Recent advancements in the development of organoid cultures and

organ-on-chip models for studying COVID-19 infections

Mina Zarea, Saptashwa Datta a, Seeram Ramakrishna a*

a
Center for Nanotechnology and Sustainability, Department of Mechanical Engineering, National
University of Singapore, Singapore 117581, Singapore

* Corresponding author seeram@nus.edu.sg

ABSTRACT

COVID-19 is a multi-systemic disease caused by the infection of the SARS-CoV-2 virus. The
infections caused by these viruses have affected many people throughout the world causing a
worldwide pandemic and has resulted in thousands of deaths. In such conditions it is very important
for to develop various models for studying the disease and screen drugs for them. Cell-based
disease models have been prevalently used for studying infectious diseases and screening drugs for
them due to their ease of maintenance compared to animal models. Moreover, results can be more
rapidly obtained using such models. In the past decade, organoids and organ-on-chip models have
been some of the greatest discoveries in cell-based models. They provide a much closer mimic of
the natural physiology than routine two dimensional cell culture models. Multiple articles have been
published on the usage of various in-vitro and in-vivo disease models for comprehending SARS-
CoV-2 infections and for screening drugs against the virus. Organoids and organ-on-chip based
models have been some of the most widely used systems for studying this particular virus. They
have been used to generate insights ranging from understanding the receptors involved in
pathogenesis, immune response against the virus, understanding integrity of cellular barriers on
viral infection, to understanding transcriptome variations between the infected and control cells. In
this review, we elucidate on the recent advances in the development of organoids and organ-on chip
models for studying COVID-19 infections. Furthermore, the various insights about SARS-CoV-2
pathogenesis derived from various organoid studies and the applications of such organoids in drug
discovery and repurposing were discussed.

Keywords: SARS-CoV-2; Organoids; Organ-on-chips; Drug screening; Disease models

1. Introduction

Respiratory viruses have wreaked havoc all over the world throughout the years [1,2]. One such
group of viruses known as the coronaviruses are a batch of enveloped and highly pathogenic
single stranded RNA viruses that are known to reside in multiple host organisms. Many of these
viruses cause severe respiratory disorders in humans resulting in high mortality rates. Moreover,
another dangerous aspect of the virus is that it has the ability of cross species transmission. There
have been multiple outbreaks of these viruses in different countries. Two of the most well-known
viral epidemics in the past due to coronaviruses took place in China and the middle east. These
viruses were named as the SARS-CoV (Human Severe Acute Respiratory syndrome-
Coronavirus) and the MERS-CoV (Middle east respiratory syndrome coronavirus) respectively
and had caused multiple deaths in these two regions [3]. A member of beta-coronaviruses named
as the 2019-nCoV (also known as SARS-CoV-2) infected a lot of people worldwide after its
initiation in China in the month of December of the year 2019. From the beginning of 2020, this
virus has spread throughout the world resulting in a highly infectious and dangerous global
pandemic that has taken multiple lives and infected a huge portion of the total world population.
The disease which has been termed as COVID-19 is characterised by a sudden onset of
respiratory distress. The major cause of mortality due to COVID-19 infections is progressive
respiratory failure due to alveolar damage. This results in hypoxia and can lead to death of tissues
in the body [4-6]. Other than affecting the respiratory system, this virus has also been seen to
have pathogenic effects on a plethora of other organs like the brain, liver, kidney, heart, intestines
and the eyes. The hyper-production of certain pro-inflammatory cytokines like IL-6, IL-8 and
TNF- can lead to the impairment in the normal functioning of the coagulation signalling pathway
leading to thromboembolism and haemorrhages in various organs. This virus is known to enter
the cells by binding to the Angiotensin Converting Enzyme 2 (ACE2) receptors which are present
on the tubular epithelium cells of various organs[7]. Recently some studies have also revealed the
presence of Coronavirus infections in discharges like tears and conjunctival secretions from
patients in various countries like Italy and China. However, the probability of samples obtained
from the ocular region testing positive for SARS-COV-2 is very low[8-10]. Figure 1 illustrates a
brief on the organs effected by the SARS-CoV-2. The virus has been reported to induce damage
in multiple organs including lungs, brain, eyes, heart, kidney, liver, and intestines. These organs
can be systematic targets for studying SARS-CoV-2 pathogenesis. It has been observed that cells
which have high expressions of ACE2 and TMPRSS2 are infected by this virus. The viral S1
spike protein has been observed to bind to ACE2. A better understanding of the damage induced
by the coronavirus on each of the organs can help tailor target specific drugs. The pathogenesis of
the virus and its effects on the various organs are still not very well understood. Hence it is
important to develop and use various disease models to study the pathogenesis of the virus.
Besides, it is essential to customise such models so that they can be used to study and screen for
various drugs. One of the most commonly used preclinical tools nowadays are organoid cultures.
Although there is a huge heterogeneity in the definitions of organoids, it is generally referred to
as progenitor or induced pluripotent stem cell derived cultures with multiple cellular lineages and
can show the organisation and characteristics of in-vivo tissue environments[11]. Over the past
decade, organoid cultures have been popular for studying host pathogen interactions for both
bacteria and viruses. These organoids are mostly used for studying parts of the cycle of infection
in specific tissues. This cycle includes modes of transmission, mechanism of entry and exit from
the cells, the reservoir of pathogens and the susceptibility of the host to the pathogen[12]. These
organoid cultures have been used extensively for screening drugs and studying their toxicity and
efficacy. Although most of the available techniques try to create common organoid models for
testing drug toxicity and efficacy, many of these models fail to provide results that can be
replicated in-vivo. Hence it is a good idea to customise such models, so that can be used to screen
for specific biomarkers that suits the particular necessary parameters of the drug and the
disease[13]. Another major model for rapid drug repurposing and understanding host-pathogen
interactions are organ-on-chip models. These models consist of microfluidic chips where two
dimensional cells are cultured on different surfaces to mimic in-vivo conditions. These models
have been used extensively for understanding how to pathogens behave in a mobile in-vitro
environment. Multiple organ-on-chip models have been developed for mimicking the function of
organs from different parts of the body. These models have been used extensively for
understanding the pathogenesis of a wide number of viral diseases as well as screening drugs for
this disease[14]. In this review, we discuss in details about the various organoid and organ-on-
chip models that have been engineered and developed for studying the mechanisms of SARS-
CoV-2 pathogenesis along with screening and testing drugs for the disease caused by this virus.

2. Organoid cultures for studying SARS-CoV-2 pathogenesis

Animal models have been used classically over the years to study host-pathogen interactions.
However, over the years it has been observed a lot of variation between the studies carried out in
these model animals when compared to humans. Moreover, another huge disadvantage is the
associated costs of maintaining the animals for the studies. Over the years monolayer cell cultures
derived from various human cells have been used to study these host pathogen interactions.
However, due to the lack of a three dimensional physiological environment consisting of multiple
types of cells interacting and communicating with each other, the translational potential of
monolayer cultures still remains quite low. Over recent years, three dimensional cell culture models
called organoids which take the best out of both the worlds have been introduced for studying host-
microbe interactions. These three dimensional in vitro cultures consist of cells from different
lineages and are able to replicate normal physiological functions till a certain extent. A number of
viral infections have been studied over the years using organoids cultures including Epstein Barr
virus, Norovirus, Rotavirus, Zika virus, Hepatitis A, B, C and E, Chikungunya virus, Japanese
encephalitis virus, Influenza, Rhinovirus and Rous sarcoma virus. These infections have been well
studied using a variety of organoids including intestinal organoids, colon organoids, brain
organoids, stomach organoids, liver organoids and lung organoids[12,15]. As discerened in figure 1,
COVID-19 has been observed to affect multiple organs, and hence studying the pathogenesis of
SARS-CoV-2 on these various organs is important[7]. It requires a lot of time, animals and money
to study the pathogenesis of this virus on different organs in animal models. Hence, cell culture
models prove to be a better option for studying the multi-systemic nature of COVID-19. However,
as monolayer cultures do not mimic the physiological conditions, much of the results obtained may
have limited translational potential. Another great drawback of monolayer cultures is that there is a
lack of cellular heterogeneity and cultures derived from multiple types of cells would be required to
study the pathogenesis of the virus on a specific organ. Hence, organoids can be great models to
study the disease, especially the molecular mechanisms of infection and pathogenesis of the virus in
specific systems. All the more, individual cell lineages can be isolated from the organoids. Various
host animal derived organoids have been used for studying the pathogenesis of viruses. The
intestinal organoids derived from the SARS-CoV host horseshoe  bat (Rhinolophus sinicus) have
been used to study the pathogenesis of SARS-CoV[16]. Over the past few months, there have been
multiple studies involving the use of a wide variety of organoids for studying SARS-CoV-2
pathogenesis. Table 1 lists the various organoid cultures that have been employed for studying
COVID-19 infections and the insights that have been generated from these studies. The primary
studies that have been performed using various organoids are to understand whether or not the
SARS-CoV-2 viruses are able to infect and replicate in various types of organoids.

2.1 SARS-CoV-2 pathogenesis in lung organoid models

One of the primary organs that have been observed to be affected by the SARS-CoV-2 virus are the
lungs[38-40]. Hence, various types of lung organoids like alveospheres, human alveosphere
organoids, human bronchial organoids, and human airway organoids have been generated over the
past few months to study the process of viral infection in the lungs. From various previous studies,
it has been noted that type 2 transmembrane serine protease (TMPRSS 2) and Angiotensin
converting enzymes are two primary entry factors that are required for SARS-COV-2 infections
[41-43]. In the various studies involving lung organoids, it was observed that these two genes were
highly expressed in the cells that got prevalently infected. In a study involving the fabrication of
human airway organoids from adult human stem cells, it was observed that ACE2 and TMPRSS2
are expressed in human airway cells in the organoid. This study revealed that these human airway
organoids were readily infected by SARS-CoV-2 virus and the viral titres were very high[30]. In a
similar study it was found that SARS-CoV-2 successfully infected both human alveolar organoids
and human airway organoids derived from human embryonic stem cells. Moreover, both types of
organoids were observed to have cells that expressed both ACE2 and TMPRSS2[22]. Organoid
cultures can be used to understand which population of cells in the organoids are more susceptible
to the viral infection. Human alveolar Type 2 cells that were derived from human alveospheres were
noticed to get easily infected by the SARS-CoV-2 virus. About 40% of these cells were observed to
express ACE2 whereas 80% of the total cellular population was noted to express TMPRSS2 [18].
Basal cells were found to express both TMPRSS2 and ACE2 in bronchial organoids cultured from
human adult bronchial cells. Subsequent experiments involving the introduction of the virus to the
organoid showed that the viral markers were found to localise in these cells suggesting that the virus
infected and replicated only in these cells[20]. However, in a similar study involving lung organoids
derived from human embryonic stem cells it was observed that the virus infected a majority of
ciliated cells and a few club cells but not any basal cells and goblet cells[22]. Notwithstanding the
possibility of bias or inconsistent experimentation, these results can suggest the possibility that lung
organoids derived from embryonic and adult stem cells can have a difference in gene expression.
Further studies like single cell transcriptomics of organoids derived from different sources can help
to identify if there is a heterogeneity in gene expression.Another study done in apical out mixed
distal lung organoids derived from distal airway cells identified club cells as the most infected
cellular population. They reported that ciliated cells and basal cells were not infected in these
organoids[28] . Ciliated cells were the most infected cell population in human alveolar organoids
that were established from a mixture of distal lung epithelial cells and MRC5 human lung fibroblast
cells. However, it was observed that a few mucin Mu5ac producing cells were infected by the
virus[32] . Another study where human airway organoids are established from human bronchial
airway stem cells also reports ciliated cells as the most predominantly affected cell population [33].
These organoids have been used to study the pathogenic effect of the viral on cells. Transmission
electron microscopy (TEM) analysis reveals a lot of pathological changes in cells of the alveolar
and bronchial organoids derived from alveolar type 2 cells. Particular aggregates of the viral
proteins were found near the nuclei of the human alveolar organoids. The various cells of the
alveoli had vacuoles that were quite large in size. Other than this various surfactant proteins and
secretory vesicles were observed to be localised in the cytoplasm. At early stages of the infections,
it was observed that the cells contained vesicles which had two membranes (also known as double
membraned vesicles or viral replication sites). These were seen to be present near the zippered
endoplasmic reticulum (ER) which is one of the favourable places for viral assembly and replication
[44]. The virions were interspersed in the cytoplasmic area either freely or enclosed in vesicles.
Secretions from the infectious virus was noted on the apical sides of the AT2 cells[30]. On
performing immunostaining procedures, it was found that virus infected alveosphere cells tested
positive for the marker caspase 3 which shows that apoptosis took place in cells that were infected
when compared to the controls [18]. Pathological experiments in another study involving bronchial
organoids showed that there was a condensation of chromatin (also known as pyknosis) in the
infected cells, which is a sign of the cells becoming apoptotic [20]. TEM analysis in another study
identified that the virion particles localise inside the lamellar bodies inside AT2 cells of the
organoids. The SARS-CoV-2 virion particles were present throughout the organoids in their lateral,
basolateral as well as apical side. The study observed that there were virion particles with dead cell
debris bound by late endosomes. Immunostaining for apoptotic cell marker caspase3 showed that
there was a lot of apoptotic cells. The human alveolar organoids had more apoptotic cells than
human airway organoids. Other than this previously noted features like double membraned vesicles,
large vacuoles, protein aggregates etc. were observed in these infected organoids [22]. Figure 2
shows SARS-CoV-2 infections in alveospheres and lung organoids(Adapted from [18] and [30])

2.2 SARS-CoV-2 pathogenesis in brain organoid models

The effect of viral infection on the nervous system is one of the most widely discussed topics.
Patients with SARS-CoV-2 infection show various symptoms related to the neurological system.
These symptoms include encephalopathy, strokes and loss of various olfactory functions like taste
and smell [45]. Hence, it is important to understand whether or not SARS-CoV-2 is neurotrophic
and have direct implications in the damage of the nervous system. Cell culture and organoid models
can prove to be a great asset in understanding the SARS-CoV-2 pathogenesis in the brain. There are
several studies to understand SARS-CoV-2 pathogenesis using brain organoids. Dorsal forebrain
organoids were observed to have a constant expression of ACE2 throughout their development. It
was observed that 10 % of the neurons were affected by the pseudo SARS-CoV-2 virus [25]. It was
observed that the SARS-CoV-2 could successfully infect induced pluripotent stem cell derived
brain organoids. Further it was noticed that these viruses could infect neural progenitor cells and
neuronal cells from the cortical region [33]. In another study involving the use of mature 3D brain
organoids derived from induced pluripotent stem cells it was observed that these viruses infect
mostly neuronal cells. The virus is then again used for infecting an organoid that was cultured in an
alternate way allowing the neuronal regions to mature and be more viable. It was observed in these
organoids that there was not increase in the amount of viral mRNA in these organoids following
infection suggesting that active replication of the virus may not take place in the neurons. Another
experiment in this same study was performed to understand the localisation of the microtubule
associated protein tau which localises generally in axons of the neurons. It was observed using
confocal microscopy, that the localisation of tau in infected neurons changed from axons to somas
of the mature neurons. This mislocalization of tau proteins have been associated with tau related
diseases. Hyper phosphorylation of the protein is associated with various tau related diseases. It was
hypothesised that this hyperphosphorylation may be a result of premature phosphorylation at
multiple previous steps. It was observed that threonine 231 was phosphorylated in the tau proteins
(pT231tau) localised in the soma of infected neurons. This phosphorylated tau species has been
observed to localise in the axons. Terminal deoxynucleotidyl transferase dUTP nick end labelling
(TUNEL) assays helps to locate DNA fragmentation in dead cells. From this assay it was observed
that a number of neurons were TUNEL positive in the infected neurons. It was observed that a
certain number of cells in the infected neurons stained positive for caspase 3, which as mentioned
earlier is used as a marker for cell death. These caspase positive cells were observed to have
localisation of pT231tau inside the somas. These results suggested that there may be altered
phosphorylation and localisation of tau proteins that are induced by the SARS-CoV-2 virus which
can result in increased stress on the neurons finally leading upto death of the cells[36]. However,
although the previous study suggests that active viral replication may not take place in the neurons.
Another study electron microscopic analysis of induced pluripotent stem cell derived organoids
show the emergence of virion particles from the ER suggesting the possibility of viral replication.
However, based on the previous studies it may be hypothesised that the rate of replication of the
viruses may be low. Further studies should be done to understand the nature and rate of replication
of these viruses in neuronal cells. In contrast to the previously mentioned study, it was noted in this
study that lot of TUNEL stained cells were not infected by the virus. It was observed that non
infected cells in areas with high density of infectious cells showed higher number of TUNEL
positive cells (apoptotic cells) [27]. One of the other main things to study is whether or not the virus
affects the structural integrity of the cellular barriers in the brain. In a study involving choroid
plexus brain organoids derived from human induced pluripotent stem cells it has been observed in
that the epithelial cells of the choroid plexus are the predominantly infected cells. These results
however showed a large contrast with previously mentioned studies. Only when tenfold the amount
of virus required to infect choroid plexus cells were supplied, the virus was observed to infect the
neuronal cells. This suggests that choroid plexus cells have a higher infectivity than other types of
brain cells. Further elegant experiments performed in the study showed that the junction (tight
junction) between cells were heavily disrupted. It was found that the infected organoids had an
aberrant morphology and there was noticeable decrease in fluid inside the organoids. Moreover, it
was observed that the media became diluted suggesting the leakage of fluid into the media. This
study suggests that the choroid epithelium maybe disrupted allowing the leakage of fluids and also
allowing the passage of cytokines, cells of the immune system and pathogens into the brain [16]. In
a study building up on the previously mentioned results, various different types of human brain
organoids like cortical organoids, hippocampal, hypothalamic and midbrain organoids have been
derived from induced pluripotent stem cells to understand the pathogenesis of the SARS-CoV-2
virus in the brain choroid plexus. The virus was found to predominantly infect the choroid plexus
epithelial cells and a small population of neuronal and GFAP + astrocyte cells. The choroid plexus
cells derived from the organoids were found to express high levels of ACE2, NRP1 and TMPRSS2.
Immunohistology coupled with confocal microscopy showed the presence of fused cells with
multiple nuclei (synctia) and this has been previously noted in multiple published studies and
suggested that this may be a result of interactions in-between the SARS-CoV-2 viral spike protein
and the ACE2 receptor. This fusion of cells is suggested to be a major method of viral transmission.
This study noted that the infections caused cell death in both infected and uninfected cells
[23].Figure 3 is an illustration of infection, damage to barrier integrity and mislocalisation of tau
protein induced by SARS-CoV-2 in brain organoids

2.3 SARS-CoV-2 pathogenesis in intestinal organoid models

Diarrhoea has been a commonly observed infection in patients with COVID-19 infections. Hence it
has been widely suggested and shown in literature that the virus infects and replicates in the gut and
hence has enteric manifestions[46-49]. Intestinal organoids derived from intestinal crypt stem cells
isolated from Rhinolophus sinicus (horseshoe) bats and humans have been utilised for studying the
enteric effects of the virus. The cells in the human organoids that were infected by the SARS-CoV-
2 coronavirus were found to be positive for the cell marker villin suggesting that enterocytes maybe
the most commonly infected cells inside the enteroids[24]. It has been observed that many bat-
borne viral infections have been observed to infect humans over the years[50]. Besides, it has been
suggested in literature that the SARS-CoV-2 infection may have been transmitted to humans from
bats [51]. The SARS-CoV-2 was able to infect and replicate freely within the intestinal enteroids
derived from the horseshoe bat. Hence models like this can be used to study the origin and
evolution of viruses. In addition, studying the differences between infections in the human derived
and bat derived enteroids can help to understand and analyse about the transmission of such viruses
from bats to humans[24]. These findings were well backed up by another study which involved the
usage of human small intestinal organoids derived from primary gut epithelial stem cells. This study
noted that human enterocytes had a stable expression of ACE2 and were among the most infected
cells amongst the whole cellular population of the organoids. TEM analysis of the infected cells of
the organoids showed similarity to the lung organoid cells that were infected by the virus. The
virion particles are found to be interspersed in the cellular lumen. Double membrane bound vesicles
also known to be centres for viral replication were observed in the cells[34]. Figure 4 shows an
overview of infection of intestinal organoids by SARS-CoV-2.

2.4 SARS-CoV-2 pathogenesis in liver organoid models

Although there have been no significant correlations between liver dysfunction and COVID- 19
disease, it has been observed clinically that many people with the infection have abnormal levels of
alanine and aspartate aminotransferase enzymes. Elevation in levels of cholangiocyte damage
associated biomarkers have been observed in COVID-19 patients. Also, examinations of the
pathology of liver samples from infected patients have shown the presence of virus in the liver [52-
54]. Hence, cholangiocyte and hepatocyte organoids have been employed to understand SARS-
CoV-2 pathogenesis in the liver. A significant cellular population of the cholangiocyte and infected
hepatocyte organoids cultured from human pluripotent stem cells were observed to be infected by
the SARS-CoV-2 virus [19]. Another study involving the use of cholangiocyte organoids derived
from primary bile ducts showed that a specific number of cells in the organoids were infected but
robust viral replication took place in the organoids. On visualisation of the infected cells of the
organoids it was observed that the cells had merged and there was the presence of synctium. Further
analysis showed using real time PCR showed that there was a downregulation in claudin 1, apical
sodium-dependent bile acid transporter (ASBT) and cystic fibrosis transmembrane conductance
regulator (CFTR). The disruption of claudin1 marker suggests that there was disruption in the
integrity of the cholangiocyte barrier. ASBT and CFTR are proteins that helps in transportation of
bile. Disruption of the expression of these genes suggest that infection by SARS-CoV-2 virus may
induce damage to the cholangiocyte barriers and may cause impairement of bile transportation
[29].Figure 5 briefly illustrates the infection of cholangiocyte and hepatocyte organoids by SARS-
CoV-2

2.5 SARS-CoV-2 pathogenesis in tonsil organoid models

One of the primary barriers of defence of our body against pathogenic organisms are the palatine
tonsils. Some clinical studies have noticed tonsil enlargement in a small population of patients
suggesting a possibility of infection of the tonsils[55]. In a study human tonsil organoids generated
from human tonsil tissue to understand whether the possibility of infection of tonsils by SARS-
CoV-2 virus exists or not. Expression of SARS-CoV-2 viral receptor proteins TMPRSS2 and ACE2
was found in certain cellular populations of the organoids. On maturation of the organoids, it was
observed that expression of TMPRSS2 was upregulated whereas ACE2 expression was not
upregulated. Expression analysis of viral mRNA using quantitative PCR revealed that there was
active replication in the organoids. Further immonstaining and TEM analysis shows the presence of
viral particles in the basal region of the organoids. Aggregates of viral particles were observed near
the outer side of the apical membrane of the cells of the basal region [21].

2.6 SARS-CoV-2 pathogenesis in kidney organoid models

Acute Kidney injury has been associated with SARS-CoV-2 infection and as tubular cells of the
kidney have been observed to express ACE2, it is hypothesised that SARS-CoV-2 Infects kidney
cells[56-58]. Kidney organoids cultured from human embryonic stem cells were observed to get
infected by the SARS-CoV-2 virus. The virus was observed to replicate in the cells of the organoid.
Moreover, treatment with soluble recombinant human ACE2 reduced the viral infection in the
organoid, showing that ACE2 is an important cell entry receptor for the virus and it is produced in
kidney organoids [26]. Figure 6a) is an illustration of SARS-CoV-2 infection in kidney organoids

2.7 SARS-CoV-2 pathogenesis in blood capillary organoid models

Formation of clots in the blood vessels also known as vascular thrombosis and inflammation of
vascular cells have been commonly observed phenotypes in patients infected with the SARS-CoV-2
virus[59, 60]. SARS-CoV- 2 was observed to infect and replicate in capillary organoids cultured
from human induced pluripotent stem cells. Experimentation with soluble human recombinant
ACE2 showed that there was a significant reduction in infection in capillary organoids, showing
that ACE2 is an important entry receptor in them[25]. Figure 6b) is an illustration of SARS-CoV-2
infection in vascular organoids.These results have been quite consistent with studies from human
cohorts. In people with SARS-CoV-2 infection it has been noticed that there are vascular injuries
associated with the infection[61].

2.8 SARS-CoV-2 pathogenesis in ocular organoid models

Discharges from the ocular regions of some patients have been shown to be positive for the
presence of the novel coronavirus. Additionally, as the eyes are one of the well exposed areas of the
body, there are certain implications in viral spread through the eyes and can necessitate the use of
eye protection gear[9]. Hence to test the hypothesis, eye organoids were cultured from human
embryonic stem cells. Infection with the SARS-CoV-2 virus showed robust infection of the
organoids by the virus. Further analysis showed that corneal cells, cells of the limbus, cells of the
sclera and the retinal pigment epithelial cells were all infected by the virus. These cells have all
been observed to have significant ACE2 expression. The limbus had the most robust viral infection
and was observed to express high levels of the viral entry receptors TMPRSS2 and ACE2. As the
limbal region is the site of conjunctival and corneal stem cells, it is suggested by the authors that
there is a chance that this virus may exploit the ability of stem cells in this region to proliferate[31].

2.9 Transcriptomics reveals valuable insights in modulation of cellular pathways in infected

organoids

Transcriptomics has widely been used to study the pathways and genes involved in the response of
the organoids to the viral infections. Similarly, in most of the studies differential gene expression
has been studied in infected organoids with respect to uninfected controls. It has been observed that
genes related to cytokine pathways, chemokines pathways, interferon related pathways,
inflammatory pathways, NF-kB signaling pathway, and TNF signaling pathway were usually
upregulated in the SARS-CoV-2 infected organoids. Among the interferon pathway IFN gamma
related genes were highly upregulated in the organoids whereas there was slight or no induction of
expression genes related to interferon type 1 and interferon type 3 [18,19,20,22,23,24,31,32]. Genes
related to barrier integrity was downregulated in the cholangiocyte organoids There was a decrease
in the expresson of the enzymes ASBT and CFTR as mentioned earlier[29]. In alveosphere genes
related to the Th1 and Th2 activation pathways were downregulated[32]. Table 2 summarises the
various genes that are upregulated and downregulated commonly in cells of various types of
organoids. Hence, we can see from this section that organoids have been used to study multiple
facets of infection by SARS-CoV-2. Some of the important studies on SARS-CoV-2 pathogenesis
using organoids include i) studying about whether or not the viral infects cells of the organoids and
replicates in them, ii)studying about the integrity of the cellular barrier iii) studying about
expression of genes like TMPRSS2 and ACE2 which allow entry of the virus into the cells iv)
studying about the population of cells in the organoid that are infected by the virus v) observing the
morphological changes induced by the virus in the infected cells vi) learning about the death of cells
in the organoids induced by the infection vii) studying about upregulated and downregulated genes
and pathways using transcriptomics. Figure 7 displays a short overview of the timeline for the
utilisation of organoids for research. The progenitor cells, stem cells or induced pluripotent stem
cells are initially taken from previously established cultures or are isolated from tissues. The
pathways in the pluripotent cells are the activated, followed by embedding in matrices and
differentiation whereas adult or stem cells can be directly embedded and placed in differentiation
medium. The organoids are then infected with the SARS-CoV-2 virus or pseudo-virus. Various
experiments like immunocytochemistry, transmission electron microscopy, RNA sequencing etc.
are performed to understand several aspects of the viral infection.
Organoids can serve as robust models for gaining new insights into mechanisms entailing the
infections. Further studies should be carried out using organoids derived from other organs like the
heart which have been observed to be affected by SARS-CoV-2. Analysis of proteomes of the
infected organoids would be a great way to gain further insights into the process of infection [62].

3. Organ on Chip models for studying COVID-19

One of the main drawbacks of organoid systems are the lack of an integrated immune system.
Hence, it is difficult to study and understand the immune responses to infections. One of the recent
advancements in in-vitro cell culture models is the development of organ-on-chip technologies for
studying diseases. These provide an avenue to study circulating immune cell response in infectious
diseases [63]. These organ-on-chip models consist of a microfluidic device where cells are cultured.
They generally consist of two types of tissues patched by a porous ECM coated interface. Various
sensors and micro devices can be integrated into these devices to monitor certain conditions like
pH, electrical resistance and can be used to image the cells. There have been various organ-on-chip
devices including liver-on-chip, gut-on-chip, nervous system-on-chip, kidney-on-chip, and lung-on-
chip models that have been developed for studying a consortium of viruses[64,65]. Similarly, there
have been multiple studies that have employed these organ-on-chip devices for studying SARS-
CoV-2 pathogenesis. Table 2 briefs on all the organ-on-chip models used for studying COVID-19
infections. Most of the studies that reported the use of lung-on-chip models for studying the
infections. These devices consisted of three main parts i) upper alveolar epithelial channel, ii) a
porous membrane, iii) lower pulmonary endothelial channel. The porous membrane is usually made
up of polydimethylsiloxane (PDMS) and is coated with an extracellular matrix. However recent
studies have shown that membranes made up of other materials like nitrocellulose and the polyester
membrane can show a good viability of the cells cultured on it and also the cells adhere nicely to
the membrane [69]. Cells are cultured on both sides of the membrane. Usually alveolar epithelial
cells are cultured on the membrane and face towards the upper endothelial layer. The bottom layer
of cells grown towards the endothelial channel consists of pulmonary microvascular endothelial
endothelial cells. The upper channel is usually the air interface layer whereas the bottom channel is
used for introducing fluids and circulating immune cells[66,67,69,70,71]. In some studies
macrophages were cocultured with the epithelial cells on the membrane towards the upper layer
[70,71]. Human intestine-on-chip model which has been utilised for understanding the viral
infection had a similar structure and design but with different types of cells in the two channels. On
the upper channel intestinal epithelial cells and intestinal cells secreting mucin were co-cultured
whereas on the other hand vascular endothelial cells were cultured towards the upper channel.
Figure 8 shows a schematic representation of the organ on chip models devised for SARS-CoV-2
infections. The device consists of three layers i) apical channel, ii) porous membrane and iii)
vascular channel. SARS-CoV-2 diluted in liquid media is introduced to the apical channel for
infection. After the virus is allowed to infect the cells towards the apical layer are washed with PBS
and the excess fluid is removed so as to restore the air-liquid interface. Circulating immune cells
and fluids are introduced through the bottom vascular channel. Human alveolus on chip models
have been used extensively for studying SARS-CoV-2 virus infections. Both the epithelial
(HPAEpiC) and endothelial cell populations (HULEC-5a) were observed to express the entry
receptors for the viruses ACE2 and TMPRSS2. Subsequently on infection with SARS-CoV-2 it was
observed that there was a greater expression of viral proteins in the epithelial cells than in the
endothelial cells in the modelling and these cells were more susceptible to the virus. Besides, after a
few days there was an increase in expression of the viral proteins in the epithelial cells when
compared to the endothelial cells, suggesting that replication of the virus took place mostly in the
epithelial cells. Further RNA sequencing helped to recapitulate the previously mentioned results. It
was further observed from the study that both the cells types showed significant transcriptomic
changes when compared to the controld. Genes in the pathways related to apoptosis, cell division
and mitotic cell cycle were upregulated in the epithelial cells whereas the genes related to
cytokinesis, transcriptional factor activity and chemotaxis were upregulated in the endothelial cells.
Further introduction of circulating PBMC’s isolated from blood were introduced through the
bottom vascular channel to study how immune cells may contribute to the damage and
pathogenesis. It was observed that infection was present in the epithelial cell layer and the
expression of cadherin in both the cells types were deranged, suggesting that the infection of the
vcirus in presence of circulating immune cells affected the barrier integrity. Moreover, cellular
detachment and disruption was observed in the vascular cells. CD14+ monocytes were one of the
most predominantly recruited cellular populations in the endothelium layer. Administration of the
interferons IL-6 and IL-8 through the vascular channel increased the recruitment and adherence of
cells of the immune system towards the vascular cellular layer, thus suggesting that these may play
a key role in the increased immune response of the cells. Many cytokines including IL-1β, IL-6, IL-
8 and TNF-alpha had an increase in expression following the infection by the virus, especially there
was very marked increase in the expression of IL-1β and IL-6 from the layer of endothelial cells
from the vascular region. The epithelial layer present in the upper apical channel was observed to
secrete these cytokines[66]. Similar results were recapitulated in two other studies using alveolus-
on-chip models. In both the studies the epithelial cell layer was found to be infected whereas thet
endothelial layer had slight or no viral infection and there was a robust release of interferons
following the infection[70,71]. However unlike in most of studies, one of them observed that that
the virus also propagated basolaterally from the epithelial to the endothelial layer. However, this
type of transmission has usually not observed in SARS and SARS-CoV-2. The authors suggested
that this may be property that is unique to the epithelial cells of the alveolar region or this may have
occurred due to the contact and interactions between the cell in this particular model[70]. The cells
were imaged using scanning electron microscopy to understand the morphologies of the infected
and uninfected cells. There were residues of dead cells and dead cells that were present on the
epithelial side of the model. The number of dead cells were significantly higher compared to the
controls. Virion particles were found attached to the surface of dead cells[71]. In the intestine-on-
chip model, the upper epithelial layer was found to be predominantly infected by the virus. In
contrast to studies in the alveolus-on-chip models where disruption of integrity of tight junctions in
epithelial cells took place only following administration of immune cells, it was observed that a
severe disruption in expression of cadherins took place in the epithelial layer of the intestinal
region. The endothelial layer was found to have a severe disruption of adherent junctions. Further
experiments involving immunostaining of mucin secreting cells showed that there was a scattering
in the clonal distribution of these cells suggesting hampering of the mucus cellular layer.
Transcriptome studies showed that both the type of cells had an upregulation in the expression of
genes related to RNA and Protein metabolism pathways. Genes in pathways related to the immune
system like NF-kappa B and TNF-alpha pathways were significantly upregulated in the cells of the
endothelial layer [68]. This has been observed in human alveolus-on-chip models whereas NF-
kappa B pathway was significantly upregulated in the endothelial cells [71]. Further studies should
be carried out in intestinal-on-chip models by infusion of immune cells to understand whether and
how immune cells are recruited to the cellular region following the infection and whether or not it
has a pathogenic effect on the cells like it was shown in human alveolus-on-chip models[66].Figure
9 is an illustration of SARS-CoV-2 infection of alveolus-on-chip models in the presence and
absence of circulating immune cells. Similar studies in organ-on-chip from other regions like the
liver, kidneys and the neural region can help understand the pathogenesis of the virus better. Also,
they can help in better understanding of the role of circulating immune cells in the infection. Further
recently it has been observed that there have been variations in the fecal microbiota of the SARS-
CoV-2 infected patients [72]. Gut-on-chip models have been developed over the years for studying
gut microbiota and the use of such models can help in understanding the effect of SARS-CoV-2
infections on the gut microbiome [73]. Overall organ-on-chips provide a stable and reliable model
for studying SARS-CoV-2 infections, especially for understanding the effect of the virus on cellular
barriers and the process of recruitment of immune cells.

4. Organoid and organ on chip models and their role in screening and repurposing of drugs
One of the most important requirements during this viral pandemic is developing platforms for
rapid screening of drugs. Organoids and organ-on-chip technologies can both serve as excellent and
robust models for screening of drugs and testing their toxicities[13,74-76] . EC 50 values based on
cell viability have been the universally used measure for testing toxicity of drugs but these values
provided limited information about the actual toxicity of the drugs. As organoids can mimic the
three dimensional architecture and physiology, more standardised and robust toxicity models based
on specific toxicity markers can help in better understanding of the drug toxicity. In addition, these
models can help to study chronic effects of drugs on cells [13]. Both organoids and organ-on-chip
models have been used extensively for screening of drugs against COVID-19 disease. Table 4
briefly summarises the various organoid and organ-on chip studies that have been employed for
discovery and screening of drugs for COVID-19. Most of the drugs that have been screened using
these models are broad spectrum antiviral drugs that have been approved by the Food and drug
administration (FDA) and those that have been observed to show a good activity on decreasing viral
infections in two dimensional cell culture models. These drugs also predominantly belong to the
category of drugs that inhibit the viral replication. Lung organoids and lung-on-chip models are the
most commonly employed models that have been used for drug discovery. This can be attributed to
the fact that the lung is one of the most affected organs during COVID-19 [79]. Camostat [80],
Remedesivir [81], hydroxychloroquine[82] have been some of the candidate drugs that have been
found to have a beneficial effect on patients with COVID-19. Hence these were some of the
primary drugs that were tested using organoid models. In one of the studies 10 micromolar of
camostat was administered to the infected organoid for a period of five days. It was observed from
the study that the transcriptome of the infected organoid treated with camostat was closer to the
infected organoid than the control. However, it was observed that treatment with camostat help to
alleviate the hyperactivated expression of interferon-I related genes which were induced upon
infection [20]. Betastatin, Remedesivir and Camostat was screened using airway and alveolar
organoids in another study. 10 μM of all the three drugs were administered to the organoids
following two hours after the viral infection. Realtime PCR analysis of the treated organoids
showed that Remedesivir had a robust antiviral effect in both airway and alveolar organoids
whereas camostat on the other hand helped to decrease the viral load slightly in only human airway
organoids [22]. Another study using alveolar organoids recapitulated the findings of the previous
study. Administration of 10 μM of Remdesivir showed a significant amount of decrease in viral
titers (9log fold decrease). 10 μM of Hydroxychloroquine was found to have effect on decrease of
viral titers but it was significantly less than the results achieved using Remdesivir [32]. In a study
involving the use of human lung-on-chip models, similar results were observed. Administration of
1μM Remdesivir showed significant decrease in viral titres. Besides, in the earlier section it was
mentioned how the introduction of immune cells led to a significant loss of barrier integrity.
Treatment with remedesivir was observed to restore the integrity of the barrier in epithelial cells
[66]. On administering remdesivir to SARS-CoV-2 infected tonsil organoids, an inhibition in viral
infection was observed. Doses of the drug at and above 1 μM showed a great efficiency in the
reduction of viral infection in the tissues [21]. Enzalutamide is a drug which has been suggested as a
potential drug that can be repurposed for use in COVID-19. However, studies on lung organoid
models revealed that this drug wasn’t successful in inhibiting infections in this model although it
was able to inhibit infections in prostate cells. Furthermore, the molecular mechanisms underlying
this were further elucidated by studies. It was observed that there was a variation in patterns of
binding of Androgen receptor with TMPRSS2 in prostate cells and lung cells. The androgen
receptor has no direct binding with the TMPRSS2 in the lung cells and hence enzalutamide does not
work successfully. In a study a high-throughput chemical assay was performed using lung
organoids to discover novel compounds that inhibited viral replication. The library that was
screened consisted of multiple FDA approved drugs also collectively known as the Prestwick
library. The high-throughput screening platform consisted of 384 10% matrigel coated wells, each
containing 10k cells and 40 μl of medium. 10 microliters of each of the drugs were added to the
wells. Three compounds i.e imatinib, mycophenolic acid (MPA), and quinacrine dihydrochloride
(QNHC) were identified to be good inhibitors of viral entry from the study [77]. Airway-on-chip
high-throughput platforms have also been employed for screening multiple compounds. A library of
compounds consisting of modiaquine, toremifene, clomiphene, chloroquine, hydroxychloroquine,
arbidol, verapamil and amiodarone was screened for activity against SARS-CoV-2 infections. The
drugs were administered to the assay using a concentration ranging from 1μl 5 μl through the
vascular channel for a period of twenty four hours. From the library three of the drug were found to
be effective in reducing the viral load in the infected model. The screened drugs consisted of
amodiaquine, which was the most effective followed by toremiphene or clomiphene in order of
effectivity. The three drugs were found to have minimal cytotoxic effects on the cell-based model.
Further the drug Amodiaquine rapidly turned into its active metabolite form desethylamodiaquine
after administration. This showed that the metabolites derived from that drug could be potential anti
SARS-CoV-2 compounds [67]. Drugs against SARS-CoV-2 infections have been screened using
intestinal organoids. From the previously mentioned studies in this section we saw that remdesivir
was able to inhibit viral replication effectively in lung organoids. In this study the drugs remdesivir
and famotidine were administered to intestinal organoids to test their efficacy. 5 μM concentration
of remdesivir was found to almost completely abolish the infection in the organoids whereas 500nM
of the drug was observed to terminate the viral load in almost 86% of the infected cells. However,
famotidine was not able to inhibit infections significantly even at concentrations as high as 2.5 μM
[78].Figure 10 shows the effect of remedisivir and famotidine on intestinal organoids.Hence, we can
see that these organoid and organ-on chip models can serve as robust models for screening of drugs
against SARS-CoV-2. They can be used to develop high-throughput assays to screen large
compound libraries and can help in checking organ or tissue specific effectivity and toxicity of the
drugs. Further as observed from studies earlier we can concur that these models can be used for
determining the molecular mechanisms underlying the activity of the drugs. Organ-on chip models
provide a good platform for understanding drug responses in a mobile environment where immune
cells are being recruited post infection. However, toxicity of the drugs is still studied using
cytotoxicity as a primary measure. Further studies should involve the use of other more relevant
biomarkers for toxicity testing which can help better understand the effects of the drugs on the cells.
Integration of biosensors into the microfluidic organ-on-chip systems can help better understand
drug responses [83].

5. Conclusion and future perspective

The wide varieties of organoids and organ-on-chip models have been developed and utilized for
drug repurposing, screening, and studying the molecular mechanisms underlying infections. One of
the major problems with organoids research is the inconsistency in studies involving similar kinds
of organoids.. The organoids from similar regions and organs were found to be highly
heterogeneous and hence there were many contradictory and inconsistent findings. Hence one of the
huge gaps in the use of organoids for research in today’s world is the lack of standardized
nomenclature, definitions, and protocols. It is therefore very important to create a standardised
catalogue for the organoids. These catalogues can be made on basis of cellular heterogeneity in the
organoids, transcriptome or proteome profiles, and the source of the organoid (e.g Embryonic stem
cells, induced pluripotent stem cells, adult stem cells etc.). However, heterogeneity is not always
bad, as a “one model for all” usually sets up a path towards failure. However, there should be a
well-defined nomenclature system for these organoids and there should be model standards that
provide a point of reference to these organoids. In recent years with the advent of various tools like
CRISPRR-Cas for engineering biological systems, these organoids can be engineered to manifest
different diseased states [84]. Additionally, there have been huge and rapid developments in
technologies for studying organoids like high resolution, three dimensional imaging of the
organoids [85]. Overall organoids provide a good but still somewhat unreliable and inconsistent
model for studying viral infections. Further studies need to be carried out to understand the
heterogeneity of organoids. Organ-on-chip models have been efficient in understanding the cellular
replication of the virus and also understand how the viral infection affects the barrier epithelial cells
and endothelial cells of the vascular regions. Moreover, they have helped in understanding how the
immune cells are recruited in a mobile environment and how it reacts to the infection. However, the
studies that can be performed using organ-on-chip models still remain quite limited and provide
limited information about infection and drug response. Recent developments in microfluidic based
platforms like organoids-on-chip [86] and multi-organ micro-physiological models [87] can prove
to be excellent models for studying infections, screening of drugs and, understanding the drug
response in COVID-19.

Author information

Affiliations
Mina Zare, Saptashwa Datta, Seeram Ramakrishna
Center for Nanotechnology and Sustainability, Department of Mechanical Engineering, National
University of Singapore, Singapore 117581, Singapore

Author Contributions
Saptashwa Datta and Mina Zare researched data for the article contributed substantially to the
discussion of the content and wrote, reviewed, and edited the manuscript before submission.Seeram
Ramakrishna approved the final version to be published and supervised the work.
The authors contributed to the finalization of the manuscript.

Corresponding author

Correspondence to Seeram Ramakrishna.

Ethics declarations
Competing Interests: The authors declare no competing interests.
Funding: The authors received no specific funding for this work.
Notes: The authors declare no competing financial interest.

Acknowledgments
The authors are grateful to for providing tremendous facilities and support to carry out the work.

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Tables

Table 1 summarizes the various studies involving organoid models derived from various
organs for studying COVID -19

Type of Source of Cultivatio Insights generated from the study References

Organoid organoid culture n
material /
scaffold
Telencephal Human embryonic Matrigel 1) Cells of the choroid plexus have [17]
ic cerebral stem cells higher susceptibility towards viral
organoids infection
 2) Destruction of epithelial barrier of
the choroid plexus
Mouse and Mouse and Matrigel 1)Viral replication took place in the [18]
Human Human alveolar alveospheres
Alveospher epithelial cells 2)Transcriptomics reveals the
es type 2 expression of interferon pathways in
alveospheres infected with SARS-
CoV-2
3)Loss or decrease in surfactant
protein expression in the infected
alveospheres
4)Prevalence of apoptotic cells in the
infected alveospheres
5)Similar in expression of genes like
those expressing chemokines,
Interferon targets, Cell death
pathway genes between the
alveosphere and samples taken from
lungs of patients with COVID-19
6)Pretreatment with interferon alpha
and interferon gamma led to
significant decrease in the viral load
in the alveospheres

Human Human Matrigel 1) A significant population of cells [19]

Hepatocyte pluripotent stem from both the hepatocyte and
organoids cells cholangiocyte organoid cultures
were infected by the SARS-CoV-2

Human Human Matrigel

2) Both the organoids had up
Cholangioc pluripotent stem
regulation of genes from cytokine-
yte cells
cytokine receptor interaction and IL-
organoids
17 signaling pathways

Human Human adult Matrigel 1) There was positive expression of [20]

Bronchial bronchial spike protein along the outer edge of
organoids epithelial cells the bronchial organoids
2) Infection and replication in basal
cells of the organoid buty not in
ciliated cells and club cells
Tonsil Human tonsil Matrigel 1) SARS-CoV-2 infects tonsil [21]
organoids tissue organoids and robust replication
takes place in these organoids

Human Human embryonic Matrigel 1)Both the types of organoids were [22]
airway stem cells infected with SARS-CoV-2. Virion
organoids particles were observed to infect the
Human Human embryonic Matrigel apical, lateral and basolateral sides
alveolar stem cells
organoids 2)Human alveolar type 2 cells are
the most commonly infected cells in
human alveolar

3)Human alveolar organoids had

more cell death when compared to
human airway organoids

4)Up-regulation of cytokine and

interferon pathway genes in both the
organoids

5)Angiotensin converting enzyme 2

downregulation took place in the
organoids

Cortical Human induced Matrigel 1)Most of the cells that were [23]
organoid pluripotent stem infected in the organoids were
cells neurons. Some of the Glial fibrillary
Midbrain Human induced Matrigel acidic protein positive (GFAP+)
organoid pluripotent stem astrocytes were infected
 cells
Hippocamp Human induced Matrigel 2)SARS-CoV-2 infection takes place
al organoids pluripotent stem in choroid plexus epithelium
cells
Hypothala Human induced Matrigel
mic pluripotent stem
organoid cells
Choroid Human induced Matrigel
plexus pluripotent stem
organoids cells
Bat enteroid Stem cells of gut Matrigel 1)Predominantly enterocytes were
crypts taken from infected in the human enteroids [24]
Rhinolophus 2)Interferon lambda 2 and Interferon
sinicus lamba 3 were highly expressed in
the human enteroids
Human Small and large Matrigel 3) In the human organoids,
enteroid intestinal mucosa expression of some genes were
upregulated like CCR1, CCR8,
IL16, IL3 and CXCL10 (IP10). The
expression of genes CCR2, CCR5
and IL5 were downregulated

Dorsal Human embryonic Matrigel The Pseudo SARS-CoV-2 affects [25]

Forebrain stem cells neuronal cells expressing ACE2
organoids
Kidney Human embryonic None 1)Viral infection is obseved in the [26]
organoids stem cells organoid and it is observed to
Blood Human induced Matrigel:C progressively increase from day 3 –
capillary pluripotent stem ollagen day 5
organoids cells (1:1) 2)Viral infection takes place in
ACE2 producing cells in the kidney
organoid
3)Administration of soluble
recombinant human ACE2 results in
inhibition of infection in both the
 blood capillary organoids and
kidney organoids. No significant
cytotoxicity is observed in both the
organoids .

Cerebral Induced Matrigel 1)Neurons, Radial glia and neural [27]

organoids pluripotent stem progenitor cells were infected in the
cells organoids. Neuronal cells were the
most prevalently type of infected
cells
3)Increased death of cells near to
infected cells
4)Genes from different pathways are
regulated in SARS-CoV-2 infected
organoids when compared to
transcriptome of zika virus infected
organoids
5)Significant inhibition of SARS-
CoV-2 infection in brain organoids
when treated with ACE2 antibodies
show that ACE2 promotes infection
in brain organoids

Distal lung Distal airway cells Extracellul 1)SARS-CoV-2 readily infects both [28]
organoids ar matrix basal organoids and “apical out”
organoids
2)Club cells were infected by the
SARS-CoV-2 virus

Human Primary bile ducts Matrigel 1)Viral infection was observed in [29]
cholangiocy multiple places in the organoids
te organoids 2)Expression of claudin 1 was
disrupted suggesting that the
infection may disrupt the integrity of
the cholangiocyte barrier.
 Expression of two bile acid
transporter genes were highly
downregulated in infected cells

Human Human alveolar Matrigel 1)The human alveolar organoids had [30]
alveolar type 2 cells greater than 100 times the viral titre
organoids of human bronchial organoids
2)Human alveolar organoids
Human Human alveolar Matrigel infected with the virus was observed
Bronchial type 2 cells to have several pathological changes
organoids including
2a)Alveolar cells with enormous
vacuoles
2b)Double membrane vesicles
present near the zippered
endoplasmic reticulum
2c) Viral particles were well spread
out in the cytoplasm or were inside
vesicular structures
2d) Various types of secretions by
the virus on the apical surface of the
organoid
3)A lot of significant changes were
found in the infected organoids
when compared to the uninfected
controls and they include
3a) Downregulation in the
expression of keratinization,
cytoskeleton and cell-cell adhesion
genes.
3b)Upregulation of genes related to
interferons and innate cellular
immunity
The transcriptional changes were
more significantly observed in
 human alveolar organoids than in
human bronchial organoids

SEAM Eye Human embryonic Matrigel 1)Eye organoids are infected by [31]
organoids stem cells SARS-CoV-2
2)The limbus in the ocular organoids
are most vulnerable to infection.
3)Inflammatory response was
elicited in the eye organoids

Alveolar Distal lung Matrigel 1)Proximal airway epithelial cells [32]

organoids epithelial cells + were infected by the virus
MRC5 human 2)Ciliated cells were observed to
lung fibroblast have a higher chance of getting
cells infected. Some of the mucin Muc5ac
producing goblet cells were affected
3)The viral infection causes
structural changes in human AT2
epithelial cells
4)Cytokine associated genes were
upregulated. Especially genes in the
interferon associated pathway were
upregulated.

3D Brain Induced None 1)SARS-CoV-2 infects cortical [33]

organoids pluripotent stem neurons and neural progenitor cells
cells in brain organoids

Human Primary gut None 1)Infected ciliated cells of the [34]

small epithelial stem airway organoids
intestinal cells 2)Intestinal organoids were infected
organoids and the virus titre in the organoids
Human Human bronchial Matrigel didn’t drop till 60 hours after the
airway airway stem cells infection. Enterocytes were among
organoids the most infected lineage of cells
3)Cells of the intestinal organoids
show pathological changes like
disruption of cellular morphology,
formation of double membraned
vesicles and rounded nucleii,
4)Up-regulation of several genes in
multiple interferon associated
pathways

Colonic Colonic stem cells Matrigel 1)Enhanced expression of ACE2 in [35]

organoids the organoids from the hypertensive
from rats when compared to wistar rats
hypertensiv 2)Greater amount of enterocytes and
e rats and goblet cells were present in the
Wister organoids from the hypertensive rats
Kyoto rats when compared to wistar rats

Brain Induced Matrigel 1)Neuronal cells are amongst the [36]

organoids pluripotent stem most infected cells in the organoids
cells 2)Alteration of localization of tau
proteins took place in the infected
organoids. Somas of infected
neurons showed higher levels of tau
3)SARS-CoV-2 infection promoted
death of neuronal cells in the
organoid

Proximal Matrigel 1)The cells of the airway are [37]

and Distal responsible for sustaining viral
airway infection
organoids 2)Alveolar cells from the distal
region is important for studying the
host response
Table 2 summarizes some of the important upregulated and downregulated genes in the organoids
following SARS-CoV-2 infection

Organoid Method used Relevant upregulated genes Relevant References

Alveosphere RNA
s sequencing
(RNA seq)
in infected organoids downregulated genes
in infected organoids
1) FNA7, IFNB1, and IFNE 1)PCNA, TOP2A, [18]
2) IFNL1, IFNL2, and IFNL3 MCM2, CCNB2
IFNAR2 and IFNGR2
3) Several IFI’s and IFIT’s
4) CXCL10, CXCL11, and
CXCL17
5)TNFSF10, CASP1,
CASP4, CASP5, and CASP7
6)STAT1 and STAT2
7)canonical targets of IFN
gamma-response mediators

Human RNA seq 1) CXCL1, CXCL3, CXCL5, 1)CYP7A1, CYP2A6, [19]

hepatocyte CXCL6, CCL20 CYP1A2, and
organoids 2)Genes from cytokine- CYP2D6
cytokine receptor interaction
3)genes from IL- 17
signaling
4)genes from chemokine
signaling pathway
5) genes from TNF signaling
6 genes from NF-kB
signaling pathway

Human RNA seq 1)CXCL1, CXCL2, CXCL3,

cholangiocy CCL2
te organoids 2) genes from IL- 17
 signaling
3)genes from cytokine-
cytokine receptor interaction

Human RNA seq 1)Genes involved in positive [20]

bronchial regulation of immune
organoids effector process
2) genes involved in
regulation of inflammatory
response
3)genes involved in
interferon-gamma production
4)genes involved in positive
regulation of acute
inflammatory response

Tonsil Microarray OAS1, IL-6, CXCL8, FOS, [21]

organoids IL21R and BMP6

Human RNA interleukin (IL)-6, tumor ACE2 [22]

airway sequencing necrosis factor (TNF),
organoids CXCL8, CXCL2, CXCL3,
CXCL10, CXCL11, NF-kB
Human related mRNA NFKB1,
alveolar NFKB2, RELB, ATF3,
organoids GEM, IFITM3, MX1

Brain RNA CCL7, interleukin-32 (IL- AQP1, AQP4, and [23]

organoids sequencing 32), CCL2 (MCP1), IL-18, SLC22A8
IL-8 NPPB, VCAN

Human Real time IFNL2, IFNL3, CCR1, CCR2, CCR5, IL5 [24]
Intestinal PCR(qPCR) CCR8, IL16, IL3 and
Organoids CXCL10
Cholangiocy qPCR Claudin1, ASBT, [29]
te organoids CFTR

SEAM eye RNA 1)NFΚBIA, NFKBIZ and [31]

organoids sequencing CXCL1
2) genes involved in cell-
cycle and in inflammation
and response to viral
infection

Alveolar 1)IFNB1, OAS1, OAS2, 1)genes from Th1 and [32]

organoids ISG15 and MX1 Th2 activation
2)genes from Interferon pathways
(IFN) signaling, NF-kB
activation, TLR signaling and
IL1 pathways

Table 3 summarizes the various studies involving organ-on-chip models derived from various
organs for studying COVID -19
Type of organ- Brief description of the system Objective of the References
on-chip system

Lung-on-chip Consists of three main parts 1)Understanding [66]

a)Upper alveolar epithelial channel lung injury induced
b)Porous polydimethylsiloxane by SARS-CoV-2
 (PDMS) membrane and immune
c)Lower pulmonary microvascular responses generated
endothelial channel in response to it
The porous membrane contains the
alveolar-capillary interface with 2)Screening of drugs
alveolar epithelial cells (HPAEpiC)
present towards the upper epithelial
channel and the pulmonary
microvascular endothelial cells
(HULEC-5a) present towards the
lower pulmonary microvascular
endothelial channel. Immune cells are
supplied to the bottom endothelial
channel during the course of infection.
SARS-CoV-2 is introduced into the
upper epithelial layer.

Human-airway- The device consists of two main 1)Studying immune [67]

chip channels separated by a porous response and
membrane that is made up of PDMS. pathogenesis of
Airway epithelial cells are cultured on virus
the top layer of the membrane whereas
endothelial cells were cultured at the 2) Screening of
bottom of the membrane. Immune cells drugs
resuspended in media can be
introduced through the lower vascular
channel. The upper channel can be
used

Human-intestine- The device consists of three main parts 1)Analysis of [68]

on-chip a)Upper intestinal epithelial channel changes to the
b)Extracellular matrix coated PDMS various types of
membrane cells brought about
c)Lower microvascular endothelial by covid-19
channel
 Intestinal epithelial cells and intestinal infections
mucin secreting cells were co-cultured 2)Characterization
towards the upper channel on the of immune
membrane. Vascular endothelial cells responses to the
were cultured towards the lower infection
channel on the membrane. Immune 3)Screening of drugs
cells are introduced from the bottom
channel

Human lung-on- This device consists of three main 1) Designing a [69]

chip parts device that can be
a)Upper epithelial channel used for studying
b)Porous membrane SARS-CoV-2
c)Lowe endothelial channel pathogenesis
d)side vaccum channels 2)Screening and
This microfluidic device consisted of repurposing of drugs
an upper channel where air can be
intoduced. The epithelial cells are
cultured on the PDMS porous
membrane and are towards the upper
layer. A layer of endothelial cells are
grown on the membrane and face the
lower layer of the device. Side vaccum
channels are introduced to mimic the
contraction and relaxation of the lung.

Human-lung-on- It consists of a porous membrane and 1)Analysis of [70]

chip two channels, one at the top and one at SARS-CoV-2
the bottom. Epithelial cells are cultured infections
on the top of the membrane whereas
endothelial cells are cultured at the 2)Screening and
bottom. CD14+ macrophages are studying effects of
added to the epithelial layer drugs

Human alveolus- This organ-on-chip model consists of 1)Analysis of barrier [71]

on-chip epithelial cells and macrophages integrity following
cocultured inside the top channel on a SARS-CoV-2
porous membrane. The human infections
vascular endothelial cells are grown
towards the bottom channel. The
circulating immune cells are
introduced from the bottom channel

Table 4 lists about the various drugs that have been screened using organ-on-chip and organoid
models

Drug of interest/ Screened Organoid Important discoveries from Referenc

library of compounds /Organ-on-chip this study es
culure used
Enzalutamide Lung organoids 1)The drug could not prevent [35]
infection in lung organoids
Various compounds that 1)Significant decrease in viral [67]
have been approved by the Human airway on entry when amodiaquine,
FDA and are known to have a chip toremiphene or clomiphene
a broad spectrum antiviral are administered. No
activity(amodiaquine, significant cytotoxicity was
toremifene, clomiphene, observed on administration
chloroquine, 2)Amodiaquine was the most
hydroxychloroquine, effective drug in inhibiting
arbidol, verapamil and viral infections. Rapid
amiodarone) transformation of this drug
into its active metabolite form
desethylamodiaquine took
place. It is hence suggested
that metabolites derived from
amodiaquine can serve as
potential drugs against the
viral infection.
Remdesivir Lung-on-chip 1) Prevention of viral [66]
replication and disruption of
integrity of the alveolar
barrier
Camostat Human Bronchial 1)Administration of Camostat [20]
organoids resulted in reduction of viral
load
Remdesisvir Tonsil organoids 1) Administration of [21]
Remdesisvir led to decrease in
expression of viral genes
Betastatin, Remdesisvir, Human alveolar 1)Remedesivir inhibited viral [22]
Camostat organoids and replication in both the
Human airway organoids whereas camostat
organoids showed a slight a slight
activity in stopping viral
replication in only human
airway organoids
Hydroxychloroquine and Human alveolar 1)Remedisivir showed a high [32]
Remedisivir organoids activity in the reduction of
viral infection in the
organoids. A lower and
variable antiviral activity was
observed when
hydroxychloroquine was
administered to the organoids
Library of FDA-approved Lung organoids Imatinib, quinacrine [77]
drugs (the Prestwick dihydrochloride and
collection) mycophenolic acid led to
significant decrease in viral
load in the organoids
Remdesivir and famotidine Intestinal 1)Remedisivir significantly [78]
organoids reduces the infection in the
intestinal organoids.
2)Famotidine doesnt reduce
 infection in the organoids

Figures

Graphical Abstract
Figure 1. A brief illustration of the organs affected by the SARS-CoV-2. The virus has been
reported to induce damage in multiple organs including lungs, brain, eyes, heart, kidney, liver, and
intestines. These organs can be systematic targets for studying SARS-CoV-2 pathogenesis. It has
been observed that cells which have high expressions of ACE2 and TMPRSS2 are infected by this
virus. The viral S1 spike protein has been observed to bind to ACE2. A better understanding of the
damage induced by the coronavirus on each of the organs can help tailor target specific drugs.
Figure 2:SARS-CoV-2 infection of alveospheres and lung organoids. 2a) SARS-CoV-2 virus
infections in the cells were visualised using wide field microscopy(I). The The cells of the
alveospheres were observed to produce both ACE2(green) and TMPRSS2(red)(A).
2b)Alveospheres infected with low and high concentrations of viruses were immunostained 72hours
after the infection(C). Quantification of the infected alveospheres were performed(B,E). Caspase
3(red), a cell death marker and Ki67(gray), a proliferation marker were immunostained in the
SARS-CoV-2 positive alveospheres(green)(F). The immunostained cells for the cell death marker
and the proliferation marker were quantified(G and H)(adapted from [18] with permission from
Elsevier).2c) Transmission microscopy images of the infected organoids. Viral aggregates and large
vacuoules(Vc) were observed(A). Blue arrows indicate the lamellar bodies(B).Red arrows indicate
the virion particles that are found to be spread out throughout the cytoplasm(C). Red arrows show
the a plethora of virion encapsulated in vesicular structures(D,G,H).Lumen containing virion
particles which were secreted out (E,J). Vesicles with double membranes observed in the vicinity of
the zippered endoplasmic reticulum(adapted from [30] under CC-BY-4.0 license).
Figure 3: Illustration of infection, damage to barrier integrity and mislocalisation of tau protein
induced by SARS-CoV-2 in brain organoids 3a)Confocal images of infection of cerebral organoids
with SARS-CoV-2. 3b) Infection of cells of choroid plexus epithelium by the SARS-CoV-2
pseudovirus.3c) Immunostaining shows disruption in Claudin 5(F).There was a significant decrease
in the fluid inside the organoids indicating damage of cellular barrier(G) High expression of
neuronal fluid protein marker APOJ in the medium for damaged organoids(I)(adapted from [17]
under CC-BY-4.0 license).3d)Tau protein is localised in the cortical neuron’s axons(A).Tau is
mislocalized to the somas in SARS-CoV-2 infected cortical neurons(B). Immunostaining with
antibody AT180 showed threonine 231 phosphorylated tau proteins(D).3e) Control organoids donot
show cell death(A) Tunnel postive cells(magenta) in the SARS-CoV-2 infected organoids(B). Some
of SARS-CoV-2 positive cells with mislocalized tau proteins are Tunel positive whereas some stain
for caspase-3 marker(yellow)(C)(adapted from [36] under CC-BY-4.0 license)
Figure 4:Infection of intestinal organoids by SARS-CoV-2. 4a) Illustration of SARS-CoV-2
infection of cells that are proliferating and enterocytes in human intestinal organoids.
Immunofluorescent staining revealed virus infected cells in these organoids(A) Apolipoprotein A1
positive cells(red) and KI67 positive cells(red) are infected by the SARA-CoV-2(B)(adapted from
[34] under CC-BY-4.0 license).4b) Increase in caspase 3(green) positive cells was observed in
SARA-CoV-2 infected human intestinal organoids(D,E)(adapted from [34] under CC-BY-4.0
license)
Figure 5: Infection of cholangiocyte and hepatocyte organoids by SARS-CoV-2. Viral mRNA
quantification(A), confocal microscopy images(B) and percentage of SARS-CoV-2 positive cells
(C) in hepatocytes. Viral mRNA quantification(D), confocal microscopy images(E) and percentage
of SARS-CoV-2 positive cells (F) in human cholangiocytes. Gene expression and transcriptome
profile of infected organoids (G,H,I,J,K,L,M,N)(Adapted from [19] with permission from Elsevier)
Figure 6: Illustration of SARS-CoV-2 infection kidney and vascular organoids. 6a) SARS-CoV-2
infection of kidney organoids. Light microscopy and confocal image of kidney
organoid(A).Recovery and quantfication of viral mRNA from organoid(B).Progeny virus can infect
VeroE6 cells(C).Human recombinant ACE2 inhibits infection in kidney organoid(D).6b) SARS-
CoV-2 infection of vascular organoids. Light microscopy and confocal image of vascular
organoid(A).Progeny virus infects Vero E6 cells(B). Recovery and quantification of viral mRNA at
3rd and 6th day of infection when infection is introduced at different concentrations(C). Human
recombinant ACE2 inhibits infection in vascular organoids(D)(Adapted from [26] with permission
from Elsevier).
Figure 7: A short overview of the timeline for the utilisation of organoids for research. The
progenitor cells, stem cells or induced pluripotent stem cells are initially taken from previously
established cultures or are isolated from tissues. The pathways in the pluripotent cells are the
activated, followed by embedding in matrices and differentiation whereas adult or stem cells can
be directly embedded and placed in differentiation medium. The organoids are then infected with
the SARS-CoV-2 virus or pseudo-virus. Various experiments like immunocytochemistry,
transmission electron microscopy, RNA sequencing etc. are performed to understand several
aspects of the viral infection
Figure 8: A schematic representation of the organ on chip models devised for SARS-CoV-2
infections. The device consists of three layers i) Apical channel, ii) Porous membrane and iii)
vascular channel. SARS-CoV-2 diluted in liquid media is introduced to the apical channel for
infection. After the virus is allowed to infect the cells towards the apical layer are washed with
PBS and the excess fluid is removed so as to restore the air-liquid interface. Circulating immune
cells and fluids are introduced through the bottom vascular channel.
Figure 9: Illustration of SARS-CoV-2 infection of alveolus-on-chip models in the presence and
absence of circulating immune cells.9a)Infection and replication on SARS-CoV-2 infected
alveolus-on-chip. Image of alveolar epithelium(E-cadherin) and pulmonary vascular
endothelium(VE-cadherin) obtained by confocal microscopy(A).Side view of the cellular setup(B).
Front and side views of alveolar capillary barrier image after 3 rd day of infection obtained by
confocal microscopy(C,D).SARS-CoV-2 infection of alveolar epithelium and pulmonary vascular
endothelium(E).Confluency of both the cell types with and without SARS-CoV-2 infection.9b)
Responses by both the type of cells following introduction of circulating immune cells. Infection of
the epithelium and endothelium layers are visualised by confocal microscopy both in the presence
and absence of circulating immune cells(Peripheral Blood Mononuclear cells(PBMC))
(A).Quantification of the confluency of the cellular layers in the absence and presence of circulating
immune cells(B,C).Images obtained using confocal microscopy shows the recruitment and adhesion
of PBMCs to the endothelial layer(D). CD14+ cells are also recruited and attached to the
endothelial layer(E). Quantification of various inflammatory cytokines including IL‐1β, IL‐6, IL‐8,
and TNF‐α from the culture medium in the vascular channel(F-I)(adapted from [66] under CC-BY-
4.0 license)..

Figure 10: Effect of remedisivir and famotidine on intestinal organoids. 10a)SARS-CoV-2 infected
human intestinal organoids treated with remdesivir(C). Remdesivir treated SARS-CoV-2 infected
human intestinal organoids showed a marked decrease in viral infection(D). IC50 value of
remdesivir was 3222 nM(F). 125 micromolar concentration of the drug was found to retain upto
more than 80% of cell viability.10b). Famotidine doesnot inhibit viral replication in intestinal
organoids(C,D,E,F) (adapted from [34] under CC-BY-4.0 license)