In vitro organ culture models of asthma

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In vitro organ culture models of asthmaSong Huang1, Ludovic Wiszniewski, Jean-Paul Derouette, Samuel Constant*Epithelix Sarl, 14 Chemin des aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland

Drug Discovery Today: Disease Models Vol. 6, No. 4 2009

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Andrew McCulloch – University of California, SanDiego, USA

Asthma and respiratory diseases

It has been long recognized that, in addition to its

barrier function, the airway epithelium is also

involved in modulating innate and adaptive immune

responses. The recent discovery of TSLP’s function in

Th2-mediated allergic responses has further rein-

forced the central position of the airway epithelium

in pathogenesis of asthma. This paradigm justifies the

development and use of in vitro cell models of the

airway epithelium in asthma research and in drug

development.

Introduction

Allergic diseases like allergic asthma are multi-factorial dis-

eases involving complex interactions between genes and the

environment. Despite of intensive research efforts, the mole-

cular and cellular mechanisms of pathogenesis of allergic

asthma are still not fully understood. Allergic asthma is

characterized by abnormal immune responses. The environ-

mental triggers, pollen, mites and chemicals, among others,

usually benign, can induce severe and exaggerated inflam-

matory reaction in genetically susceptible individuals. Ana-

lyses of inheritance patterns in families and twins allowed to

identify some genetic factors linked to pathogenesis of bron-

chial asthma [1,2]. As example, the chromosome 5q, espe-

cially in 5q31-334-6 where the Th2 cytokine genes are

clustered, seems to be associated to allergic asthma [3–5].

Indeed, abundant clinical evidence and studies in animal

models clearly pinpointed the involvement of Th2-mediated

immune response in allergic asthma, characterized by the

production of key cytokines like IL-4, IL-5 and IL-13 that

*Corresponding author: : S. Huang ([email protected]),S. Constant ([email protected])

1 www.epithelix.com.

1740-6757/$ � 2009 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2009.08.002

Section Editor:Michelle Epstein – Department of Dermatology, DIAID,Experimental Allergy, Medical University of Vienna, Vienna,Austria

regulate the synthesis of allergen-specific IgE and airway

inflammation and remodeling [6,7].

Pathogenesis of asthma

As all adaptive immune reactions, the pathogenesis of asthma

consists of at least three distinct stages: induction, early

asthmatic reactions and late asthmatic reactions (Fig. 1) [7].

A. During the induction phase of the allergic immune

response, the dendritic cells are considered as a key player:

upon antigen stimulation, dendritic cells (DC) are acti-

vated, and then migrate into the local lymph nodes to

present the antigen epitopes to helper T cells (usually

CD4+ cells). By virtue of the cytokines that DC cells make,

the T lymphocytes could differentiate into either Th1-

type or Th2-type. Th1 pathway usually results in cell-

mediated immunity, whereas Th-2 pathway leads to anti-

gen-specific IgE production, which is the signature of

allergic reactions.

B. The early phase of asthmatic reaction is mediated by

mast cells and basophils: these cells express high affinity

receptors (FceRI) for IgE antibody. Cross-linking of FceRI

receptors by IgE-allergen, upon re-exposure, induces

the activation of these effector cells, resulting in degralu-

nation and release of inflammatory mediators like

histamine, leukotrienes and cytokines. These inflamma-

tory mediators provoke symptoms including cough,

bronchospasm, smooth muscle constriction and mucus

secretion.

137

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Drug Discovery Today: Disease Models | Asthma and respiratory diseases Vol. 6, No. 4 2009

Figure 1. (modified according to Fig. 1 of Verstraelen et al. [7]): Classical view of the asthma pathogenesis and the different types of cells involved. In the

induction phase, inhaled antigens are captured by dendritic cells (DCs), professional Ag presenting cells (MHC II positive). The activated DCs will migrate to

the lymph nodes and promote there the differentiation of naive T helper cells (Th0) into type 2 T-help cells (Th2) pathway. In turn, the Th2 cells induce the B

cell activation and maturation, leading to IgE production following isotype switch. Upon the crosslink of IgE receptors, the effector cells (mast cells,

basophils, eosinophils, among others) are activated and release a large panel of inflammatory mediators, causing characteristic asthma symptoms, such as

bronchoconstriction, mucus hypersecretion and airway remodeling.

C. The cytokines released in the early phase of asthma recruit

more leukocytes into the lung and initiate a more intense

and stronger inflammatory reaction. This late phase of

asthmatic attack is mainly mediated by eosinophils, but

with the participation of other cell types such as baso-

phils, neutrophils and T cells. These cells secrete, in turn, a

large spectrum of inflammatory mediators, free radicals,

proteases, leading to the airway obstruction and injuries.

Over the years, the chronic inflammation impairs the lung

function of the asthmatic patients with observable struc-

ture change of the airway tracts.

TSLP: a key player in allergic asthma

For a long time, it has been recognized that allergic reactions,

in particular the asthma, are mediated by Th2 lymphocytes.

But what triggers the differentiation of the Th2 cells

remained elusive. Recent advances suggest that a cytokine

named Thymic Stromal Lymphopoietin (TSLP) might play a

key role in biasing the Th0 cells to Th2 differentiation path-

way [8–11]. TSLP was discovered as a growth factor produced

by Z210R.1 thymic stromal cells, which support proliferation

138 www.drugdiscoverytoday.com

and survival of the NAG8/7 pre-B cells [12]. TSLP is structured

as four a-helical bundles similar to type I cytokines like

Interleukin 7 (IL-7). TSLP signals via a receptor complex

composed of IL-7Ra and a subunit similar to common cyto-

kine receptor gamma chain. Even though the detailed mole-

cular mechanism of signal transduction for TSLP is not fully

elucidated yet, it has been shown that TSLP activates Stat5

transcription factor and expression of downstream genes

[13].

Importantly, expression of TSLP is elevated in the bron-

chial biopsies from the asthmatic patients compared to that

of healthy donors [14]. What is more, TSLPR-knock-out mice

failed to develop lung inflammation upon ovalbumin chal-

lenge [15]. Conversely, the overexpression of TSLP in mice

induces spontaneous airway inflammation and atopic der-

matitis [16,17], suggesting that TSLP is an important factor

necessary and sufficient for the initiation of allergic airway

inflammation. The implication of TSLP in Th2 response is

further confirmed by studies of other allergic diseases such

as the skin atopic dermatitis [16] and intestinal immune

homeostasis [18].

Vol. 6, No. 4 2009 Drug Discovery Today: Disease Models | Asthma and respiratory diseases

Figure 2. Local activation of Th2 cells by allergen-activated DCs under the influence of TSLP produced by the airway epithelial cells. Upon allergen/antigen

challenge, the native T helper cells are directed to either Th1 or Th2-differentiation pathway in a local environment rich in antagonist cytokines like IL-12

and TSLP. In asthmatic patients, because of genetic predispositions or because of external insults (viral or bacterial infections, cigarette smoke, among

others), the differentiation of Th0 is biased to Th2 by increased TSLP production of epithelial airway cells. In the chronic phase of asthma, the effector cells

such as mast cells, basophils, eosinophils and memory T cells amplify and perpetuate the inflammatory responses, leading to airway remodeling and airway

hyper-responsiveness (AHR), mucus hypersecretion, forming a vicious circle.

The airway epithelium: central to the pathogenesis of allergic

asthma

One of the major sources of TSLP is the epithelial cells

including the airway epithelial cells [19]. As consequence,

the airway epithelial cells occupy a central position initiating

and modulating the allergic immune responses [20,21]. In

fact, it has been long recognized that the airway epithelium is

more than just a barrier: it synthesizes and releases a large

panel of chemokines, cytokines, lipids, growth factors, pro-

teases, protease inhibitors, for example, IL-8, IL-6, IL-17F and

TGFs [22–24]. And the expression of these cytokines and

chemokines are induced and modulated by various external

insults like viral and bacterial infections, cigarette smokes

[25] and are associated with disease conditions like asthma

[26]. Furthermore, unlike the classic adaptive immune

responses, the Th2 cells might be activated locally in the

lung rather than in the lymph nodes [27,28]. Indeed it has

been demonstrated that the resident lung antigen presenting

cells have the capacity to promote Th2 T cell differentiation in

situ, and migration of the antigen-loaded antigen-presenting

cells (APC) into secondary lymphoid organs is not crucial for

T-cell priming to occur [29]. Moreover, local blockade of the

TSLP signaling with a neutralizing antibody alleviated allergic

disease by regulating airway dendritic cells [11]. Taken

together, epithelial cells and its adjacent local environment,

so-called epithelial–mesenchymal trophic unit (EMTU) [30],

contains all the ingredients for initiating either Th1, or Th2, or

immune tolerance, the final outcome of an allergen challenge

will be the results of subtle balance between the antagonistic

cytokineshighlightedbyIL-12andTSLP (Fig. 2). Thisparadigm

provides a theoretic framework for developing more relevant in

vitro cells models of allergic asthma, especially the in vitro 3D

cell models of the human airway epithelium.

The experimental models

In vitro cell models

1. The airway epithelia

The airway epithelia constitute the first line of defense

against the external insults. It has a pseudo-layer structure

consisted of three main types of cells: ciliated epithelial cells,

mucus cells and basal cells. The mucus cells synthesize and

secrete mucin-rich mucus that traps most of the inhaled

particles, virus and bacteria, the later are eliminated from

the body by muco-cilliary clearance via the cilia-beating

(Fig. 2). All the three cell types contribute to the asthma

www.drugdiscoverytoday.com 139


pathogenesis, for example, asthma induction, mucus hyper-

secretion and airway remodeling [6–7].

A. Cell lines

Cell lines of airway epithelia have been established, such as

BEAS-2B, 16HBE14o- and Calu3. The characteristics and uses

of these cell lines have been nicely reviewed by Verstraelen

et al. [7]. For asthma research, these cells are very useful for

studying the cellular and molecular mechanisms of gene

expression, signal transduction, among others. However,

the results obtained from the cell lines need to be confirmed

in more physiological situation (organ culture, or in animal

models), because

1. These cells have been transformed by oncogenes one way

or other, thus certain signal transduction networks have

been deregulated.

2. These cell lines cannot give rise to fully differentiated

airway epithelial phenotypes such as cilia formation,

mucus secretion, epithelium repair and remodeling.

B. Fully differentiated 3D human airway epithelial models

A more realistic in vitro cell model is the 3D-culture model

of the airway epithelium, reconstituted with primary human

epithelial cells freshly isolated from the nasal or bronchial

biopsies [31,32] or cryo-conserved NHBE cells with or without

retinoic acid.

Fully differentiated and ready-to-use 3D model of human

airway epithelium are commercialized by Epithelix [33]

Table 1. A comparison of existing 3D models of human airway

Company/Academia

Epithelix

Product name MucilAirTM

Availability Commercially

Cell types Primary human cells

Anatomical origin Bronchial, Nasal and Tracheal

Differentiation Fully differentiated

Shelf-life Up to one year

Disease versions Asthma, COPD, CF, allergic

rhinitis, smoker

Applications Acute, chronic and long-term toxicity tests

Drug delivery

Immune responses

Respiratory diseases

Bacterial and viral infections

Nanotoxicology

Electrophysiology

Cilliogenesis


and MatTek [34]. The Air–Liquid Interface model, so-called

MucilAirTM, is not only morphologically and functionally

differentiated, but also can be maintained in a homeostatic

state for a long period of time (over a year). These features of

MucilAirTM make it possible to

- Study the initial and chronic aspects of asthma disease such

as the airway remodeling.

- Study the interaction between different types of cells:

epithelial cells, mesenchyme, dendritic cells, T cell, mast

cells, eosinophils, among others.

- Search for the genetic determinants for asthma diseases.

Such possibility is illustrated by a study of the polymorphism

ofTSLP transcripts: a long splice form ofTSLP is foundamong

the Japanese population and its expression is highly induced

by poly (I:C) [35]. But the correlation between the expression

of this long splice form of TSLP and the occurrence of asthma

disease has not been established. It is also possible to study

the whole process of asthma pathogenesis from induction

phase to early and even late phase of asthma.

- Screen and test potential drugs.

2. Effector cells

Dendritic cells: The DC cells play a key role in the induction

phase of asthma; therefore, it is important to develop the in

vitro culture model of DC cells. Several protocols have been

established to generate human DC in vitro. Starting with

blood or bone marrow-derived CD34+ hematopoietic pro-

genitor cells (HPC), DC can be generated under various

culture conditions with a cocktail of specific cytokines.

Despite of the progress made in the field, it is still difficult

epithelia

MatTek Academic

EpiAirwayTM Home-made

Commercially Not available

Primary human cells Cell lines and primary human cells

Bronchial Bronchial

Fully differentiated Yes/no

One month One month

Asthma, COPD Asthma, CF

Acute toxicity

Drug delivery

Immune responses



Nanotoxicology

Electrophysiology

Immune responses



Nanotoxicology


to obtain sufficient amount of primary DC cells for basic or

clinic research. Therefore, the use of cell lines such as THP-1,

KG-1, especially the MUTZ-3, proves to be invaluable

(Table 1) [36].

Mast cells, eosinophils, neutrophils and basophils, are consid-

ered as effector cells, which are involved in early and late

phases of asthma by releasing a plethora of inflammatory

mediators. Their roles in broncho-constriction, mucus secre-

tion and airway remodeling have been clearly defined. Many

therapeutics are targeting these effector cells and associated

key molecules [37]. These cells can be isolated from the blood

or cord blood and cultured in vitro. Their behaviors such as

migration, free radical production, viability and apoptosis, can

be assessed after stimulation by allergen and cytokines [38–40].

Coculture systems: With the concept of EMTU and local

activation of CD4+ T cells, it makes sense to simulate the

asthma pathogenesis by coculturing different cells involved

in vitro. Such experiments have been carried out using estab-

lished cell lines: for example, BEAS-2B are cocultured with

human lung fibroblasts (HFL-1 or WISTAR-38) [41], human

umbilical vein endothelial cells (ECV304) [42], or eosinophils

[43], and primary human BECs with alveolar macrophages

[44]. In such culture systems, the two different types of cells

were cultured in submerged conditions: either on semi-porous

membrane or in a traditional culture dish. The adherent cells

like fibroblasts can be placed on one side of the membrane and

the airway epithelial cells on another side of the membrane

[41]. For the nonadherent cells like eosinophils, the cells are

simply put on top of a layer of confluent BEAS-2B cells [43]. A

more elaborated version of the coculture systems is a three-

dimensional coculture systeminwhichmyofibroblastsderived

from human bronchial wall were maintained in collagen gels

and a human bronchial epithelial cell line, 16HBE14o-, was

grown on the surface of the gels [45]. These coculture systems

constantly demonstrated the regulatory role of the airway

epithelial cells in immune response and airway remodeling

by releasing growth factors. Using 3D cell models like

MucilAirTM and freshly isolated immune cells, more reliable

and faithful coculture systems could be developed for studying

the asthma disease and for drug development. Ideally, the

culture models should mimic the disease development and

progression.For example, the effector cells such as eosinophils,

basophils,mast cells orother relevant cells, couldbeembedded

in an agarose gel and on top of the gel an insert with the semi-

porous membrane (a transwell insert, e.g.), could be added: on

one side (bottom) of the insert myofibroblast could be placed,

and on another side, the fully differentiated airway epithelial

cells. Such a model could be used for studying the recruitment

and activation of the effectors in asthma.

Explants

Organ explants: part of animal or human lung was excised,

sliced and cultured in vitro for a period of days or weeks.

Because the lung is rather fragile, it is necessary to perfuse the

lung in situ with 1.5% low-melting agarose gel before cutting

into 200–400 mm-thick slices using tissue slicer. The lung

slices can be cultured for 1–3 days at 37 8C, 5% CO2 and

100% air humidity under standard cell culture conditions. As

for the trachea or bronchi, they are simply dissected and

excised from surrounding connective tissues under sterile

conditions, then sectioned into four tubular segments of

desired length. These tracheal or bronchial rings can also

be maintained in appropriate culture media for several days.

In most of cases, the lung tissues come from the laboratory

animals like mice, rats and guinea pigs. Occasionally, human

lung could also be obtained and studied. The effects of

cytokines or chemical compounds on broncho-constriction

have been studied [46]. Using explants models it is possible to

assess immunomodulatory effects [47] and understand sig-

naling processes involved in tissue hyper-responsiveness

related to asthma [48]. Precision Cut Lung Slides (PCLS) are

now used for studying the effects of sensitizers. PCLS mirrors

the complex interplay of different cell types in the living

organ and allows physiological processes to be mimicked

[49]. Compared to the epithelial models, the major advantage

of the organ explants is their complexity: they contain almost

all the cell types involved in asthma pathogenesis. Indeed,

Henjakovic et al. demonstrate that the effects of low mole-

cular weight allergens, like TMA and DNCB, on ex vivo lung

functions tested in PCLS reflect the in vivo situation in terms

of IgE synthesis and cytokine/chemokine releases.

But the explants suffer from several drawbacks:

1. The explants degrade and degenerate very rapidly in cul-

ture. As example, the PCLS has to be used within the 24

hours after being made. By contrast, the in vitro cell models

of the airway epithelium can last for months.

2. It is difficult to routinely make explants of the human

lungs, whereas the in vitro human epithelial models are

commercially available.

Animal models

Animal model of asthma is indispensable to our understanding

of the disease development. Naturally, because of the econom-

ical and technical reasons, the small rodents like mice and rats

are the most popular and widely used animal models. Besides,

guinea pig, cats, dogs, sheep and monkeys are also used to

study the pathogenesis of allergic asthma [50]. Except some

naturally occurring allergic animals, such as Ascaris suum-

allergic sheep and flea-allergic dog, most of the asthmatic

symptoms are artificially induced by immunizing and challen-

ging the respiratory tracts with antigen or allergen. Even

though some asthmatic symptoms like eosinophil infiltration,

mucus hypersecretion, airway hyper responsiveness and ele-

vated IgE production, can be observed, the lung inflammation

is often transient rather than chronic as in human being.



Transgenic mice, knock-out or overexpression of targeted

genes, are powerful means for dissecting the molecular

mechanisms underlying the asthma pathogenesis. For exam-

ple, the implication of TSLP in allergic asthma pathogenesis

has been clearly demonstrated in mouse models [51]. Huma-

nized animal models have also been used to explore the

pathogenesis of allergic asthma: the human peripheral blood

mononuclear cells were isolated and injected into T and B

lymphocyte deficient severe combined immunodeficiency

(SCID) mice, creating a human-SCID mouse model [52–54].

Because the molecular and cellular mechanisms seem to be

conserved during the evolution; the results obtained from

animal models can be extrapolated to humans. However, none

of these animal models can reflect the human genetic hetero-

geneity which is the key to understand the allergic asthma.

In silico models: virtual cells, virtual organs and virtual

patients

With increasing calculation capacity and better software, it is

now possible to simulate the biological processes at all levels:

molecular interaction (ligand–receptor), virtual organs, and

even virtual patients [6]. The in silico approach allows to

rapidly integrating new data and novel knowledge, and it

can get better and better with time. As example, G-protein-

coupled receptors constitute privileged therapeutic targets for

treating asthma [55]. In silico virtual screening of GPCRs

allows to identify ligands and drug leads [56].

At organ level, the biological phenomenon is too compli-

cated to be entirely simulated in silico. Therefore, only a

Table 2. Comparison summary table

In vitro models In vivo models

Pros Appropriate for studying the

induction phase of asthma

Easy to use

Human tissues

High-throughput screening

of drug leads

Mechanistic studies

Genetic heterogeneity

Possibility

Replicate most of

Possibility to stud

of asthma pathoge

Mechanistic studie

Drug screening an

Cons Do not reflect in vivo situations,

especially in the chronic phase

of asthma

Lack genetic heter

Ethical issues

Best use of model Study of molecular mechanism

induction phase of asthma

pathogenesis

High-throughput screening

of drug candidates

Early as well as ch

of asthma diseases

Mechanistic study

Preclinical develop

Reference [31–49] [50–54]


specific aspect of asthma pathology is simulated. For exam-

ple, broncho-constriction during asthmatic attack is a major

problem of asthma disease, and many efforts have been made

to understand the cause and the mechanism of this phenom-

enon [57]. It has been known that the smooth muscle cell

shows a particularly pronounced and complex mechanical

response. Using a hybrid approach, namely an in silico model

connected to real airway smooth muscles in which the mus-

cles were subjected to a virtual load created by a servo-con-

trolled lever system, Latourelle et al. were able to

quantitatively estimate the effect of tidal breathing and deep

inspirations on airway caliber [58]. Furthermore, using the

same hybrid approach, Oliver et al. found that the smooth

muscle mass is the functionally dominant cause for excessive

airway narrowing [59]. To improve drug delivery efficiency in

asthmatic patients, Martonen et al. built an in silico model of

the lung, based on 3D images acquired by magnetic and

nuclear medical techniques. This in silico model, with the

potential to be customized, allows the clinical investigators to

predict the spatial distribution of an inhaled pharmacolo-

gic drug in the lungs [60]. To understand the global con-

sequences of the interactions between allergens and

immune system, Llop-Guevara et al. [61] have developed

an in silico (computational) view of the aeroallergens and

the host. In this model, the impact of dose and length of

aeroallergen exposure on allergic sensitization and allergic

disease outcomes (airway inflammation, lung dysfunction

and airway remodeling) has been analyzed [61]. Similarly, a

virtual asthma patient has been developed by Entelos

In silico models

the important symptoms

y the whole process

nesis

s

d testing

Data integration

Hypothesis driven

Provide insight about mechanistic at all levels

Identification and validation of drug candidate

ogeneity Special expertise is needed

Often patent-protected

The complexity of asthma disease cannot

be faithfully simulated

The results have to be checked in real models

ronic phase

ment of drugs

Allows integrating all the relevant data sets for

global as well as focal analysis of asthma

diseases from a single molecule to the

whole body

For ligand identification

Prediction of drug effects

[57–61]


(http://www.entelos.com). It has been reported that this

virtual asthma patient has saved Aventis millions of dollars

by predicting that an anti-IL-5 antibody therapy for treat-

ing allergic asthma disease did not have the expected

beneficial effects on the virtual patient. It turned out to

be also the case in clinical trial with real patients (by

another company!).

Model comparison

Each model has its strengths and weaknesses and there is no

perfect asthma model. Depending on the goal or on applica-

tion, one model could be better than another (Table 2).

Because of its central role in asthma pathogenesis, the in

vitro cell models of the human airway epithelium deserve

more attention in the future. In vitro cell models are suitable

for studying the induction phase of asthma disease: the

influence of bacterial or viral infection on the synthesis

and release of inflammatory mediators by the airway epithe-

lium, in particular TSLP. By sampling hundreds and even

thousands of asthmatic patients, it is possible to search for the

genetic determinants predisposing susceptible individuals to

allergic asthma. The in vitro cell models can easily be stan-

dardized and used in high-throughput screening and testing.

Obviously, a lot of important players are missing in the in vitro

cell models. As consequence, the complexity of asthma dis-

ease cannot be recapitulated.

The strength of the animal models is their ability to simu-

late most, if not all, the symptoms of asthma pathology.

Because the cellular and molecular mechanisms of allergic

reactions are more or less conserved, the results obtained

from the animal models can be extrapolated and verified in

human being, vice versa. Transgenic mice allow functional

analysis of a specific target gene by loss-of function or by

overexpression. However, animal models do not and cannot

reflect the genetic heterogeneity of human populations,

which is essential component of asthma etiology [1,2].

At the molecular level, in silico models are powerful and

highly predictive, because of our advanced knowledge in

chemistry and physics. In silico approach is now routinely

used in drug design and drug development. Because of the

complexity and the heterogeneity of biological phenom-

enon, it is less predictive at organ and organism level, like

the asthmatic lung and patient. But, with the enrichment of

our knowledge databases, in silico models have the potential

to simulate more realistically the pathogenesis of asthma

diseases.

Conclusion

As other scientific endower, asthma research needs experi-

mental models. But making good disease models is a challen-

ging task and tedious. Most of the models described in this

review, such as the animal models, have been developed for a

long time. But they are still invaluable and indispensable for

elucidating the molecular and cellular mechanisms under-

lying the asthma pathology. Up to now, the main focus of

asthma researches has been the immune cells. With the

recognition of the central role of the airway epithelium in

asthma pathogenesis, especially with the discovery of TSLP’s

function in allergic reactions, the airway epithelium will

come into the limelight. Fully differentiated 3D in vitro cell

models of the human airway epithelium will provide us with

a powerful means to extrapolate and deepen our knowledge

obtained with animal models to human beings. The 3D in

vitro cell models constitute also an ideal platform for screen-

ing and identifying potential drug targets for treating allergic

asthma.

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