Meeting
March 15, 2018

2. Disclaimer: This presentation does not
necessarily reflect EPA policy. Mention of
trade names or commercial products does
not constitute endorsement or
recommendation for use

3. No ALI Exposure Standards
• Inconsistency in published literature
Exposure conditions
Biological results
Monitoring of test chemical/particle
• Difficult to evaluate and compare biological
results
• Various ALI systems exists but cannot be
compared based on existing studies.

4. Example
C
lean
A
ir
O
zone
C
lean
A
ir
O
zone
0
2
4
6
8
10
12
**
9 hours
24 hours
LDH
(FoldChangeOverAirCt.)
C
lean
A
ir
O
zone
C
lean
A
ir
O
zone
0
250
500
750
1000
*9 hours
24 hours
*
IL-8(pg/mL)
Exposure System: GIVES
Membrane Inserts: 12-mm Snapwells
(6-well format)
Pollutant: Ozone
Concentration: 0.4 ppm
Exposure Length: 4 hours
Cell Type: A549
Results: ↑ cytotoxicity, ↑ pro-inflammatory cytokine secretion
Zavala et al. Inhal Toxicol., 2016, 28(6), 251-259
Exposure System: VITROCELL
Membrane Inserts: 24-mm Transwells
(6-well format)
Pollutant: Ozone
Concentration: 4.0 ppm
Exposure Length: 4 hours
Cell Type: A549
Results: NO cytotoxicity, NO changes in pro-inflammatory cytokines
Anderson et al. Toxicol In Vitro., 2013 Mar; 27(2): 721-730

5. Comparison of ALI Exposure Systems
Study Objective: How well does each system deliver gases
and particles to the target area (i.e., cell inserts).

6. •Fluorescent polystyrene latex (PSL) spheres were used
as a surrogate for PM.
Evaluation of Particle Delivery to Culture Inserts
•PSL spheres were nebulized and delivered to the in
vitro systems.
•Deposition was quantified by dissolving particles in
ethyl acetate and using a spectrofluorometer.
•This method involves NO CELLS.
•Filters were placed on cell
culture inserts filled with DPBS
in the basolateral side to
simulate the conductive culture
medium.

7. Evaluation of Gas Delivery to Culture Inserts
• We used a fluorescence-
based method with 125 ppb
O3 as a test gas and measured
its reactivity on an indigo dye-
impregnated filter inside each
well.
• The method involves NO CELLS; measuring chemical
reaction
• Normalized data to an impinger system operated at
250 mL/min as a positive control.

8. VITROCELL® Systems
Model 6 CF
Principle of Operation
Gases: Diffusion
Particles: Diffusion/Sedimentation
Flow Rate: 2-10 mL/min
• Air flow is perpendicular to
the cells
• Air flow is perpendicular to the cells
• System relies on the particle’s natural
electrical charge for ESP to work.
• System uses an isokinetic sampler
instead of manifold.
Model 6 CF
Principle of Operation
Gases: Diffusion
Particles: Electrostatic Precipitation (ESP)
(Positive or Negative Polarity)
Flow Rate: 2-10 mL/min

9. VITROCELL® Systems Performance
• In 6 CF system, carbon-impregnated
silicone tubing reduces tubing loses with
50-nm particles.
• ESP on 6/3 CF relying on particle’s natural
charge enhances deposition.
• In 6 CF system, Teflon
tubing, while
impractical, improves
gas delivery.
• 6/3 CF system has very
poor gas delivery.
• Tubing and nozzles
were investigated to
determine their role.

10. • This chamber has 2 modes of operation
EPA’s Cell Culture Exposure System (CCES)
Principle of Operation
Gases: Diffusion
Particles: Thermophoresis (THP)
(Thermal Gradient)
Flow Rate: 25-50 mL/min
• CCES with 6-well format uses both modes.
• CCES with 24-well format uses Mode 1 only.

11. EPA’s CCES Performance
1 µm
• In both formats format, flow rate of 25
mL/min/well is better than 5 mL/min/well.
• Regardless of multi-well format, gas is
delivered at similar relative efficiencies.
• In 6-well format,
thermophoresis
enhances deposition
for 50 nm particles, but
not 1 µm.
• In 6-well format, flow
rate of 25 mL/min is
better than 5 mL/min.
50 nm

12. Gas In Vitro Exposure System (GIVES)
Principle of Operation
Gases: Diffusion
Particles: Diffusion/Sedimentation
Flow Rate: > 1 L/min
• Particles < 1 µm do not induce
biological effects.
• Virtually a “gas-only” system
Ebersviller et al. Atmos. Chem. Phys., 2012, 12277-12292

13. GIVES Performance
• Flow rates of 1 and 2 L/min were
tested for gas delivery.
• Small increase in delivery observed at
2 L/min.
• A high relative efficiency was
observed.
• Only flow rate of 1 L/min tested.
• As expected, particle loading is very
poor regardless of particle size.
• Residence time of particles inside GIVES
too short for gravitational setting to have
an effect.

14. Principle of Operation
Gases: Diffusion
Particles: Electrostatic Precipitation (ESP)
(Positive Electric Field)
Flow Rate: 2.2 L/min
• Corona wire allows unipolar
charging of particles to enhance
the particle deposition.
• Air flow is horizontal to the cells
Gillings Sampler
Zavala et al. Chemico-Biological Interactions., 220 (2014), 158-168

15. Gillings Sampler Performance
• Only 1 operating condition was tested for gas delivery
as flow rate and multi-well format were fixed.
• A high relative efficiency was observed.
• 50 nm, 500 nm, and 1 µm
particles tested since ESP
is good for wide range of
particles.
• Particle loading is
consistent regardless of
particle size.
• Using 1 um particles, the
effect of unipolar charging
with ESP was investigated.

16. Ranking of Systems
Under Normal Operating Conditions
Gas Delivery

17. Particles
• An external force, such as THP or ESP, is needed to enhance
particle deposition.
— Relying on sedimentation/diffusion will produce little/no
effect on cells exposed unless very high concentrations are
used (e.g. several mg/m3)
• Unipolar charging of particles significantly increases deposition
• Gillings Sampler is most effective at delivering particles.
Gases
• Selecting the appropriate flow rate and tubing is critical.
• The Gillings Sampler is most effective for delivering gases.
Characterization of systems is needed prior to their
use to understand their advantages and limitations
Overall Observations

18. Acknowledgements
EPA
• Mark Higuchi
• Allen Ledbetter
• Earl Puckett
• Todd Krantz
Funding
• Oak Ridge Institute for Science and
Education (ORISE)
• Intramural research programs at the
U.S. EPA
UNC
• Will Vizuete (Gillings Sampler)
Health Canada
• Paul White (Vitrocell 6 CF)
VITROCELL
• Tobias Krebs (Vitrocell 6/3 w ESP)

19. José Zavala, PhD
ORISE Postdoctoral Fellow
NHEERL, EPHD, Inhalation Toxicology Facilities Branch
Mentor: Mark Higuchi
Email: zavala-mendez.jose@epa.gov
SOT Annual Meeting
March 15, 2018
Questions?