1

Supporting Online Material for

Hanging drop: an in-vitro air toxic exposure

model using human lung cells in 2D and 3D

structures Faye F. Liu*

1, 2, Cheng Peng

1, 2, Beate I. Escher

1, Emmanuelle Fantino

3, Cindy Giles

4,

Stephen Were4, Lesley Duffy

5, Jack C. Ng*

1, 2

RECEIVED DATE (to be automatically inserted after your manuscript is accepted)

*To whom correspondence should be addressed: 1. Faye F. Liu, Phone: +61 7

32749060, Fax: + 67 32749003, Email: faye.liu@uq.edu.au; 2. Jack C. Ng, Phone: +61

7 32749020, Fax: + 67 32749003, Email: j.ng@uq.edu.au.

This PDF file includes:

Materials and methods

Figures S1 to S4

Table S1

References S1 to S4

2

1 IN SITU MTS, CELLTITER-GLO® AND CELLTITER-BLUE®

ASSAY

Three types of cell viability assays were used for cell viability monitoring in this paper due

to differences in reaction volume and the need of higher surface tension of the reaction drops.

The assays were: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay

(Promega # G5430), CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7571)

and CellTiter-Blue® Cell Viability Assay (Promega #G8081).

1.1 MTS assay

Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation (MTS) assay kit was

used for diffusion air exposure and the protocol was in accordance to the manufacturer’s

instructions with modifications to perform in situ reaction in the following three steps: 1)

pipette 400 µL MTS agent into a 20 mL VOA vial containing 1.6 mL of DMEM with

HEPES; 2) transfer the vial into a 37oC constant temperature room for 45 min, and 3) transfer

150 µL of the reaction mix into each well of a 96 well plate, for absorbance reading @

490nm on a Tecan Infinite plate reader (M200, Tecan group Ltd, Switzerland).

1.2 CellTiter-Glo® Luminescent Cell Viability Assay

CellTiter-Glo® Luminescent Cell Viability Assays were used in monitoring cell viability of

Hanging drop cultures and it was performed according to the manufacturer’s instructions with

modifications to perform in-situ cell lysis reaction: After the exposure, the VOA vials were

opened in the laminar flow cabinet with the lid facing up. 20 µL of CellTiter-Glo® agent

were pipette onto the 20 µL hanging drop culture on the septum of the lid. These lids were

3

then placed in a tray, covered with a transparent cover to prevent evaporation and at the same

time to allow luminescence to develop under light. This tray was then placed on a platform

shaker to shake for 12 min at room temperature, at a speed that did not disturb the drops.

When finished, an aliquot of 25 µL of the reaction mixture was then transferred into a clear

bottom 384 well plate (Corning #3712) for luminescence measurement on an Infinite M200

plate reader (TECAN, Switzerland).

1.3 CellTiter-Blue® Cell Viability Assay

In situ CellTiter-Blue®

Cell Viability assays were performed according to manufacturer’s

recommendation with modifications. Briefly: 1 plate of HD cultures were opened, inverted

to have the drops on top of the lid in the biohazard hood, water from the bottom tray was

drained on a tissue; 20 µL of CellTiter-Blue® reagent was then added on top of each drop of

10 randomly selected drops; the drops were then capped with the bottom of the tray on top

and placed back into the incubator for 2 hours before the drops were transferred into a 384

well plates (Corning #3712) for florescent measurement on an Infinite M200 plate reader

(TECAN, Switzerland) with the gain manually set at 100.

2 HISTOLOGY

For histology studies, A549 cell 3D structures were fixed in 10% formalin, confined into a

cell button for easy handing with 3% low melting point agarose, processed, embedded in wax

in Cytology Laboratory in Queen Elizabeth II Hospital and stained in the Queensland Health

Forensic and Scientific Services (QHFSS) Histology Laboratory.

As have shown in microscopic and histological studies illustrated in Figure S1, although

A549 cells in a 2D format do not differentiate, in 3D spheroid form, H and E stains of day 10

HD showed that the culture underwent different stages of differentiation and evidently ducts

4

resembling alveolar sacs were starting to form on day 10. No evidence of necrosis was found

for all of the 3D cultures. The growth of HD spheroids is illustrated in Figure S2. Although as

illustrated in Figure S2, no trend of changes in spheroid diameter was found between days 5

to 14 cultures, a trend of increase in cell viability via CellTiter-Blue® assay was found from

day 1 up until day 10.

3 CELL GROWTH

Because CellTiter-Glo® assay reagent has a weak surface tension, which makes the

neighboring reaction drops to run into each other hence loose the reaction. CellTiter-Blue®

Cell Viability Assay (Promega #G8081) were chosen instead of CellTiter-Glo® assay to

monitor cell viability changes of non exposed HD cultures over 20 days. To set up the

cultures, 20 µL hanging drop (HD) cultures at 1 x 104 cells/mL were inoculated on the lids of

each of 60-well plates (Thermo Scientific #439225) using a multichannel pipette. 1 mL of

water was added into the well side of the 60-well plate for form individual humidity

chambers. All of the plates with HD cultures were then packed further into plastic humidity

chamber before placed into 37ºC incubator until cell viability assay time. The HD cell

viability is assayed on day 1, 2, 4, 5, 6, 8, 10 and 17 to monitor cell growth of HD cultures.

Cell viability of the HDs was plotted against culture age as shown in Figure S2. As have

shown in Figure S2, HD cell viability, as represented by florescent intensity, continued to

grow from day 1 until day 10. The florescent intensity at day 17 falls to the same level as in

day 4.

5

Since the cells of the HD system would grow and subdivide, its cell number in the HD

system has been modelled using the cell viability data at growth phase (up to day 10) which

have been illustrated in Figure S2 and the semi-log linear regression is illustrated in Figure

S3.

4 HD SYSTEM PARAMETERS AND BIOAVAILABILITY

CALCULATIONS OF THE INJECTED BENZENE

As shown in Figure 1 of the main text, our HD system consists of 1 mL H2O, and 20 µL

culture drop in a 20 mL VOA (volatile organic analysis) vial. A nominated amount of VOC

(volatile organic compound) was injected through the septum of the vial. After complete

volatilisation, the amounts (or proportions) of VOCs will distribute among air, water, and

cells following Henry’s Law at a given temperature. The bioavailability of VOAs in the vial

was calculated using a four-compartment mass balancing model [1-3]where Henry’s

partitioning co-efficient Kair-water (Kaw), of Koctanol-water (Kow) were taken from recent

publications [1] with temperature adjustments.

[1]

Hence the bioavailability, which is the cell exposure fraction, would be

Since , and

Therefore

,

6

And

Hence

[2]

[3]

` [4]

7

Where fw and fa are fractions in water and in air (head space) respectively; Kaw, KFBSw,

Kcellw, Klipw and Kproteinw are partitioning coefficients between air-water, FBS- water, cell-

water, lipid-water, and protein-water, respectively; Va and Vw are volumes of air and water,

respectively; mFBS and mcell are FBS and cell contents from dry weight in kilograms,

respectively; Fw, fa and fcell are factions in water, air and cell, respectively.

An experimental value of log Kaw of 0.65 at 25ºC was adjusted to 0.85 at 37ºC [4] and a

calculated value of logKow of 2.17at 25ºC [4] together with an experimental value of

logKBSAw of 1.58 at 37ºC[1] were used to estimate the Klipw and Kproteinw values. Since it is

well accepted that BSA can be used as a generic protein protein-chemical interactions, KBSAw

value is used as Kproteinw for the calculations. Following equations were derived from Esher

et. al[2]:

[5]

[6]

[7]

In order to extrapolate the KFBSw and Kcellw values, routine dry weight fraction values of

FBS and rat hepatoma H4IIE cells were adopted in house and their values are used to

estimate the mass balance of benzene.

Based on Supplementary Figure S3, cell numbers and hence mcell in HD system have been

extrapolated and tabulated as in Table S1. The HD cell numbers of after day 10 were

calculated using their actual level of florescence at that age. The calculations were made

assuming the single cell dry weight is 8 x 10-13kg, which is adopted from the rat hepatoma

H4IIE cells.

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Hence at its complete volatilization, the mass distribution of the benzene was calculated

using above formulas [1]-[7] and the results and system parameters are presented in Table 1

of the main text.

5 BENZENE PERSISTANCE IN VOA VIALS

The stability of benzene concentration after one 10 µL Hamilton needle pierce of the VOA

vial was monitored by GC-MS. A Trace GC Ultra with DSQ single quadruple GC-MS from

Thermo Scientific equipped with a Phenomenex ZB-624 capillary column (20 m x 0.18 mm

id, 1µm film) was used based on manufactures recommendations. For analyte identification

and quantification, the temperature program was: 40oC held for 1 min, then ramped at

10oC/min to 120oC. The carrier gas was helium at 1 mL/min. The mass spectrometer detector

was operated in SIM (selected ion monitoring) mode monitoring ions at 78, 77 and 51

m/z. 10 µL samples were injected directly through the septum using a 50 µL air tight

Hamilton syringe with inlet temperature of 250oC, splitless mode for 0.5 min and split flow

50 mL/min. Peak areas corresponding to the benzene retention time obtained were plotted

against the days of free standing at room temperature to approximate potential benzene loss

during the cell exposure period.

The persistence of benzene concentration in VOA vials over several days was monitored

through GC-MS in order to determine the stability of air exposure doses. As shown in Figure

S4, there was an approximately 30% decrease in benzene concentration over 24 h.

9

Table S1. Calculated mcell value of the HD air exposure system

HD exposure type starting cultural age ( days) Cell Numbers Mcellsa

Benzene 2D 1h 2 1600 1.28 x 10-09 Benzene 2D24h 3 2273 1.82 x 10-09 Benzene day 7 3D 1h 8 3900 3.12 x 10-09 Benzene day7 3D 24h 8 3900 3.12 x10-09 Benzene chronic 1day 2 1600 1.28 x10-09 Benzene chronic 7days 8 3900 3.12 x10-09 Benzene chronic 14days 15 2750 2.20 x10-09 Benzene chronic 20days 21 2750 2.20 x10-09

saAssuming the single cell dry weight is 8 x 10-13kg, which is adopted from the rat hepatoma H4IIE cells

Figure S1a. A549 cell culture (x100) under stero-microscope. Cell boundaries are clearly

defined for Day 2 Hanging drop (HD) culture whilst from day 5 on, individual cells are

indistinguishable. Bar: 200 µm.

10

Figure S1b. A549 Hanging drop (HD) H & E stain. No sign of necrosis found up to day 15.

Bar: 200 µm.

11

Figure S1c. H & E stains for A549 Alvetex® 3D cultures (x200). All exposures were performed on day 13 cultures. While cells were still attached to the matrix for the 1 h 5 µL benzene exposures, they gradually started to detach for the 24 h 5 µL and the 1 h 10 µL benzene exposures. All of the A549 cells were completely detached from the matrix for the 24 h 10 µL benzene exposure.

12

0 5 10 150.300.350.400.450.500.55

A549 HD culture age (days)

A

Dia

metr

e

(mm

)

0 5 10 15 200

10000

20000

30000

40000

50000

A549 HD culture age (days)

B

Cell v

iab

ilit

y u

nit

via

Tit

reB

LU

E

Figure S2. A: Illustrates the size variation in diameter of the HD spheroids with minimum

10 randomly selected samples for each culture age; B: Illustrates the cell viability changes

over 17 days.

13

0.0 0.5 1.0 1.50

10000

20000

30000

40000

50000

y=35537x+4183

Log of culture age (days)

Cell v

iab

ilit

y v

ia T

itre

BL

UE

Figure S3. Linear regression: A549 HD cell number versus Log of cultural age (p<0.0001).

14

D: 1h HD vs scaffold

Control 5 L 10 L0

50

100

150Hanging drop2D roller mixer- control3D scaffold roller mixer

Benzene vol (uL)

% C

ell v

iabi

lity

via

MTS

Control 5 L 10 L0

50

100

150

200

C: 48h air exposure comparison

Benzene vol (L)

E: 24h HD vs scaffold

Control 5 L 10 L0

50

100

150Hanging drop2D roller mixer - control3D scaffold

Benzene vol (uL)

% r

ecov

ery

via

MTS

Control 5 L 10 L0

50

100

150Hanging drop1mL roller mixer1mL media diffusion2mL media diffusion4mL media diffusion

A: 1h Air exposure comparison

1 2 30

50

100

150 B: 24h Air exposure comparison

control 5 L 10L

%C

ell v

iail

ity

via

Titr

eGlo

Figure S4. A-C: Comparison of HD air exposure method with the roller mixer and media

diffusion methods; D-E: Comparison of benzene toxicity to A549 cells in 3D spheroid and

3D scaffold cultures. Cell viability of the Alvetex® 3D culture was monitored by MTS assay

while HD spheroid benzene sensitivity was accessed using the CellTiter-Glo® assay. 2D

roller mixer exposure was used as exposure methodology control in this experiment. All tests

were performed in 5 replicates. Only 1 mL of media was used for the roller mixer exposure

and some error bars were too small to be visible on the graph and no roller mixer exposure

was carried out for 48 h exposure.

15

0 1 2 3 4 50

5.0107

1.0108

1.5108

2.0108

Days of free standing

Ben

zen

e c

on

cen

trati

on

@ G

C-M

S m

ass v

alu

e

Figure S5. Concentration reduction of benzene represented by mass value over time with an

initial introduction of 10 µL of benzene in 40 mL VOA vials. Mass value was measured

using a Trace GC Ultra with DSQ single quadrupole GC-MS from Thermo Scientific

equipped with a Phenomenex ZB-624 capillary column (20m x 0.18mm id, 1µm film).

References [1] S. Endo, K.-U. Goss, Serum albumin binding of structurally diverse neutral organic compounds: data and models, Chem Res Toxicol, 24 (2011) 2293-2301. [2] B.I. Escher, C.E. Cowan-Ellsberry, S. Dyer, M.R. Embry, S. Erhardt, M. Halder, J.-H. Kwon, K. Johanning, M.T.T. Oosterwijk, S. Rutishauser, H. Segner, J. Nichols, Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals, Chem Res Toxicol, 24 (2011) 1134-1143. [3] M. Gülden, H. Seibert, In vitro–in vivo extrapolation: estimation of human serum concentrations of chemicals equivalent to cytotoxic concentrations in vitro, Toxicol, 189 (2003) 211-222. [4] R.P. Schwarzenbach, P.M. Gschwend, D.M. Imboden, Environmental Organic Chemistry, 2nd ed., John Wiley & Sons, Inc. Hoboken, New Jersey, Canada, 2003.