Airway and Alveoli Organoids as Valuable Research Tools in COVID-19

Abstract

The coronavirus disease 2019 (COVID-19), caused by the novel coronavirus, SARS-CoV-2, affects tissues from different body systems but mostly the respiratory system, and the damage evoked in the lungs may occasionally result in severe respiratory complications and eventually lead to death. Studies of human respiratory infections have been limited by the scarcity of functional models that mimic in vivo physiology and pathophysiology. In the last decades, organoid models have emerged as potential research tools due to the possibility of reproducing in vivo tissue in culture. Despite being studied for over one year, there is still no effective treatment against COVID-19, and investigations using pulmonary tissue and possible therapeutics are still very limited. Thus, human lung organoids can provide robust support to simulate SARS-CoV-2 infection and replication and aid in a better understanding of their effects in human tissue. The present review describes methodological aspects of different protocols to develop airway and alveoli organoids, which have a promising perspective to further investigate COVID-19.

Full text

Airway and Alveoli Organoids as Valuable Research Tools in COVID-
19
Miriane de Oliveira, Maria T. De Sibio, Felipe A. S. Costa, and Marna E. Sakalem*
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ABSTRACT: The coronavirus disease 2019 (COVID-19), caused
by the novel coronavirus, SARS-CoV-2, affects tissues from
different body systems but mostly the respiratory system, and
the damage evoked in the lungs may occasionally result in severe
respiratory complications and eventually lead to death. Studies of
human respiratory infections have been limited by the scarcity of
functional models that mimic in vivo physiology and pathophysi-
ology. In the last decades, organoid models have emerged as
potential research tools due to the possibility of reproducing in
vivo tissue in culture. Despite being studied for over one year,
there is still no effective treatment against COVID-19, and
investigations using pulmonary tissue and possible therapeutics are
still very limited. Thus, human lung organoids can provide robust
support to simulate SARS-CoV-2 infection and replication and aid in a better understanding of their effects in human tissue. The
present review describes methodological aspects of different protocols to develop airway and alveoli organoids, which have a
promising perspective to further investigate COVID-19.
KEYWORDS: lung, virions, SARS-CoV-2, respiratory disorders, coculture, 3D cell models
■INTRODUCTION
For the past year, the world has been facing the new
coronavirus pandemic, caused by the Severe Acute Respiratory
Syndrome-CoronaVirus-2 (SARS-CoV-2), which leads to the
Coronavirus Disease 2019 (COVID-19) in approximately 40−
45% of infected patients.
1,2
Ever since its outbreak in
December 2019, in Wuhan, China, SARS-CoV-2 has rapidly
spread throughout the country, reaching the first epicenter in
the city of Hubei and, shortly thereafter, the globe.
3−6
The
clinical outcomes observed in patients infected with SARS-
CoV-2 are variable, ranging from asymptomatic cases to acute
respiratory discomfort and multiorgan failure.
7
Among the
most common COVID-19 symptoms are cough, fatigue,
headache, loss of taste and smell, myalgia and sputum, and
diarrhea (mild outcome); and cyanosis, dyspnea, thoracic pain,
shortness of breath, hypoxemia, severe pneumonia, pulmonary
edema, and multiple organ failure (severe outcome).
7−9
One
prominent feature once patients undergo thorax tomography is
the ground-glass opacity observed in the lungs, affecting both
lungs in the periphery of inferior lobes; this occurs even in
asymptomatic patients.
7,10
In addition to the commitment of
lungs, other systems may be severely affected, in particular,
digestive, cardiovascular, epithelial, renal, and central nervous
system.
6
Nevertheless, pulmonary tissue involvement is of
greatest concern since it is the one involved in the
development of acute respiratory distress syndrome (ARDS)
and the risk of death.
11,12
The main structural viral proteins that constitute SARS-
CoV-2 are small envelope glycoprotein (E), membrane
glycoprotein (M), nucleocapsid protein (N), and spike
glycoprotein (S), along with some accessory proteins.
13
The
S glycoprotein is particularly important for the development of
COVID-19, since it is a transmembrane protein located in the
external part of the virus that directly binds to angiotensin-
converting enzyme 2 (ACE2), expressed in human host cells,
in particular, pulmonary cells,
14
including epithelium alveolar
cells type 2 (AT2).
15
Additionally, other host factors enable
and/or facilitate viral entry, such as transmembrane protease
serine 2 (TMPRSS2), which acts on protein S priming; furin
paired basic amino acid cleaving enzyme (FURIN), which
activates protein substrates and pathogenic agents; and
neuropilin-1 (NRP1), involved in angiogenesis and highly
expressed on vascular cells and epithelia facing external
Received: March 5, 2021
Accepted: July 9, 2021
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environment, and which participates in tissue infiltration.
16−20
Although most research on virus entry focuses on ACE2, it is
understandable that the availability of virus receptors and the
interaction among cofactors help determine infectivity; in the
case of SARS-CoV-2, cells with low ACE2 expression are also
infected, which could be explained by the involvement of
cofactors.
16,17
In addition, diverse genes have been reported to
be upregulated in patients that develop severe COVID-19,
possibly due to a direct correlation with the viral cycle in the
human organism; these include ADAM metallopeptidase
domain 10 (ADAM10), Toll-like receptor 3 (TLR3), histone
acetyltransferase 1 (HAT1), histone deacetylase 2 (HDAC2),
lysine demethylase 5B (KDM5B), sirtuin 1 (SIRT1), member
renin-angiotensin system (RAS) oncogene family (RAB1A),
and FURIN.
18,21
Importantly, there is a link between
upregulation of these genes and ACE2 expression, and
consequently virus entry.
21
After this cleavage, the virus
manages to infect the host cell and releases mRNA into the
cells, which is then translated into protein and generates new
viruses.
22
Ever since the first genomic sequencing to this date,
a considerable number of mutations of the SARS-CoV-2 have
been observed throughout the world, mainly concerning S
proteins, which can miscarry the effectiveness of vaccines, in
addition to favoring the rise of new pathogenicities.
23,24
In the
past months, several new strains have emerged and raised
concern, since they usually present a significantly higher
transmission rate possibly due to improved host−cell receptor
interaction, and what results in a more effective infection
establishment and propagation; these events make it more
challenging to contain the virus spread.
25
At the initial infectious stages, the viral replication occurs
rapidly, leading to death of pulmonary cells, especially AT2
cells; as the virus keeps on infesting more cells, there is
subsequent vascular leaking, which in turn triggers an
exacerbated inflammatory response and leads to an event
called storm of cytokines and chemokines.
26,27
The cytokines
are possibly related to an elevated concentration of hyperactive
neutrophils in the affected pulmonary areas; acting together,
they trigger neutrophil extracellular traps (NETs), which lead
to extensive tissue damage.
28
NETs, extracellular webs of
protein, are released by neutrophils in the aim of fighting
ongoing infections; nevertheless, when not properly regulated,
they originate thromboinflammatory states, including the ones
underlying respiratory failure.
29,30
Moreover, blood plasma
from deceased and hospitalized patients indicates a significant
correlation between NET levels and clinical outcomes;
remarkably, there are significant differences in NET levels
among intubated and non-intubated patients, suggesting that
this parameter could be considered for evaluating the chance of
survival and clinical outcome prognosis.
29
Post-mortem
investigations indicate the presence of microvascular thrombi
and active neutrophils, with NET release, together with
Figure 1. Pathophysiology of SARS-CoV-2 in human alveolar cells. SARS-CoV-2 infects human respiratory cells through the S protein linkage to
host cell entry mediators; after cleavage, the virus delivers mRNA into the cell and the viral replication begins. Once the new virions are released,
inflammatory response takes place, and cytokine storm and NETs release start, leading to clinical features. Abbreviations: SARS-CoV-2: Severe
Acute Respiratory Syndrome-CoronaVirus-2; ACE2: angiotensin-I-converting enzyme-2; NPR1: neuropilin-1; Furin: furin paired basic amino acid
cleaving enzyme; TMPRSS2: transmembrane serine protease 2; NETs: neutrophil extracellular traps; COVID-19: CoronaVirus Disease 2019.
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platelets, in particular, in patients with rapid disease
progression.
29,31
Cytokine storm has been demonstrated to
be followed by infection in sera from patients;
32
thus, increases
in cytokine and NETs levels, one related to the other,
associated with vascular disorders, are important to predict
prognosis and possible treatment interventions. Besides, it has
been suggested that the negative regulation of ACE2 evoked by
cell death can impair the anti-inflammatory function of RAS.
This effect increases the vascular permeability of pulmonary
arterioles and further aggravates the inflammatory re-
sponse.
26,33
Such events can result in exudate leaking in the
alveolar aerial spaces and end in pulmonary edema and
consolidation,
34
alveolar cell desquamation, and hyaline
membrane development.
35
On top of that, the healing process
from the pulmonary inflammation can evolve to fibrosis, which
worsen the patient’s prognosis.
34
A scheme containing the
respiratory cell infection and the clinical outcomes is described
in Figure 1.
Because of the viral infection through ACE2, SARS-CoV-2
transmission depends mostly on respiratory droplets, and the
primary viral replication is considered to occur in the human
respiratory tract, with the distal airway, lung included, being
the most vulnerable target.
36
Due to the complicated and not
yet completely understood characteristics of COVID-19,
reliable models that allow a substantial evaluation of SARS-
CoV-2 infection in the pulmonary tissue are required, along
with consistent methods in the constant search for possible
therapeutic agents, effective antiviral drugs, and vaccines.
■RESEARCH MODELS FOR RESPIRATORY DISEASE
The first models used to investigate human pathology were
animal-based.
37
Such investigations enabled incredible ad-
vances in life sciences and are of obvious importance to this
date, but present some downsides and considerations in regard
to translatability, since some human aspects and disease
characteristics are not correspondent in laboratory animals.
38
Under the respiratory spectrum, for instance, the presence of
excessive mucus production, observed in some chronic
pulmonary diseases as a key symptom, cannot be replicated
in rodents due to the anatomical structures of the bronchial
glands of mice and rats and their difference from the human
respiratory anatomy.
39
Monolayer cell cultures, consisting mostly of one cell type,
present uniformity and allow patterned and consistent
investigations that rely on morphological, genetic, and
physiological aspects. This traditional cell culture method is
easily replicated, low on financial demand, usually of fast
development and data collection, easily interpreted, and
sustainable for long-term cultures, which leads bidimensional
(2D) cultures to be, to date, the most commonly used lab
method.
40
They have aided in the discovery of many biological
and disease processes; however, they are unable to realistically
simulate complicated microenvironment cells experience in
vivo.
40,41
In humans, responses to infection, inflammation, cell
recruitment, tissue remodeling, and regulation of homeostasis,
including in the respiratory system, are complex events
involving different types of cells.
42
Overall, studies of human
respiratory infections have been limited by the scarcity of
functional models that reproduce physiology and pathophysi-
ology in vivo. In addition, drug testing from both animal and in
vitro models yields low success rates for new medicines; less
than half of the new drugs under testing fail due to lack of
efficacy or concerns on safety before hitting the market.
43,44
In
this aspect, tridimensional (3D) culture models represent a
more consistent alternative method, since they allow the
integration of diverse cell types that spontaneously organize,
and cell−cell and cell−matrix interactions; therefore, they
replicate key histological and functional aspects of the target in
vivo organ.
45
Although the majority of these 3D cultures are
organoids, more recent models, such as organs-on-a-chip, are
rapidly gaining popularity, and so far, there have been reports
of 3D cell culture models for almost every human tissue and
organ, including of the respiratory system.
15,46
In particular,
while both spheroids and organoids usually spontaneously
organize themselves once primary or stem cells are allocated
on a nonadherent surface or on top of scaffold,
47
for 3D tissue
models,usually3D-printed,thescaffolding structure is
designed and engineered separately, and then the cell cultures
are developed on the scaffold to form the final model.
48
While
the former can usually “freely”grow in resemblance to the
natural development, manipulated by the growth medium, the
latter are artificially orchestrated into growing in an
architecture determined by the researchers using physical
templates.
49,50
As an example, organs-on-a-chip combine 3D
cell culture and microfluidic workflow, resulting in a dynamic
biomimetic device, in contrast to the more static cultures.
47
The greatest advantage of using spheroids and organoids over
organs-on-a-chip relies on the level of complexity of the
culture; although more complex in comparison to 2D cell
cultures, organoids are still much less complex to establish than
organs-on-a-chip; and for institutions to obtain a patterning on
organ-on-a-chip technology, they usually first go through
spheroid and organoid experimentation. This can be observed
by a recent publication investigating metrics on organoid and
organ-on-a-chip publications: while over 2000 institutions were
working and publishing with organoids, only 811 were
researching organs-on-a-chip.
51
As of a comparison between
spheroids and organoids, spheroids are usually more suitable
for replicating tissues and simplified versions of organs, while
organoids offer the possibility to mimic multiple tissues within
an organ, recapitulating the morphogenesis in vitro,
47
making
it plausible to understand the quantity of research using
organoids as a model in comparison to other 3D tissue models.
Each of the previously mentioned approaches presents
indications of use, as well as advantages and limitations; a brief
summary with the most prominent characteristics are shown in
Table 1. Taking all these aspects into consideration, models
that mimic human physiology and pathophysiology, such as
organoid models, could be explored and are of great value due
to the urge evoked by zoonotic infections, such as the ones
caused by Severe Acute Respiratory Syndrome (SARS),
Middle East Respiratory Syndrome (MERS), influenza A
virus (IAV) pandemics,
52
as well as the new SARS-CoV-2.
SARS-CoV-2 may affect diverse human body systems, such
as digestive, cardiovascular, and urinary; however, so far data
indicate that the most drastically affected system is the
respiratory.
11
Zhao and collaborators
15
showed through the
use of human liver ductal organoids that the ex vivo virus
infection of liver tissue evokes severe damage, demonstrating
that there is a need for human organoids to deeply investigate
SARS-CoV-2-related tropism and pathogenesis in other tissues,
which could also be valuable to treat COVID-19 patients.
Besides, Monteil and colleagues
11
developed human blood
vessel and kidney organoids, and showed that SARS-CoV-2
successfully infected the in vitro culture. In these models, the
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authors proved that a higher expression of a recombinant form
of ACE2, the protein used as a receptor for the virus in
mammalian cells, significantly reduced the cell infection rate by
the virus. Yet, this was only achieved if delivered during the
earlier stages of infection. Since both studies model other
tissues than the ones in the respiratory system, and considering
that the lung is the most affected organ during a SARS-CoV-2
infection, it is of upmost importance to evaluate the virus
behavior in the lung tissue, preferably in in vitro models that
are able to mimic in vivo conditions, such as organoids.
This review investigated and summarized the most recent
publications that present airway and alveoli organoid protocols,
in order to present different methodologies that might be of
great help in better understanding and eliciting critical aspects
related to SARS-CoV-2 infection and the resulting COVID-19.
■GENERATING LUNG ORGANOIDS
The starting point to create an organoid can vary considerably.
In the models used to date, the tissue structure is mainly
obtained from embryonic stem cells, adult cells/stem cells/
progenitors collected from human biopsy of the target organ,
or adult tissue-derived induced stem cells. Therefore, the cell
types that can be used to initiate an organoid are (1)
pluripotent stem (PS) cells, cells with the capacity to
differentiate into every cell type present in the organism, and
that include embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs); (2) multipotent stem (MS)
cells, which are able to differentiate into every cell type within a
germ layer, and include cells obtained from fetal tissue (9
weeks of development and on) and cells collected and isolated
from some specific tissues, such as adipose tissue; and (3)
adult tissue (AT) cells, which possess restricted differentiation
capacity, oligo or unipotent, usually of one or two cell lineages
present in the organ in which they are found, and can be
isolated from adult tissue, mostly through biopsy of the target
organ. Despite the cell lineage used as a starting point, all the
mentioned cells are suitable for organoid generation because of
their apparent infinite expansion potential in culture.
53
With respect to lung organoids, several research groups have
already shown the successful generation of 3D models from
pluripotent stem cells or primary respiratory cells,
37,54−56
and
most studies are based on the protocol by Miller and
colleagues, published in 2019.
57
The resulting structure
presents a similar cell organization to what is observed in a
living lung, consisting of diverse cell layers and cell types,
making lung organoids a highly sophisticated in vitro model to
study developmental, homeostatic, and pathological pro-
cesses.
37
These lung organoids that present multiple lung cell lineages
are very attractive as research models due to their potential use
in developmental and regeneration investigations, as well as
more complex studies involving inflammatory and toxicological
effects to particles and inhalants. Furthermore, they support
the seductive possibility of developing transplantable material
for patients suffering from pulmonary diseases that are, so far,
incurable, such as fibrosis and degenerative lung diseases in
general,
58−60
in addition to viral infections, such as the one
evoked by SARS-CoV-2
11
that could leave substantial sequelae
in the recovered patient.
61
Thus, regenerative medicine
approaches, aimed at repairing, regenerating, and restoring
missing function or tissue through isolated cells or lab-
constructed 3D cell models, could aid in the treatment of
COVID-19.
12
Table 1. Evolution of Research Models in Life Sciences
time of
method
introduction type characteristics
first reported
use animal model contribution/possibilities of use advantages limitations
500 B.C. Mainly rodent models
(murine + rat)
Provided much of what is now known in life sciences. Pharmacology,
behavior, toxicology
Low cost; easy access Some aspects cannot be studied due to
significant differences to human
cell culture
XX (1950) Bidimensional (2D) Enabled uncountable advances ever since the first cell lineage isolation,
HeLa cells. Understanding molecular, chemical and physiological
processes
Uniformity, patterned, consistent. Easily replicable, low cost, fast Unable to mimic microenvironment of
in vivo
XX and XXI Tridimensional (3D);
spheroids and
organoids
Represents in vivo more consistently. Investigating normal function,
disease, infection, etc.
Allows cell−cell and cell-matrix interactions; controlled environment, structure
and aspects; more accurate representation of histological and functional aspects
of tissue
High cost; sometimes difficult to
replicate; usually represents isolated
tissue or organ
XXI Tridimensional (3D);
organs-on-a-chip
Allows microfluidic circulation, intersystem interactions; intrasystem
interaction is possible (i.e., trachea + bronchus)
Similar to 3D stated above; in additions, allows continuous fluid circulation;
representation of several different structures combined is possible
High cost, and can be difficult to pattern
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In order to replicate the extracellular matrix, most organoids
are placed into a scaffold to which they interact and get
support to form the cell agglomeration, which induces the 3-
dimensionality. The first scaffolds used were microporous
filters applied on metal, collagen, or sponge grid; they were
gradually preferably replaced by porous membrane inserts.
62
There are naturally derived scaffolds, such as collagen, gelatin,
elastin, fibrin, and fibrinogen; synthetic polymers; or hybrids
containing biomaterials and synthetics.
63
Any selected scaffold
aims to reconstruct the physical properties of the extracellular
matrix
64
and elicit cultivated cells to exhibit biofunctionalized
characteristics of the in vivo cells.
63
The micro architecture resulting from porosity, permeability,
and mechanical stability enables biophysical and biochemical
interaction among cells. Thus, the addition of a scaffold into
the organoid culture allows the regulation of the spatial
configuration, as well as cell migration, differentiation, and
proliferation. There are two main configurations using a
scaffold: the organoid may grow immersed in the scaffold, or it
may grow on top of scaffold; in the latter case, culture media is
added to help cultivate and nurture the cells.
65
The choice of
scaffold depends on the cell type cultivated and the intent of
the study, and diverse growth as well as inhibitory factors
might be added to the culture in order to regulate and control
the organoid development.
63,64
In the past decade, because of
the increased research with organoid and 3D cultures in
general, advances of cultivation techniques led to the mass
commercialization of scaffold input, such as Matrigel, one of
the most used scaffolds observed in the latest published
research.
66
The exposure of organoids to the external environment is
still limited, and recent approaches have proposed the contact
of lung organoids to air, which enables the complete
differentiation of the adult airflow cells, and an even more
realistic investigation of the effects of interaction with
pollutants such as toxic gases or toxic micro- and nano-
particles,
67
including viral particles. This method is known as
the air−liquid interface (ALI), in which the organoids are
exposed to air by removing or partially removing the culture
media, so that at least a part of the organoid is exposed to air. It
is also possible to convert organoids into a monolayer by
dissociating and seeding the cells on transwells in order to
obtain 2D ALIs.
68
The advantage of such a technique is the
rapid generation of multiple homogeneous monolayers,
consisting of the same diverse cell types previously present
in the 3D cell culture, but with easier access to apical and basal
surfaces, and regular exposure to medium.
69
Airway cells
maintained under these conditions self-organize in a more
natural manner, replicating more consistently the in vivo
respiratory epithelium, with the development of ciliated
pseudostratified cylindrical epithelium and goblet cells.
70,71
A
simplified visual description of the main steps to generate an
organoid considering the starting point, cultivation methods,
and 3D induction is in Figure 2.
To date, there are diverse organoid models to represent the
respiratory system, including cultures to mimic proximal and
distal airway, and alveoli. The most recent organoid methods
used in the pneumological field and based on human tissue are
described below, according to the origin cell lineage used to
start the culture (PS, MS, or adult stem cells/progenitors).
Figure 2. Organoid starting cell types and cultivation development. Human distal airway and alveoli organoids can be developed from different
origin cells, and cultivation include particular steps. Abbreviations: ESCs: embryonic stem cells; iPSCs: induced pluripotent stem cells; MS:
multipotent stem; AT cells: adult tissue cells.
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■AIRWAY AND LUNG ORGANOID MODELS
1. PS Cell-Derived Organoids. Pluripotent stem cell-
derived organoids include organoids developed from both
ESCs and iPSCs. Either way, lung organoids derived from
human PS cells (hPSCs) possess complex tissue structure in
vitro, with epithelium and supporting tissue (cartilage, smooth
muscle, fibroblasts). Yet, they reflect the fetal airway, and adult
airway-like structures only appear after in vivo transplanta-
tion.
72
This has been shown for several other organoids and
hPSC-based systems.
37,73
Considering organoids originating from ECSs, Zhao and
colleagues
74
used human lung bud organoids derived from
human embryonic stem cells (hESCs) to test viral infection.
Bud tip progenitor cells generate all murine lung epithelial
lineages and are found in the developing human lung, and
although their certain role in development of human tissue is
not completely clear, they might be involved.
75
The steps used
to achieve the organoid passed through definitive endoderm
differentiation with activin A, then induction of the anterior
foregut until the formation of a lung bud organoid. The
resulting lung bud organoid was embedded in a scaffold
(Matrigel) in transwell inserts in order to induce an air−liquid
interface (ALI). The authors demonstrated with this research
that lung organoid derived from hESCs is able to simulate the
infection of influenza virus to human, suggesting the clinical
perspective of these organoid model,
74
suggesting that this
model most probably is replicable for investigating other viral
infections. In late October 2020, Han and colleagues
76
successfully showed that lungand colonicorganoids,
derived from hESCs, were permissive to SARS-CoV-2, and
also performed a drug screening of FDA-approved medicine,
proving that organoids are valuable models to investigate
SARS-CoV-2 infection and COVID-19.
76
In another recent research study, Dye and collaborators
77
demonstrated that organoids derived from ESCs mimic early
stages of fetal development but are able to mature at the
molecular and structural levels when transplanted into
immunocompromised mice. They transplanted hPSCs-derived
lung organoids allocated in different biomaterial scaffolds to
immunodeficient mice and observed that after some weeks in
vivo, organoids presented improved tissue structure and
cellular differentiation. Their aim was to define the
physicochemical biomaterial properties that maximally en-
hance transplant efficacy and concluded that, while micro-
porous scaffolds of poly(ethylene glycol) (PEG) hydrogel
inhibited growth and maturation (they report that organoids
remained consisting mostly of immature lung progenitors),
scaffolds of polylactide coglycolide (PLG) or polycaprolactone
(PCL) allowed maturation to tube-like structures that
resembled the structure and cellular diversity of adult airways.
Since PS cell-derived organoids usually derive from fetal-like
structures, it is important to understand the best scaffold to be
used in case a transplantation in vivo of lung organoids is
required, in order to induce further maturation.
Other researchers working with ECSs proposed replication
of genetically related lung diseases, such as fibrosis, in
organoids. To date, there are organoid models with the
introduction of mutation in diverse genes with the help of
lentivirus or plasmid inserts, proving a resulting organoid that
replicates alterations observed in vivo.
46,74,78
The model
presented by Strikoudis and colleagues
78
replicated the
human airway and alveoli, composed of epithelial and
mesenchymal cells, in particular, a large fraction of AT2
cells, and importantly presented a branching organization, with
a configuration that resembles the second trimester of human
development. AT2 cells synthesize, secrete, and recycle all
components of the surfactant; dysfunctions in its metabolism
result in diseases (including distress syndrome and interstitial
lung disease
79
). Such models allow genetic manipulation in
order to replicate specific lung diseases, with the possibility of
investigating potential drug targets and medical treatments for
specific diseases.
Still, in the case of ECSs-derived organoids, Porotto and co-
workers
80
developed a model containing pulmonary mesoderm
and endoderm, and branching airway and early alveolar
structures after plating in scaffold, such as Matrigel.
46
The
authors investigated viral infections common to infants that
affect the distal airway. This method enables investigations
related with viral pathogenesis in the developing or infant
lung.
80
Another possibility to work with PS cells besides using
embryonic cells is to induce pluripotency from adult cells,
resulting in iPSCs.
81
Organoids derived from these cells are
very similar to those developed from ECSs, representing early
stages (pseudo glandular and canalicular) of lung development.
In a publication from 2019, Leibel and colleagues
79
also
studied lung diseases with a genetic background, related to
surfactant protein B deficiency. This deficiency leads to a fatal
disease due to problems in surfactant production and depends
on AT2 cells. The authors developed lung organoids from
patient biopsy material that presented epithelial and mesen-
chymal cell populations of the proximal and distal airways,
including AT2 cells. These, however, due to the genetic
patient-specific configuration, presented alterations related to
surfactant metabolism. Later, the researchers inserted a wild-
type, normal gene using a lentivirus. The “corrected”organoids
presented normal lamellar bodies and secretion of surfactant,
similar to a healthy lung. This publication corroborates the
idea that it is possible to mimic genetically related lung diseases
in vitro, and in addition, it is possible to alter the genetic
background of organoids. It is important to mention that the
previously described PS cell-derived organoids are induced to
3-dimensionality at the anterior foregut stage; this work
postulates that, by using the lung progenitor stage as the
starting point, the organoid differentiation was shorter, and the
culture presented cells in different stages of lung develop-
ment.
79
Huang and colleagues
82
used commercially available iPSCs
to generate AT2 cells (referred to as iAT2), which presented
the ability to endlessly propagate inside a 3D culture. The
iAT2 cells were first cultivated as alveolospheres; then, cells
were dissociated and further maintained in 2D ALI, with help
of transwells. These 2D ALI cultures were permissive to viral
infection and, thus, able to simulate apical viral infection by
SARS-CoV-2. Although models using ESCs usually replicate
immature and fetal stages of development, gene expression
analyses demonstrated the presence of maturation genes,
including surfactant proteins. In addition, air exposure seems
to be important to induce further cell maturation, since iAT2
cells in 2D ALI presented maturation markers. After incubating
the organoid with SARS-CoV-2, it was proven that the lamellar
bodies, responsible for extracellular surfactant release and
present inside iAT2 cells, were infected by virions; this result
might further contribute to the current knowledge of the
pathophysiology of COVID-19 and proves that this model is
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representative for investigations involving SARS-CoV-2 and a
reliable platform for drug screening.
2. MS Cell-Derived Organoids. Multilineage Cells.
Skardal and co-workers
83
developed lung organoids modeled
on the structure and cellular organization present in the
airways by mounting them in three layers: in the lower layer,
they added lung microvasculature endothelial cells, commer-
cially acquired; in the middle layer, there were airway stromal
mesenchymal cells, donated; and the upper layer was made up
of bronchial epithelial cells. Thus, although they used
multipotent isolated cells, they combined them in order to
create a multigerm line-resulting 3D culture, which resembles
PS cell-derived organoids but mimicks adult tissue. The
purpose of the study was to perform a drug screening from
commercial drugs removed from the market by the Food and
Drug Administration (FDA) due to toxicity in humans. In
addition to the lung organoids, they developed six other
bioengineered models and articulated them in a microfluidic
methodology (further mentioned in Future Perspectives in
COVID-19).
Fetal Bud Progenitors. Organoids can also be derived from
fetal tissue (as already mentioned, 9+ weeks of development)
or fated cells originated from PS cells. In this case, the cells
isolated are no longer pluripotent but multipotent. Miller and
colleagues
57,75
successfully generated lung organoids from PS
cells induced as bud tip progenitors, that presented in vivo
similarities, including airway expansion, only observed after
maturation inducement with transcription and growth factors.
Although not all organoids survived the induction, the
remaining ones presented multiepithelial cell lineages, such
as secretory, multiciliated, club, goblet, and neuroendocrine
cells, along with the remaining progenitor cells, and thus
represented the developing prenatal lung epithelium.
75
This
model is very valuable to study human lung development in
vitro. By the end of 2020, researchers from the same laboratory
managed to expose the human lung organoids to SARS-CoV-2
infection.
84
Samuel and colleagues investigated whether anti-
androgenic drugs could have an effect on the viral infection;
the results indicated that there was a decrease in ACE2 levels,
and a consequent reduction of the number of cells infected by
the virus, showing that the anti-androgenic property presents a
protective effect against SARS-CoV-2.
Another study, also working with fetal bud progenitors, was
able to show that, with a specific combination of signal ligands
and inhibitors, a fetal-like organoid possessing AT2 cells can be
generated.
85
In addition, besides working with AT2 pop-
ulations, the authors showed that alveolar epithelial type I cells,
that constitute a thin wall for gas exchange in lung, can also be
cultured in vitro. In order to do this, differently from Miller et
al.,
57,75
they used cocultures from primary alveolar cells and
magnetically activated cell sorting, and then further sorted into
wells with a scaffold. It is important to mention that fibroblasts
were removed from all cultures, and by doing so, the resulting
organoid presented alveolar cells (AT2, mainly) and not only
epithelial cells; in addition, there were organoids with and
without empty cavities, resembling the airway. When
fibroblasts were present, tracheospheres were generated. The
authors also succeeded in inducing retroviral genetic
modification to evoke fibrosis-associated alterations in organo-
ids, suggesting that this method can also be used to induce
diverse genetic-associated lung diseases. This indicates the
possibility of use of this model for genetic alterations.
Lamers and colleagues
86
adapted a protocol from Miller et
al.,
87
based on lung bud tip organoids, and started at fetal tissue
(15−20 weeks) to derive bronchioalveolar organoids. The
resulting model presented diverse cell types, including club,
goblet, and ciliated cells, with more than 90% of alveolar-like
cluster cells of which 46% were AT2 cells. After the organoid
was established, cells were dissociated and seeded on transwells
for ALI-monolayer cultivation, as previously explained. Then,
the model was exposed to SARS-CoV-2 and proven to be
susceptible to viral infection. An important point shown in this
article is that AT2 cells seem to present low ACE2 expression,
and in accordance with studies using human tissues, SARS-
CoV-2 infection might start by other cell populations, such as
ciliated cells, indicating a systematic infection by the virus.
88
The authors also used the model to perform drug screenings
and presented results that indicate that interferon treatment
with low doses was sufficient to reduce viral replication and
tissue infection. Thus, alveolar-like and airway models present
the potential to be used for further investigations on SARS-
CoV-2 infection and COVID-19 therapeutics.
Mesenchymal Stem Cells. Afinal possibility when working
with MS stem cells for lung organoids is to use mesenchymal
stem cells as the starting point. Wang and collaborators
89
developed an alveolar organoid with epithelial stem/progenitor
cells from mesenchymal cells obtained and isolated from the
postnatal human lung, obtained from pediatric patients
undergoing elective surgery for some airway abnormalities.
This method may be used to study congenital lung lesions and
alterations, and although this publication used pediatric-
derived tissue, the use of adult isolated cells might replicate
adult in vivo tissue.
3. AT Cell-Derived Organoids. Finally, lung organoids
can be developed from stem and progenitor cells isolated from
adult tissue (AT). The convenience of this method is that adult
cell-organoids present the physiological dynamic consistent
with adult in vivo tissue, which was not possible in stem cell-
derived models.
76
In research from 2019, Kim and colleagues
90
successfully reconstituted the lung cancer morphology and
histological features of original tissues in an organoid model
starting from primary lung cancer tissues. In order to do so,
cells isolated from patient biopsies were paired with non-
neoplastic airway tissues, creating a biobank of 80 lung cancer
organoid lines from five subtypes of lung cancer and five
normal bronchial organoids. The aim of the study was to
perform an anticancer drug screening, but the model can also
be used for predicting individual patient responses to different
drugs. As organoids developed, tubule-like structures were
observed, and the morphology was maintained for subsequent
passages (over 10 passages). The model presented a
pseudostratified epithelium composed of basal cells, a type of
progenitor cell, and luminal cells including secretory and
ciliated cells. The authors confirm that normal bronchial
generated organoids maintain the histological and genetic
characteristics of their respective parental tissues, and thus
have potential for use in patient-specific drug trials and proof-
of concept studies on targeted therapy and resistance
mechanisms.
Also starting from biopsy material, Bui and colleagues
91
generated organoids from differentiated primary human
bronchial epithelial cells and type-I-like alveolar epithelial
cells from nontumoral residual tissues of patients undergoing
surgical resection. The resulting airway organoid was positive
for a variety of lung epithelial cells, such as goblet, basal, and
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ciliated cells, along with the presence of mucus within the
lumen and active ciliary beating. The model was used to study
viral infection, and the authors show that the virus infected the
organoid cells, demonstrating a similar tropism to what is
observed in the in vivo human airway. This indicated the
possible use of this method to study viral infections in general.
Considering studies that already managed to investigate the
SARS-CoV-2 infection in organoids, Salahudeen and col-
leagues
71
used biopsy-derived human lung distal pulmonary
tissue in order to generate AT2 and basal organoids, which
were later exposed to SARS-CoV-2 (and H1N1) in order to
better investigate the viral tropism in the pulmonary tissue.
The authors discovered that, in relation to SARS-CoV-2, the
virus targets in particular club cells, while ciliated cells seem
not to be infected.
Wang and colleagues used club and AT2 cells isolated from
adult mice to generate lung organoids, which were tested to
investigate drug efficacy in SARS-CoV-2 infections. Although
the effect of drugs on organoids cannot be fully translated to
clinic expectations, this first glimpse using organoids exposed
to SARS-CoV-2 is very useful in the search for new
therapeutics.
92
Similarly, Youk and colleagues
93
also used AT2 cells, but
isolated from biopsy material from human lung tissue. The
results indicate that AT2 cells serve as stem cells in the model,
generating a feeder-free human 3D alveoli-like organoid,
suitable for self-maintenance for at least 6 months.
Interestingly, authors also managed to infect the model with
SARS-CoV-2 and showed that alongside AT2-infected cells,
viral transcripts were also found in the supernatant, suggesting
secretion of viral particles by infected cells. These findings
prove that the model successfully replicated key aspects of
SARS-CoV-2 infection and simulate the viral propagation
within the organ and to outer structures similarly to the
circulatory viral spread observed in human patients. In
addition, after transcriptome investigation, it was shown that
both genes that regulate interferons and proinflammatory
genes were overexpressed, indicating that the model is able to
show an endogenous innate immune response after viral
infection. Likewise, Katsura and collaborators
94
isolated AT2
cells from human lung biopsies and generated alveolospheres
for subsequent SARS-CoV-2 infection. These authors also
developed mouse-derived alveolospheres for comparison, as
well as clinical analyses from human patients. The results
indicate that 3D-cultured AT2 cells expressed ACE2 and were
permissive to SARS-CoV-2 infection, and the culture presented
an inflammatory state post-infection, indicating a delayed
innate immune response, consistent with the investigation by
Youk et al.
93
In addition, there was a significant down-
regulation of surfactant proteins release in spheroids, similarly
to what is observed in human patients and that underlies the
alveolar collapse. In addition, the model was used to investigate
preinfection treatment with interferons, which was shown to
present prophylactic effects against SARS-CoV-2 infection, and
should be further investigated with regard to its potential
against the disease. Overall, the model is representative of
diverse responses observed in human in vivo lungs and also in
drug and therapeutic screenings for COVID-19.
More recently, Mulay and colleagues
95
also developed
alveolospheres, but in addition, they developed proximal
airway cultures, which are important for representing the
initial site of viral infection. The starting points were tracheal
and upper bronchial human tissue collected from deceased
organ donors.
95
For both types of culture, isolated cells were
cultivated with fibroblasts, and proximal airway culture was
maintained in ALI. After establishment, 3D cultures were
exposed to SARS-CoV-2, and the infection was heterogeneous:
at first stages, the virus predominantly targeted ciliated cells, in
accordance with previous investigations using in vitro models
and patient biopsies;
88,96
besides, proximal cultures were more
easily infected by the virus than distal cultures, which had to be
gently “opened”to be permissive. This step had to be
performed so that the apical cellular membrane, usually facing
inward, was exposed and allowed viral infection and
replication. Moreover, after AT2 cells were infected, a
proinflammatory response was observed, along with interferon
Figure 3. Airway organoid model possibilities. Airway organoid models can be derived to represent either proximal or distal airway. For cultivation
methods, they can be maintained in 3D configuration using scaffold, nonadherent surface, or transwell, or dissociated and kept as 2D monolayer for
easier pathogen exposure, for example; both methods can also be exposed to air using ALI. Organoids can be used to further explore the
pathophysiology of a SARS-CoV-2 lung infection, and to screen for therapeutics and prophylactics. Abbreviations: ALI: air liquid interface.
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Table 2. Recent Human Lung Organoid Models
a
differentiation
potential cell line origin initial 2D culture 3D induction
cell types in orga-
noid representation possible uses limitations refs
PS cells ESCs Standardized
commercial cell
lines (NIH)
PSCs induced to endoderm,
induced to anterior fore-
gut, induced to spheroids
Transferred into scaffold
+ Transwell insert
(ALI)
Endoderm cells Distal airway Viral Infection, due to air exposure Fetal-like lung model Zhao et al.,
2020
74
Transferred into scaffold
+ in vivo transplanta-
tion for maturation
Airway epithelial
cells + support
tissue
(fetal + adult)
airway
Adult-like lung model Necessity of vivo trans-
plantation (use of ani-
mals); only airway repre-
sentation
Dye et al.,
2020
77
Transferred into scaffold Epithelial + mesen-
chymal cells;
large fraction of
AT2 cells
Airway + al-
veoli
Mimic lung diseases (fibrosis) Fetal-like lung model Strikoudis et
al., 2019
78
Mesoderm + endo-
derm cells
Distal airway
+ alveoli
Respiratory viral pathogenesis in
infant lung
Porotto et
al., 2019
80
iPSCs Skin biopsy (fi-
broblast)
iPSCs induced to endoderm,
induced to anterior fore-
gut, induced to lung pro-
genitors
Transferred into scaffold Epithelial + mesen-
chymal cells
Proximal and
distal airway
+ alveoli
Disease-specific targeting; Surfac-
tant metabolism
Fetal-like lung model; not
vascularized
Leibel et al.,
2019
79
MS cells Mesenchymal
Endothelial
Epithelial
Stroma donation;
Standardized
commercial cell
lines
Separated cell lineages culti-
vated
Transfer all cultures
transferred into micro-
fluidic semipermeable
membrane
Stromal, endothe-
lial, epithelial
cells
Airway Effects of drugs; interaction with
other tissues (6 tissues)
Necessity of a chip tech-
nology; only airway rep-
resentation
Skardal et
al., 2020
83
PS cells - Fetal
bud tip pro-
genitors
Human lung fetal
tissue (+12
weeks)
Bud tip progenitors isolated
and cultivated
Transferred into scaffold Endoderm, meso-
derm, epithelial
cells
(fetal + adult)
airway
Study human lung; regenerative
medicine, tissue engineering, and
pharmaceutical safety and efficacy
testing
Need for vivo transplanta-
tion (use of animals);
only airway representa-
tion
Miller et al.,
2019
57
and
2020
75
AT2 cells isolated and culti-
vated
AT2 cells mainly Alveoli Induce and test genetic manipula-
tion
Fetal-like lung model Shiraishi et
al., 2019
85
AT2 and fibroblasts coculti-
vated
AT2 + fibroblasts Trachea Fetal-like lung model; only
airway representation
Mesenchymal Adult tissue (bi-
opsy) (pedia-
tric patients)
Tissue dissociation; mesen-
chymal cells isolated; cul-
tivated in monolayer
Transferred into scaffold Mesenchymal cells Alveoli Study congenital lung lesions and
COPD
Postnatal lung model; not
yet known if adult-derived
tissue evokes adult-like
tissue
Wang et al.,
2020
89
AT cells Bronchial pro-
genitor cells
Adult tissue (bi-
opsy)
Tissue dissociation; cell iso-
lation; monolayer cultiva-
tion
Transferred into scaffold Epithelial cells Distal airway
+ alveoli
Develop patient-specific drug trials Absence of stromal and
immune cells
Kim et al.,
2019
90
Bronchial and
type-I-like al-
veolar cells
Bronchial progeni-
tor cells + AT1-
like cells
Airway Viral infection (Influenza B) Absence of stromal and
immune cells; only airway
representation
Bui et al.,
2019
91
a
Pluripotent stem (PS) cells); ESCs (embryonic stem cells); induced pluripotent stem cells (iPSCs); multipotent stem (MS) cells; adult tissue (AT) cells; AT1 (alveolar type 1) cells; AT2 (alveolar type
2) cells.
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Table 3. Recent Human Lung Organoids Models Used to Study SARS-CoV-2
a
differentiation
potential cell line origin initial 2D culture 3D induction cell types in organoid representation
applications in
study limitations refs
PS cells PS cells -
Fetal bud
tip progeni-
tors
Standardized
commercial
cell lines
(NIH)
PSCs induced to endoderm,
induced to anterior foregut,
induced to spheroids
Transferred into scaffold + in vivo
transplantation for maturation
Airway epithelial
cells
Airway + al-
veoli
Exposure to
SARS-CoV-2
and drug
screening
Necessity of vivo transplanta-
tion (use of animals); only
airway representation
Han et al.,
2021
76
iPSCs Standardized
commercial
cell lines
(SPC2)
iPSCs induced to iAT2 iAT2 cells cultivated as alveolospheres;
then, cells were dissociated and
further maintained in 2D ALI
iAT2 cells Alveoli Exposure to
SARS-CoV-2
and drug
screening
Lack of AT1, mesenchymal
and immune cells
Huang et al.,
2020
82
MS cells PS cells -
Fetal bud
tip progeni-
tors
Standardized
commercial
cell lines
(NIH)
PSCs induced to endoderm,
induced to anterior foregut,
induced to spheroids
Transferred into scaffold + Transwell
insert (ALI)
Lung epithelial cells
(diverse subtypes;
specially AT2)
Distal airway Exposure to
SARS-CoV-2
and drug
screening
Fetal-like lung model. Lack
mesenchymal and immune
cells
Samuel et al.,
2020
84
Fetal bud tip
progenitors
Human lung
fetal tissue
(+12weeks)
Mostly alveolar cells AT2 cells cultivated as organoids; then,
cells were dissociated and further
maintained in 2D ALI
Club, globet, ciliated
and alveoli-like
cells
Alveoli Exposure to
SARS-CoV-2
and drug
screening
Lack of mesenchymal and
immune cells
Lamers et al.,
2021
86
AT cells Lung cells Adult tissue (bi-
opsy)
Tissue dissociation; cell iso-
lation; monolayer cultivation
AT2 cells cultivated as organoids; then,
cells were dissociated and further
maintained in 2D
AT2 Alveoli Exposure to
SARS-CoV-2
Absence airway and single
type cellular representation
Youk et al.,
2020
93
AT2 cells cultivated as alveolospheres;
then, cells were dissociated and
further maintained in 2D
AT2 Alveoli Exposure to
SARS-CoV-2
and treatment
Absence airway and single
type cellular representation
Katsura et al.,
2020
94
Transferred into scaffold Club cells, ciliated
cells and AT2 cells
Distal lung H1N1 or SARS-
CoV-2
Absence of stromal and im-
mune cells
Salahudeen et
al., 2020
71
Airway tissue Tissue dissociation and
monolayer cultivation
Cell cultivates with fibroblast, proximal
airway maintained in ALI
Proximal airway
cells; distal airway
cells
Proximal and
distal airway
Exposure to
SARS-CoV-2
Lack immune cells Mulay et al.,
2020
95
Cells cultivated as organoids; then, cells
were dissociated and further main-
tained in 2D ALI
Small airway epithe-
lial cells
Airway Exposure to
SARS-CoV-2
and drug
screening
Lack of mesenchymal and
immune cells
Lamers et al.,
2021
86
a
Pluripotent stem (PS) cells); ESCs (embryonic stem cells); induced pluripotent stem cells (iPSCs); multipotent stem (MS) cells; adult stem (AS) cells; AT1 (alveolar type 1) cells; AT2 (alveolar type
2) cells; iAT2 (induced alveolar type 2) cells; ALI (air−liquid interface).
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pathway upregulation, in accordance with other authors
investigating lung organoid SARS-CoV-2 infection;
93
this
response was followed by an increase in proteins associated
with cell-autonomous and non-cell-autonomous apoptosis,
which contributes in the long run to alveolar injury.
In addition to the human fetal bud tip lung organoids,
Lamers and colleagues
86
also derived airway organoids from
adult human donors. Following a similar protocol, after
organoid generation, cells were dissociated and seeded on
transwells to induce 2D ALI; these cultures were compared to
the fetal-derived culture, already described. The model
represents the small airway epithelium, also very valuable to
investigate viral infection.
A systematic example of organoid cultivations mentioned in
the articles present in this review and used in COVID-19
investigations are listed in Figure 3. A summary of the
mentioned models suitable for infectious diseases can be seen
in Table 2, and airway models specifically for COVID-19
investigations are listed in Table 3.
Future Perspectives in COVID-19. Due to the possibility of
reproducing the in vivo tissue in culture, human lung organoids
can provide robust support to simulate SARS-CoV-2 infection
and replication in humans. The virus tropism in the organism
tissues is not completely understood, due to the lack of suitable
research models that allow this investigation; thus, mechanisms
of SARS-CoV-2 pathogenesis mainly depend on clinical
characteristics and autopsy reports and bioinformatics
analysis.
35
Although it seems that alveoli are the most affected
structures during severe COVID-19, it has already been
demonstrated that the distal airway as a whole is more
drastically affected, because SARS-CoV-2 can significantly
infest a higher number of cells, due to an increased expression
of ACE2 proteins in cells of the inferior respiratory tract; in
addition, ciliated cells from the superior respiratory tract tend
to direct the viral load to the esophagus (digestive system),
while viruses that reach the larynx (inferior respiratory tract)
continue in the descendant path toward the alveoli, indicating
that once SARS-CoV-2 manages to infect the inferior tract, it
can replicate and continue to infect respiratory cells.
8,97
Previous studies indicate a high prevalence of type II
pneumocytes, bronchial epithelial cells, and AT2 cells as
major targets of SARS-CoV-2 infection.
98,99
In the present
review, the most recent protocols to develop human airway
organoids are listed and briefly described, and they include
models that replicate alveoli in specific and distal airways, and
also the proximal airway; in addition, these models can be
developed from diverse origin cells, from PS such as ESCs and
iPSCs, MS such as mesenchymal but also fetal cells, to AS cells.
Thus, such organoid models can be adapted and employed to
help better understand COVID-19 and SARS-CoV-2 behavior
in the lung and, as a consequence, provide insights on possible
interventions such as treatments and vaccines.
In addition, according to Elbadawi and Efferth,
100
as recently
reviewed, the coculture of alveolar cells with cells from
different tissues would be ideal for COVID-19 investigations,
such as immune cells, which would deliver an overview of the
immunological response to the virus and effects of
immunomodulatory drugs. Adding vascular cells such as
endothelial lineages to the organoid model could also represent
an interesting approach, since the virus reaches the circulatory
flow through intimate contact with the pulmonary capillary
bed, as also reported for SARS-CoV
101
as well as SARS-CoV-
2.
102−104
This could be more easily modeled using a transwell
or by adding the ALI system, inducing mucus production,
formation of stratified epithelium, and functionality of
pulmonary cells for diverse pathogen-induced pulmonary
diseases such as tuberculosis.
105,106
These particularities will
allow further investigation of viral effects on human pulmonary
tissue, virus tropism, and cytokine release and permit
investigation of therapeutic drugs, with the help of
complementary methods, such as polymerase chain reaction
(PCR), Western blot, and immunohistochemistry.
100
Thus,
lung organoids are very helpful for high-throughput assays for
host−pathogen interaction and outcome characterization, such
as proteomics, phosphoproteomics, and global transcriptome,
among others.
107
These approaches enable the achievement of
tissue-specific results, without the interference of systemic
factors, and provide insight on the physiology and pathophysi-
ology of the target organ, such as the lungs.
In addition to the pulmonary tissues, several researchers
have shown that COVID-19 patients showed multiorgan
damage and dysfunction, possibly also leading to multiorgan
failure.
5,7,96,108
In view of the need to understand the tropism
of SARS-CoV-2 in the human organism, other 3D approaches
than lung organoids, including microfluidic on-a-chip tech-
nologies, that mimic in vitro the interaction of different organs,
must be explored. As an example, Zhao and colleagues
15
(already described under PS Cell-Derived Organoids)
developed an integrated system with six different organoids
and simulated a circulatory system among them. Considering
what is now known for SARS-CoV-2 infection and how the
virus spreads throughout the body to induce COVID-19-
related alterations, a more detailedand integratedin vitro
replication such as these could give a more complete
perspective on how we could overcome COVID-19 and the
SARS-CoV-2 pandemic.
Considering all the possible respiratory tract organoid
models to investigate COVID-19, either representing proximal
or distal airway, it is important to consider that in spite of the
advantages, there are some important limitations. A major
downside is the absence of vasculature and immune cell
components in the majority of the mentioned protocols,
crucial for the systemic understanding of a viral infection. In a
physiologically relevant in vitro model of study, in such cases, it
is important to ensure that all prominent cellular components
are present in the cultivation.
109
In the will to overcome this
barrier, some researchers have developed 3D cell culture
models using commercially available cell lines and artificially
combining AT2, endothelial, macrophages, and mast cells,
cultivated in ALI.
110
Such tetra-culture can also be very
valuable for COVID-19 investigation, since it represents
mature alveolar tissue. Similarly, there is increasing inves-
tigation using artificially assembled microfluidic approaches
that enable the cocultivation with capillary-like structures, as
well as exposure to immune cells, with the advantage of also
presenting exposure to “blood”flow.
111,112
To date, there are
reports of airway microfluidic cultures, cocultivated with
vascular and immune cells, for posterior exposure to
pathogens, including SARS-CoV-2.
38,102−104
The microfluidic
upside is that the blood-like tissue is pumped throughout the
culture system, using a fluid flow, simulating the in vivo
situation.
111,112
There are reports of the establishment of distal
airway microfluidic models, or specifically alveolar cultures,
cocultivated with microvasculature
102,104
and immune
cells
102−104
on a chip, for further SARS-CoV-2 exposure and
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investigation. The results indicate the important involvement
of immune responses through the alveolar barrier, showing that
the microfluidic system resembles in vivo situations and could
be valid for research that requires interaction to blood and
immune cells.
A combination of organoid and chip technology was
proposed in 2010,
113
and since then further developed.
Organoids-on-a-chip improve the efficiency and reproducibility
of organoid cultures.
114
In this approach, diverse cultivation
parameters can be precisely controlled by the chip technology,
with the cell development and spontaneous arrangement
typical from organoid culture.
114
Considering that microfluidic
devices provide long-term culture system, they are able to
deliver nutrients, metabolites, and gas exchanges to the
organoids via laminar flow.
115
Furthermore, it is possible to
induce vascularization in the organoids by the microfluidic chip
and, with this, increase representability and organ function-
ality.
116
Though promising, this type of model presents some
limitations, in particular in relation to the necessity of external
pumps and connectors to correctly operate, which reduces the
patterning and elevates the costs.
114
Still, it is expected that, in
the future, such models could be more easily replicated and
accessible to a higher number of researchers.
■CONCLUSION
In the current scenario, where humankind is confronted with a
new and highly infectious virus, with so many aspects yet to be
discovered, the access to models that consistently represent the
human organs on a dish is undoubtedly urgent. Organoids and
other 3D models are valuable complementary tools to
investigate COVID-19 pathophysiology and the effects of
SARS-CoV-2 in human tissue and further enable the
development and validation of therapeutics and prophylactics.
■AUTHOR INFORMATION
Corresponding Author
Marna E. Sakalem −Department of Anatomy, CCB, State
University of Londrina (UEL), 86057-970 Londrina,
Parana, Brazil; orcid.org/0000-0002-3143-4093;
Email: marna7@gmail.com,marna@uel.br
Authors
Miriane de Oliveira −Department of Internal Clinic,
Botucatu Medicine School, São Paulo State University
(UNESP), 18618-000 Botucatu, São Paulo, Brazil
Maria T. De Sibio −Department of Internal Clinic, Botucatu
Medicine School, São Paulo State University (UNESP),
18618-000 Botucatu, São Paulo, Brazil
Felipe A. S. Costa −São Paulo State University (UNESP),
School of Agricultural Sciences, Department of Bioprocesses
and Biotechnology, Central Multiuser Laboratory, 18610-
034 Botucatu, Sao Paulo, Brazil
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsbiomaterials.1c00306
Author Contributions
All authors performed the literature search, drafted sections of
the manuscript, and prepared figures and tables. M.E.S.
supervised the manuscript and reviewed the language. All
authors subsequently revised the manuscript.
Funding
This study was financed in part by the Coordenaçãode
Aperfeiçoamento de Pessoal de Ni ́
vel Superior (CAPES) −
Brazil −Finance Code 001 (Process number 88887.503344/
2020-00).
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
All Images were created with Biorender.com.
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