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www.bioscience.lonza.com© 2023 Lonza Walkersville, Inc.

B-ALITM Immune-Airway Co-Culture Model: Normal Human
Bronchial Epithelial Cells and Peripheral Blood MononuclearCells
Instructions for use

Safety Statements
These products are not for use in GMP
manufacturing, nor human or animal in vivo use,
including use as a diluent or as an excipient, or for
diagnostic use.
These products are for research use only.
WARNING: LONZA PRIMARY CELLS CONTAINHUMAN SOURCE MATERIAL; TREAT AS
POTENTIALLY INFECTIOUS. Each donor is tested
and found non-reactive by an FDA-approved method
for the presence of HIV-I, hepatitis B virus andhepatitis C virus. Where donor testing is notpossible, cell products are tested for the presence ofviral nucleic acid from HIV-1, hepatitis B virus, andhepatitis C virus. Testing cannot offer complete
assurance that HIV-1, hepatitis B virus, and hepatitis
C virus are absent. All human-sourced products
should be handled at the biological safety level 2 tominimize exposure to potentially infectious products,
as recommended in the CDC-NIH manual, Biosafetyin Microbiological and Biomedical Laboratories, 5thedition. If you require further information, pleasecontact your site safety officer or Scientific Support.
Preparation of reagents
All work should be performed in a laminar flow hood.
Decontaminate the external surfaces of all
supplement vials and the medium bottles with ≥70%
ethanol or isopropanol.
1. B-ALITM Growth Medium and B-ALITM
Differentiation MediumPrepare Growth Medium and Differentiation
Medium according to instructions in the B-ALITM
Bronchial Air-Liquid Interface Medium
Instructions for Use guide found on the Lonza

website. NOTE: the Retinoic Acid and Inducer
supplements are light sensitive. Do not add
inducer to the Differentiation Medium until you
are ready to use the medium (must be thawedfrom frozen single aliquots and prepared freshfor each media change).
2. 50/50 Co-Culture Medium
a. Determine the total volume of 50/50 Coculture Medium needed (e.g., 500 µL per
well in a 24-well Corning® Transwell® plate;Corning® P/N 3470). You will need tochange medium at least once duringcoculture.
i. This protocol was tested to 4 days of coculture. We recommend changing media
every 2 days.
b. Prepare all media on day of co-culture
establishment and refrigerate surplus media
for up to 2 weeks at 4°C in aliquots for lateruse.
c. Create the required amount of 50/50 basal
medium by combining X-VIVO® 15 Serumfree Hematopoietic Cell Medium andB-ALITM Differentiation Basal Medium(medium without SingleQuots® added) ina 1:1 ratio.
d. Add B-ALITM SingleQuots® supplements
except for the Inducer to 500 mL of the50/50 basal medium following theinstructions in the table below:

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SingleQuots®
Supplements

Volume to add
to 500 mL 50/50
basal medium

BPE

2.0 mL

Insulin

0.5 mL

Hydrocortisone

0.5 mL

GA-1000

0.5 mL

Retinoic Acid

0.5 mL

Transferrin

0.5 mL

Triiodothyronine

0.5 mL

Epinephrine

0.5 mLhEGF

0.5 mLi. Adjust volumes of supplements
according to the total volume of 50/50
basal medium used.
e. Resuspend 10 µg IL-2 IS in 1000 µL PBS fora final concentration of 500X IL-2.
i. Final concentration in 50/50 Co-Culture
Medium should be 50 – 100 IU/mL;
adjust stock concentration as needed.
f. Add an appropriate volume of 500X IL-2 tothe 50/50 medium to make a final
concentration of 1X IL-2.
g. Aliquot medium to be used for future media
changes and store at 4°C for up to 2 weeks.
h. To the remaining medium, add 20 µL
B-ALITM Inducer supplement (found in theB-ALITM SingleQuots® Kit) per 10 mL 50/50
Co-Culture Medium.
i. For each media change, use a single aliquotof 50/50 Co-Culture Medium and add theinducer as above immediately prior to use.
Normal Human Bronchial Epithelial (NHBE) Cell
Culture and Airlift
NOTE: All work is to be performed in a laminar flowhood.
1. Add 25 mL of B-ALITM Growth Medium to a T-75
flask and allow the vessel to equilibrate in a37°C, 5% CO2 humidified incubator for at least
30 minutes.
2. Wipe cryovial of NHBE cells with ethanol orisopropanol before opening. In a sterile hood,briefly twist cap to relieve pressure and then retighten. Thaw the cryovial in a 37°C water bath.
DO NOT SUBMERGE. Remove when a sliver ofice remains (takes less than 2 minutes).
3. Resuspend the cells in the cryovial.
4. Pipette all cell suspension into the T-75 flask
from step 1. Gently rock to distribute cells andreturn to incubator.

a. DO NOT CENTRIFUGE CRYOVIAL. Thisdamages the cells more than DMSO residuein culture.
5. Change media on Day 1, using 25 mL prewarmed B-ALITM Growth Medium.
6. Prepare collagen-coated plates on the day ofNHBE harvest (when confluence is between
50 – 90%; 2 – 4 days after seeding).
a. Dilute stock collagen with PBS to make a
30 µg/mL collagen solution. Make enoughsolution to add 100 µL per Transwell®.
b. Coat plate inserts with 100 µL of collagensolution per well. Place plate in the incubatorfor 45 minutes.
c. Aspirate the collagen solution from eachwell.
d. Gently wash each well with 150 µL PBS andaspirate off liquid.
e. Leave the dry plate in the hood until cells
are added. Do not let the plate sit overnight.
7. Harvest NHBE cells according to theinstructions below:
a. Warm 5mL trypsin/EDTA to 37°C in a waterbath.
b. Allow 10 mL HEPES buffered saline(HEPES-BSS) to come to roomtemperature.
c. Allow 5 mL of trypsin neutralizing solution(TNS) to come to room temperature.
d. Warm up 15 mL of B-ALITM Growth medium
to 37°C in the water bath.
i. Do not warm up the entire bottle, justwarm 15 mL for each 24-well plate.
e. Aspirate medium from the T-75 flask with
NHBE cells.
f. Rinse cells with 10 mL room-temperature
HEPES BSS.
g. Aspirate the HEPES-BSS from the flask.
h. Cover cells with 5 mL trypsin/EDTA.
i. When 90% of cells have rounded up (takes
2 – 6 minutes), gently tap the flask against
the palm of your hand to release the majorityof cells.
i. If cells do not detach, wait another 30
seconds and tap again. Repeat until cells
detach.
j. Add 5 mL trypsin neutralizing solution.
k. Quickly transfer detached cells to a 15 mL
centrifuge tube.
i. If, after 6 minutes, the majority of cells
haven’t detached, harvest detached cells
and then repeat steps h-k.
l. Rinse flask with a final 2 mL of HEPES-BSS
to collect residual cells and add rinse to the
15 mL centrifuge tube.

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m. Centrifuge harvested cells at 200xg for
5 minutes.
n. Aspirate supernatant except for
100 – 200 µL.
8. Resuspend pellet in 1 mL B-ALITM Growth
Medium and count a 20 µL aliquot with Trypan
Blue 0.4% and a hemocytometer. We
recommend using a 1:2 dilution of Trypan Blue(e.g., 20 µL sample + 20 µL Trypan Blue).
9. Dilute cells to an appropriate concentration
using B-ALITM Growth Medium (e.g., 500,000cells/mL) and then seed on the collagen-coatedCorning® 24-well Transwell® membrane plate ata density of 50,000 cells/well.
10. Add 500 µL B-ALITM Growth Medium to thebasal chamber and culture in the incubator for3 – 4 days.
a. Change media after 1 day and again on day
3 if cells are not showing the morphology
displayed in Figure 1.
b. Check morphology and confluence daily.
Proceed to airlift step on day 3 or day 4,
whenever the cell layer displays the propermorphology (Figure 1).
c. Airlift step: On day 7 – 8 (3 – 4 of Transwell®
culture), remove media from both sides ofthe membrane. Add 500 µL B-ALITM
Differentiation Medium with freshly
thawed inducer to the basal media
chamber underneath the membrane.

Figure 1: NHBE morphology before airlift,
day 3-4.
11. Culture for 14 more days at 37°C, 5% CO2,performing media changes (basal mediachamber only) every 2 – 3 days.
12.
NHBE: PBMC Co-Culture Establishment
1. On day 14 after airlifting the NHBE cells, thaw1 x 108 Lonza Peripheral Blood Mononuclear
Cells (PBMCs) at 37°C until a sliver of ice
remains.

a. Lonza offers ampules of 5 x 107 (CC-2702)
and 1 x 108 (CC-2703) PBMCs.
b. Thaw more or fewer cells depending on thetotal # needed – each plate well will receive
5 x 105 PBMCs.
2. Pipette 25.5 mL of pre-warmed X-VIVO® 15
Serum-free Hematopoietic Cell Medium into a
50 mL conical tube.
3. Pipette cells into 50 mL conical tube ofX-VIVO® 15 from step 2.
4. Wash cell vial(s) with 1 mL X-VIVO® 15 and add
wash to 50 mL conical tube.
5. Centrifuge cells at 300xg for 10 minutes at room
temperature.
6. Aspirate supernatant without disturbing cells andresuspend pellet in 2-5 mL 50/50 Co-Culture
Medium (remember to add inducer – seereagent preparation steps).
7. Count an aliquot of cells using Trypan Blue
(0.4%) and a hemocytometer (1:2 dilution; e.g.,
20 µL sample + 20 µL Trypan Blue).
8. Dilute cells to a final concentration of 1 x 106
cells/mL.
9. Remove media from the basal chambers of theTranswell® plate.
10. Add 500 µL of PBMC cell suspension (5 x105 PBMCs) to the basal chamber of each wellwith desired treatment conditions added to themedia.
a. For a positive T cell activation control, add
25 µL of ImmunoCultTM anti-CD3/CD28
antibody (STEMCELLTM Technologies) per
mL of media to the 50/50 Co-Culture media
before resuspending cells and adding
500 µL per positive control well.
11. Incubate co-culture at 37°C, 5% CO2 for 4 days
(counting day of co-culture establishment).
12. Change medium 2 days after plating:a. Remove medium and suspended PBMCs
from each well and place in a separate,labeled microcentrifuge tube.
b. Refill basal chamber of each well with
500 µL PBS.
c. Spin each tube at 300xg for 5 minutes.
d. Remove supernatant from eachmicrocentrifuge tube without disturbing cells.
e. Resuspend cells in 500 µL prewarmed
50/50 Co-Culture Medium with inducer,
treatment conditions, and ImmunoCultTM
anti-CD3/CD28 antibody stimulator added
where appropriate.
f. Remove PBS from the basal chamber of
each well and replace PBMCs back inappropriate wells.

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13. A variety of analyses can be performed on thisco-culture to examine impacts of experimental
treatments:a. NHBE membrane health can be assessedby:i. TransEpithelial Electrical Resistance(TEER) – a measure of membrane
integrity.
ii. Mucin staining (mucin production; ameasure of NHBE cellular differentiation).
iii. β-tubulin staining (a measure ofmembrane integrity and cilia formation).
iv. Membrane sectioning and hematoxylin +eosin (H&E) staining (to measuremembrane thickness and assess overall
integrity visually).
b. PBMC population viability and functionality
can be assessed via immunophenotyping(flow cytometry). For example, a panel ofantibodies including CD3, CD4, CD8, CD25,
CD45-RA and CD45-RO can be used toanalyze the T cell population for T cell
activation and assess sub-population sizesof differentiated T cells (helper, cytotoxic,etc.)
14. Applications for this model include effects ofinflammation, respiratory diseases, drugs, andenvironmental contaminants on airway healthand functioning.
a. NOTE: In this model, PBMCs do not comeinto full contact with NHBE cells because ofthe Transwell® membrane. Therefore, thismodel is more suited to study necrotic andinflammatory effects of coculture thaneffects induced by T-cell receptor binding.

Ordering Information
Catalog No.
Description

00193514

B-ALITM Bronchial
Air-Liquid Interface
Medium BulletKit®

Contains Growth
Basal Medium,Differentiation
Basal Medium,and SingleQuots®
supplement kit

04-418Q,

X-VIVO® 15
Serum-freeHematopoietic CellMedium

1 L, complete with
L-Glutamine,gentamicin, andphenol red;
xenofreeCC-5024

HEPES Buffered
Saline Solution(HEPES-BSS)

500 mL bottle

CC-5012

Trypsin/EDTA

100 mL bottle

CC-5002

Trypsin
Neutralizing
Solution (TNS)

100 mL bottle

02-060Q (EU andROW)

THESE PRODUCTS ARE FOR RESEARCH USE
ONLY.
Not approved for human or veterinary use, for application tohumans or animals, or for use in clinical or in vitro procedures.
Not for use in diagnostic procedures.
PBS without Calcium or Magnesium (ThermoFisher Scientific10010023) mentioned is a product of GIBCOTM.
IL-2 IS (Miltenyi 130-097-743) mentioned is a product of Miltenyi
Biotec.
Collagen (rat tail collagen type I; Corning® 354236) mentioned isa product of Corning®.
Trypan Blue 0.4% (ThermoFisher 15250061) mentioned is a
product of GIBCOTM.
24-well Transwell® plates (Corning® 3470) mentioned are aproduct of Corning®.
ImmunoCultTM CD3/CD28 antibody (STEMCELLTM Technologies
10991) is a product from STEMCELLTM Technologies.

Size

CC-2540S

Cryopreserved
Human Normal
Bronchial
Epithelial (NHBE)
Cells for B-ALITM
culture≥ 500,000 cells

CC-2702,
CC-2703,
CC-2705

Human Peripheral
Blood
Mononuclear
Cells (HPBMC)≥ 50 million cells,≥100 million cells,≥25 million cells

00193516

B-ALITM Growth
Basal Medium

250 mL

00193517

B-ALITM
Differentiation
Basal Medium

500 mL

00193515

B-ALITM
SingleQuots®
Supplement Kit

1 kit

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www.lonza.com/legal.©2023 Lonza. CD-OP016 07/23

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