Enabling a Healthier World

Modeling Lung
Inflammation in 3D
A Physiologically Relevant Modelfor Studying Interactions Between
Immune Cells and the Lung
Epithelium Using Lonza's Bronchial
Air-Liquid Interface (B-ALI™) System

Ethan Shelkey1, Scott Kelsey1, Kevin Grady2,
Aurita Menezes1, and Anshika Sharma1
1

Lonza – Durham, NC, USA; 2Lonza – Walkersville, MD, USA

In recent years, the compounding effects of widespread
upper-respiratory viruses, such as COVID-19, influenza and
RSV, have highlighted the dire need for physiologically
relevant models that accurately represent interactions
between multiple cellular systems, such as immune cells
and the lung epithelium. Such models are an essential
component of effective therapeutic testing and mechanistic analysis.
Here we discuss how we developed and optimized a
co-culture model where immune cells (peripheral bloodmononuclear cells; PBMCs) were co-cultured alongsiderespiratory pathway cells (normal human bronchial epithelial cells; NHBEs) using Lonza’s B-ALI™ (Bronchial Air-LiquidInterface) system. We demonstrate that our optimized
model supports physiologically functional epithelial membranes on the air interface, as NHBE cells polarized anddifferentiated to form cilia and tight junctions. In addition,
the immune cell population in the liquid compartment was
shown to behave as expected, with the T cell population
exhibiting an activation response upon stimulation.
We then validated this co-culture as a physiologically
relevant model for virally induced inflammation by stimulating the T cell population with viral antigens. The resulting
co-culture demonstrated inflammatory responses by the
T cell population through increased CD69 expression as
well as an increase in the inflammatory factors granzyme
B, TNF-α, and IL-6. Furthermore, we observed that NHBE
membranes visibly thinned when co-cultured alongside
PBMCs stimulated with viral antigens, which suggests thatthe inflammatory response by the PBMC fraction may havetriggered necrosis in the epithelial lining above.
Taken as a whole, this model provides a straightforward,
physiologically relevant system for studying interactions
between the immune system and the lung epithelium.

Introduction
The recent national and global outbreaks of upper respiratory viruses, such as COVID-19, influenza, and RSV, alongwith the FDA’s shifting emphasis towards xeno-free models
for drug discovery research, have drastically increased
the demand for culture systems that can better mimic thecomplex physiology of respiratory system damage than
traditional 2D culture total submersion models.1
3D Air-Liquid Interface (ALI) culture using primary cells
allows for the differentiation and unique polarization of human airway epithelial cells into the physiologically relevant
cell types (e.g., mucin-producing goblet cells or ciliatedcells) that allow for the proper functioning of the respiratory system in vivo.1,2 This system provides the unique benefit
of approximating the actual environmental conditions
in which human airway cells exist in vivo, with the liquidcompartment beneath the cells representing the bloodstream. This compartment contains a medium that induces
2 | Modeling Lung Inflammation in 3D

differentiation and polarization of the seeded epithelial
cells, causing them to mature into mucin- and cilia-producing cells and form tight junctions between cells like thosefound in the lining of the airway epithelium.1,2
Importantly, the use of primary human airway cells in an
ALI culture system has a distinct advantage over immortalized cells, cancer cell lines, and even animal models inthat they maintain the closest gene expression and diseasesusceptibility to in vivo conditions in the human body.1-3 For
example, both cancer cells and immortalized cells may lack
the receptor expression found in their primary cell counterparts, which can change their susceptibility to diseases
such as influenza.3
In addition, ALI culture allows for the co-culture of differentiated airway cells and immune cells, providing a systemcapable of modeling how respiratory capacity could be
harmed by immune-mediated inflammatory effects during
an infection or toxicological exposure.1 For these reasons,
ALI culture models using primary human airway cells serveas an excellent, physiologically relevant tool for exploring
both disease pathology as well as drug discovery andtoxicology.
One of the key challenges in developing a 3D ALI airway-immune cell co-culture model is balancing the media in sucha way that airway cells can form a healthy, differentiatedmembrane with cilia and high integrity (i.e. with tight junctions) while also supporting the expansion, differentiation,
and activation of the immune fraction residing in the basal
chamber of the Transwell® plate.
Here, we present an easy-to-use airway-immune cell
co-culture model using NHBE cells and PBMCs based in
Lonza’s B-ALI™ Media System. We show that the membranes developed using this protocol are healthy, robust,and differentiated, while the immune cell population
displays appropriate proliferative and functional responses
over the course of a 72-hour co-culture. Finally, we tested
the inflammatory response of our co-culture by stimulating
the immune (PBMC) fraction using viral antigens. PBMCs
displayed a strong inflammatory reaction to the addition ofviral antigens, while the NHBE membranes above thinned
markedly compared to control conditions, suggesting thatthe underlying inflammatory immune response induced
necrotic effects in the NHBE layer above. Overall, our
NHBE-PBMC B-ALI™ Co-culture System is a highly-relevant
model of lung physiology that can be utilized in a variety of
applications from disease research to drug discovery.

Methods
3D B-ALI™ NHBE-PBMC Co-culture
A detailed protocol for establishing an optimized 3D B-ALI™
NHBE-PBMC co-culture can be found on the Lonza website.4 In brief, Lonza NHBE cells (Lonza part no. CC-2540S;

n = 3 donors) were thawed in a 37°C water bath until only asliver of ice remained, gently resuspended, and then pipetted into a T-75 flask with 25 mL of B-ALI™ Growth Mediumthat had been allowed to equilibrate to 37°C. This plate wasincubated at 37°C for 2 – 4 days and cells harvested at 50 –90% confluence, with a medium change on day 1.
On the day of NHBE harvest, Corning® Transwell® plate inserts were coated with 30 µg/mL collagen solution, washedwith PBS, and then left to dry prior to cell plating.4 NHBE
cells were then trypsinized, harvested, counted, and thendiluted using B-ALI™ Growth Medium in order to seed themat a density of 50,000 cells/well. Cells were then incubatedfor 3 – 4 days in 100 µL of B-ALI™ Growth Medium in the top
chamber and 500 µL of B-ALI™ Growth Medium in the basal
chamber. The medium was changed on day 1 and again on
day 3 if proper morphology was not yet obtained. Oncethe cell layers displayed the proper morphology on day 3
or 4 (filling in the surface of the well, Figure 1), cells were
exposed to air by removing media from the top chamberof the Transwell® above the membrane. The B-ALI™ Growth
Medium in the basal chamber was replaced with 500 µL of
B-ALI™ Differentiation Medium and then cells were cultured
for another 14 days at 37°C, with medium changes every
2 – 3 days.

Figure 1.
NHBE morphology before airlift, day 3 – 4 of culture. Imaged at 10x
magnification.

We established co-cultures with PBMC’s (Lonza part no.
CC-2703; n = 3 donors; allogenic with NHBEs; Donors 1 – 3
hereafter) 14 days after airlifting the NHBE cells. The
PBMCs were thawed, counted, and resuspended into oneof three media treatments (B-ALI™ Differentiation Medium,
X-VIVO® 15, or a 50/50 mix). We seeded PBMCs into thebasal chamber for all co-culture wells or an empty 24-wellplate for a PBMC-only control at a density of 5x105 cells/
well (1x106 cells/mL). NHBE-only controls were also maintained alongside all treatments with no added PBMCs.
The B-ALI™ Inducer was added to all media conditions to
support NHBE differentiation, and all B-ALI™ SingleQuots®
Supplements were added at full strength to all media conditions. Viable T cell activation was established by adding
IL-2 and anti-CD3/CD28 antibody to the PBMCs and medium after counting but before seeding for all treatments.

We incubated the co-cultures for 72 hours at 37°C, changing medium at 2 days (accomplished by removing the
PBMCs and medium from each well, centrifuging andremoving the medium, then resuspending the PBMCs infresh medium and returning to the appropriate well). NHBE
membrane measurements included trans-epithelial electrical resistance (TEER; a measure of tight junction formation
or membrane integrity), mucin production (via Alcian blue
staining), and membrane thickness (via sectioning andhematoxylin and eosin (H&E) staining). We assessed PBMC
population viability and functionality via immunophenotyping using flow cytometry.

Co-culture Inflammatory Response
to Viral Antigen Stimulation
We used a different set of n = 4 unique NHBE and n = 4
unique PBMC donors (allogenic pairs; Donors 4 – 7 hereafter) to examine model responses to viral inflammation.
PBMCs were pre-screened for responsiveness to thecytomegalovirus, Epstein-Barr, and influenza (CEF) peptide
pool. We then we repeated the above protocol using thefollowing modifications. First, we used only the 50/50 medium, as we found this to have the best balance between
an optimal membrane and PBMC population.4 Second,
when the PBMC co-culture was established, we added
CEF MHC Class 1 Peptide to the cell culture medium ineach inflammation treatment well (“Virus”), bringing thefinal concentration of each peptide up to 0.5 μg/mL.
Finally, in order to establish both positive and negative
controls for T cell stimulation, we cultured PBMCs without
NHBEs, and then exposed both PBMCs and NHBE-PBMCs
to the following treatments: no stimulants (IL-2 alone; negative control), IL-2 + Virus, and IL-2 + Virus + anti-CD3/CD28
antibody (positive control; “CD3/28”).
The same endpoints were measured in these trials as thefirst set of experiments; however, results from Donor 4 led
us to also include additional endpoints to further clarify
the inflammatory response of viral-activated PBMCs. For
Donors 5 – 7, we also assessed inflammatory factors (granzyme B, TNF-α, and IL-6) released by the T cell population
via ELISA to determine whether or not the T cell population
exhibited an inflammatory response to the viral peptides.

Results
Optimizing the 3D B-ALI™
Co-culture Model
We undertook three rounds of optimization and found thatusing a 50/50 mix of B-ALI™ Differentiation Basal Medium
with X-VIVO® 15 Serum-free Hematopoietic Cell Medium(with all B-ALI™ Supplements and Inducer added at fullstrength to the mix) and performing a medium change the
day after co-culture initiation produced the best balance
between a robust membrane and a healthy, functional
Modeling Lung Inflammation in 3D | 3

Figure 2.
Representative images of H&E stained NHBE membrane sections fromDonor 1 (A), Donor 2 (B), and Donor 3 (C) for the 50/50 medium treatment.
This treatment formed multiple layers of cells, with visible ciliation (seepanel B in the top left of the membrane). Disconnected membrane fragments (e.g., panel C) are the results of fragmentation during sectioning in
preparation for H&E staining. Membranes were harvested after 72 hours of
co-culture. All images taken at 40x magnification.

4 | Modeling Lung Inflammation in 3D

4000
3000
2000

550 Ω*cm2

Donor 2

Donor 1

Day 0
Day 1
Day 2
Day 3

Day 0
Day 1
Day 2
Day 3

Day 0
Day 1
Day 2
Day 3

0

Donor 3Figure 3.
TEER values of NHBE membranes for all three donors grown in co-culture
with PBMCs using the 50/50 media treatment. Membranes were harvested
after 72 hours of co-culture (day 0 – day 3). The black bar indicates theminimum passing threshold TEER level for healthy membrane integrity(550 Ω*cm2).
A

B

Donor 1 CD8+ CD69+ Fraction

Donor 2 CD8+ CD69+ Fraction

+
+
Donor
Fraction
Day 0 3 CD8DayCD69
1Day
1 Control

Day 3

2.0

1. 0
0.5

Day 0

Day 1

Day 1 Control

X-VIVO® 15

PBMC Control

50/50

50/50 w/ Change

B-ALI™ Differentiation

X-VIVO® 15

PBMC Control

0.050/50 w/ Change

PBMC Control

50/50

X-VIVO® 15

PBMC Control

B-ALI™ Differentiation

50/50

X-VIVO® 15

Day 0

C

B-ALI™ Differentiation

0

Day 0
Day 1
Day 1 Con
Day 3

50/50

1

Day 0

2

Day2.50
Day 1
2.0
Day 1 Control
Day 3
1. 5

B-ALI™ Differentiation

Fold Change (Da y 1 P BMC CRL )

3

Day 3

Day 0
Day 1
Day 1 Control
Day 3

1. 5
1. 0
0.5

PBMC Control

X-Vivo 15

PBMCs w/o Supp®

50/50 w/ Change

B-ALI™ Differentiatation

PBMC Control

PBMCs w/o Supp

X-Vivo® 15

0.050/50 w/ Change

C

5000

B-ALI™ Differentiatation

B

6000

1000

Fold Change (Day 1 PBMC CRL)

A

7000

Day 0

T cell populations exhibited a healthy activation response
across the 72-hour co-culture (Figure 4), with the highest
levels of T cell Activation (population of CD8+CD69+ PBMCs)
in the X-VIVO® Medium treatment, followed by the 50/50
medium treatment for all three donors. There was nodifference in either lymphocyte viability (ranging between
45 – 83% viability for 50/50 medium) or cytotoxic T cell
population fractions (ranging between 16 – 46% CD8+ expression) among treatments, indicating that all media werecapable of supporting a healthy population of lymphocytes
and that any differences between experiments were aresult of donor variation (data not shown).

Resistance (Ω *cm2)

The 50/50 medium produced the thickest membranes
with the most visible cilia (Figure 2) for all three donors
compared to either B-ALI™ Differentiation Medium or
X-VIVO® 15 Medium alone (data not shown). While, in somecases, membranes formed with X-VIVO® 15 alone hadhigher overall transepithelial/transendothelial electricalresistance (TEER) values (data not shown), the TEER values
for all three donors cultured with the 50/50 medium were
well above the passing threshold value of 550 Ω*cm2 for aphysiologically healthy membrane (Figure 3). TEER values
were also improved by performing a medium change the
day after co-culture initiation (tested with Donor 2; utilized
in Donor 3 as the only 50/50 media treatment). No difference in mucin production was seen among treatments(data not shown).

Fold Change (Da y 1 P BMC CRL )immune cell population over the course of a 72-hour
co-culture.

Day 4.
0
Day 1
Day 1 Control
Day 3
FigureT cell population activation for each donor (graphs A – C) from day 0 (initiation of NHBE-PBMC co-culture) to day 3 (72 hours of co-culture) for eachmedium. Data consists of fold change for each population relative to the
PBMC monoculture control population size on culture day 1 (Day 1 PBMC
Control). T cell population was determined by flow cytometric detection of
the PBMC population subset expressing CD8+CD69+, indicating a cytotoxic
T cell population that has been stimulated by the anti-CD3/CD28 antibody
addition. Note that, for Donor 2, we added an additional media change
for 50/50 medium after one day (panel B) for comparison. As it improved
endpoints, we implemented this media change as standard procedure for
Donor 3 (panel C).

8

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Donor 6

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Expression (%)
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Donor 760

Expression (%)

Expression (%)

60

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PB
40

100 + CTL
CD69

CD69+ C

+
CD25
CTL
80

CD25+ C

60
40

20

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Similarly, the production of inflammatory factors byimmune cells was higher in co-culture when compared
to PBMCs in monoculture, and was typically higher whenviral peptides were added versus the negative control(NHBE-PBMC co-culture + IL-2 only; Figure 6). The releaseof inflammatory cytokines and lytic enzymes generally reflected the size of the initial T cell population of the PBMCs,
with the highest concentrations (and most significant differences between viral-activated co-culture and controls)
released by Donor 5 (highest initial T cell population, Table
1) and decreasing with Donor 6 and 7 (Figure 6).

CD25+ C

40

PB

CD25+ expression, a later-stage indicator of activation in
CD8+ cytotoxic T cells, followed a similar trend, though theeffect was not consistent across all four donors (Figure 5).
Positive controls (CD3/28 antibody) for T cell stimulation
and activation were consistently much higher for both activation markers when PBMCs were co-cultured with NHBEs
when compared to PBMCs in monoculture (Figure 5).

CD69+ C

+
CD25
CTL
80

20

PB

When stimulating the PBMC population using viral peptides(CEF peptides; primarily impact the cytotoxic MHC-I restricted T cell population), CD69+ expression, an early-stage
indicator of CD8+ cytotoxic T cell activation, increased
when PBMCs were co-cultured with NHBEs versus unstimulated controls (NHBE-PBMC co-culture + IL-2 only; Figure 5).
This same pattern of increased activation in the co-culture
was seen in comparison to PBMCs cultured in monoculture
and exposed to viral particles for all four donors used inthis study (Figure 5).

100 + CTL
CD69

40

2

Table 1.
Initial percent of PBMC population expressing CD3+ (T cells) for each of
the four donors co-cultured with NHBEs in this trial. Note the wide rangeof variation, with Donor 7 being especially depleted in comparison to theother three donors.

60

C

52.1
27.3

80

Vi

Donor 6
Donor 7

100

Vi

79.7

Donor 5+

Donor 5

B

Donor 42

Day 0 % CD3 Expression (T cells)
72.6

A

IL

PBMC Donor
Donor 4++We used four different allogenic donor pairs of NHBEs
and PBMCs in the viral inflammation experiments. PMBC
donors were selected by prescreening for response to the
CEF peptide pool to ensure effects of inflammation were
present to be observed. Initial T cell population size varied
between these 4 donors (Table 1).

four days for co-culture treatments that were exposed to
the viral peptides (Figure 7, column 3 vs. 1) or viral peptides
and the anti-CD3/CD28 positive control (Figure 7, column
4 vs. 1). This trend was less noticeable in Donor 7, which
overall produced a thin, poor-quality membrane (Figure
7, row D). No significant differences were seen between
treatments for TEER or mucin production (data not shown),
though it should be noted that at no point did the TEER values for Donor 7 exceed the 550 Ω*cm2 threshold indicatinga healthy, intact membrane.

Expression (%)

The Effect of Viral-Induced
Inflammation on 3D B-ALI™
NHBE-PBMC Co-culture

CD25+ CTL

Figure 5.
Percent expression for CD69+ and CD25+ among the CD8+ populationof PBMCs (cytotoxic T lymphocytes; CTL) after 72 hours of monoculture(PBMC) or co-culture (Co-culture) for all four donors (graphs A-D, respectively) and for all treatments (IL-2, IL-2 + viral antigens (“Virus”), and IL-2 +Virus + CD3/28 antibody positive control). CD69+ is a marker of early CD8+
T cell activation, while CD25+ is a late-stage indicator of activation. Higher
expression of either marker indicates an inflammatory immune response.

When comparing membrane quality across all four
treatments (Co-culture + IL-2, Co-culture + IL-2 + Virus,
Co-culture + IL-2 + Virus + CD3/28, and NHBE monoculture)
between the first and last day of co-culture, it was noticeable that membranes had thinned over the course of theModeling Lung Inflammation in 3D | 5

Concentration (pg/mL)

60000
45000
30000
400
300
200
100

10000
450
NS

100
NS

50

2000
250

NS

200
150
NS

50
0

50
25

15
10
5

100
20

5

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB cul
o
C

300
200
100
40
30
NS

20
10

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB cul
o
C

NS

100
80
60
40
30
20
10

NS
NS
NS NS NS0
2 s 2 s0 BE IL2 us IL2 rus 28 28yy 0 BE IL iru IL iru /28 /28i/ /ir
Da NH C + + V re + + V D3 D3
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
M 2 tu L2 + C + C
PB + IL cul + I us us
PB + IL cul + I us us
r
r
e
i
i
C
C o- ure Vir Vir
M Co ltur + V + V
BM C ult 2 + 2 +
u
PB
P
2
2
-c IL IL-c IL ILCo C + re +Co C + re +
u
M
M tu
t
PB culPB culooCC
Figure 6.
is directly reflective of the size of the initial CD3+ T cell population. Donor
Intracellular inflammatory cytokine and lytic enzyme concentrations for5 had the highest T cell population of the donors tested and displays theDonors 5 – 7 (rows A – C, respectively) for each treatment. Day 0 values
highest level of inflammatory factors while the reverse is true with Donor 7.
were measured upon initiation of co-cultures. All other values were measNote that cytokine release was not measured for Donor 4. The maximum
ured after 72 hours. Statistical differences indicated were determined byintracellular concentration found in healthy individuals is shown by the
T tests comparing the viral stimulated co-culture (Co-culture + IL2 + Virus;
horizontal black line in each panel.5–7 NS = not significantly different fromlight-colored) against all other treatments except the two positive controls
viral-stimulated co-culture, * p<0.05, ** p<0.01, *** p<0.001.
(treatments with CD3/28 antibody addition). The overall response profile
2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
- e
ir ir
C
M Co ltur + V + V
uPB2-c IL IL2
Co C + re +M tu
PB culoC

6 | Modeling Lung Inflammation in 3D

020

IL-6 Donor 7

150

10

40

0

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB culoCNS60

IL-6 Donor 6

300
250
200
150
20

15

800
600
400

0

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB culoC

TNF-α Donor 7

4000

100

NS

0

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB culoC

Grz B Donor 7
Concentration (pg/mL)

C

75

TNF-α Donor 6

20000

0

1000

Concentration (pg/mL)

Concentration (pg/mL)

Grz B Donor 6

Concentration (pg/mL)

B

1500

0

2 s 2 sy 0 BE IL iru IL iru /28 /28
Da NH C + + V re + + V D3 D3
M 2 tu L2 + C + C
PB + IL cul + I us us
C o- ure Vir Vir
M C lt+ +u
PB-c IL2 IL2
Co C + re +M tu
PB culoCConcentration (pg/mL)

0IL-6 Donor 5
Concentration (pg/mL)

TNF-α Donor 5

Concentration (pg/mL)

Grz B Donor 5
Concentration (pg/mL)

A

1

2

3

4

5

A

B

C

D

Figure 7.
H&E stained membrane sections for Donors 4 – 7 (rows A – D, respectively)
collected on day 0 (initiation) of co-culture (column 1), and day 3 (fourth
day of co-culture) for the following treatments: column 2: Co-culture + IL-2;
column 3: Co-culture + IL-2 + viral antigens; column 4: Co-culture + IL-2 +Discussion
Our results demonstrate the successful creation of a
co-culture model using Lonza’s B-ALI™ System with primary
NHBEs and PBMCs. The model mimics the inflammatory
responses seen in the respiratory system when triggeredby viral antigens and driven by cytotoxic T lymphocytes.
In our first set of experiments, we found that, overall, a50/50 mix of B-ALI™ Medium and X-VIVO® 15 medium (specifically formulated to support immune cells) best supported the co-culture model. To formulate the 50/50 media
mix, we chose to incorporate the B-ALI™ SingleQuots® Supplements at full strength (adding to the combined 50/50
medium rather than adding supplements to the B-ALI™
Differentiation Medium base and then splitting) in order tobest support the quality of the NHBE fraction.
These supplements contain components, such as epinephrine and hydrocortisone, which could suppress inflammatory responses. Indeed, we found some evidence that cytotoxic T cell activation could have been partially influenced

anti-CD3/CD28 antibody (positive control); and column 5: NHBE monoculture. Disconnected membrane fragments (e.g., panel A5) are the results offragmentation during sectioning in preparation for H&E staining. All images
taken at 40x magnification.

by the B-ALI™ Supplements, as the PBMC monoculture
control had lower activation when compared to the PBMC
monoculture without the B-ALI™ Supplements (Figure 4C;
only tested in Donor 3).
However, in all cases, the interactive effects of co-culturing
PBMCs with NHBE cells was demonstrated by the high degree of cytotoxic T cell activation when the PBMC fraction
was stimulated with anti-CD3/CD28 antibody in the presence of the NHBE membrane (Figure 4), near 100% in somecases. This indicated that chemical crosstalk between
the NHBE and PBMC fractions primed immune cells for
activation and negated any dampening effect by the B-ALI™
Supplements.
Whether donor mismatch between NHBE cells and PBMCs
played a part in activating cytotoxic T cells is not clear, but
due to the nature of the Transwell® Membrane plate, theimmune cells are prevented from coming into contact with
the NHBE cells, thus making an allogenic response lesslikely.8,9Modeling Lung Inflammation in 3D | 7

Overall, when all endpoints were weighed against eachother, the thickness and degree of differentiation of themembrane produced with the 50/50 medium (Figure 2)
indicated that the 50/50 medium was the best medium touse for this co-culture, as it also promoted high membrane
integrity (TEER) (Figure 3) as well as a healthy, functionally-responsive T cell population (Figure 4).
Our second set of experiments validated the performanceof the co-culture as a model for virus-induced inflammatory effects on the lung epithelium. Here we used a different
set of donors that had been pre-screened for responsiveness to the CEF peptide pool. Once again, we observed ageneral “priming” effect of NHBEs on PBMCs in co-culture,as the expression of both early (CD69) and late (CD25)
CD8+ cytotoxic T cell markers (Figure 5) as well as inflammatory factor production (Figure 6) tended to be higher in
co-culture than when PBMCs were in monoculture.
Regardless, stimulation of co-cultures with viral antigens
generally increased T cell activation markers (Figure 5)
and the release of inflammatory factors (Figure 6) whencompared to co-cultures with IL-2 alone, though there wasvariation across donors for these effects that correlatedwith the initial T cell population sizes (Table 1). For example,
Donor 5 had the highest T cell population of the donors
tested and displayed the highest level of inflammatory factors while the reverse is true with Donor 7 (Table 1, Figure6). This evidence suggests that an inflammatory response
is occurring within the PBMC population as a result of viralstimulation, which is further supported by the fact that
NHBE membranes co-cultured with PBMCs induced by CEF
peptides thinned noticeably over the entire course of theco-culture (Figure 7 column 3) when compared to NHBE
membranes cultured without PBMCs (Figure 7 column 5).
Membranes from viral-stimulated co-cultures most closely
resembled those from co-cultures activated by the anti-CD3/CD28 antibody control (Figure 7 column 4), which
stimulated drastic inflammatory responses across all
immune cell endpoints (Figures 5 and 6). Of note, Donor
7 could be considered a “low responder” as the PBMC
population that had the lowest starting T cell population
size (Table 1). To further examine models of minimal inflammation, PBMC donor lots can be pre-screened for a lackof response to the CEF peptide pool. Donors with minimal
response will not produce the same heightened levels
of inflammatory factors or display the same activation
markers in a monoculture pre-screen environment. Utilizingthese lots will create an environment with minimal necrotic
effects.
Similar to our results, other studies have shown that inflammatory, pro-apoptotic cytokines released by activatedimmune cells lead to necrotic tissue death of the surrounding tissue.10 It should be noted that, in any ALI model usinga Transwell® Plate with sub-cell sized pores, any inflammatory-driven tissue necrosis would only be accomplishedthrough chemical messengers such as cytokines, as the
8 | Modeling Lung Inflammation in 3D

membrane itself bars immune cells from infiltrating anddirectly contacting the epithelial tissue layer above.

Conclusions
We present a 3D NHBE-PBMC B-ALI™ Co-culture systemthat is both easy to implement and successfully models thephysiologically relevant pathological effects of inflammation on the airway epithelium when stimulated with common viral peptides.
Lung epithelial membranes developed using this protocol
are healthy, robust and differentiated. We demonstrated
that this unified system undergoes noticeable shifts inphenotype for both lung and immune compartments after
antigen administration.
We tested the inflammatory response of our co-culture bystimulating the immune (PBMC) fraction using viral antigens. In response, the PBMCs displayed a strong inflammatory reaction and the NHBE membranes thinned markedly
compared to control conditions, suggesting that the underlying inflammatory immune response induced necrotic
effects in the NHBE layer above.
The use of differentiated, primary NHBE cells in the B-ALI™
System better represents in vivo conditions and patientphysiology than 2D submerged systems, reducing the needfor animal models by offering increased translational valueof experiments across a number of applications including
immunotoxicity, vaccine development, small molecule therapies, and biologic therapies.1-3
Overall, our NHBE-PBMC B-ALI™ Co-culture system has
proven to be a highly relevant model of lung physiologythat has numerous important applications from diseaseresearch to drug discovery.

References:
1.

Rijsbergen, L. C., et al. 2021. In vitro modeling of respiratory virus infections in human airway epithelial cells – a systematic review. Frontiers in
Immunology 12: 683002. doi: 10.3389/fimmu.2021.683002.

2. Baldassi, D. et al. 2021. Air-liquid interface cultures of the healthy anddiseased human respiratory tract: promises, challenges and future
directions. Advanced NanoBiomed Research 1: 2000111. doi: 10.1002/
anbr.202000111.
3. Xia, S. et al. 2020. Coupled CRC 2D and ALI 3D cultures express receptors of emerging viruses and are more suitable for the study of viral
infections compared to conventional cell lines. Stem Cells International
2020: 2421689. doi: 10.1155/2020/2421689.
4. B-ALI™ Immune-Airway Co-culture Model: Normal Human Bronchial
Epithelial Cells and Peripheral Blood Mononuclear Cells. Link.
5. Qiao, J., et al. 2020. Elevated serum granzyme B levels are associatedwith disease activity and joint damage in patients with rheumatoid
arthritis. Journal of International Medical Research 48(11): 1-10. doi:
10.1177/0300060520962954.
6. Arican, O., et al. 2005. Serum levels of TNF-α, INF-γ, IL-6, IL-8, IL-12, IL-17,
and IL-18 in patients with active psoriasis and correlation with disease
severity. Mediators of Inflammation 5: 273-279. doi: 10.1155/MI.2005.273.
7.

Said, E. A., et al. 2021. Defining IL-6 levels in healthy individuals: a meta-analysis. Journal of Medical Virology 93(6): 3915-3924. doi: 10.1002/
jmv.26654.

8. Irekeola, A. A., et al. 2020. Technical considerations in ex vivo humanregulatory T cell migration and suppression assays. Cells 9(2): 487. doi:
10.3390/cells9020487.

Contact Us

9. Ghaffarian, R., and S. Muro. 2013. Models and methods to evaluate
transport of drug delivery systems across cellular barriers. Journal of
Visualized Experiments 80: e50638. doi: 10.3791/50638.

Customer Service: + 1 800 638 8174 (toll free)order.us@lonza.com
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10. Connors, T. J., et al. 2016. Airway CD8+ T cells are associated with lunginjury during infant viral respiratory tract infection. American Journal of
Respiratory Cell and Molecular Biology 54(6): 822-830.

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Ordering Information
Catalog No.

Description

Size

CC-2540S

Cryopreserved Human Normal Bronchial Epithelial (NHBE) Cells for B-ALI™
Culture≥ 500,000 cells

CC-2703

Cryopreserved Human Peripheral
Blood Mononuclear Cells (PBMCs)≥ 100 million cells

00193516

B-ALI™ Growth Basal Medium

250 mL

00193517

B-ALI™ Differentiation Basal Medium

500 mL

00193515

B-ALI™ SingleQuots® Supplement Kit

SingleQuots®

00193514

B-ALI™ Bronchial Air-Liquid Interface
Medium BulletKit®

BulletKit®

02-060Q

X-VIVO® 15 Serum-free Hematopoietic
Cell Medium, w/ L-Glutamine, gentamicin, and phenol red

1 Liter

CC-5024

HEPES Buffered Saline Solution

500 mL

CC-5012

Trypsin/EDTA

100 mL

CC-5002

Trypsin Neutralizing Solution

100 mL

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