APPLICATIONNOTEHigh-throughput in-vitro
airway modelling withThinCert® 96 Well HTS Insert
www.gbo.com /bioscience1. INTRODUCTION
Growing cells in an air-liquid interface (ALI)
is a popular method to generate a stable andfunctional model of the respiratory tract byrecreating a pseudostratified epithelium invitro. ALI cell cultures are frequently adopted
to investigate different processes associatedto lower airways including disease modelling [1,2], drug testing, cell-to-cell signalling
and air pollutant-induced pulmonary toxicity studies [3]. To establish an ALI cell culture, normal human bronchial epithelial cells(NHBEs) are cultivated on a porous membrane insert (ThinCert® 96 well HTS insert).
In the initial phase the cells are grown undersubmerged conditions to form a confluent
cell layer. Once this is achieved, the media
is removed from the membrane insert andthe cells are transferred to the air-liquid interphase. Due to the two-component designof the ThinCert® HTS insert, the basal sideof the cells are still in direct contact with thecell culture medium while the apical side isexposed to air. In this way the cells undergo amorphological and functional differentiation
similar to that found in intact tissues. This
includes the formation of tight junctions, thedevelopment of cilia and mucus and an established cellular polarity.
ALI cell cultures have attracted even more
attention in course of COVID-19 disease modelling and are frequently applied for SARSCoV-2 related research. Single permeable
membrane inserts such as ThinCert® cellculture insert have proven to be an essential tool for modeling airway-epithelia in vitro [4, 5, 6, 7]. With the addition of an automation-friendly permeable cell culture support
specifically optimised for high-throughput
assays, the ThinCert® 96 well HTS insert expands and enhances the well-established
Greiner Bio-One ThinCert® portfolio. In contrast to single inserts, the 96 well HTS insert
can be handled as a single unit significantly
reducing the manual effort.

2. MATERIALS AND
METHODS
Item

Manufacturer

Item No.

Cells
Normal Human Bronchial Epithelial Cells

Lonza

CC-2540S

Cell culture media kits
PneumaCult-Ex Plus culture medium

Stemcell Technologies

05040

PneumaCult-ALI Medium for differentiation

Stemcell Technologies

05001

Hydrocortisone, 2 x 10 -4 M

Stemcell Technologies

07925

Heparin, 2 mg/ml / 0,2 %Stemcell Technologies

07980

Accutase cell detachment solution

Stemcell Technologies

07920

Rat Tail Collagen Coating Solution, 50 µg/ml

Sigma Aldrich

122-20Trypsin/EDTA solution (0.05 %/0.02 %)PAN Biotech

P10-023100

Cell culture chemicals

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Application NoteItem

Manufacturer

Item No.

Immunofluorescent staining
Mouse anti-ß-Tubulin IV

Sigma-Aldrich

T7941-.2ML

Alexa 488 goat anti-mouse

Life Technologies

A11001

Rabbit anti-ZO1

Life Technologies

40-2200

Alexa 546 goat anti-rabbit

Life Technologies

A11010

Alcian blue staining
Alcian blue 8GX

Sigma Aldrich

A3157-10G

Sigma-Aldrich

D8417-10MG

DAPI staining
DAPI

3. ASSAY PRINCIPLE
3.1 SEEDING PREPARATION(GENERAL CELL CULTURE)
1. NHBE cells are cultivated according tostandard cell culture protocols (e.g., 60 %confluence)
2. Remove and discard the cell culture
media and rinse with PBS
3. Add Accutase solution to initiate celldetachment
4. Transfer the cell suspension to a tube andcentrifuge at 350 X g for 5 min.
5. Resuspend the cell pellet in pre-warmed
cell culture media
6. Dilute the solution to a seeding density ofapproximately 150,000 cells/cm²(420,000 cells/ml)

3.3 SEEDING CELLS IN
THINCERT® 96 WELL HTS INSERT(SUBMERGED EXPANSIONPHASE)
1. Add 50 µl of the above prepared cellsuspension to each well (approx.
21,000 cells/well)
2. Add 200 µl cell culture media to each wellof the receiver plate
3. Place the ThinCert® 96 well HTS insert intothe receiver plate
4. Place the plate in the incubator (37 °C,5 % CO2)
5. Cultivation for 5 days until a confluent celllayer has formed
6. Perform media change after 48 h

3.2 COATING WELLS WITH
COLLAGEN
1. Add 28 µl of rat tail collagen stock
solution (10 µg/cm²) to each well of themembrane plate
2. Incubate the collagen at room
temperature for 1 h
3. Rinse with PBS

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3.4 MEDIA CHANGE
1. Remove media from the wells of thereceiver plate
2. Remove media from the wells of the insert
plate (upper chamber wells)
3. Add media to the wells of the receiverplate
4. Add media to the wells of the insert plate(upper chamber wells)
General remark: The media change can beperformed through the access ports (seefigure 1), e.g., with a multi-channel pipette.
Alternatively, the insert plate can be lifted
and the media change of the receiver plate
can be performed accordingly.
Useful hint:
Please handle the plate carefully to avoid
excessive movement of the medium in thereceiver plate.

3.5 AIR-LIFT CELL CULTURE(DIFFERENTIATION PHASE)
1. Remove media from the wells of thereceiver plate
2. Remove media from the wells of the insert
plate (upper chamber wells)
3. Add 150 µl differentiation media to thewells of the receiver plate
4. The wells of the insert plate remain empty
to facilitate the ALI cell culture
5. Exchange the differentiation media of thereceiver plate every second day
6. Wash the cells in the insert plate with
100 µl PBS buffer once a week to remove
the mucus
7. Carefully aspirate the PBS
Note: Do not damage the cell layer tomaintain the integrity of the epithelial barrier.

8. The cells are cultivated over a period of
20 to 25 days
Note: If media diffuses from the receiver wellto the upper chamber, remove it carefully.

Figure 1: Access port is located next to the well andis accessible for (multi-channel) pipettes, automatedliquid handling robots as well as for electrodes
for Transepithelial Electric Resistance (TEER)
measurements

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Application Note4. AIR-LIFT CULTURE

1Insert plate preparation
Coat membrane with collagen2

Submerged cell culture (expansion)
Add cell suspension to each well ofthe insert plate

Collagen

5 days (media change every 48 h)

NHBE

3

Air-lift culture (differentiation)
Add differentiation media to thewells of the receiver plate andremove media from the well of theinsert plate
20-25 days media change every 48 h
Wash with PBS buffer once a week

Differentiation
media

4

Air-lift culture (differentiation)
Cultivate the cells for 20-25 days

Mucus
Ciliated cells

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4.1 DIFFERENTIATION MARKER
To assess the differentiation performance,
anti-ß-tubulin IV was used to detect ciliatedcells that make up most of the cells found in
the airway epithelium. The presence of mucus-producing goblet cells was proven by analcian blue dye which stains sulfated and carboxylated polysaccharides found in the mucus layer of the respiratory tract. Both cells
play a major role in mucociliary clearance, aself-clearing mechanism and first-line defence against particles and pathogens. To
prove that tight junctions have formed as aselective barrier on the porous membrane
the tight junction protein-1 (ZO-1) was marked
with the respective rabbit antibody.

4.2 IMMUNOFLUORESCENTSTAINING WITH ANTI-β-TUBULIN IV
1. Fix the cells on the apical side with cold 10 %formalin for 15 min at room temperature
2. Wash the wells 2 x with 100 µl PBS buffer
3. Permeabilise the cells with 50 µl 0.1 %Triton X-100 in PBS buffer for 5 min atroom temperature

4.3 IMMUNOFLUORESCENTSTAINING WITH ANTI-ZO-1
1. Fix the cells on the apical side withcold 10 % formalin for 15 min at roomtemperature
2. Wash the wells 2 x with 100 µl PBS
3. Permeabilise the cells with 50 µl 0.1 %Triton X-100 in PBS for 5 min at room
temperature
4. Wash the wells with 100 µl PBS
5. Use blocking buffer (PBS + 5% goat serum)
and incubate for 1 h
6. Wash the wells with 100 µl PBS
7. Incubate primary antibody (rabbit antiZO-1) in blocking buffer for 1 h at room
temperature or overnight at 4 °C
8. Wash the wells 3 x with 100 µl PBS
9. Incubate secondary antibody (Alexa 546
goat anti-rabbit) in blocking buffer for 1 hat room temperature in the dark
10. Wash the wells 3 x with 100 µl PBS buffer
11. Cut out membranes with a scalpel

4. Wash the wells with 100 µl PBS buffer

12. Place the membrane on a microscopy
slide and mount in properly

5. Use blocking buffer (PBS + 5 % goatserum) and incubate for 1 h

4.4 ALCIAN BLUE STAINING

6. Wash the wells with 100 µl PBS buffer
7. Incubate primary antibody (mouse antiß-Tubulin IV) in blocking buffer for 1 h atroom temperature or overnight at 4 °C
8. Wash the wells 3 x with 100 µl PBS buffer
9. Incubate secondary antibody (Alexa 488
goat anti-mouse and DAPI if needed
10 µg/ml) in blocking buffer for 1 h at room
temperature in the dark
10. Wash the wells 3 x with 100 µl PBS buffer
11. Cut out membranes with a scalpel
12. Place the membrane on a microscopeslide and mount in properly

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Application Note

Preparation of alcian blue solution: 10 ml HCl+ 90 ml H2O demin. + 1 g alcian blue (powder)
1. Fix the cells on the apical side withcold 10 % formalin for 30 min at room
temperature
2. Wash the wells 3 x with 100 µl PBS
3. Incubate 50 µl alcian blue solution for30 min at room temperature
4. Wash the wells 3 x with 0.1 N HCl
5. Apply distilled water and microscope (noneed to cut out the membrane here)5. RESULTS
In this application note the ThinCert® 96 well
HTS insert was used to generate an in-vitro
model of the respiratory epithelium. Therefore, NHBEs were cultivated and subjectedto ‘air-lift’.

5.1 IMMUNOFLUORESCENTSTAINING
To analyse the differentiation status of thepseudostratified epithelium, the ALI cultures
were fixed and stained with anti-ß-tubulin IVand anti-ZO-1 to detect ciliated cells and thecharacteristic tight junctions. A DAPI staining was used to visualize the nuclei and to assess gross cell morphology.
The immunocytochemistry shows an intactmonolayer of epithelial cells that have developed tight junctions (Fig. 4) as a primaryboundary between the apical and basolateralregion. As a major cilia protein, ß-tubulin IV isan excellent differentiation marker to prove
the presence of a differentiated cell composition (Fig. 2).

Figure 4: Anti-ZO-1 staining of tight junctions (red) on
the membrane of the ThinCert® HTS insert (63 x)

5.2 HISTOCHEMICAL STAINING
After 25 days post air-lift, the fixed cells
were stained with the alcian blue dye (Fig.
5) to mark the typical polysaccharides andglycoproteins found in the mucus of thepseudostratified epithelium. The staining
acts as a further indicator for the differentiation under air-liquid conditions.

Figure 5: Staining of mucus polysaccharides withalcian blue (10x)Figure 2: Anti-tubulin IV staining (green) of ciliatedcells on the membrane of the ThinCert® HTS insert(63 x)

Figure 3: DAPI staining (blue) of cell layer on themembrane of the ThinCert® HTS insert (20 x)

Figure 6: Unfixed NHBE cells after culturing in air-liftfor 25 days (10x)

The ThinCert® 96 well HTS insert enables invivo-like growth conditions in vitro and facilitate the reconstruction of epithelia fromindividual cells. The ALI cultures develop
tissue-specific features, such as tight junctions, cellular polarity and barrier function,and may be used to study tissue-specificphenomena.
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PRODUCTOVERVIEW

ThinCert® 96 Well HTS Insert (Membrane plates and receiver plates)
Growth area: 14 mm², Working volume (well of membrane plate): 15 - 160 µl,Working volume (well of receiver plate): 120 – 300 µl, Lid: yes, condensation rings
Pore densityØ Pores

Optical featuresof membrane

Surface treatment

SterileQty. inner / outer

6556401 x 108/cm2

0.4 µmtranslucent

TC+

1/5655641

2 x 10 7/cm2

0.4 µmoptimisedtransparency

TC+

1/5

Item no.

Receiver plates for ThinCert® 96 Well HTS Insert
Working volume (well of receiver plate): 120 – 300 µl, Lid: yes, condensation rings
Item no.

Growth area

655169-655167

53 mm

Surface treatment

SterileQty. inner / outer

non-treated+

8 / 32

TC+

8 / 32

2REFERENCES
1. Bluhmki, T., Bitzer, S., Gindele, J.A. et al.
Development of a miniaturized 96-Transwell
air–liquid interface human small airway epithelial
model. Sci Rep 10, 13022 (2020)
2. Schögler, A., Blank, F., Brügger, M. et al.
Characterization of pediatric cystic fibrosis
airway epithelial cell cultures at the air-liquidinterface obtained by non-invasive nasal cytology
brush sampling. Respir Res 18, 215 (2017)
3. Upadhyay S, Palmberg L. Air-Liquid Interface:
Relevant In Vitro Models for Investigating Air
Pollutant-Induced Pulmonary Toxicity. Toxicol Sci.
2018 Jul 1;164(1):21-30
4. Lukassen, Soeren, et al. "SARS‐CoV‐2 receptor
ACE 2 and TMPRSS 2 are primarily expressed inbronchial transient secretory cells." The EMBO
journal 39.10 (2020): e105114.

Greiner Bio-One GmbH
Maybachstraße 2 / 72636 Frickenhausen / Germany
PHONE +49 7022 948-0 / FAX +49 7022 948-514 / E-MAIL info@de.gbo.com

5. Djidrovski, Ivo, et al. "SARS-CoV-2 infects an upper
airway model derived from induced pluripotent
stem cells." Stem Cells 39.10 (2021): 1310-1321.
6. Le, Nguyen Phan Khoi, et al. "Comparative AntiInflammatory Effects of Salix Cortex Extracts
and Acetylsalicylic Acid in SARS-CoV-2 Peptide
and LPS-Activated Human In Vitro Systems."
International journal of molecular sciences 22.13(2021): 6766.
7. Schulze, Jessica, et al. "Analysis of SARS-CoV-2
replication in explant cultures of the human upperrespiratory tract reveals broad tissue tropism ofwild-type and B. 1.1. 7 variant viruses." The Journalof Infectious Diseases (2021).

8/8F073140 Application Note Air-Lift Culture ThinCert® 96 Well HTS Insert en [01.2023]